EIGHTH

EDITION FOOD CHEMICALS CODEX

FCC 8 By authority of the United States Pharmacopeial Convention.

Prepared by the Council of Experts and published by the
Board of Trustees

THE UNITED STATES PHARMACOPEIAL CONVENTION

12601 Twinbrook Parkway, Rockville, MD 20852
 / 1

NOTICE AND WARNING

Compliance with Federal Statues and Other Laws

The fact that an article appears in the Food Chemicals Codex or its supplements does not exempt it from compliance with
requirements of acts of Congress, with regulations and rulings issued by agencies of the United States Government under
authority of these acts, or with requirements and regulations of governments in other countries as relevant.

Concerning U.S. Patent or Trademark Rights

The inclusion in Food Chemical Codex of a monograph on any article in respect to which patent or trademark rights may exist
shall not be deemed, and is not intended as, a grant of, or authority to exercise, any right or privilege protected by such
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exercise the same without express permission, authority, or license secured from such patent or trademark owner.

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Attention is called to the fact that FCC text is fully copyrighted. Authors and others wishing to use portions of the text should
request permission to do so from the Legal Department of the United States Pharmacopeial Convention.

Copyright © 2012 The United States Pharmacopeial Convention

12601 Twinbrook Parkway, Rockville, MD 20852

All rights reserved.

ISBN 978-1-936424-05-4
ISSN 2153-1412 (print)
ISSN 2153-1455 (online)

Printed in the United States by United Book Press, Inc., Baltimore, MD

FCC 8
Preface
FCC 8
This section provides general information about the Eighth
Edition of the Food Chemicals Codex (FCC) and background
information on the United States Pharmacopeial Convention
(USP). Additional information about the specific uses of this
compendium is provided in the General Provisions and
Requirements section (page 1).
MISSION
FCC is published in continuing pursuit of the mission of USP:
To improve the health of people around the world through
public standards and related programs that help ensure the
quality, safety, and benefit of medicines and foods.
HISTORY
FCC began after the passage of the 1958 Food Additives
Amendment to the United States (U.S.) Federal Food, Drug,
and Cosmetic Act. Although the U.S. Food and Drug
Administration (FDA) had, by regulations and informal
statements, defined in general terms the quality
requirements for food additives, food colors, substances
generally recognized as safe for use in foods (GRAS) and
other food chemicals in the US market prior to 1958 (prior-
sanctioned articles), these requirements were not sufficiently
specific to serve as release, procurement, and acceptance
specifications for manufacturers and users of food chemicals.
Therefore, regulators, industry and other interested parties
recognized the need for a compendium of standards
designed especially for food chemicals, comparable to the
United States Pharmacopeia for drugs and the National
Formulary for excipients, which would define the quality of
food-grade chemicals in terms of identity, strength, and
purity. The National Academy of Sciences (NAS) was
requested to develop this compendium and published the
first edition of the FCC in 1966. Subsequent editions were
published by the NAS in 1972, 1981, 1996, and 2003,
through the Food and Nutrition Board of the Institute of
Medicine (IOM), which formed a Committee on Food
Chemicals Codex to elaborate the FCC.
The scope of FCC has expanded with each new edition.
Substances included in the first edition were limited to
chemicals added directly to foods to achieve a desired
function. Subsequent editions added: (a) processing aids
such as enzymes, extraction solvents, filter media, and boiler
water additives; (b) foods, such as fructose and dextrose;
and (c) functional ingredients that affect not the foods to
which they are added, but the human body when the food
is consumed. Over the years, FCC has become a
comprehensive compendium of standards for these articles,
collectively termed food ingredients. The introduction of
new food ingredients as well as constant changes in
manufacturing processes and advances in analytical and
metrological sciences lead to a need for continuous revision
of the FCC. Because of its regulatory status in countries
other than the United States, and its worldwide use, the FCC
Preface / v
Front Matter
contains monographs for ingredients that may not be
currently marketed in the United States.
USP acquired FCC from the NAS in 2006 and assumed
responsibility for its ongoing development and publication.
To continue the work of the Food and Nutrition Board of
IOM, USP formed a Food Ingredients Expert Committee
within its Council of Experts. This Expert Committee is
responsible for approving all new and revised standards in
FCC.
FCC 8
The Eighth Edition of FCC (FCC 8) includes more than
1,100 monographs. It also contains more than 150 General
Tests and Assays, providing procedures frequently cited in
monographs, sometimes with acceptance criteria, in order
to avoid repetition of this text. Additionally, FCC 8 offers a
chapter with up-to-date relevant informational materials on
method validation and various analytical techniques,
reference tables and information on current Good
Manufacturing Practices. Additions, deletions, and other
revisions of text from the FCC Seventh Edition are indicated
on page xix in the Admissions section. The FCC and its
Supplements become effective 90 days from the official date
of publication, unless otherwise noted.
Monograph Elements
Each FCC 8 monograph represents the documentary stan-
dard for an article, manifested by specifications that speak
to the quality and safety of the food ingredient. Each mono-
graph includes, when available, the following: empirical
formula, structural formula, and formula weight; description
of the substance, including physical form, odor (flavoring
agents only), and solubility (see the descriptive terms for
solubility in the General Provisions and Requirements section);
function; packaging and storage; labeling; identification; as-
say (or a quantitative test to serve as an assay); impurities
(inorganic and organic); specific tests; and other require-
ments. The specifications provided, taken together, repre-
sent a compositional understanding of the substance.
PUBLICATION OF FCC REVISIONS
FCC revisions are published biennially in new editions, in
Supplements published in intervening years and, when
circumstances warrant, as Expedited Standards or Immediate
Standards.
Supplements
The First Supplement to FCC 8 will be published in Septem-
ber 2012 and will become effective 90 days from the official
date of publication, unless otherwise noted. The Index in
each Supplement is cumulative and includes citations to the
biennial revision. The contents of the Supplement are inte-
grated into the following edition of FCC, along with new
revisions that have been adopted since the Supplement to
the previous compendium.
 vi / Preface
Front Matter
Expedited Standards
Expedited Standards are revisions that the Food Ingredients
Expert Committee determines, for public health or other
reasons, should become effective prior to publication of the
next edition of the FCC or Supplement. Proposed expedited
standards are posted on the FCC Forum website for a com-
ment period of 90 days. If there are no significant com-
ments, they become effective on the date posted on the
USP website, unless otherwise noted. These revisions will be
incorporated into the next published edition of the FCC or
Supplement.
Immediate Standards
Immediate Standards are revisions that the Food Ingredients
Expert Committee determines should be made available im-
mediately because of an urgent public health need. These
standards are posted as final on the USP website without
prior public notice and comment and are effective upon
website publication unless a delayed effective date is speci-
fied. These standards will be incorporated into the next
published edition of the FCC or Supplement.
Errata
Errata are text published in the FCC or its Supplements that
do not accurately reflect the intended standards as ap-
proved by the Food Ingredients Expert Committee. A list of
errata and corresponding corrections to an edition of the
FCC or to a Supplement are published on USP‘s website, and
incorporated into the next published edition of the FCC or
Supplement. Errata shall not be subject to public notice and
comment.
Print and Electronic Presentations
The FCC and its Supplements are available in print form and
in an Internet version that allows individual registered users
to access the FCC online. The Internet format provides ac-
cess to FCC content, along with extensive search options. It
is continuously and cumulatively updated to integrate the
content of Supplements. For users of the print edition, the
Supplements are included with the purchase of the FCC.
Users of the FCC print edition must retain the Supplements
and review the FCC portion of the USP website in order to
have up-to-date information.
Symbols
Indicating change to effective text, symbols identify the be-
ginning and end of each revision. The following table sum-
marizes the types of symbols and the associated subscripts
used in FCC publications:
Revision Type Symbol Subscript
Text Deletion Adopted as Effective Date
an Expedited or Immedi- ••
ate Standard
Text Deletion Adopted in 1S, 2S, 3S (FCC biennial
 
a Supplement edition)
Text Deletion Adopted in FCC biennial edition
 
FCC
New Text Adopted as an Effective Date
Expedited or Immediate •new text•
Standard
FCC 8
Revision Type Symbol Subscript
New Text Adopted in a 1S, 2S, 3S (FCC biennial
 new text
Supplement edition)
New Text Adopted in FCC  new text FCC biennial edition
The following table shows symbols and effective dates for
FCC 8 and its Supplements:
Supplement Effective Date Symbols
FCC 8 June 1, 2012  and FCC8
1 December 1, 2012  and1S(FCC8)
2 June 1, 2013  and2S(FCC8)
3 December 1, 2013  and3S(FCC8)
FCC REVISION PROCESS
The FCC is revised on an ongoing basis in accordance with
USP Policies and Rules and Procedures. Users of the FCC are
requested and encouraged to submit suggestions for
updating and improving the specifications and general
analytical methods, and to review and comment upon
proposed revisions through the processes discussed below.
Food Ingredients Expert Committee
The Food Ingredient Expert Committee (FIEC) is part of
USP‘s Council of Experts and is the scientific decision-
making body for the FCC. Its principal functions include the
following:
• To propose means by which FCC standards may be kept
current in reflecting food-grade quality on the basis of
ingredient safety, good manufacturing practices, and
advances in analytical capabilities.
• To provide information on issues relating to standards for
particular substances and analytical test procedures.
• To recommend the establishment of Expert Panels
consisting of a committee member and other experts or
specialists to address specific issues relevant to
monograph development and to report their findings and
advisory recommendations to the full committee.
• To evaluate comments submitted by interested parties on
any aspect of proposed FCC standards.
• To approve final standards before their publication in the
FCC or its Supplements.
• To consider and act on any other issues concerning the
development and publication of standards for new and
existing food-grade ingredients.
The FIEC meets regularly to discuss food ingredients topics,
including technical and policy issues relevant to the FCC.
Public Participation in FCC Revisions
Although the FIEC is the ultimate decision-making body for
FCC standards, these standards are developed by an excep-
tional process of public involvement and substantial interac-
tion between USP and its stakeholders, both domestically
and internationally. Participation in the revision process re-
sults from the support of many individuals and groups and
also from scientific, technical, and trade organizations.
FCC 8 Preface / vii

Front Matter
Figure 1. Public Review Process

Requests for revision of monographs, either new opportunity to comment on the development and revision
monographs or those needing updating, contain informa- of FCC standards. All proposals will have a 90-day comment
tion submitted voluntarily by manufacturers and other inter- period. Figure 1 shows the public review and comment pro-
ested parties. At times, USP staff may develop information cess and its relationship to standards development.
to support a monograph through a Request for Revision. USP
has developed a document titled Guideline for Submitting Re-
Working with Government Agencies
quests for Revision to FCC, which is available at www.usp.org.
USP works in many ways with government agencies in the
To facilitate the continuous revision of FCC and ensure an
United States and abroad, including the FDA, to promote
open, transparent, and participatory revision process, USP
good communications and optimal interactions. The USP
solicits and encourages public comment on FCC
Government Liaison Program allows government representa-
monographs, General Tests and Assays, and other draft doc-
tives to participate in FIEC meetings, enabling continuing
uments via the FCC Forum. The Forum is available free of
interactions between the regulators‘ scientific staff and Ex-
charge. For more information, visit www.usp.org/fcc.
pert Committee activities. Staff in the FDA Centers, who are
Comments received are considered by the FIEC, who de-
responsible for review of USP compendial activities, provide
termine whether changes should be made to the proposed
specific links and opportunities for exchange of comments.
revisions based on those comments. Proposed standards are
The Center for Food Safety and Applied Nutrition is the
finalized when the FIEC votes to make them effective text in
center that links FDA and USP in the areas of food ingredi-
FCC. Thus, the USP standards-setting process gives those
ents and FCC.
who manufacture, regulate, and use food ingredients the
 viii / Preface
Front Matter
LEGAL RECOGNITION OF FCC
STANDARDS
The FCC has earned international recognition by
manufacturers, vendors, and users of food chemicals. FCC
standards serve as the basis for many buyer and seller
contractual agreements.
In the United States, the first edition of FCC was given
quasi-legal recognition in July 1966 by means of a letter of
endorsement from FDA Commissioner James L. Goddard,
which was reprinted in the book. The letter stated that “the
FDA will regard the specifications in the Food Chemicals
Codex as defining an ‘appropriate food grade’ within the
meaning of Sec. 121.101(b)(3) and Sec. 121.1000(a)(2) of
the food additive regulations, subject to the following
qualification: this endorsement is not construed to exempt
any food chemical appearing in the Food Chemicals Codex
from compliance with requirements of Acts of Congress or
with regulations and rulings issued by the Food and Drug
Administration under authority of such Acts.”
Subsequently, various additional specifications from
previous FCC editions were also incorporated by reference in
the U.S. Code of Federal Regulations to define specific safe
ingredients under Title 21, in various parts of Sections 172,
173, and 184. It is anticipated that FDA will from time to
time continue to update its regulatory references to the FCC.
USP will work diligently to assure that the FCC contains
monographs for all substances added to foods in the United
States, including all ingredients that are marketed as food
additives and color additives under an FDA regulation
following a successful petition of FDA, ingredients that are
affirmed to be GRAS, and ingredients that are marketed
under approvals issued prior to the 1958 Food Additive
Amendments (prior-sanctioned items).
In Canada, in the absence of national specifications, the
Fourth edition of the FCC, as amended from time to time, is
officially recognized in the Canadian Food and Drug
Regulations under Section B.01.045(b) as the reference for
specifications for food additives.
For Australia and New Zealand, the Food Standards
Australia New Zealand recognizes the Seventh Edition of the
FCC as a primary source of identity and purity specifications
for substances added to food in Standard 1.3.4 Identity and
Purity of its Food Standards Code.
In Israel, the Public Health Regulations state that those
who produce, import, market, or store a food additive must
comply with the requirements established in the latest
edition of FCC or in the latest edition of the Compendium of
Food Additive Specifications published by the Joint FAO/WHO
Expert Committee on Food Additives (JECFA).
GENERAL INFORMATION REGARDING
USP
USP GOVERNANCE, STANDARDS-SETTING, AND ADVI-
SORY BODIES
USP’s governing, standards-setting, and advisory bodies in-
clude the USP Convention, the Board of Trustees, the Coun-
cil of Experts and its Expert Committees, Expert Panels (for-
merly known as Advisory Panels), and staff. Additional
volunteer bodies include Stakeholder Forums, Project Teams,
FCC 8
and Advisory Groups, which act in an advisory capacity to
provide input to USP’s governing, standards-setting, and
management bodies.
USP Convention
The composition of the USP Convention membership is
designed to ensure a global representation from all sec-
tors of health care, with an emphasis on practitioners,
given USP’s practitioner heritage (see the History section).
Voting Delegates of Convention member organizations
elect USP’s President, Treasurer, other members of the
Board of Trustees, and the Council of Experts. They also
adopt resolutions to guide USP’s strategic direction and
amend USP’s Bylaws. The 2010 meeting of the USP Con-
vention occurred in April 2010 in Washington, DC. A list-
ing of all current Voting Delegates of the USP Conven-
tion is included in the People section.
Board of Trustees
USP’s Board of Trustees is responsible for the manage-
ment of the business affairs, finances, and property of
USP. During its 5-year term, the Board defines USP’s stra-
tegic direction through its key policy and operational de-
cisions. A listing of the members of the 2010–2015 Board
of Trustees is included in the People section.
Council of Experts
The Council of Experts is the standards-setting body of
USP. For the 2010–2015 cycle it is composed of 21
members, elected to 5-year terms by USP’s Convention,
each of whom chairs an Expert Committee. These Chairs,
in turn, elect the members of their Expert Committees.
The Expert Committees are responsible for the content of
USP’s official and authorized publications (see Figure 2).
The Executive Committee of the Council of Experts in-
cludes all Expert Committee Chairs and provides overall
direction, is an appeals body, and performs other func-
tions that support the Council of Experts’ operations.
Expert Panels to the Council of Experts
The Chair of the Council of Experts may appoint Expert
Panels to assist the Council of Experts by providing advi-
sory recommendations to particular Expert Committees
in response to a specific charge consistent with the Ex-
pert Committee’s Work Plan. Expert Panels are continu-
ously formed; their topics and membership appear in the
People section.
Stakeholder Forums and Project Teams
USP may form several domestic and international Stake-
holder Forums and Project Teams during the 2010–2015
cycle, including the Food Ingredients and Dietary Supple-
ments Stakeholder Forums, to exchange information and
receive comments on USP’s standards-setting activities.
Depending on the topic, a Stakeholder Forum may create
Project Teams to work on selected topics. USP also holds
Standards and Science Symposia in various regions
throughout the world to promote scientific exchanges on
topics relating to USP compendia.
International Standards and Science Symposia
• North America
• India/West Asia
• China/East Asia
• Latin America
FCC 8
Figure 2. 2010–2015 USP Council of Experts
• Europe
• Middle East/North Africa
Staff
USP maintains a staff of over 700 scientists, professionals,
and administrative personnel at its Rockville, Maryland
headquarters and throughout the world, including an ac-
count management office in Basel, Switzerland, and labo-
ratory facilities in Hyderabad, India; Shanghai, China; and
São Paulo, Brazil.
USP POLICIES, RULES, AND PROCEDURES
Governing Documents
USP’s Articles of Incorporation, its Constitution and Bylaws,
and the Rules and Procedures of the 2010–2015 Council of
Experts are available on USP’s website (www.usp.org). Col-
lectively, these documents serve USP volunteers and staff as
the governing principles for USP’s standards-setting
activities.
Conflicts of Interest
USP’s Conflict of Interest provisions require all members of
the Council of Experts, its Expert Committees, Expert Panels,
Board of Trustees, and key staff to disclose financial or other
interests that may interfere with their duties as USP volun-
teers. Members of the Board of Trustees, Council of Experts,
and its Expert Committees are not allowed to take part in
the final discussion or vote on any matter in which they
Preface / ix
Front Matter
have a conflict of interest or there is the appearance of a
conflict of interest. Members of Expert Panels may partici-
pate and vote, so long as any conflicts have been ade-
quately and promptly disclosed and are communicated to
the relevant Expert Committee along with any Expert Panel
recommendations.
Confidentiality and Document Disclosure
Members of the Council of Experts, Expert Committees, and
Expert Panels sign confidentiality agreements, in keeping
with USP’s Confidentiality Policy and the confidentiality pro-
visions of the Rules and Procedures of the Council of Experts.
The USP Document Disclosure Policy, available on USP’s
website, contributes to the transparency of the standards-
setting process by making information available to the pub-
lic, yet provides protection to manufacturers and others
who submit confidential information to USP.
OTHER USP PUBLICATIONS
United States Pharmacopeia and the National For-
mulary— The United States Pharmacopeia (USP) and Na-
tional Formulary (NF) are compendia of science-based stan-
dards for drug and biologic dosage forms, drug substances,
excipients, medical devices, and dietary supplements. These
standards are set by Expert Committees following public no-
tice and opportunity for comment through publication in
 x / Preface
Front Matter
the free Pharmacopeial Forum. The USP and NF are recog-
nized as official compendia of the United States in the Fed-
eral Food, Drug, and Cosmetic Act, and also are recognized
in the laws of many countries around the world. The USP
and the NF are separate compendia although they are pub-
lished in the same volume.
Chromatographic Columns— This comprehensive refer-
ence, previously titled Chromatographic Reagents, provides
detailed information needed to conduct chromatographic
procedures found in USP–NF. Chromatographic Columns lists
the brand names of the column reagents cited in every pro-
posal for new or revised gas- or liquid-chromatographic ana-
lytical procedures that have been published in PF since
1980. Chromatographic Columns also helps to track which
column reagents were used to validate analytical procedures
that have become official. The branded column reagents list
is updated bimonthly and maintained on USP’s website.
USP Dictionary— The USP Dictionary of USAN and Inter-
national Drug Names provides, in a single volume, the most
up-to-date United States Adopted Names of drugs; official
USP–NF names; nonproprietary, brand, and chemical names;
graphic formulas; molecular formulas and weights; CAS reg-
istry numbers and code designations; drug manufacturers;
and pharmacologic and therapeutic categories. The Diction-
ary helps to ensure the accuracy of the following: product
labeling; reports, articles, and correspondence; FDA regula-
tory filings; and pharmaceutical package inserts. It is pub-
lished annually and is recognized by FDA as the official
source for established drug names. (See Nomenclature.)
USP Dietary Supplements Compendium— The Dietary
Supplements Compendium combines, in a single volume,
USP–NF standards for dietary supplements, standards and in-
formation from the Food Chemicals Codex, regulatory and
FCC 8
industry documents, and other tools and resources. It is
published every two years, as a hardcover print edition.
USP Medicines Compendium— The USP Medicines Com-
pendium (MC) includes monographs, general chapters, and
reference materials for suitable chemical and biological
medicines and their ingredients approved by national regu-
latory authorities. The purpose of the MC is to help ensure
that these medicines are of good quality by providing up-
to-date, relevant public standards and reference materials.
MC standards are available to manufacturers, purchasers, na-
tional regulatory authorities, and others to ensure conform-
ity of a medicine to MC standards through testing. The MC
does not include standards for foods or for traditional
medicines/dietary supplements.
USP Catalog— Use of official USP Reference Standards pro-
motes uniform quality of drugs, food ingredients, and dietary
supplements and supports first-, second-, and third-party test-
ing of all manufactured and compounded articles. The publi-
cation listing the collection of official USP Reference Standards
can be accessed on the USP website at www.usp.org
and is available in print form by contacting USP Sales and
Marketing staff at 301-816-8237. The listing identifies new
items, replacement lots, lots of a single item that are simul-
taneously official, lots deleted from official status, and a pre-
view of items eventually to be adopted. Purchase order in-
formation is included, and the names of distributors who
can facilitate international availability of these items are sug-
gested. The USP Reference Standards program benefits from
the widespread voluntary contribution of suitable materials
and test data from manufacturers. USP advances this unoffi-
cial material to official status via careful characterization
studies and collaborative testing, followed by review and ap-
proval by the appropriate Expert Committee.
FCC 8 Contents / iii

Contents
PREFACE .............................................................................................................................................................. v

PEOPLE ............................................................................................................................................................... xi

ADMISSIONS .................................................................................................................................................. xviii

ANNOTATED ..................................................................................................................................................... xix

GENERAL PROVISIONS AND REQUIREMENTS APPLYING TO SPECIFICATIONS,

TESTS, AND ASSAYS OF THE FOOD CHEMICALS CODEX .............................................................................. 1

MONOGRAPH SPECIFICATIONS........................................................................................................................ 9

PROVISIONAL MONOGRAPH SPECIFICATIONS......................................................................................... 1209

GENERAL TESTS AND ASSAYS.................................................................................................................... 1213

Appendix I: Apparatus for T est and Assays ................................................................................................. 1217
Appendix II: Physical T ests and Determinations .......................................................................................... 1221
A. Chromatograhy............................................................................................................................... 1221
B. Physicochemical Properties ............................................................................................................. 1230
C. Others ............................................................................................................................................ 1242
Appendix III: Chemical T ests and Determinations ...................................................................................... 1262
A. Identification Tests .......................................................................................................................... 1262
B. Limit Tests ....................................................................................................................................... 1264
C. Others ............................................................................................................................................ 1279
Appendix IV: Chewing Gum Base .............................................................................................................. 1298
Appendix V: Enzyme Assays ....................................................................................................................... 1303
Appendix VI: Essential Oils and Flavors ...................................................................................................... 1336
Appendix VII: Fats and Related Substances ................................................................................................ 1341
Appendix VIII: Oleoresins ........................................................................................................................... 1357
Appendix IX: Rosins and Related Substances .............................................................................................. 1360
Appendix X: Carbohydrates (Star ches, Sugars, and Related Substances) .................................................... 1364
Appendix XI: Flavor Chemicals (Other Than Essential Oils) ........................................................................ 1375
Appendix XII: Microbiological T ests............................................................................................................ 1381
Appendix XIII: Adulterants and Contaminants in Food Ingredients ............................................................ 1384
Appendix XIV: Markers for Authenticity T esting ......................................................................................... 1388

SOLUTIONS AND INDICATORS ................................................................................................................... 1393

GENERAL INFORMATION ............................................................................................................................ 1409

INDEX............................................................................................................................................................ 1613
 FCC 8
Monographs
. Acceptance criteria: 99.0%–101.0% of C4H4KNO4S, on
Acesulfame Potassium
First Published: Prior to FCC 6
Last Revision: FCC 7
Acesulfame K
6-Methyl-1,2,3-oxathiazine-4(3H)-one-2,2 Dioxide Potassium
Salt
C4H4KNO4S Formula wt 201.24
INS: 950 CAS: [55589-62-3]
UNII: 23OV73Q5G9 [acesulfame potassium]
DESCRIPTION
Acesulfame Potassium occurs as a white, free-flowing
crystalline powder. It is freely soluble in water and very
slightly soluble in ethanol.
Function: Non-nutritive sweetener; flavor enhancer
Packaging and Storage: Store in well-closed containers
in a cool, dry place.
IDENTIFICATION
• A. PROCEDURE
Sample solution: 0.3 g in 1 mL of glacial acetic acid
and 5 mL of water
Analysis: Add a few drops of sodium cobaltinitrite TS to
the Sample solution.
Acceptance criteria: A yellow precipitate forms.
• B. ULTRAVIOLET ABSORPTION
Sample solution: 0.01 mg/mL
Acceptance criteria: The Sample solution shows an
absorption maximum at 227 ± 2 nm.
• C. INFRARED ABSORPTION, Spectrophotometric Identification
Tests, Appendix IIIC
Reference standard: USP Acesulfame Potassium RS
Sample and standard preparation: K
Acceptance criteria: The spectrum of the sample
exhibits maxima at the same wavelengths as those in
the spectrum of the Reference standard.
ASSAY
• PROCEDURE
Sample: 200–300 mg, previously dried at 105° for 2 h
Analysis: Dissolve the Sample in 50 mL of glacial acetic
acid in a 250-mL flask. [NOTE—Dissolution may be
slow.] Add 2 or 3 drops of crystal violet TS, and titrate
with 0.1 N perchloric acid to a blue-green endpoint
that persists for at least 30 s. [CAUTION—Handle
perchloric acid in an appropriate fume hood.] Perform a
blank determination (see General Provisions), and make
any necessary correction. Each mL of 0.1 N perchloric
acid is equivalent to 20.12 mg of C4H4KNO4S.
Monographs / Acesulfame Potassium / 9
the dried basis
IMPURITIES
Inorganic Impurities
• FLUORIDE, Fluoride Limit Test, Method III, Appendix IIIB
Monographs
Sample: 4 g
Acceptance criteria: NMT 3 mg/kg
• LEAD, Lead Limit Test, Appendix IIIB
Sample solution: 2 g in 20 mL of water
Control: 2 µg Pb (2 mL of Diluted Standard Lead
Solution)
Acceptance criteria: NMT 1 mg/kg
Organic Impurities
• ORGANIC IMPURITIES
Mobile phase: Acetonitrile and 0.01 M tetrabutyl
ammonium hydrogen sulfate (40:60, v/v)
Standard: 4-hydroxybenzoic acid ethyl ester
Sample solution: 10 mg/mL
Dilute sample solution: 0.2 mg/L
Chromatographic system, Appendix IIA
Mode: High-performance liquid chromatography
Detector: UV or diode array (227 nm)
Column: 25-cm × 4.6-mm (id) stainless steel, or
equivalent, packed with 3- to 5-µm reversed phase
C18 silica gel, or equivalent
Flow rate: About 1 mL/min
Injection volume: 20 µL
Elution: Isocratic
System suitability
Suitability requirements: The resolution, R, between
acesulfame potassium and 4-hydroxybenzoic acid
ethyl ester is NLT 2.
Analysis: Inject the Sample solution into the
chromatograph and obtain the chromatogram. If peaks
other than that caused by acesulfame potassium
appear within three times the elution time of
acesulfame potassium, carry out a second analysis
using the Dilute sample solution.
Acceptance criteria: The sum of the areas of all peaks
eluted in the analysis of the Sample solution within
three times the elution time of acesulfame potassium,
except for the acesulfame potassium peak, does not
exceed the peak area of acesulfame potassium in the
analysis of the Dilute sample solution (NMT 20 µg/g of
UV-active compounds).
SPECIFIC TESTS
• LOSS ON DRYING, Appendix IIC: 105° for 2 h
Acceptance criteria: NMT 1.0%
• PH, pH Determination, Appendix IIB
Sample solution: 10 mg/mL
Acceptance criteria: Between 5.5 and 7.5
 10 / Acetaldehyde Diethyl Acetal / Monographs FCC 8

. Solubility in Alcohol, Appendix VI: One mL dissolves in 1

Acetaldehyde Diethyl Acetal mL of 95% ethanol.
First Published: Prior to FCC 6 Function: Flavoring agent

IDENTIFICATION
Acetal • INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.
Monographs

C6H14O2 Formula wt 118.17 ASSAY

FEMA: 2002 • PROCEDURE: Proceed as directed under M-1b, Appendix
UNII: 5G14F9E2HB [acetal] XI.
Acceptance criteria: NLT 97.0% of C6H14O2
DESCRIPTION
Acetaldehyde Diethyl Acetal occurs as a colorless to pale SPECIFIC TESTS
yellow liquid. • REFRACTIVE INDEX, Appendix II: At 20°
Odor: Ethereal, fruity Acceptance criteria: Between 1.379 and 1.384
Solubility: Soluble in propylene glycol, vegetable oils; • SPECIFIC GRAVITY: Determine at 25° by any reliable
slightly soluble in water method (see General Provisions).
Boiling Point: ∼102° Acceptance criteria: Between 0.821 and 0.827

Acetaldehyde Diethyl Acetal

. C2H4O Formula wt 44.05

Acetaldehyde FEMA: 2003
First Published: Prior to FCC 6 UNII: GO1N1ZPR3B [acetaldehyde]
Last Revision: First Supplement, FCC 6
DESCRIPTION
Acetaldehyde occurs as a flammable, colorless liquid. It may
Acetic Aldehyde contain a suitable antioxidant.
Ethanal Odor: Pungent, ethereal
Solubility: Miscible in alcohol, organic solvents, water
Boiling Point: ∼21°
Function: Flavoring agent
 FCC 8 Monographs / Acetanisole / 11

IDENTIFICATION SPECIFIC TESTS

• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.
ASSAY
• PROCEDURE: Proceed as directed under M-2b, Appendix
XI.
• ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
OILS), M-15, Appendix XI
Acceptance criteria: NMT 5.0
• SPECIFIC GRAVITY: Determine at 0° ± 0.05° by means of a
hydrometer calibrated to give the apparent specific
gravity at 0°/20° (see General Provisions).
Acceptance criteria: Between 0.804 and 0.811
OTHER REQUIREMENTS

Monographs
Acceptance criteria: NLT 99.0% of C2H4O • RESIDUE ON EVAPORATION, M-16, Appendix XI
Acceptance criteria: 0.006%

Acetaldehyde

. Boiling Point: ∼153° (26 mm Hg)

Acetanisole Solubility in Alcohol, Appendix VI: One g dissolves in 5
First Published: Prior to FCC 6 mL of 50% alcohol.
Function: Flavoring agent
4-Acetylanisole IDENTIFICATION
p-Methoxyacetophenone • INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

C9H10O2 Formula wt 150.18 ASSAY

FEMA: 2005
UNII: 0IRH2BR587 [4-acetylanisole]
DESCRIPTION
Acetanisole occurs as a colorless to pale yellow fused solid.
Odor: Hawthorn
Solubility: Soluble in most fixed oils, propylene glycol;
insoluble or practically insoluble in glycerin
• PROCEDURE: Proceed as directed under M-1b, Appendix
XI.
Acceptance criteria: NLT 98.0% of C9H10O2
OTHER REQUIREMENTS
• CHLORINATED COMPOUNDS, Appendix VI
Acceptance criteria: Passes test
 12 / Acetanisole / Monographs FCC 8

• LEAD, M-9, Appendix XI

Acceptance criteria: 10 mg/kg
Monographs

Acetanisole

. Analysis: Transfer the Sample into a tared, glass-

Acetic Acid, Glacial stoppered flask and weigh. Add 40 mL of water and
First Published: Prior to FCC 6 phenolphthalein TS and titrate with 1 N sodium
hydroxide. Each mL of 1 N sodium hydroxide is
equivalent to 60.05 mg of C2H4O2.
Acceptance criteria: NLT 99.5% and NMT 100.5%
C2H4O2 by weight

IMPURITIES
C2H4O2 Formula wt 60.05 Inorganic Impurities
INS: 260 • LEAD, Lead Limit Test, Atomic Absorption Spectrophotometric
FEMA: 2006 Graphite Furnace Method, Method I, Appendix IIIB
CAS: [64-19-7] Acceptance criteria: NMT 0.5 mg/kg
UNII: Q40Q9N063P [acetic acid]
SPECIFIC TESTS
DESCRIPTION • NONVOLATILE RESIDUE
Acetic Acid, Glacial, occurs as a clear, colorless liquid. It boils Sample: 19 mL (20 g)
at about 118°. When well-diluted with water (e.g., 1:100), Analysis: Evaporate the Sample in a tared dish on a
it has a vinegar odor and taste. It is miscible with water, steam bath and dry at 105° for 1 h.
with alcohol, and with glycerin. Acceptance criteria: NMT 0.005%
Function: Acidifier; flavoring agent • READILY OXIDIZABLE SUBSTANCES
Packaging and Storage: Store in tightly closed Sample: 2 mL
containers. Analysis: Dilute the Sample with 10 mL of water in a
glass-stoppered container and add 0.1 mL of 0.1 N
IDENTIFICATION potassium permanganate.
• ACETATE, Appendix IIIA
Acceptance criteria: The pink color does not change to
Sample solution: 333 mg/mL
brown within 2 h.
Acceptance criteria: Passes tests
• SOLIDIFICATION POINT, Appendix IIB
ASSAY Acceptance criteria: NLT 15.6°
• PROCEDURE
Sample: 2 mL
 FCC 8 Monographs / Acetoin Monomer / 13

. .

Acetoin Dimer Acetoin Monomer

First Published: Prior to FCC 6 First Published: FCC 6
Last Revision: Second Supplement, FCC 7

Acetyl Methyl Carbinol

Dimethylketol
3-Hydroxy-2-butanone

Monographs
C8H16O4 Formula wt 176.21 C4H8O2 Formula wt 88.11
FEMA: 2008 FEMA: 2008
UNII: BG4D34CO2H [acetoin] UNII: BG4D34CO2H [acetoin]

DESCRIPTION DESCRIPTION
Acetoin Dimer occurs as a white to pale yellow powder. Acetoin Monomer occurs as a colorless to pale yellow liquid.
Odor: Odorless It can contain some variable amount of its dimer.
Solubility: Soluble in hot propylene glycol; slightly soluble Odor: Buttery
in weak alkali; insoluble or practically insoluble in most Solubility: Miscible in alcohol, propylene glycol, water;
solvents insoluble or practically insoluble in vegetable oils
Function: Flavoring agent Boiling Point: ∼148°
Function: Flavoring agent
ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix IDENTIFICATION
XI. • INFRARED SPECTRA, Spectrophotometric Identification Tests,
Acceptance criteria: NLT 96.0% of C4H8O2 Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix
XI.
Acceptance criteria: NLT 96.0% of C4H8O2
 14 / Acetoin Monomer / Monographs FCC 8
Monographs

Acetoin Monomer

ASSAY
Acetone • PROCEDURE
First Published: Prior to FCC 6 Sample solution: 1 mg/mL
Analysis: Place 10 mL of the Sample solution into a
2-Propanone glass-stoppered flask, add 25 mL of sodium hydroxide
Dimethyl Ketone TS, and allow the mixture to stand for 5 min. Add 25
mL of 0.1 N iodine, stopper the flask, allow the
contents to stand in a cold, dark place for 10 min, and
add 30 mL of 1 N sulfuric acid. Titrate the excess iodine
with 0.1 N sodium thiosulfate, using starch TS as the
indicator. Perform a blank determination (see General
C3H6O Formula wt 58.08
CAS: [67-64-1] Provisions) and make any necessary correction. Each mL
UNII: 1364PS73AF [acetone] of 0.1 N iodine is equivalent to 0.9675 mg of C3H6O.
Acceptance criteria: NLT 99.5% and NMT 100.5%
DESCRIPTION C3H6O, by weight
Acetone occurs as a clear, colorless, volatile liquid. It is
miscible with water, with alcohol, with ether, with IMPURITIES
chloroform, and with most volatile oils. Inorganic Impurities
Function: Extraction solvent • LEAD, Lead Limit Test, Atomic Absorption Spectrophotometric
Packaging and Storage: Store in tight containers remote Graphite Furnace Method, Method I, Appendix IIIB
from fire. Acceptance criteria: NMT 1 mg/kg
[CAUTION—Acetone is highly flammable.] Organic Impurities
• ALDEHYDES (AS FORMALDEHYDE)
IDENTIFICATION Sample solution: 2.5 mL of sample and 7.5 mL of
• PROCEDURE water
Sample: 0.1 mL Standard solution: 40 µg formaldehyde in 10 mL of
Analysis: Mix the Sample with 10 mL of water, add 5 water
mL of 1 N sodium hydroxide, warm, and add 5 mL of Analysis: To both the Sample solution and 10 mL of the
iodine TS. Standard solution, add 0.15 mL of a 5% solution of 5,5-
Acceptance criteria: A yellow precipitate of iodoform dimethyl-1,3-cyclohexanedione in alcohol, and
forms. evaporate on a steam bath until the Acetone is
volatilized. Dilute both to 10 mL with water and cool
quickly in an ice bath while stirring vigorously.
FCC 8
Acceptance criteria: Any turbidity produced by the
Sample solution does not exceed that produced by the
Standard solution (NMT 0.002%).
• METHANOL
Sample solution: 100 µL/mL
Control solution: 40 µg/mL methanol
Analysis: Add 0.2 mL of 10% phosphoric acid and 0.25
mL of 50 mg/mL potassium permanganate solution to
1 mL of each Control solution and Sample solution.
Allow the mixtures to stand for 15 min, then add 0.3
mL of 100 mg/mL sodium bisulfite solution to each,
and shake until colorless. Slowly add 5 mL of ice-cold
80% sulfuric acid, keeping the mixtures cold during
the addition. Add 0.1 mL of 10 mg/mL chromotropic
acid solution, mix, and digest on a steam bath for 20
min.
Acceptance criteria: Any violet color produced by the
Sample solution does not exceed that produced by the
Control solution (NMT 0.05%).
• PHENOLS
Sample: 3 mL
Analysis: Evaporate the Sample to dryness at 60°. Add 3
drops of a solution of 100 mg of sodium nitrite in 5
mL of sulfuric acid to the residue, allow the mixture to
stand for about 3 min, and then carefully add 3 mL of
2 N sodium hydroxide.
Acceptance criteria: No color appears.
SPECIFIC TESTS
• ACIDITY (AS ACETIC ACID)
Sample: 38 mL
Analysis: Mix the Sample with an equal volume of
carbon dioxide-free water, add 0.1 mL of
phenolphthalein TS, and titrate with 0.1 N sodium
hydroxide.
Acceptance criteria: NMT 0.1 mL is required to
produce a pink color (NMT 0.002%)
• ALKALINITY (AS AMMONIA)
Sample: 23 mL
Analysis: Add 1 drop of methyl red TS to 25 mL of
water, add 0.1 N sulfuric acid until a red color just
appears, then add the Sample, and mix.
Acceptance criteria: NMT 0.1 mL of 0.1 N sulfuric acid
is required to restore the red color (NMT 10 mg/kg)
• DISTILLATION RANGE, Appendix IIB
Acceptance criteria: Within a range of 1°, including
56.1°
• NONVOLATILE RESIDUE
Sample: 125 mL (∼100 g)
Analysis: Evaporate the Sample to dryness in a tared
dish on a steam bath, dry the residue at 105° for 30
min, cool, and weigh.
Acceptance criteria: NMT 10 mg/kg
• REFRACTIVE INDEX, Appendix IIB
[NOTE—Use an Abbé or other refractometer of equal or
greater accuracy.]
Acceptance criteria: Between 1.358 and 1.360 at 20°
• SOLUBILITY IN WATER
Sample: 38 mL
Analysis: Mix the Sample with an equal volume of
carbon dioxide-free water.
Monographs / Acetone Peroxides / 15
Acceptance criteria: The solution remains clear for at
least 30 min.
• SPECIFIC GRAVITY: Determine by any reliable method (see
General Provisions).
Acceptance criteria: NMT 0.7880 at 25°/25°
(equivalent to 0.7930 at 20°/20°)
• SUBSTANCES REDUCING PERMANGANATE
Sample: 10 mL
Analysis: Transfer the Sample into a glass-stoppered
cylinder, add 0.05 mL of 0.1 N potassium
Monographs
permanganate, mix, and allow to stand for 15 min.
Acceptance criteria: The pink color does not entirely
disappear.
• WATER, Water Determination, Appendix IIB
Analysis: Use freshly distilled pyridine instead of
methanol as the solvent.
Acceptance criteria: NMT 0.5%
.
Acetone Peroxides
First Published: Prior to FCC 6
INS: 929 CAS: [1336-17-0]
UNII: 3O959710YK [acetone peroxide]
DESCRIPTION
Acetone Peroxides, usually mixed with an edible carrier such
as cornstarch, occur as a fine, white, free-flowing powder.
They are a mixture of monomeric and linear dimeric
acetone peroxides (mainly 2,2-hydroperoxypropane), with
minor proportions of higher polymers.
Function: Bleaching agent; maturing agent; dough
conditioner
Packaging and Storage: Store in tightly closed
containers in a cool, dry place, preferably below 24°.
[CAUTION—Acetone Peroxides are strong oxidizing agents.
Avoid exposure to the skin and eyes.]
IDENTIFICATION
• PROCEDURE
Analysis: Dissolve 20 mg of sample in 5 mL of 1:10
sulfuric acid, allow to stand for a few minutes, and add
a drop of potassium permanganate TS.
Acceptance criteria: The pink color disappears.
ASSAY
• PROCEDURE
Sample: 200 mg
Analysis: Transfer the Sample into a 250-mL beaker, add
50 mL of 10% sulfuric acid, allow to stand for at least 3
min, stirring occasionally, and titrate with 0.1 N
potassium permanganate to a light pink color that
persists for at least 20 s. Calculate the total peroxides,
P, as g of hydrogen peroxide equivalents per 100 g of
the sample, by the equation:
P = V × N × 0.017 × 100/W
V = volume of the potassium permanganate
(mL)
 16 / Acetone Peroxides / Monographs FCC 8

N = normality of the potassium permanganate Odor: Very sweet, pungent

0.017 = milliequivalent weight of hydrogen peroxide
W = weight of the sample (g) taken
Multiply the value P so obtained by 1.6 to convert to
percent acetone peroxides.
Acceptance criteria: A sample yields an amount of
hydrogen peroxide equivalent to NLT 16.0% of acetone
peroxides.
Solubility: Very soluble in most fixed oils, propylene glycol;
soluble in alcohol, chloroform, ether; slightly soluble in
water; insoluble or practically insoluble in glycerin
Boiling Point: ∼202°
Solubility in Alcohol, Appendix VI: One mL dissolves in 5
mL of 50% alcohol.
Function: Flavoring agent

IMPURITIES IDENTIFICATION
Monographs

Inorganic Impurities • INFRARED SPECTRA, Spectrophotometric Identification Tests,

• LEAD, Lead Limit Test, Appendix IIIB
Sample solution: Prepare as directed for organic
compounds.
Control: 4 µg Pb (4 mL of Diluted Standard Lead
Solution)
Acceptance criteria: NMT 4 mg/kg
.
Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.
ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix
XI.
Acceptance criteria: NLT 98.0% of C8H8O

Acetophenone SPECIFIC TESTS

First Published: Prior to FCC 6 • REFRACTIVE INDEX, Appendix II: At 20°
Acceptance criteria: Between 1.533 and 1.535
Acetylbenzene • SPECIFIC GRAVITY: Determine at 25° by any reliable
Methyl Phenyl Ketone method (see General Provisions).
Acceptance criteria: Between 1.025 and 1.028

OTHER REQUIREMENTS
• CHLORINATED COMPOUNDS, Appendix VI
Acceptance criteria: Passes test
• SOLIDIFICATION POINT, Appendix IIB
C8H8O Formula wt 120.15 Acceptance criteria: NLT 19°
FEMA: 2009
UNII: RK493WHV10 [acetophenone]

DESCRIPTION
Acetophenone occurs as a practically colorless liquid above
20°.
 FCC 8 Monographs / 3-Acetyl-2,5-dimethyl Furan / 17

Monographs
Acetophenone

. Function: Flavoring agent

3-Acetyl-2,5-dimethyl Furan
IDENTIFICATION
First Published: Prior to FCC 6
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
2,5-Dimethyl-3-acetylfuran Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
• PROCEDURE: Proceed as directed under M-1a, Appendix
XI.
C8H10O2 Formula wt 138.17
Acceptance criteria: NLT 99.0% of C8H10O2
FEMA: 3391
UNII: 798V2T7ZBV [3-acetyl-2,5-dimethylfuran] SPECIFIC TESTS
• REFRACTIVE INDEX, Appendix II: At 20°
DESCRIPTION Acceptance criteria: Between 1.484 and 1.492
3-Acetyl-2,5-dimethyl Furan occurs as a yellow liquid.
• SPECIFIC GRAVITY: Determine at 25° by any reliable
Odor: Powerful, slightly roasted, nutty
method (see General Provisions).
Solubility: Soluble in alcohol, most fixed oils, propylene
Acceptance criteria: Between 1.027 and 1.048
glycol; slightly soluble in water
Boiling Point: ∼83° (11 mm Hg)
 18 / 3-Acetyl-2,5-dimethyl Furan / Monographs FCC 8
Monographs

3-Acetyl-2,5-dimethyl Furan

Analysis: Transfer the Sample into a glass-stoppered flask

N-Acetyl-L-Methionine
.

and add 100 mL of water, 5 g of dibasic potassium

First Published: Prior to FCC 6 phosphate, 2 g of monobasic potassium phosphate, and
2 g of potassium iodide. Mix well to dissolve, add 50.0
mL of 0.1 N iodine, stopper the flask, and mix. Allow to
N-Acetyl-L-2-amino-4-(methylthio)butyric Acid
stand for 30 min, add starch TS indicator, and then
titrate the excess iodine with 0.1 N sodium thiosulfate.
Perform a residual blank titration. Each mL of 0.1 N
iodine is equivalent to 9.563 mg C7H13NO3S.
Acceptance criteria: NLT 98.5% and NMT 101.5%
C7H13NO3S, calculated on the dried basis
C7H13NO3S Formula wt 191.25 IMPURITIES
CAS: [65-82-7] Inorganic Impurities
UNII: 9J12WX5B6A [n-acetylmethionine]
• LEAD, Lead Limit Test, Appendix IIIB
DESCRIPTION Sample Solution: Prepare as directed for organic
N-Acetyl-L-Methionine occurs as a colorless or lustrous, compounds.
white, crystalline solid or a white powder. It is soluble in Control: 5 µg Pb (5 mL of Diluted Standard Lead
water, in alcohol, in alkali solutions, and in dilute mineral Solution)
acids, but practically insoluble in ether. Acceptance criteria: NMT 5 mg/kg
Function: Nutrient SPECIFIC TESTS
Packaging and Storage: Store in tightly closed, light- • LOSS ON DRYING, Appendix IIC: 105° for 2 h
resistant containers. Acceptance criteria: NMT 0.5%
IDENTIFICATION • OPTICAL (SPECIFIC) ROTATION, Appendix IIB
• INFRARED ABSORPTION, Spectrophotometric Identification Sample: 20 mg/mL (sample previously dried), made to
Tests, Appendix IIIC 100 mL
Sample preparation: Mineral oil mull Acceptance criteria: [α]D20 between −18.0° and
Acceptance criteria: The spectrum of the sample −22.0°, on the dried basis
exhibits relative maxima at the same wavelengths as • RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
those of the spectrum below. Sample: 1 g
Acceptance criteria: NMT 0.1%
ASSAY
• PROCEDURE
Sample: 250 mg
 FCC 8 Monographs / 2-Acetyl Thiazole / 19

Monographs
N-Acetyl-L-Methionine (Mineral Oil Mull)

. Function: Flavoring agent

2-Acetyl Thiazole
IDENTIFICATION
First Published: Prior to FCC 6
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
C5H5NOS Formula wt 127.17 • PROCEDURE: Proceed as directed under M-1b, Appendix
FEMA: 3328 XI.
UNII: 16IGS5268I [2-acetylthiazole] Acceptance criteria: NLT 98.0% of C5H5NOS

DESCRIPTION SPECIFIC TESTS

2-Acetyl Thiazole occurs as a colorless to pale yellow liquid.
Odor: Popcorn
Solubility: Soluble in propylene glycol, vegetable oils;
insoluble or practically insoluble in water
Boiling Point: ∼89° (12 mm Hg); ∼91° (1 mm Hg)
Solubility in Alcohol, Appendix VI: One mL dissolves in 1
mL of 95% ethanol.
• REFRACTIVE INDEX, Appendix II: At 20°
Acceptance criteria: Between 1.542 and 1.552
• SPECIFIC GRAVITY: Determine at 25° by any reliable
method (see General Provisions).
Acceptance criteria: Between 1.219 and 1.226
 20 / 2-Acetyl Thiazole / Monographs FCC 8
Monographs

2-Acetyl Thiazole

. Function: Emulsifier; coating agent; texture-modifying

Acetylated Monoglycerides agent; solvent; lubricant
First Published: Prior to FCC 6 Packaging and Storage: Store in well-closed containers.

IMPURITIES
Acetylated Mono- and Diglycerides Inorganic Impurities
Acetic and Fatty Acid Esters of Glycerol • LEAD, Lead Limit Test, Flame Atomic Absorption
Acetoglycerides Spectrophotometric Method, Appendix IIIB
Sample: 10 g
Acceptance criteria: NMT 2 mg/kg

INS: 472a
UNII: 5Z17386USF [diacetylated monoglycerides]
DESCRIPTION
Acetylated Monoglycerides occur as clear, thin liquids or
solids, ranging in color from white to pale yellow. They
consist of partial or complete esters of glycerin with a
mixture of acetic acid and edible fat-forming fatty acids.
They may be manufactured by the interesterification of
edible fats with triacetin and glycerin in the presence of
catalytic agents, followed by molecular distillation, or by
the direct acetylation of edible monoglycerides with acetic
anhydride and without the use of a catalyst or molecular
distillation. They are insoluble in water, but are soluble in
alcohol, in acetone, and in other organic solvents, the
extent of solubility depending on the degree of
esterification and the melting range.
SPECIFIC TESTS
• ACID VALUE, Method II, Appendix VII
Acceptance criteria: NMT 6
• FREE GLYCERIN, Free Glycerin or Propylene Glycol, Appendix
VII
Acceptance criteria: The result should conform to the
representations of the vendor.
• IODINE VALUE, Appendix VII
Acceptance criteria: The result should conform to the
representations of the vendor.
• REICHERT-MEISSL VALUE, Appendix VII
Acceptance criteria: Between 75 and 200
• SAPONIFICATION VALUE, Appendix VII
Acceptance criteria: The result should conform to the
representations of the vendor.
 FCC 8 Monographs / 2-Acetylpyrrole / 21

IDENTIFICATION
3-Acetylpyridine • INFRARED SPECTRA, Spectrophotometric Identification Tests,
First Published: Prior to FCC 6 Appendix IIIC
Acceptance criteria: The spectrum of the sample
Methyl Pyridyl Ketone exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
• PROCEDURE: Proceed as directed under M-1a, Appendix
XI.

Monographs
Acceptance criteria: NLT 98.0% of C7H7NO
C7H7NO Formula wt 121.14
FEMA: 3424 SPECIFIC TESTS
UNII: 00QT8FX306 [3-acetylpyridine] • REFRACTIVE INDEX, Appendix II: At 20°
Acceptance criteria: Between 1.530 and 1.540
DESCRIPTION • SPECIFIC GRAVITY: Determine at 25° by any reliable
3-Acetylpyridine occurs as a colorless to yellow liquid. method (see General Provisions).
Odor: Sweet, nutty, popcorn Acceptance criteria: Between 1.100 and 1.115
Solubility: Soluble in acids, alcohol, ether, water
Boiling Point: ∼230° OTHER REQUIREMENTS
Function: Flavoring agent • WATER, Water Determination, Method I, Appendix IIB
Acceptance criteria: 0.5%

3-Acetylpyridine

. FEMA: 3202
2-Acetylpyrrole UNII: 9K28W7PM6N [2-acetylpyrrole]
First Published: Prior to FCC 6
DESCRIPTION
2-Acetylpyrrole occurs as a white to pale brown fine
Methyl 2-Pyrrolyl Ketone crystal.
Odor: Bready
Solubility: Insoluble or practically insoluble in propylene
glycol, vegetable oils, water
Boiling Point: ∼220°

C6H7NO Formula wt 109.13

 22 / 2-Acetylpyrrole / Monographs FCC 8

Solubility in Alcohol, Appendix VI: One g dissolves in 6 FEMA: 3126

mL of ethanol. UNII: GR391IBU5C [2-acetylpyrazine]
Function: Flavoring agent
DESCRIPTION
ASSAY 2-Acetylpyrazine occurs as colorless to pale yellow crystals.
• PROCEDURE: Proceed as directed under M-1a, Appendix Odor: Popcorn
XI. Solubility in Alcohol, Appendix VI: One g dissolves in 20
Acceptance criteria: NLT 98.0% of C6H7NO mL of 95% alcohol.
Function: Flavoring agent
OTHER REQUIREMENTS
• MELTING RANGE OR TEMPERATURE DETERMINATION, Appendix IDENTIFICATION
Monographs

IIB • INFRARED SPECTRA, Spectrophotometric Identification Tests,

Acceptance criteria: Between 88° and 92°
• RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
Acceptance criteria: NMT 0.3%
Appendix IIIC
Sample preparation: Mineral oil mull
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
.

2-Acetylpyrazine • PROCEDURE: Proceed as directed under M-1a, Appendix

First Published: Prior to FCC 6 XI.
Acceptance criteria: NLT 99.0% of C6H6N2O
Methyl Pyrazinyl Ketone
OTHER REQUIREMENTS
• MELTING RANGE OR TEMPERATURE DETERMINATION, Appendix
IIB
Acceptance criteria: Between 75° and 78°

C6H6N2O Formula wt 122.13

2-Acetylpyrazine (Mineral Oil Mull)

 FCC 8
. Sample stock solution: Dissolve sample, as needed
Acid Hydrolysates of Proteins
First Published: Prior to FCC 6
Last Revision: Second Supplement, FCC 7
Acid-Hydrolyzed Proteins
Hydrolyzed Vegetable Protein (HVP)
Hydrolyzed Plant Protein (HPP)
Hydrolyzed (Source) Protein Extract
Acid-Hydrolyzed Milk Protein
DESCRIPTION
Acid Hydrolysates of Proteins occur as liquids, pastes,
powders, or granules. They are composed primarily of
amino acids, small peptides (peptide chains of five or fewer
amino acids), and salts resulting from the essentially
complete hydrolysis of peptide bonds in edible
proteinaceous materials, catalyzed by food-grade acids and
/or heat. Cleavage of peptide bonds typically ranges from
a low of 85% to essentially 100%. In processing, the
protein hydrolysates may be treated with safe and suitable
alkaline materials. The edible proteinaceous materials used
as raw materials are derived from corn, soy, wheat, yeast,
peanuts, rice, or other safe and suitable vegetable or plant
sources, or from milk.
Function: Flavoring agent; flavor enhancer
Packaging and Storage: Store in well-closed containers.
[NOTE—Perform all tests on the dried basis. Evaporate
liquid and paste samples to dryness in a suitable tared
container; then, as for the powdered and granular forms,
dry to constant weight at 105°. (See General Provisions.)]
ASSAY
• TOTAL NITROGEN, Nitrogen Determination, Appendix IIIC
Acceptance criteria: NLT 4.0%
IMPURITIES
Inorganic Impurities
• LEAD, Lead Limit Test, Appendix IIIB
Sample solution: Prepare as directed for organic
compounds.
Control: 3 µg Pb (3 mL of Diluted Standard Lead
Solution)
Acceptance criteria: NMT 3 mg/kg, on the dried basis
Organic Impurities
• 3-CHLOROPROPANE-1,2-DIOL (3-MCPD)
Standard stock solution: 125 µg/mL of reagent-grade
3-chloropropane-1,2-diol (3-MCPD) in ethyl acetate
Diluted standard solution: 6.25 µg/mL of 3-MCPD in
ethyl acetate from the Standard stock solution
Internal standard solution: 10 µg/mL of 1-chlorotet-
radecane in ethyl acetate
Standard solution A: 2 mL of Diluted standard solution
and 2.5 mL of Internal standard solution diluted to 25
mL with ethyl acetate (contains 0.5 µg/mL 3-MCPD)
Standard solution B: 8 mL of Diluted standard solution
and 2.5 mL of Internal standard solution diluted to 25
mL with ethyl acetate (contains 2.0 µg/mL 3-MCPD)
Standard solution C: 16 mL of Diluted standard solution
and 2.5 mL of Internal standard solution diluted to 25
mL with ethyl acetate (contains 4.0 µg/mL 3-MCPD)
Monographs / Acid Hydrolysates of Proteins / 23
with 20% aqueous sodium chloride, to obtain a
solution with a solids content of 36%.
Sample preparation: Transfer a 20-g aliquot of the
Sample stock solution into a 20-mL Extrelut NT column
(EM Science, Gibbstown, NJ), or equivalent, and allow
it to equilibrate for 15 min. Elute the column with 150
mL of ethyl acetate, collecting the eluent in a 250-mL
short-neck, round-bottom flask with a 24/40 joint.
Using a rotary evaporator at 50°, concentrate the
Monographs
eluent to a volume of approximately 3 mL. Add 0.5 mL
of Internal standard solution to the eluent, transfer this
mixture to a 4-dram screw-cap vial, and dilute to a
volume of 5.0 mL.
Chromatographic system, Appendix IIA
Mode: Gas chromatography
Detector: Electrolytic conductivity detector. [NOTE—
Operate the detector in the halogen mode.]
Column: 30-m × 0.53-mm (id), fused-silica column, or
equivalent, coated with 1-µm Supelcowax 10 or an
equivalent bonded carbowax column fitted with a
50-cm retention gap of 0.53-mm, deactivated, fused
silica, or equivalent
Temperature
Column: Hold at 170° for 5 min, then increase at
5°/min to 250°, hold at 250° for 10 min
Injector: 225°
Detector reactor: 900°
Detector base: 275°
Carrier gas: Helium
Reactant gas: Hydrogen
Solvent: 1-Propanol
Flow rate
Helium: 8 mL/min
Hydrogen: 30 mL/min
1-Propanol: 0.5 mL/min through the cell or at the
manufacturer’s specified flow rate for the optimum
operation of the detector
Injection volume: 1.0 µL
Injection type: Use a capillary injector operated in the
splitless mode or a purged, packed injector with a
glass insert.
[NOTE—Minimize contamination of the reaction tube
by venting flow from the column at all times, except
for the time during which compounds of interest
elute.]
Analysis: Separately inject Standard solution A,
Standard solution B, Standard solution C, and the
Sample preparation into the chromatograph and
record the resulting chromatograms. Calculate the
area ratios of 3-MCPD to the Internal standard
solution for each Standard solution. Plot the area ratios
versus the µg of 3-MCPD in each Standard solution to
obtain the standard curve. From the chromatogram
of the Sample preparation, measure the area ratio of
3-MCPD to the Internal standard solution and, using
the standard curve, determine the amount of 3-
MCPD, in µg, in the 20-g aliquot of Sample stock
solution taken.
Acceptance criteria: NMT 1 mg/kg, on the dried
basis
 24 / Acid Hydrolysates of Proteins / Monographs
• 1,3-DICHLORO-2-PROPANOL (DCP)
Diluent: Pentane and diethyl ether (85:15) (v/v)
Stock solution: 1 mg/mL of reagent-grade 1,3-
dichloro-2-propanol (DCP) in Diluent
Diluted standard solution: 1 µg/mL of DCP in Diluent
made from the Stock solution
Internal standard solution: 1 µg/mL of
trichlorobenzene in Diluent
Standard solutions: Pipet 1, 2, 3, and 4 mL portions of
Diluted standard solution, into separate 50-mL
Monographs
volumetric flasks. Add 1.0 mL of Internal standard
solution to each and dilute with Diluent to volume.
Sample solution: Dissolve 5.0 g of the sample in a
minimal volume of 20% aqueous sodium chloride
solution. Quantitatively transfer this solution to an
Extrelut NT column (EM Science, Gibbstown, NJ), or
equivalent. After 15 min, elute the column with three
20-mL portions of Diluent, and collect all of the eluate.
Carefully evaporate the eluate to less than 4 mL. Add
1.0 mL of Internal standard solution, and dilute with
Diluent, as necessary, to bring the final volume to 5.0
mL.
Chromatographic system, Appendix IIA
Mode: Gas chromatography with a split injector
Detector: Electrolytic conductivity detector
Column: 50-m × 0.2-mm (id), fused-silica column
(Carbowax 20M, or equivalent) coated with
dimethylpolysiloxane, or equivalent
Temperature
Column: Hold at 115° for 10 min, then increase at
30°/min to 200°, hold at 200° for 12 min
Injector: 250°
Detector: 300°
[NOTE—Precondition the column by heating it at
200° and the detector at 300° for 24 h.]
Carrier gas: Nitrogen
Flow rate: 8 mL/min
Injection size: 1.0 µL
Analysis: Separately inject each of the Standard
solutions and the Sample solution into the
chromatograph and record the resulting
chromatograms. Calculate the area ratios of DCP to
Internal standard solution for each Standard solution.
Plot the area ratios versus the µg of DCP in each
Standard solution to obtain the standard curve. From
the chromatograph of the Sample solution, measure
the area ratio of DCP to the Internal standard solution
and, using the standard curve, determine the amount
of DCP, in µg, in the sample taken.
Acceptance criteria: NMT 0.05 mg/kg, on the dried
basis
SPECIFIC TESTS
• α-AMINO NITROGEN, Appendix IIIC
Acceptance criteria: NLT 3.0%, on the dried basis
• α-AMINO NITROGEN/TOTAL NITROGEN PERCENT RATIO
Analysis: Calculate by the formula:
Result = 100[(AN – P)/(TN – P)]
FCC 8
AN = percentage of α-Amino Nitrogen, determined
above
P = percentage of Ammonia Nitrogen,
determined below
TN = percentage of Total Nitrogen, determined
above
Acceptance criteria: 62.0%–85.0%, when calculated on
an ammonia nitrogen-free basis
• AMMONIA NITROGEN, Appendix IIIC
Acceptance criteria: NMT 1.5%, on the dried basis
• GLUTAMIC ACID, Appendix IIIC
Acceptance criteria: NMT 20.0% as glutamic acid
(C5H9NO4) and NMT 35.0% of the total protein, both
on the dried basis
• INSOLUBLE MATTER
Sample: 5 g
Analysis: Transfer the Sample into a 250-mL Erlenmeyer
flask, add 75 mL of water, cover the flask with a watch
glass, and boil gently for 2 min. Filter the solution
through a tared filtering crucible, dry at 105° for 1 h,
cool, and weigh.
Acceptance criteria: NMT 0.5%, on the dried basis
• POTASSIUM
Standard solution: 1.91 µg/mL of potassium chloride
(corresponds to 1.0 µg/mL of potassium ion)
Sample stock solution: Transfer 1.00 ± 0.05 g of
previously dried sample into a silica or porcelain dish.
Ash in a muffle furnace at 550° for 2–4 h. Allow the ash
to cool, and dissolve in 5 mL of 20% hydrochloric acid,
warming the solution if necessary to complete solution
of the residue. Filter the solution through acid-washed
filter paper into a 1000-mL volumetric flask. Wash the
filter paper with hot water, dilute to volume, and mix.
Sample solution: 1:300 (v/v) dilution of the Sample
stock solution
Analysis: Using a suitable atomic absorption
spectrophotometer, determine the absorbance of the
Standard solution and the Sample solution at 766.5.
Acceptance criteria: The absorbance of the Sample
solution does not exceed that of the Standard solution.
(NMT 30.0%, on the dried basis)
• SODIUM
Standard stock solution: 254.2 µg/mL of sodium
chloride
Standard solution: 12.71 ng/mL of sodium chloride
made from the Standard stock solution (corresponds to
5 ng/mL of sodium ion)
Sample stock solution: Transfer 1.00 ± 0.05 g of
previously dried sample into a silica or porcelain dish.
Ash in a muffle furnace at 550° for 2–4 h. Allow the ash
to cool, and dissolve in 5 mL of 20% hydrochloric acid,
warming the solution if necessary to complete solution
of the residue. Filter the solution through acid-washed
filter paper into a 100-mL volumetric flask. Wash the
filter paper with hot water, dilute to volume, and mix.
Sample solution: 1:4000 (v/v) dilution of the Sample
stock solution
Analysis: Using a suitable atomic absorption
spectrophotometer, determine the absorbance of the
Standard solution and the Sample solution at 589.0.
 FCC 8
Acceptance criteria: The absorbance of the Sample
solution does not exceed that of the Standard solution.
(NMT 20.0%, on the dried basis)
.
Acidified Sodium Chlorite Solutions
First Published: Prior to FCC 6
DESCRIPTION
Acidified Sodium Chlorite (ASC) Solutions occur as clear,
colorless to pale yellow liquids. The ASC Solutions are
equilibrium mixtures of sodium chlorite (NaClO2) and
chlorous acid (HClO2). ASC Solutions are produced by
lowering the pH of a sodium chlorite solution with a safe
and suitable acid to achieve a pH within the range 2.3 to
3.9 depending on the intended use.
Function: Antimicrobial agent in processing water used to
spray, dip, rinse, or store food before processing, to be
followed by rinsing in potable water or by blanching,
cooking, or canning; sanitizer for hard surfaces; broad-
spectrum bactericide, virucide, fungicide, and sporicide
Packaging and Storage: Store in closed, opaque
containers. Avoid exposure to sun or ultraviolet light
because chlorine dioxide gas will generate in the solution.
IMPURITIES
Inorganic Impurities
• LEAD, Lead Limit Test, Appendix IIIB
Sample solution: 1.0 mL of sample mixed with 5 mL of
water and 11 mL of 2.7 N hydrochloric acid
Control: 10 µg of Pb (10 mL of Diluted Standard Lead
Solution)
Acceptance criteria: NMT 1 mg/kg
• MERCURY, Mercury Limit Test, Appendix IIIB
Sample preparation: Transfer 2.0 mL of sample into a
50-mL beaker; add 10 mL of water, 1 mL of 20%
sulfuric acid, and 1 mL of a 40 mg/mL potassium
permanganate solution. Cover the beaker with a watch
glass, boil for a few seconds, and cool.
Acceptance criteria: NMT 1 mg/kg
SPECIFIC TESTS
• PH, pH Determination, Appendix IIB
[CAUTION—To minimize the evolution of hazardous
chlorine dioxide gas, do not adjust the pH below 2.3.]
Acceptance criteria: Between 2.3 and 3.9
[NOTE—The pH is chosen depending on the application.
It controls the concentration of metastable chlorous
acid, which rapidly breaks down into chlorine dioxide,
chloride, and in some applications, chlorate]
• SODIUM CHLORITE
[NOTE—See 21 CFR 173.325; “Determination of Sodium
Chlorite: 50 ppm to 1500 ppm,” Alcide Corporation.]
Sample: For solutions containing 40 to 250 ppm, use a
100-g sample; for those containing 250 to 500 ppm,
use a 50-g sample; for those containing 500 to 1100
ppm, use a 20-g sample; for those containing 1100 to
1500 ppm, use a 15-g sample.
Monographs / Aconitic Acid / 25
Analysis: Transfer the appropriate Sample into a tared
250-mL Erlenmeyer flask, and record the weight to the
nearest 0.1 mg. Add a magnetic stirring bar. Add
approximately 2 g of potassium iodide, place the flask
over a magnetic stirrer, and stir until the potassium
iodide crystals dissolve (about 1 min). Add 1 mL of 6 N
hydrochloric acid, and stir for 30 s. While continuously
stirring, titrate the liberated iodine with standardized
0.025 N sodium thiosulfate (Na2S2O3). When most of
the brown iodine color has faded, add 2 mL of starch
Monographs
indicator solution, and titrate to a clear endpoint,
allowing adequate mixing time between additions of
titrant near the endpoint. Record the volume of titrant,
V, in mL. Calculate the amount of Sodium Chlorite, in
ppm, by the formula:
Result = (V × N × Mr × F)/(W × FE)
V =volume of titrant (mL)
N =normality of the sodium thiosulfate titrant
Mr =molecular weight of sodium chlorite, 90.44
F =conversion factor for mg/g to ppm, 1000
W =weight of the sample taken (g)
FE =mEq of sodium thiosulfate/mEq of sodium
chlorite, 4
[NOTE—The concentration of sodium chlorite also can
alternatively be determined using ion chromatography
by following U.S. Environmental Protection Agency
Method 300.11 or amperometrically by following
American Public Health Association Method 4500-
ClO2.2 ]
Acceptance criteria: Between 40 and 1200 ppm,
depending on the application
.
Aconitic Acid
First Published: Prior to FCC 6
Equisetic Acid
Citridic Acid
Achilleic Acid
1-Propene-1,2,3-tricarboxylic Acid
C6H6O6 Formula wt 174.11
FEMA: 2010
CAS: [499-12-7]
UNII: 93371T1BXP [aconitic acid]
1 Hautman, Daniel P. and Munch, David J. “Method 300.1: Determination of
inorganic anions in drinking water by ion chromatography, Revision 1.0.” U.S.
Environmental Protection Agency, Office of Ground Water and Drinking
Water. 1997. Online Available: http://www.epa.gov/OGWDW/methods/
sourcalt.html [accessed October 19, 2007].
2 Franson, MA, ed. 1998. Standard methods 4500-ClO , amperometric
2
method II. In: Standard Methods for the Examination of Water and Wastewater,
20th Ed. Baltimore, MD: APHA/AWWA/WEF. Pp. 4-73 and 4-79.
 26 / Aconitic Acid / Monographs
DESCRIPTION
Aconitic Acid occurs in the leaves and tubers of Aconitum
napellus L. (Fam. Ranunculaceae) and various species of
Achillea and Equisetum, in beet root, and in sugar cane. It
may be synthesized by the dehydration of citric acid by
sulfuric or methanesulfonic acid. Aconitic Acid from the
above sources has the “trans” configuration. It has a
melting point of 195° to 200° with decomposition. It is
practically odorless and has a winy taste. It is soluble in
water and in alcohol and is slightly soluble in ether.
Monographs
Function: Flavoring substance; adjuvant
Packaging and Storage: Store in tightly closed
containers.
IDENTIFICATION
• INFRARED ABSORPTION SPECTRUM
Sample preparation: Neat as a potassium bromide
dispersion
Acceptance criteria: The Sample preparation exhibits
infrared absorption bands at 3030, 2630, and
1720 cm−1.
• VISIBLE ABSORPTION SPECTRUM
Sample solution: Aqueous solution
Acceptance criteria: The Sample solution exhibits major
absorption peaks at 411 and 432 nm, with little or no
absorption at 389 nm.
ASSAY
• PROCEDURE
Sample solution: 3 g
Analysis: Dissolve the Sample in 40 mL of water, add
phenolphthalein TS, and titrate with 1 N sodium
hydroxide. Each mL of 1 N sodium hydroxide is
equivalent to 58.04 mg of C6H6O6.
Acceptance criteria: NLT 98.0% and NMT 100.5% of
C6H6O6, calculated on the anhydrous basis
IMPURITIES
Inorganic Impurities
• LEAD, Lead Limit Test, Atomic Absorption Spectrophotometric
Graphite Furnace, Method I, Appendix IIIB
Sample: 10 g
Acceptance criteria: NMT 0.5 mg/kg
SPECIFIC TESTS
• OXALATE
Sample solution: 100 mg/mL
Analysis: Neutralize 10 mL of Sample solution with 6 N
ammonium hydroxide, add 5 drops of 2.7 N
hydrochloric acid, cool, and add 2 mL of calcium
chloride TS.
Acceptance criteria: No turbidity develops.
• READILY CARBONIZABLE SUBSTANCES, Appendix IIB
Sample: 1.0 g, finely powdered
Control: Matching Fluid K
Analysis: Transfer the Sample into a 22- × 175-mm test
tube previously rinsed with 10 mL of 95% sulfuric acid
and allowed to drain for 10 min. Add 10 mL of 95%
sulfuric acid, agitate the tube until solution is complete,
and immerse the tube in a water bath at 90° ± 1° for
60 ± 0.5 min, keeping the level of the acid below the
level of the water during the heating period. Cool the
FCC 8
tube in a stream of water and transfer the acid solution
into a color comparison tube. View the tube vertically
against a white background and compare to the same
volume of the Control in a similar matching tube.
Acceptance criteria: The color of the Sample solution is
not darker than that of the Control.
• RESIDUE ON IGNITION (SULFATED ASH), Method I, Appendix
IIC
Sample: 4 g
Acceptance criteria: NMT 0.1%
• WATER, Water Determination, Appendix IIB
Acceptance criteria: NMT 0.5%
.
5′-Adenylic Acid
First Published: First Supplement, FCC 7
Adenosine 5′-monophosphate
Adenylic acid
AMP
Adenosine 5′-phosphoric acid
C10H14N5O7P Formula wt 347.23
CAS: [61-19-8]
UNII: 415SHH325A [adenosine phosphate]
DESCRIPTION
5′-Adenylic Acid occurs as colorless or white crystals, or as a
white, crystalline powder. It is very slightly soluble in
water, and practically insoluble in alcohol. It is produced
by enzymatic cleavage of yeast ribonucleic acid (RNA) with
a 5′-phosphodiesterase followed by heat treatment, further
purification steps, and washing of crystals with ethanol.
Function: Source of 5′-Adenylic Acid
Packaging and Storage: Store in tight containers
protected from light and moisture.
IDENTIFICATION
• A. INFRARED ABSORPTION, Spectrophotometric Identification
Tests, Appendix IIIC
Reference standard: USP 5’-Adenylic Acid RS
Sample and standard preparation: A
Acceptance criteria: The spectrum of the sample
exhibits maxima at the same wavelengths as those in
the spectrum of the Reference standard.
• B. PROCEDURE
Acceptance criteria: The retention time of the major
peak (excluding the solvent peak) in the chromatogram
of the Sample solution corresponds to that of the
Standard solution in the Assay.
ASSAY
• PROCEDURE
Mobile phase: 0.1 M potassium dihydrogen phosphate
(KH2PO4) in degassed water, adjusted with 0.1 M
FCC 8
dipotassium hydrogen phosphate (K2HP04) to a pH of
5.6
Standard solution: 0.02 mg/mL of USP 5’-Adenylic Acid
RS in Mobile phase. [NOTE—Ultra-sonication for 15 min
at 30° may be necessary to aid in complete dissolution.]
Sample solution: 0.02 mg/mL in Mobile phase. [NOTE—
Ultra-sonication for 15 min at 30° may be necessary to
aid in complete dissolution.]
Chromatographic system, Appendix IIA
Mode: High-performance liquid chromatography
Detector: UV 254 nm
Column: 25 cm × 4.6-mm; packed with 5-µm reversed
phase C18 silica gel1
Column temperature: Ambient
Flow rate: About 1.0 mL/min
Injection size: 50 µL
System suitability
Sample: Standard solution
Suitability requirements
Suitability requirement 1: The relative standard
deviation of the 5’-adenylic acid area responses from
replicate injections is NMT 2.0%.
Suitability requirement 2: The resolution, R,
between the 5′-adenylic acid peak and all other
peaks is NLT 2.0.
Analysis: Separately inject equal volumes of the Standard
solution and Sample solution into the chromatograph,
and measure the responses for the major peaks on the
resulting chromatograms. [NOTE—The approximate
retention time for 5′-adenylic acid is 27.5 min.]
Calculate the percentage of 5′-adenylic acid,
C10H14N5O7P, in the sample taken:
Result = (rU/rS) × (CS/CU) × 100
rU = peak area response for 5′-adenylic acid in the
Sample solution
rS = peak area response for 5′-adenylic acid in the
Standard solution
CS = concentration of 5′-adenylic acid in the
Standard solution (mg/mL)
CU = concentration of the sample in the Sample
solution (mg/mL)
Acceptance criteria: 98.0%–103.0%, calculated on the
anhydrous basis
• CADMIUM
IMPURITIES
Inorganic Impurities
• ARSENIC
[NOTE—When water is specified as a diluent, use
deionized ultra-filtered water. When nitric acid is
specified, use nitric acid of a grade suitable for trace
element analysis with as low a content of arsenic as
practical.]
Diluent: 4% nitric acid in water
Standard stock solution: 100 µg/mL of arsenic
prepared by diluting a commercially available 1000 mg
/kg arsenic ICP standard solution
1 YMC-Pack ODS-AQ (YMC Europe GmbH, Dinslaken, Germany), or
equivalent.
Monographs / 5′-Adenylic Acid / 27
Standard solutions: 0.05, 0.1, 0.2, 0.5, 1, and 2 µg/mL
of arsenic, from the Standard stock solution diluted with
Diluent
Sample: 5 g
Sample solution: Dissolve the Sample in 40 mL of 10%
nitric acid in a 100-mL volumetric flask, and dilute with
water to volume.
Spectrophotometric system, Plasma Spectrochemistry,
Appendix IIC
Mode: Inductively coupled plasma–optical emission
Monographs
spectroscopy (ICP–OES)
Setup: Use a suitable ICP–OES configured in a radial
optical alignment. [NOTE—This method was
developed using a Varian Vista MPX ICP–OES unit.]
The instrument parameters are as follows: Set the
ultra-violet detector to scan arsenic at 188.980 nm.
Set the sample read time to 20 s. Set the forward
power from the RF generator to 1150 watts. Use an
argon plasma feed gas flow of 13.5 L/min with the
auxiliary gas set to flow at 2.25 L/min. The sample is
delivered to the spray chamber by a multi-channel
peristaltic pump set to deliver the sample at a rate
of 20 rpm. Samples are flushed through the system
for 20 s prior to analysis. A 40-s read delay is also
programmed into the sampling routine to allow for
fluid flow equilibration after the high-speed flush,
prior to the first analytical read of the sample.
Between samples, the pumping system is washed by
flushing the Diluent for 20 s.
Analysis: Generate a standard curve using Diluent as a
blank and the Standard solutions. [NOTE—The
correlation coefficient for the best-fit line should not be
less than 0.999.]
Similarly, analyze the Sample solution on the ICP.
Calculate the concentration (mg/kg) of arsenic in the
Sample taken:
Result = (C/W) × F
C = concentration of arsenic in the Sample
solution determined from the standard
curve (µg/mL)
W = weight of the Sample taken (g)
F = final volume of the Sample solution, 100 mL
Acceptance criteria: NMT 2 mg/kg
[NOTE—When water is specified as a diluent, use
deionized ultra-filtered water. When nitric acid is
specified, use nitric acid of a grade suitable for trace
element analysis with as low a content of cadmium as
practical.]
Diluent: 4% nitric acid in water
Standard stock solution: 100 µg/mL of cadmium
prepared by diluting a commercially available 1000 mg
/kg cadmium ICP standard solution
Standard solutions: 0.005, 0.05, 0.1, 0.2, 0.5, 1, and 2
µg/mL of cadmium, from the Standard stock solution
diluted with Diluent
Sample: 5 g
Sample solution: Dissolve the Sample in 40 mL of 10%
nitric acid in a 100-mL volumetric flask, and dilute with
water to volume.
 28 / 5′-Adenylic Acid / Monographs
Spectrophotometric system, Plasma Spectrochemistry,
Appendix IIC
Mode: ICP–OES
Setup: Same as that described in the test for Arsenic,
but set to scan for cadmium at 228.802 nm
Analysis: Generate a standard curve using Diluent as a
blank and the Standard solutions. [NOTE—The
correlation coefficient for the best-fit line should not be
less than 0.999.]
Similarly, analyze the Sample solution on the ICP.
Monographs
Calculate the concentration (mg/kg) of cadmium in the
Sample taken:
Result = (C/W) × F
C = concentration of cadmium in the Sample
solution determined from the standard
curve (µg/mL)
W = weight of the Sample taken (g)
F =final volume of the Sample solution, 100 mL
Acceptance criteria: NMT 0.1 mg/kg
• LEAD
[NOTE—When water is specified as a diluent, use
deionized ultra-filtered water. When nitric acid is
specified, use nitric acid of a grade suitable for trace
element analysis with as low a content of lead as
practical.]
Diluent: 4% nitric acid in water
Standard stock solution: 100 µg/mL of lead prepared
by diluting a commercially available 1000 mg/kg lead
ICP standard solution
Standard solutions: 0.05, 0.1, 0.2, 0.5, 1, and 2 µg/mL
of lead, from the Standard stock solution diluted with
Diluent
Sample: 5 g
Sample solution: Dissolve the Sample in 40 mL of 10%
nitric acid in a 100-mL volumetric flask, and dilute with
water to volume.
Spectrophotometric system, Plasma Spectrochemistry,
Appendix IIC
Mode: ICP–OES
Setup: Same as that described in the test for Arsenic,
but set to scan for lead at 220.353 nm
Analysis: Generate a standard curve using Diluent as a
blank and the Standard solutions. [NOTE—The
correlation coefficient for the best-fit line should not be
less than 0.999.]
Similarly, analyze the Sample solution on the ICP.
Calculate the concentration (mg/kg) of lead in the
Sample taken:
Result = (C/W) × F
C = concentration of lead in the Sample solution
determined from the standard curve (µg/
mL)
W = weight of the Sample taken (g)
F = final volume of the Sample solution, 100 mL
Acceptance criteria: NMT 1 mg/kg
• MERCURY
[NOTE—When water is specified as a diluent, use
deionized ultra-filtered water. When nitric acid is
FCC 8
specified, use nitric acid of a grade suitable for trace
element analysis with as low a content of mercury as
practical.]
Diluent: 4% nitric acid in water
Standard stock solution: 100 µg/mL of mercury
prepared by diluting a commercially available 1000 mg
/kg mercury ICP standard solution
Standard solutions: 0.025, 0.05, 0.1, 0.2, 0.5, 1, and 2
µg/mL of mercury, from the Standard stock solution
diluted with Diluent
Sample: 5 g
Sample solution: Dissolve the Sample in 40 mL of 10%
nitric acid in a 100-mL volumetric flask, and dilute with
water to volume.
Spectrophotometric system, Plasma Spectrochemistry,
Appendix IIC
Mode: ICP–OES
Setup: Same as that described in the test for Arsenic,
but set to scan for mercury at 194.164 nm
Analysis: Generate a standard curve using Diluent as a
blank and the Standard solutions. [NOTE—The
correlation coefficient for the best-fit line should not be
less than 0.999.]
Similarly, analyze the Sample solution on the ICP.
Calculate the concentration (mg/kg) of mercury in the
Sample taken:
Result = (C/W) × F
C = concentration of mercury in the Sample
solution determined from the standard
curve (µg/mL)
W = weight of the Sample taken (g)
F = final volume of the Sample solution, 100 mL
Acceptance criteria: NMT 0.5 mg/kg
Organic Impurities
• ETHANOL
Standard solution: 10 mg/kg of ethanol in 1 N
sodium hydroxide. Add 10 mL of this solution to a 20-
mL headspace vial, and cap tightly.
Sample solution: 100 mg/g in 1 N sodium hydroxide.
Add 10 mL of this solution to a 20-mL headspace vial,
and cap tightly.
Chromatographic system, Appendix IIA
Mode: Gas chromatography equipped with pressure-
loop headspace autosampler
Detector: Flame ionization
Column: 30-m × 0.53-mm (id) capillary column with
a 6% cyanopropylphenyl–94% dimethylpolysiloxane
stationary phase and a 3.00-µm film thickness2
Column temperature: 20 min at 40°; increase to
240° at 10°/min; maintain at 240° for 10 min
Injection port temperature: 140°
Detector temperature: 250°
Carrier gas: Nitrogen
Flow rate: 2.5 mL/min
Headspace unit: 2.5 mL/min
Equilibration temperature: 80°
Equilibration time: 60 min
Loop temperature: 85°
2 CP-Select 624 CB (Varian-Chrompack, Palo Alto, CA), or equivalent.
FCC 8
Transfer temperature: 90°
Pressurization time: 0.5 min
Loop fill time: 0.1 min
Injection time: 1 min
Injection size: 1 mL of headspace
System suitability
Sample: Standard solution
Suitability requirement: The relative standard
deviation of the ethanol peak area responses from
replicate injections is NMT 5.0%.
Analysis: Separately inject equal volumes of the
Standard solution and Sample solution into the
chromatograph, record the chromatograms, and
measure the peak responses. [NOTE—The approximate
retention time for ethanol is 11 min.]
Acceptance criteria: The peak area from the Sample
solution does not exceed that from the Standard
solution (NMT 100 mg/kg).
• OTHER RIBONUCLEOTIDES
Mobile phase and Chromatographic system: Prepare
as directed in the Assay.
Sample solution: 1.0 mg/mL. [NOTE—Ultra-sonication
for 15 min at 30° may be necessary to aid in complete
dissolution.]
Standard solution: Mixture of USP Disodium 5′-
Uridylate RS, USP 5′-Adenylic Acid RS, USP 5′-Cytidylic
Acid RS, USP Disodium Guanylate RS, and USP
Disodium Inosinate RS, each at 0.02 mg/mL in Mobile
phase
Suitability requirements
Sample: Standard solution
Suitability requirement 1: The relative standard
deviation of the 5’-adenylic acid peak area responses
from replicate injections is NMT 2.0%.
Suitability requirement 2: The resolution, R, between
the 5’-adenylic acid peak and all other nucleotide
peaks is NLT 2.0.
Analysis: Separately inject equal volumes of the
Standard solution and Sample solution into the
chromatograph, and measure the responses for all
nucleotide peaks on the resulting chromatograms,
except the peak from 5’-adenylic acid. [NOTE—The
approximate retention times are 4.6 min (5′-cytidylic
acid), 6.2 min (5′-uridylic acid), 10.3 min (5′-guanylic
acid), 11.5 min (5′-inosinic acid), and 27.5 min (5′-
adenylic acid).] Separately calculate the percentage of
each analyte (5′-cytidylic acid, 5′-guanylic acid, 5′-
inosinic acid, and 5′-uridylic acid) in the sample taken:
Result = (rU/rS) × (CS/CU) × 100
rU = peak area of the analyte from the Sample
solution
rS = peak area of the analyte from the Standard
solution
CS = concentration of analyte in the Standard
solution (mg/mL)
CU = concentration of analyte in the Sample
solution (mg/mL)
Acceptance criteria: The sum of the percentages for all
nucleotide impurities is NMT 0.5%, calculated on the
anhydrous basis.
Monographs / Adipic Acid / 29
SPECIFIC TESTS
• PH, pH Determination, Appendix IIB
Sample solution: 0.05 mg/mL
Acceptance criteria: 3.3–4.3
• WATER, Water Determination, Method I, Appendix IIB
Acceptance criteria: NMT 6.0%
• BILE-TOLERANT GRAM-NEGATIVE BACTERIA, Appendix XIIC
Sample preparation: Proceed as directed using a 10-g
sample and incubating at 30–35° for 18–24 h.
Acceptance criteria: Negative in 10 g
Monographs
• ENTEROBACTER SAKAZAKII (Cronobacter Spp.), Appendix XIIC
Sample preparation: Proceed as directed using a 10-g
sample and incubating at 30–35° for 18–24 h.
Acceptance criteria: Negative in 10 g
• SALMONELLA SPP., Appendix XIIC
Sample preparation: Dissolve 25 g of the sample at a
sample/broth ratio of 1/8, and proceed as directed.
Acceptance criteria: Negative in 25 g
• TOTAL AEROBIC MICROBIAL COUNT, Method I (Plate Count
Method), Appendix XIIB
Acceptance criteria: NMT 1,000 cfu/g
• TOTAL YEASTS AND MOLDS COUNT, Method I (Plate Count
Method), Appendix XIIB
Acceptance criteria: NMT 100 cfu/g
.
Adipic Acid
First Published: Prior to FCC 6
Last Revision: FCC 7
Hexanedioic Acid
1,4-Butanedicarboxylic Acid
C6H10O4 Formula wt 146.14
INS: 355 CAS: 124-04-9
UNII: 76A0JE0FKJ [adipic acid]
DESCRIPTION
Adipic Acid occurs as white crystals or a crystalline powder.
It is not hygroscopic. It is freely soluble in alcohol, soluble
in acetone, and slightly soluble in water.
Function: Buffer; neutralizing agent
Packaging and Storage: Store in well-closed containers.
IDENTIFICATION
• INFRARED ABSORPTION, Spectrophotometric Identification
Tests, Appendix IIIC
Reference standard: USP Adipic Acid RS
Sample and standard preparation: K
Acceptance criteria: The spectrum of the sample
exhibits maxima at the same wavelengths as those in
the spectrum of the Reference standard.
ASSAY
• PROCEDURE
Sample: 1.5 g
 30 / Adipic Acid / Monographs
Analysis: Mix the Sample with 75 mL of recently boiled
and cooled water contained in a 250-mL glass-
stoppered Erlenmeyer flask, add phenolphthalein TS,
and titrate with 0.5 N sodium hydroxide to the first
appearance of a faint pink endpoint that persists for at
least 30 s, shaking the flask as the endpoint is
approached. Each mL of 0.5 N sodium hydroxide is
equivalent to 36.54 mg of C6H10O4.
Acceptance criteria: 99.6%–101.0% of C6H10O4,
calculated on the anhydrous basis
Monographs
IMPURITIES
Inorganic Impurities
• LEAD, Lead Limit Test, Flame Atomic Absorption
Spectrophotometric Method, Appendix IIIB
Sample: 5 g
Acceptance criteria: NMT 2 mg/kg
SPECIFIC TESTS
• MELTING RANGE OR TEMPERATURE DETERMINATION, Appendix
IIB
Acceptance criteria: Between 151.5° and 154°
• RESIDUE ON IGNITION
Sample: 100.0 g
Analysis: Transfer the Sample into a tared 125-mL
platinum dish that has been previously cleaned by
fusing with 5 g of potassium pyrosulfate or bisulfate,
followed by boiling in 2 N sulfuric acid and rinsing with
water. Melt the sample completely over a gas burner,
then ignite the melt with the burner. After ignition
starts, lower or remove the flame to prevent the sample
from boiling and to keep it burning slowly until it is
completely carbonized. Ignite at 850° in a muffle
furnace for 30 min or until the carbon is completely
removed, then cool and weigh.
Acceptance criteria: NMT 0.002%
• WATER, Water Determination, Appendix IIB
Acceptance criteria: NMT 0.2%
.
Agar
First Published: Prior to FCC 6
INS: 406 CAS: [9002-18-0]
UNII: 89T13OHQ2B [agar, unspecified]
DESCRIPTION
Agar is commercially available as white to pale yellow
bundles consisting of thin, membranous agglutinated
strips, or in cut, flaked, granulated, or powdered forms.
Agar is a generic name given to a group of related
molecules with a repeating unit of agarobiose formed
basically by D-and L-galactose units interlinked with α-1,3
and β-1,4 linkages. Approximately every tenth
D-galactopyranose unit contains a sulfate ester group. It is
extracted from the cellular walls of agarophyte seaweed,
considering as such the red seaweed from phylum
Rodophyta, which belong to the Gelidiceae, Gracilariaceae,
and Ahnpheltiaceae families. It is insoluble in cold water,
but it is soluble in boiling water.
FCC 8
Function: Stabilizer; emulsifier; thickener
Packaging and Storage: Store in well-closed containers.
IDENTIFICATION
• A. PROCEDURE
Analysis: Place a few fragments of unground sample or
a small amount of the powder on a slide, add a few
drops of water, and examine microscopically.
Acceptance criteria: The sample appears granular and
somewhat filamentous. A few fragments of the spicules
of sponges and a few frustules of diatoms may be
present.
• B. PROCEDURE
Sample: 1 g
Analysis: While stirring continuously, boil the Sample
with 65 mL of water for 10 min, and adjust to a
concentration of 1.5%, by weight, with hot water.
Acceptance criteria: A clear liquid results that congeals
between 32° and 39° to form a firm, resilient gel that
does not liquefy below 85°.
IMPURITIES
Inorganic Impurities
• ARSENIC, Arsenic Limit Test, Appendix IIIB
Sample solution: Prepare as directed for organic
compounds.
Acceptance criteria: NMT 3 mg/kg
• LEAD, Lead Limit Test, Appendix IIIB
Sample solution: Prepare as directed for organic
compounds.
Control: 5 µg Pb (5 mL of Diluted Standard Lead
Solution)
Acceptance criteria: NMT 5 mg/kg
SPECIFIC TESTS
• ASH (ACID-INSOLUBLE), Appendix IIC
Acceptance criteria: NMT 0.5%, calculated on the dried
basis
• ASH (TOTAL), Appendix IIC
Acceptance criteria: NMT 6.5%, calculated on the dried
basis
• GELATIN
Analysis: Dissolve 1 g of sample in 100 mL of boiling
water and cool to 50°. Add 5 mL of trinitrophenol TS
to 5 mL of the solution.
Acceptance criteria: No turbidity forms within 10 min.
• INSOLUBLE MATTER
Sample: 7.5 g
Analysis: Add sufficient water to the Sample to make
500 g, boil for 15 min, and readjust to the original
weight. Add hot water to 100 g of the mixture to make
200 mL, heat almost to boiling, filter while hot through
a tared filtering crucible, rinse the container with several
portions of hot water, and pass the rinsings through the
crucible. Dry the crucible and its contents at 105° to
constant weight, cool, and weigh.
Acceptance criteria: The weight of the residue does not
exceed 15 mg. (NMT 1.0%)
• LOSS ON DRYING, Appendix IIC: 105° for 5 h
Sample preparation: Cut unground sample into 2- to
5-mm pieces before drying.
Acceptance criteria: NMT 20.0%
 FCC 8 Monographs / DL-Alanine / 31

• STARCH Sample preparation: Mineral oil mull

Analysis: Boil 100 mg of sample in 100 mL of water,
cool, and add a few drops of iodine TS.
Acceptance criteria: No blue color appears.
• WATER ABSORPTION
Sample: 5 g
Analysis: Place the Sample in a 100-mL graduated
cylinder, fill to volume with water, mix, and allow to
stand at about 25° for 24 h. Pour the contents of the
cylinder through moistened glass wool, allowing the
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.
ASSAY
• PROCEDURE
Sample: 200 mg
Analysis: Dissolve the Sample in 3 mL of formic acid and
50 mL of glacial acetic acid. Add 2 drops of crystal

water to drain into another 100-mL graduated cylinder.
Acceptance criteria: NMT 75 mL of water is obtained.
.
Monographs
violet TS and titrate with 0.1 N perchloric acid to a
blue-green endpoint. Perform a blank determination
(see General Provisions), and make any necessary
correction. Each mL of 0.1 N perchloric acid is
equivalent to 8.909 mg of C3H7NO2.
[CAUTION—Handle perchloric acid in an appropriate

DL-Alanine
fume hood.]
First Published: Prior to FCC 6 Acceptance criteria: NLT 98.5% and NMT 101.5% of
C3H7NO2, calculated on the dried basis
DL-2-Aminopropanoic Acid
IMPURITIES
Inorganic Impurities
• LEAD, Lead Limit Test, Appendix IIIB
Sample preparation: Prepare as directed for organic
compounds.
C3H7NO2 Formula wt 89.09 Control: 5 µg Pb (5 mL of Diluted Standard Lead
CAS: [302-72-7] Solution)
UNII: 1FU7983T0U [alanine, dl-] Acceptance criteria: NMT 5 mg/kg

DESCRIPTION SPECIFIC TESTS

DL-Alanine occurs as a white crystalline powder. It is freely • LOSS ON DRYING, Appendix IIC: 105° for 3 h
soluble in water, but sparingly soluble in alcohol. The pH Acceptance criteria: NMT 0.3%
of a 1:20 aqueous solution is between 5.5 and 7.0. It melts • RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
with decomposition at about 198°. It is optically inactive. Sample: 1 g
Function: Nutrient Acceptance criteria: NMT 0.2%
Packaging and Storage: Store in well-closed, light-
resistant containers.

IDENTIFICATION
• INFRARED ABSORPTION, Spectrophotometric Identification
Tests, Appendix IIIC
 32 / DL-Alanine / Monographs FCC 8
Monographs

DL-Alanine (Mineral Oil Mull)

ASSAY
L-Alanine • PROCEDURE
First Published: Prior to FCC 6 Sample: 200 mg
Last Revision: FCC 6 Analysis: Dissolve the Sample in 3 mL of formic acid and
50 mL of glacial acetic acid. Add 2 drops of crystal
L-2-Aminopropanoic Acid violet TS and titrate with 0.1 N perchloric acid to a
blue-green endpoint. Perform a blank determination
(see General Provisions), and make any necessary
correction. Each mL of 0.1 N perchloric acid is
equivalent to 8.909 mg of C3H7NO2. [CAUTION—Handle
perchloric acid in an appropriate fume hood.]
C3H7NO2 Formula wt 89.09 Acceptance criteria: NLT 98.5% and NMT 101.5% of
CAS: [56-41-7] C3H7NO2, calculated on the dried basis
UNII: OF5P57N2ZX [alanine]
IMPURITIES
DESCRIPTION Inorganic Impurities
L-Alanine occurs as a white crystalline powder. It is freely • LEAD, Lead Limit Test, Appendix IIIB
soluble in water, sparingly soluble in alcohol, and insoluble Sample solution: Prepare as directed for organic
in ether. The pH of a 1:20 aqueous solution is between 5.5 compounds.
and 7.0. Control: 5 µg Pb (5 mL of Diluted Standard Lead
Function: Nutrient Solution)
Packaging and Storage: Store in well-closed, light- Acceptance criteria: NMT 5 mg/kg
resistant containers.
SPECIFIC TESTS
IDENTIFICATION • LOSS ON DRYING, Appendix IIC: 105° for 3 h
• INFRARED ABSORPTION, Spectrophotometric Identification Acceptance criteria: NMT 0.3%
Tests, Appendix IIIC • OPTICAL (SPECIFIC) ROTATION, Appendix IIB
Reference standard: USP L-Alanine RS Sample: 10 g, previously dried
Sample and Standard preparation: K Analysis: Dissolve the Sample in sufficient 6 N
Acceptance criteria: The spectrum of the sample hydrochloric acid to make 100 mL.
exhibits maxima at the same wavelengths as those in Acceptance criteria
the spectrum of the Reference standard. [α]D20 between +13.5° and +15.5°, on the dried basis; or
 FCC 8 Monographs / Alitame / 33

[α]D25 between +13.2° and +15.2°, on the dried basis Acceptance criteria: NLT 20% and NMT 23% of carbon
• RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC dioxide (CO2), corresponding to between 91.0% and
Sample: 1 g 104.5% of (C6H8O6)n (equiv wt 200.00), calculated on
Acceptance criteria: NMT 0.2% the dried basis.

IMPURITIES
Inorganic Impurities
• ARSENIC, Arsenic Limit Test, Appendix IIIB
.

Alginic Acid Sample solution: Prepare as directed for organic

First Published: Prior to FCC 6 compounds.

Monographs
Acceptance criteria: NMT 3 mg/kg
(C6H8O6)n Formula wt, calculated 176.13 • LEAD, Lead Limit Test, Appendix IIIB
Formula wt, actual (avg.) 200.00 Sample solution: Prepare as directed for organic
INS: 400 CAS: [9005-32-7] compounds.
UNII: 8C3Z4148WZ [alginic acid] Control: 5 µg Pb (5 mL of Diluted Standard Lead
Solution)
DESCRIPTION Acceptance criteria: NMT 5 mg/kg
Alginic Acid occurs as a white to yellow-white, fibrous
powder. It is a hydrophilic colloidal carbohydrate extracted SPECIFIC TESTS
from various species of brown seaweeds (phaeophyceae) • LOSS ON DRYING, Appendix IIC: 105° for 4 h
with dilute alkali. It may be described chemically as a linear Acceptance criteria: NMT 15.0%
glycuronoglycan consisting mainly of β-1,4 linked D- • RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
mannuronic and L-guluronic acid units in the pyranose ring Sample: 3 g
form. Alginic Acid is insoluble in water, readily soluble in Acceptance criteria: NMT 8.0%, calculated on the dried
alkaline solutions, and insoluble in organic solvents. The pH basis
of a 3:100 suspension in water is between 2.0 and 3.4.
Function: Stabilizer; thickener; emulsifier
Packaging and Storage: Store in well-closed containers. .

IDENTIFICATION Alitame
• A. PROCEDURE First Published: FCC 7
Sample solution: 1:150 in 0.1 N sodium hydroxide Last Revision: Third Supplement, FCC 7
Analysis: Add 1 mL of calcium chloride TS to 5 mL of
Sample solution. L-α-Aspartyl-N-(2,2,4,4-tetramethyl-3-thietanyl)-D-
Acceptance criteria: A voluminous gelatinous precipitate alaninamide, hydrated
forms.
• B. PROCEDURE
Sample solution: 1:150 in 0.1 N sodium hydroxide
Analysis: Add 1 mL of 2 N sulfuric acid to 5 mL of
Sample solution.
Acceptance criteria: A heavy gelatinous precipitate
forms
• C. PROCEDURE C14H25N3O4S·2.5 H2O Formula wt hydrated 376.5
Sample: 5 mg INS: 956 CAS: hydrated [99016-42-9]
Analysis: Place the Sample in a test tube. Add 5 mL of UNII: 6KI9M51JOG [alitame]
water, 1 mL of a freshly prepared 1:100 solution of
naphtholresorcinol:ethanol, and 5 mL of hydrochloric
DESCRIPTION
Alitame occurs as a white, odorless, crystalline powder
acid. Heat the mixture to boiling, boil gently for about
having an intensely sweet taste. One method of production
3 min, and then cool to about 15°. Transfer the
is through a multi-step synthesis involving the reaction
contents of the test tube into a 30-mL separatory
between two intermediates, (S)-[2,5-dioxo-(4-thiazolidine)]
funnel with the aid of 5 mL of water, and extract with
acetic acid and (R)-2-amino-N-(2,2,4,4-tetramethyl-3-
15 mL of isopropyl ether. Perform a blank
thietanyl)propanamide. The final product is isolated and
determination (see General Provisions).
purified through crystallization of an alitame/4-
Acceptance criteria: The isopropyl ether extract from
methylbenzenesulfonic acid adduct followed by additional
the Sample exhibits a deeper purple hue than that from
purification steps, and finally recrystallization from water as
the blank.
the 2.5 hydrate. It is freely soluble in water and alcohol,
ASSAY and the pH of a 5% solution is between 5.0 and 6.0.
• ALGINATES ASSAY, Appendix IIIC Function: Sweetener; flavor enhancer
Analysis: Each mL of 0.25 N sodium hydroxide Packaging and Storage: Store in tight containers in a
consumed in the assay is equivalent to 25 mg of cool place.
(C6H8O6)n (equiv wt 200.00).
 34 / Alitame / Monographs
IDENTIFICATION
• A. INFRARED ABSORPTION, Spectrophotometric Identification
Tests, Appendix IIIC
Reference standard: USP Alitame RS
Sample and standard preparation: K
Acceptance criteria: The spectrum of the sample
exhibits maxima at the same wavelengths as those in
the spectrum of the Reference standard.
• B. PROCEDURE
Sample: 10 mg
Monographs
Analysis: To 5 mL of a solution containing 300 mg of
ninhydrin in 100 mL of n-butanol and 2 mL of glacial
acetic acid, add the Sample, and heat to gentle reflux.
Acceptance criteria: An intense blue-violet color is
formed.
• C. PROCEDURE
Sample: 10 mg
Analysis: To 5 mL of a freshly prepared 0.001 M
potassium permanganate solution, add the Sample, and
mix thoroughly.
Acceptance criteria: The purple solution changes to
brown.
ASSAY
• PROCEDURE
[NOTE—In this procedure, alitame and its impurities,
alanine amide (N-(2,2,4,4-tetramethyl-3-thietanyl)-D-
alaninamide) and beta-isomer (L-β-aspartyl-N-(2,2,4,4-
tetramethyl-3-thietanyl)-D-alaninamide hydrate) [2:5]),
are measured by reverse-phase ion-pair high
performance liquid chromatography.]
Solution A: Dissolve 0.69 g of sodium phosphate
monobasic monohydrate and 4.32 g of 1-
octanesulfonate, sodium in 200 mL of water. Adjust
with 85% phosphoric acid to a pH of 2.5, then dilute
with water to 1000 mL. Pass through a 0.22-µm
Millipore filter, or equivalent.
Mobile phase: Acetonitrile and Solution A (1:4). [NOTE—
Degas by sonication under aspirator vacuum for 2 min.]
Standard solution A: Transfer 25 mg each of a suitable
alanine amide reference standard and a suitable beta-
isomer reference standard to a 500-mL volumetric flask,
using 50 mL of methanol to aid in dissolution. Dilute
with water to volume. [NOTE—Store in a refrigerator.]
Dilute standard solution A: Transfer 15.0 mL of
Standard solution A to a 50-mL volumetric flask, and
dilute with water to volume.
Standard solution B: Transfer 50 mg of USP Alitame RS
to a 10-mL volumetric flask. Add 3 mL of water to
dissolve, then add 5 mL of Dilute standard solution A,
and dilute with water to volume.
Dilute standard solution B: Transfer 5 mL of Standard
solution B to a 50-mL volumetric flask, and dilute with
water to volume.
FCC 8
Sample solution: 5 mg/mL
Dilute sample solution: 0.5 mg/mL from the Sample
solution
Chromatographic system, Appendix IIA
Mode: High-performance liquid chromatography
Detector: UV 217 nm
Column: 15-cm × 0.39-cm NovaPak C18 reverse phase
ion-pair (Waters, or equivalent)
Flow rate: 1.0 mL/min. [NOTE—Maintain the Mobile
phase at a pressure and flow rate capable of giving the
elution times listed under Analysis.]
Injection size: 100 µL
System suitability
Sample: Dilute standard solution B (three replicates)
Suitability requirement: The relative standard
deviation is NMT 2% for the alitame peak area.
Analysis: [NOTE—All injections should be done in
triplicate. The retention times for the beta-isomer,
alitame, and alanine amide should be approximately 6,
10, and 15 min, respectively. If a column of a different
make or length is used, the retention times may vary
proportionally to the times listed.]
Equilibrate the column by pumping Mobile phase
through it until a drift-free baseline is obtained. Inject
the Dilute sample solution and Dilute standard solution B
into the chromatograph, and record the
chromatograms. Calculate the average peak areas for
alitame from both chromatograms.
Calculate the weight percentage for alitame in the
sample taken:
Result = (rDU/rDS) × (CDS/CDU) × 100
rDU = peak response for alitame from the Dilute
sample solution
rDS = peak response for alitame from Dilute
standard solution B
CDS = concentration of alitame in Dilute standard
solution B, corrected for water content and
purity (mg/mL)
CDU = concentration of the Dilute sample solution,
corrected for water (mg/mL)
Acceptance criteria: 98.0%–101.0% of alitame, on the
anhydrous basis
IMPURITIES
Inorganic Impurities
• LEAD, Lead Limit Test, Atomic Absorption Spectrophotometric
Graphite Furnace Method, Appendix IIIB
Sample: 5 g
Acceptance criteria: NMT 1 mg/kg
 FCC 8 Monographs / Allura Red / 35

Organic Impurities
• ALANINE AMIDE AND BETA-ISOMER
Solution A, Mobile phase, Standard solution A, Dilute
standard solution A, Standard solution B, Dilute
standard solution B, Sample solution, Dilute sample
solution, and Chromatographic system: Proceed as
directed in the Assay. C18H14N2O8S2Na2 Formula wt 496.43
Analysis: Proceed as directed in the Assay. Inject the INS: 129 CAS: [25956-17-6]
Sample solution and Standard solution B into the UNII: WZB9127XOA [fd&c red no. 40]
chromatograph, and record the chromatograms.
DESCRIPTION

Monographs
Calculate the average peak areas for the beta-isomer
Allura Red occurs as a red-brown powder or granule. It is
and alanine amide from both chromatograms.
principally the disodium salt of 6-hydroxy-5-[(2-methoxy-5-
Calculate the weight percentage of alanine amide and
methyl-4-sulfophenyl)azo]-2-naphthalenesulfonic acid. It
beta-isomer in the sample taken:
dissolves in water to give a solution red at neutrality and in
Result = (rU/rS) × (CS/CU) × 100 acid and dark red in base. It is insoluble in ethanol.
Function: Color
rU = peak response for the analyte from the Packaging and Storage: Store in well-closed containers.
Sample solution
rS = peak response for the analyte from Standard IDENTIFICATION
solution B • PROCEDURE
CS = concentration of the analyte in Standard Sample solution: 16.4 µg/mL
solution B, corrected for water content and Analysis: Adjust the pH of three aliquots of the Sample
purity (mg/mL) solution to pH 1, pH 7, and pH 13. Measure the
CU = concentration of the Sample solution, absorbance intensities (A) and wavelength maxima of
corrected for water (mg/mL) these solutions with a suitable UV-visible
Acceptance criteria spectrophotometer.
Alanine amide: NMT 0.2% on the anhydrous basis Acceptance criteria
Beta-isomer: NMT 0.3% on the anhydrous basis pH 1: A = 0.83 at 490 nm (Both neutral and acid
solutions exhibit a shoulder at about 410 nm.)
SPECIFIC TESTS pH 7: A = 0.87 at 500 nm
• RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC pH 13: A = 0.37 at 500 nm and A = 0.41 at 450 nm
Sample: 1 g
Acceptance criteria: NMT 1.0% ASSAY
• OPTICAL (SPECIFIC) ROTATION, Appendix IIB • TOTAL COLOR, Color Determination, Methods I and II,
Sample solution: 10 mg/mL, on the as-is (undried) Appendix IIIC: Both methods must be used.
basis Method I Spectrophotometric
Acceptance criteria: [α]D25 between +40° and +50° Sample: 175 to 225 mg
• WATER, Water Determination, Appendix IIB Analysis: Transfer the Sample into a 1-L volumetric
Acceptance criteria: 11%–13% flask; dissolve in and dilute to volume with water.
Determine as directed at 502 nm using 0.052 L/
(mg·cm) for the absorptivity (a) for Allura Red.
Method II TiCl3 Titration
Sample: 0.2 g
.

Allura Red1 Analysis: Determine as directed using 8.06 as the

First Published: Prior to FCC 6 stoichiometric factor (Fs) for Allura Red.
Acceptance criteria: The average of results obtained
Allura Red AC from Methods I and II is NLT 85.0% total coloring
CI 16035 matters.
Class: Monoazo
IMPURITIES
Inorganic Impurities
• ARSENIC, Appendix IIIB
Sample solution: Prepare as directed for organic
compounds.
1To be used or sold for use to color food that is marketed in the United Acceptance criteria: NMT 3 mg/kg
States, this color additive must be from a batch that has been certified by the • LEAD, Lead Limit Test, Appendix IIIB
U.S. Food and Drug Administration (FDA). If it is not from an FDA-certified Sample solution: Prepare as directed for organic
batch, it is not a permitted color additive for food use in the United States, compounds.
even if it is compositionally equivalent. The name FD&C Red No. 40 can be
applied only to FDA-certified batches of this color additive. Allura Red is a
Control: 10 µg Pb (10 mL of Diluted Standard Lead
common name given to the uncertified colorant. See the monograph entitled Solution)
FD&C Red No. 40 for directions for producing an FDA-certified batch. Acceptance criteria: NMT 10 mg/kg
 36 / Allura Red / Monographs
Organic Impurities
• UNCOMBINED INTERMEDIATES AND PRODUCTS OF SIDE
REACTIONS, Color Determination, Method II, Appendix IIIC
Sample solution: 250 mg/mL in 0.1 M disodium borate
(Na2B4O7)
Analysis: Use an injection volume of 20 µL for the
Sample solution.
Acceptance criteria
4-Amino-5-methoxy-o-toluenesulfonic acid: NMT
0.2%
Monographs
6,6’-Oxybis(2-naphthalenesulfonic acid), Disodium
salt: NMT 1.0%;
6-Hydroxy-2-naphthalenesulfonic acid, Sodium
salt: NMT 0.3%
SPECIFIC TESTS
• COMBINED TESTS
Tests
• LOSS ON DRYING (VOLATILE MATTER), Color Determination,
Appendix IIIC
• CHLORIDE, Sodium Chloride, Color Determination,
Appendix IIIC
• SULFATES (AS SODIUM SALTS), Sodium Sulfate, Color
Determination, Appendix IIIC
Acceptance criteria: NMT 15.0% in combination
• ETHER EXTRACTS, Color Determination, Appendix IIIC
Acceptance criteria: NMT 0.2%
• SUBSIDIARY COLORS, Thin-Layer Chromatography, Appendix
IIA
Adsorbent: Silica Gel G
Developing solvent system: Acetonitrile, ethyl acetate,
isoamyl alcohol, water and ammonium hydroxide
(5:5:5:5:1)
Standard solution: 20 mg/mL of purified Allura Red
(free of subsidiary colors) and 1 mg/mL each of lower
and higher sulfonated subsidiary colors. [NOTE—Store in
the dark.]
Sample solution: 20 mg/mL
Application volume: 3 µL
Analysis: Prepare a 20- × 20-cm glass plate coated with
a 0.25-mm layer of Adsorbent. Spot aliquots of the
Sample solution and the Standard solution side-by-side 3
cm from the bottom. [NOTE—Up to seven samples and
standards may be run simultaneously.] When the plate
has air dried for 15 min, develop it in an unlined tank
equilibrated with the Developing solvent system for at
least 20 min. Allow the solvent front to reach to within
3 cm from the top of the plate. Allow the plate to dry
in a fume hood, and by visual inspection, compare the
intensities of the lower and higher sulfonated subsidiary
colors with those in the Standard solution. If the
subsidiary colors in the Sample solution appear more
concentrated than those in the Standard solution,
determine the quantity of each, using a densitometer
set to monitor the absorbance maximum of each.
Calculate the percentage of each of the subsidiary
colors, if present above 0.1%, by the formula:
Result = (A × p)/AS
FCC 8
A = area of the densitometer curve
p = percent of subsidiary color in the Standard
solution
AS = area of the densitometer curve for the
subsidiary color in the Standard solution
Acceptance criteria
6-Hydroxy-5-[(2-methoxy-5-methyl-4-
sulfophenyl)azo]-8-(2-methoxy-5-methyl-4-
sulfophenoxy)-2-naphthalenesulfonic acid, Disodium
salt: NMT 1.0%
Higher and Lower Sulfonated Subsidiary Colors (as
sodium salts): NMT 1.0% each
• WATER-INSOLUBLE MATTER, Color Determination, Appendix
IIIC
Acceptance criteria: NMT 0.2%
Allyl α-Ionone
.
First Published: Prior to FCC 6
Allyl Ionone
C16H24O Formula wt 232.37
FEMA: 2033
UNII: 8IP66F9ODG [allyl α-ionone]
DESCRIPTION
Allyl α-Ionone occurs as a colorless to yellow liquid.
Odor: Fruity, woody
Solubility: Soluble in alcohol; insoluble or practically
insoluble in water
Boiling Point: ∼265°
Solubility in Alcohol, Appendix VI: One mL dissolves in 1
mL of 90% alcohol to give a clear solution.
Function: Flavoring agent
IDENTIFICATION
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.
ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix
XI.
Acceptance criteria: NLT 88.0% of C16H24O
SPECIFIC TESTS
• REFRACTIVE INDEX, Appendix II: At 20°
Acceptance criteria: Between 1.502 and 1.507
• SPECIFIC GRAVITY: Determine at 25° by any reliable
method (see General Provisions).
Acceptance criteria: Between 0.926 and 0.932
 FCC 8 Monographs / Allyl Cyclohexanepropionate / 37

OTHER REQUIREMENTS
• ALLYL ALCOHOL, M-1b, Appendix XI
Acceptance criteria: NMT 0.1%

Monographs
Allyl α-Ionone

. Acceptance criteria: The spectrum of the sample

Allyl Cyclohexanepropionate exhibits relative maxima at the same wavelengths as
First Published: Prior to FCC 6 those of the spectrum below.

ASSAY
Allyl-3-cyclohexanepropionate • PROCEDURE: Proceed as directed under M-1b, Appendix
XI.
Acceptance criteria: NLT 98.0% of C12H20O2

SPECIFIC TESTS
• ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
C12H20O2 Formula wt 196.29 OILS), M-15, Appendix XI
FEMA: 2026 Acceptance criteria: NMT 5.0
UNII: H4W9H3L241 [allyl cyclohexanepropionate] • REFRACTIVE INDEX, Appendix II: At 20°
Acceptance criteria: Between 1.457 and 1.462
DESCRIPTION • SPECIFIC GRAVITY: Determine at 25° by any reliable
Allyl Cyclohexanepropionate occurs as a colorless liquid. method (see General Provisions).
Odor: Pineapple Acceptance criteria: Between 0.945 and 0.950
Solubility: Miscible in alcohol, chloroform, ether; insoluble
or practically insoluble in glycerin, water OTHER REQUIREMENTS
Solubility in Alcohol, Appendix VI: One mL dissolves in 4 • ALLYL ALCOHOL, M-1b, Appendix XI
mL of 80% alcohol. Acceptance criteria: NMT 0.1%
Function: Flavoring agent

IDENTIFICATION
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
 38 / Allyl Cyclohexanepropionate / Monographs FCC 8
Monographs

Allyl Cyclohexanepropionate

. Acceptance criteria: The spectrum of the sample

Allyl Heptanoate exhibits relative maxima at the same wavelengths as
First Published: Prior to FCC 6 those of the spectrum below.

ASSAY
Allyl Heptoate • PROCEDURE: Proceed as directed under M-1b, Appendix
XI.
Acceptance criteria: NLT 97.0% of C10H18O2

SPECIFIC TESTS
C10H18O2 Formula wt 170.25 • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
FEMA: 2031 OILS), M-15, Appendix XI
UNII: AU4CYG9V68 [allyl heptanoate] Acceptance criteria: NMT 1.0
• REFRACTIVE INDEX, Appendix II: At 20°
DESCRIPTION Acceptance criteria: Between 1.426 and 1.430
Allyl Heptanoate occurs as a colorless to pale yellow liquid. • SPECIFIC GRAVITY: Determine at 25° by any reliable
Odor: Sweet, pineapple method (see General Provisions).
Boiling Point: ∼210° Acceptance criteria: Between 0.880 and 0.885
Solubility in Alcohol, Appendix VI: One mL dissolves in 1
mL of 95% alcohol. OTHER REQUIREMENTS
Function: Flavoring agent • ALLYL ALCOHOL, M-1b, Appendix XI
Acceptance criteria: NMT 0.1%
IDENTIFICATION
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
 FCC 8 Monographs / Allyl Hexanoate / 39

Monographs
Allyl Heptanoate

. Acceptance criteria: The spectrum of the sample

Allyl Hexanoate exhibits relative maxima at the same wavelengths as
First Published: Prior to FCC 6 those of the spectrum below.

ASSAY
Allyl Caproate • PROCEDURE: Proceed as directed under M-1b, Appendix
XI.
Acceptance criteria: NLT 98.0% of C9H16O2

SPECIFIC TESTS
C9H16O2 Formula wt 156.22 • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
FEMA: 2032 OILS), M-15, Appendix XI
UNII: 3VH84A363D [allyl hexanoate] Acceptance criteria: NMT 1.0
• REFRACTIVE INDEX, Appendix II: At 20°
DESCRIPTION Acceptance criteria: Between 1.422 and 1.426
Allyl Hexanoate occurs as a colorless to light yellow liquid. • SPECIFIC GRAVITY: Determine at 25° by any reliable
Odor: Strong, pineapple method (see General Provisions).
Solubility: Miscible in alcohol, most fixed oils; insoluble or Acceptance criteria: Between 0.884 and 0.890
practically insoluble in propylene glycol, water
Boiling Point: ∼185° OTHER REQUIREMENTS
Solubility in Alcohol, Appendix VI: One mL dissolves in 6 • ALLYL ALCOHOL, M-1b, Appendix XI
mL of 70% alcohol. Acceptance criteria: NMT 0.1%
Function: Flavoring agent

IDENTIFICATION
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
 40 / Allyl Hexanoate / Monographs FCC 8
Monographs

Allyl Hexanoate

. Acceptance criteria: The spectrum of the sample

Allyl Isothiocyanate exhibits relative maxima at the same wavelengths as
First Published: Prior to FCC 6 those of the spectrum below.

ASSAY
• PROCEDURE: Proceed as directed under M-1a, Appendix
XI.
Acceptance criteria: NLT 93.0% of C4H5NS
C4H5NS Formula wt 99.16 SPECIFIC TESTS
FEMA: 2034 • REFRACTIVE INDEX, Appendix II: At 20°
UNII: BN34FX42G3 [allyl isothiocyanate]
Acceptance criteria: Between 1.527 and 1.531
DESCRIPTION • SPECIFIC GRAVITY: Determine at 25° by any reliable
Allyl Isothiocyanate occurs as a colorless to pale yellow, method (see General Provisions).
strongly refractive liquid. Acceptance criteria: Between 1.013 and 1.020
Odor: Irritating, acrid taste, mustard [CAUTION— OTHER REQUIREMENTS
lachrymator] • ALLYL ALCOHOL, M-1b, Appendix XI
Solubility: Miscible in alcohol, carbon disulfide, ether Acceptance criteria: NMT 0.1%
Boiling Point: ∼150° • DISTILLATION RANGE, Appendix IIB
Function: Flavoring agent Acceptance criteria: Between 148° and 154°
IDENTIFICATION • PHENOLS, M-17, Appendix XI
• INFRARED SPECTRA, Spectrophotometric Identification Tests, Acceptance criteria: Passes test
Appendix IIIC
 FCC 8 Monographs / Allyl Isovalerate / 41

Monographs
Allyl Isothiocyanate

. Acceptance criteria: The spectrum of the sample

Allyl Isovalerate exhibits relative maxima at the same wavelengths as
First Published: Prior to FCC 6 those of the spectrum below.

ASSAY
Allyl Isopentanoate • PROCEDURE: Proceed as directed under M-1b, Appendix
XI.
Acceptance criteria: NLT 98.0% of C8H14O2 (one
isomer)

C8H14O2 Formula wt 142.20 SPECIFIC TESTS

FEMA: 2045 • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
UNII: 3551Z86V7T [allyl isovalerate] OILS), M-15, Appendix XI
Acceptance criteria: NMT 1.0
DESCRIPTION • REFRACTIVE INDEX, Appendix II: At 20°
Allyl Isovalerate occurs as a colorless to pale yellow liquid. Acceptance criteria: Between 1.413 and 1.418
Odor: Fruity, apple • SPECIFIC GRAVITY: Determine at 25° by any reliable
Boiling Point: ∼155° method (see General Provisions).
Solubility in Alcohol, Appendix VI: One mL dissolves in 1 Acceptance criteria: Between 0.879 and 0.884
mL of 95% alcohol.
Function: Flavoring agent OTHER REQUIREMENTS
• ALLYL ALCOHOL, M-1b, Appendix XI
IDENTIFICATION Acceptance criteria: NMT 0.1%
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
 42 / Allyl Isovalerate / Monographs FCC 8
Monographs

Allyl Isovalerate

. Function: Flavoring agent

Allyl Phenoxy Acetate
IDENTIFICATION
First Published: Prior to FCC 6
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
C11H12O3 Formula wt 192.21 • PROCEDURE: Proceed as directed under M-1b, Appendix
FEMA: 2038 XI.
UNII: Q3P8UAF9WE [allyl phenoxyacetate] Acceptance criteria: NLT 97.0% of C11H12O3

DESCRIPTION SPECIFIC TESTS

Allyl Phenoxy Acetate occurs as a colorless to pale yellow • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
liquid. OILS), M-15, Appendix XI
Odor: Honey, pineapple Acceptance criteria: NMT 1.0
Solubility: Slightly soluble in propylene glycol; very slightly • REFRACTIVE INDEX, Appendix II: At 20°
soluble in water; insoluble or practically insoluble in Acceptance criteria: Between 1.513 and 1.518
vegetable oils • SPECIFIC GRAVITY: Determine at 25° by any reliable
Boiling Point: ∼265° method (see General Provisions).
Solubility in Alcohol, Appendix VI: One mL dissolves in 1 Acceptance criteria: Between 1.100 and 1.105
mL of 95% ethanol.
 FCC 8 Monographs / Allyl Propionate / 43

Monographs
Allyl Phenoxy Acetate

IDENTIFICATION
Allyl Propionate • INFRARED SPECTRA, Spectrophotometric Identification Tests,
First Published: Prior to FCC 6 Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.

ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix
C6H10O2 Formula wt 114.15 XI.
FEMA: 2040 Acceptance criteria: NLT 97.0% of C6H10O2
UNII: 0OYW8C5029 [allyl propionate]
SPECIFIC TESTS
DESCRIPTION • ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
Allyl Propionate occurs as a colorless to pale yellow liquid. OILS), M-15, Appendix XI
Odor: Ethereal, fruity Acceptance criteria: NMT 2.0
Solubility: Soluble in propylene glycol, vegetable oils; • REFRACTIVE INDEX, Appendix II: At 20°
insoluble or practically insoluble in water Acceptance criteria: Between 1.408 and 1.413
Boiling Point: ∼124° • SPECIFIC GRAVITY: Determine at 25° by any reliable
Solubility in Alcohol, Appendix VI method (see General Provisions).
One mL dissolves in 1 mL of 95% ethanol. Acceptance criteria: Between 0.912 and 0.917
Function: Flavoring agent
 44 / Allyl Propionate / Monographs
Monographs
Allyl Propionate
Almond Oil, Bitter, FFPA
First Published: Prior to FCC 6
Last Revision: FCC 6
Bitter Almond Oil Free from Prussic Acid
CAS: [8013-76-1]
UNII: 6TQK77W0EX [bitter almond oil]
DESCRIPTION
Almond Oil, Bitter, FFPA, occurs as a colorless to slightly
yellow liquid with a strong almond aroma and a slightly
astringent, mild taste. It is a volatile oil obtained from the
nuts of the bitter almond tree, Prunus amygdalus Batsch
var. amara (De Candolle) Focke (Fam. Rosaceae), apricot
kernel (Prunus armeniaca L.), and other fruit kernels
containing amygdalin. It is prepared by steam distillation of
a water-macerated, powdered, and pressed cake that has
been specially treated and redistilled to remove
hydrocyanic acid. It is soluble in most fixed oils and in
propylene glycol, slightly soluble in mineral oil, and
insoluble in glycerin.
Function: Flavoring agent
Packaging and Storage: Store in a cool place protected
from light in full, tight containers that are made from steel
or aluminum and that are suitably lined.
IDENTIFICATION
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
Appendix IIIC
Acceptance criteria: The spectrum of the sample
exhibits relative maxima at the same wavelengths as
those of the spectrum below.
FCC 8
ASSAY
• ALDEHYDES, Appendix VI
Sample: 1 mL
Analysis: Use 53.05 for the equivalence factor (e) in the
calculation.
Acceptance criteria: NLT 95.0% of aldehydes,
calculated as benzaldehyde (C7H6O)
SPECIFIC TESTS
• ACID VALUE (ESSENTIAL OILS AND FLAVORS), Appendix VI
Acceptance criteria: NMT 8.0
• CHLORINATED COMPOUNDS, Appendix VI
Acceptance criteria: Passes test
• HYDROCYANIC ACID
Sample: 1 mL
Analysis: Transfer the Sample into a test tube and add 1
mL of water, 5 drops of a 100 mg/mL sodium
hydroxide solution, and 5 drops of a 100 mg/mL
solution of ferrous sulfate solution. Shake the test tube
thoroughly, and acidify with 0.5 N hydrochloric acid.
Acceptance criteria: No blue precipitate or color
appears. (about 0.015%)
• OPTICAL (SPECIFIC) ROTATION, Appendix IIB: Use a 100 mm
tube.
Acceptance criteria: Optically inactive, or NMT ±0.15°
• REFRACTIVE INDEX, Appendix IIB
[NOTE—Use an Abbé or other refractometer of equal or
greater accuracy.]
Acceptance criteria: Between 1.541 and 1.546 at 20°
• SOLUBILITY IN ALCOHOL, Appendix VI
Acceptance criteria: One mL of sample dissolves in 2
mL of 70% alcohol to form a clear solution.
 Next Page

FCC 8 Monographs / Aluminum Ammonium Sulfate / 45

• SPECIFIC GRAVITY: Determine by any reliable method (see Acceptance criteria: Between 1.040 and 1.050
General Provisions).

Monographs
Almond Oil, Bitter, FFPA

. pH 4.5 Buffer solution: 77.1 g of ammonium acetate

Aluminum Ammonium Sulfate and 57 mL of glacial acetic acid diluted to 1000 mL
First Published: Prior to FCC 6 Analysis: Dissolve the Sample in 50 mL of water, add
50.0 mL of 0.05 M disodium EDTA and 20 mL of pH
4.5 Buffer solution, and boil gently for 5 min. Cool and
Ammonium Alum
add 50 mL of alcohol and 2 mL of dithizone TS. Back
AlNH4(SO4)2·12H2O Formula wt 453.32 titrate with 0.05 M zinc sulfate to a bright rose-pink
INS: 523 CAS: [7784-25-0] color. Perform a blank determination (see General
UNII: 5C36DRL9ZN [ammonium alum] Provisions) and make any necessary correction. The
volume of 0.05 M disodium EDTA consumed (in mL) is
DESCRIPTION
equivalent to 50 minus the mL of 0.05 M zinc sulfate
Aluminum Ammonium Sulfate occurs as large, colorless
used. Each mL of 0.05 M disodium EDTA consumed is
crystals, white granules, or a powder. One g dissolves in 7
equivalent to 22.67 mg of AlNH4(SO4)2·12H2O.
mL of water at 25° and in about 0.3 mL of boiling water.
Acceptance criteria: NLT 99.5% and NMT 100.5% of
Its solutions are acid to litmus. It is insoluble in alcohol,
AlNH4(SO4)2·12H2O
and is freely, but slowly, soluble in glycerin.
Function: Buffer; neutralizing agent IMPURITIES
Packaging and Storage: Store in well-closed containers. Inorganic Impurities
• FLUORIDE, Fluoride Limit Test, Method V, Appendix IIIB
IDENTIFICATION
Acceptance criteria: NMT 0.003%
• ALUMINUM, Appendix IIIA
• LEAD, Lead Limit Test, APDC Extraction Method, Appendix
Sample solution: 50 mg/mL
IIIB
Acceptance criteria: Passes tests
Acceptance criteria: NMT 3 mg/kg
• AMMONIUM, Appendix IIIA
• SELENIUM, Selenium Limit Test, Method II, Appendix IIIB
Sample solution: 50 mg/mL
Sample: 200 mg
Acceptance criteria: Passes test
Acceptance criteria: NMT 0.003%
• SULFATE, Appendix IIIA
Sample solution: 50 mg/mL SPECIFIC TESTS
Acceptance criteria: Passes tests • ALKALIES AND ALKALINE EARTHS
Sample: 1 g
ASSAY
Analysis: Completely precipitate the aluminum from a
• PROCEDURE
boiling solution of the Sample in 100 mL of water by
Sample: 1 g
 FCC 8
Provisional Monographs
Meso-Zeaxanthin
.
First Published: First Supplement, FCC 7
Last Revision: Third Supplement, FCC 7
β-β-Carotene-3,3′-diol, (3R,3′S)-
(3R,3′S-meso)-Zeaxanthin
ph l
ra a
C40H56O2
n Formula wt 568.88
s
CAS: [31272-50-1]
UNII: CV0IB81ORO [zeaxanthin]
og io
DESCRIPTION
Meso-Zeaxanthin occurs as a free-flowing, orange to pale-
yellow powder. It is the purified fraction obtained from
on vis
isomerization of lutein from Tagetes erecta L., which
contains both the (3R,3S′-meso)-zeaxanthin and the (3R,
3′R)-zeaxanthin isomers with approximate concentrations
of 94% and 6% (of total zeaxanthin), respectively.
Function: Source of meso-zeaxanthin; color
Packaging and Storage: Store in tight, light-resistant
o
containers in a cool place.
Pr
IDENTIFICATION
• A. ULTRAVIOLET ABSORPTION
Acceptance criteria: The Sample solution from the test
for Total Carotenoids shows an absorption maximum at
about 453 nm.
• B. PROCEDURE
Acceptance criteria: The retention time of the major
peak in the chromatogram of the Sample solution
M
corresponds to that in the chromatogram of the
Standard solution as obtained in the Assay for
Zeaxanthin.
• C. STEREOISOMERIC COMPOSITION
Mobile phase: Hexane, alcohol, and isopropanol
(80:5:5)
Sample solution: Transfer 10 mg of sample to a 100-mL
volumetric flask, add 50 mL of alcohol, and place the
flask in an ultrasonic bath at 60° for 2–5 min to dissolve
the sample. Remove the flask from the bath, cool to
room temperature, and dilute with hexane to volume.
Filter the solution through a 0.45-µm filter membrane.
Standard solution: Prepare a solution containing 0.1
mg/mL of USP Meso-Zeaxanthin RS as follows: dissolve
an amount of Reference Standard in an amount of
ethanol equal to 50% of the final volume of the
solution. Heat in an ultrasonic bath at 60° for 2–5 min
to dissolve the Reference Standard. Remove the flask
from the bath, cool to room temperature, and dilute
with hexane to the desired volume (the volume of
hexane used in the solution should equal the volume of
Provisional Monographs / Meso-Zeaxanthin / 1209
ethanol used to dissolve the standard). Filter the
solution through a 0.45-µm filter membrane.
Chromatographic system, Appendix IIA
Mode: High-performance liquid chromatography
Detector: 453 nm
Column: 4.6-mm × 25-cm column containing amylose
tris-3,5-dimethylphenylcarbamate-coated, porous,
spherical silica particles, 5-µm in diameter1
Column temperature: 35°
Flow rate: 0.5 mL/min
Injection size: 20 µL
System suitability
Sample: Standard solution
[NOTE—The approximate relative retention times for (3S,
3’S)-zeaxanthin, (3R,3’S, meso)-zeaxanthin, (3R,3’R)-
Provisional Monographs
zeaxanthin, and (3R,3’R,6’R)-lutein are 0.94, 1.00,
1.06, and 1.11, respectively. The chromatogram from
the Standard solution should be similar to the Reference
Chromatogram provided with the USP Meso-
Zeaxanthin RS being used.]
Suitability requirement: The resolution between each
pair peak due to (3S,3’S)-zeaxanthin, (3R,3’S, meso)-
zeaxanthin, (3R,3’R)-zeaxanthin, and (3R,3’R,6’R)-
lutein is NLT 1.5.
Analysis: Separately inject the Sample solution and the
Standard solution into the chromatograph, record the
chromatograms, and compare them to the Reference
Chromatogram provided with the USP Meso-
Zeaxanthin RS being used in order to identify the
relevant analyte peaks. Measure the peak areas and
calculate the percentage of (3S,3’S)-zeaxanthin, (3R,3’S,
meso)-zeaxanthin, and (3R,3’R)-zeaxanthin:
Result = (rU/rT) × 100
rU = peak area of the analyte of interest
rT = total peak area of the chromatogram
Acceptance criteria
(3S,3’S)-Zeaxanthin: NMT 1.0%
(3R,3’S, meso)-Zeaxanthin: NLT 85.0%
(3R,3’R)-Zeaxanthin: NMT 15.0%
ASSAY
• ZEAXANTHIN
[NOTE—Use low-actinic glassware.]
Mobile phase: Hexane and ethyl acetate (75:25);
filtered and degassed. Make adjustments if necessary.
Standard solution: 150 µg/mL of USP Meso-Zeaxanthin
RS prepared as follows: dissolve 15.0 mg of USP Meso-
Zeaxanthin RS in 10 mL of chloroform, swirling briefly,
and dilute with Mobile phase to 100 mL.
Sample solution: Transfer 15.0 mg of sample to a 100-
mL volumetric flask, add 10 mL of chloroform, and
place the flask in an ultrasonic bath at 30° for 2–5 min
to obtain a clear solution. Dilute with Mobile phase to
volume.
1 Chiralpak AD-H from Chiral Technologies, or equivalent.
 1210 / Meso-Zeaxanthin / Provisional Monographs
Chromatographic system, Appendix IIA
Mode: High-performance liquid chromatography
Detector: 453 nm
Column: 4.6-mm × 25-cm column that contains 5- to
10-µm porous silica packing2
Flow rate: 1.5 mL/min
Injection size: 10 µL
System suitability
Sample: Standard solution
[NOTE—The approximate relative retention times for
lutein and zeaxanthin are about 0.95 and 1.0,
respectively.]
Suitability requirement 1: The resolution between
zeaxanthin and lutein is NLT 1.0.
Suitability requirement 2: The tailing factor is NMT
ph l
2.
ra a
Suitability requirement 3: The relative standard
deviation for replicate injections is NMT 2.0%.
n
Analysis: Inject the Sample solution into the
s
Provisional Monographs
chromatograph, record the chromatogram, and
measure the peak area responses. [NOTE—The peak area
og io
of zeaxanthin is NLT 90.0% of the total detected area
of peaks in the chromatogram.]
Calculate the percentage of zeaxanthin in the sample
on vis
taken:
Result = T × (rU/rT)
T = percentage of Total Carotenoids determined
below
rU = peak response of zeaxanthin
o
rT = sum of the responses of all of the peaks
Acceptance criteria: NLT 74.0%
Pr
• TOTAL CAROTENOIDS
[NOTE—Use low-actinic glassware.]
Sample stock solution: Transfer 25.0 mg of the sample
to a 100-mL volumetric flask, add 20 mL of chloroform,
and place the flask in an ultrasonic bath at 30° for 2–5
min to obtain a clear solution. Dilute with cyclohexane
to volume to obtain a solution containing 250 µg/mL.
Sample solution: 2.5 µg/mL in cyclohexane from the
M
Sample stock solution
Blank: Cyclohexane
Analysis: Determine the absorbance of the Sample
solution against that of the Blank at the wavelength of
maximum absorbance at about 453 nm, with a suitable
spectrophotometer.
Calculate the percentage of total carotenoids as
zeaxanthin (C40H56O2):
Result = A/(C × F)
A = absorbance of the Sample solution
C = concentration of the Sample solution (g/mL)
F = absorptivity of zeaxanthin in cyclohexane
(2540 mL·g−1·cm−1)
Acceptance criteria: NLT 80.0%
2 Agilent Zorbax Rx-SIL, or equivalent.
FCC 8
IMPURITIES
Inorganic Impurities
• LEAD, Lead Limit Test, Flame Atomic Absorption
Spectrophotometric Method, Appendix IIIB
Sample: 10 g
Acceptance criteria: NMT 1 mg/kg
Organic Impurities
• LUTEIN AND OTHER RELATED COMPOUNDS
[NOTE—Use low-actinic glassware.]
Mobile phase: Hexane and ethyl acetate (75:25);
filtered and degassed. Make adjustments if necessary.
Standard solution: 150 µg/mL of USP Meso-Zeaxanthin
RS prepared as follows: dissolve 15.0 mg of USP Meso-
Zeaxanthin RS in 10 mL of chloroform, swirling briefly,
and dilute with Mobile phase to 100 mL.
Sample solution: Transfer 15.0 mg of sample to a 100-
mL volumetric flask, add 10 mL of chloroform, and
place the flask in an ultrasonic bath at 30° for 2–5 min
to obtain a clear solution. Dilute with Mobile phase to
volume.
Chromatographic system, Appendix IIA
Mode: High-performance liquid chromatography
Detector: 453 nm
Column: 4.6-mm × 25-cm column that contains 5- to
10-µm porous silica packing2
Flow rate: 1.5 mL/min
Injection size: 10 µL
System suitability
Sample: Standard solution
[NOTE—The approximate relative retention times for
lutein and zeaxanthin are about 0.95 and 1.0,
respectively.]
Suitability requirement 1: The resolution between
zeaxanthin and lutein is NLT 1.0.
Suitability requirement 2: The tailing factor is NMT
2.
Suitability requirement 3: The relative standard
deviation for replicate injections is NMT 2.0%.
Analysis: Inject the Sample solution into the
chromatograph, record the chromatogram, and
measure the peak area responses. [NOTE—The peak
area of lutein is NMT 9.0% of the total detected area
of peaks in the chromatogram of the Sample solution.]
Calculate the percentage of lutein in the sample taken:
Result = T × (rU/rT)
T = percentage of Total Carotenoids determined
above
rU = peak response of lutein
rT = sum of the responses of all of the peaks
Calculate the percentage of other related compounds in
the portion of the sample taken:
Result = 100 × (rO/rT)
rO = individual peak response of any other peak
in the chromatogram, excluding zeaxanthin
and lutein
rT = sum of the responses of all of the peaks
FCC 8 Provisional Monographs / Meso-Zeaxanthin / 1211

Acceptance criteria Acceptance criteria: NMT 1.0%

Lutein: NMT 8.5% • WATER, Water Determination, Appendix IIB
Other related compounds: NMT 1.0% of any other Acceptance criteria: NMT 1.0%
single related compound

SPECIFIC TESTS
• RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
Analysis: Proceed as directed, but igniting at 600 ± 50°.

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FCC 8 General Tests and Assays / Contents / 1213

General Tests and Assays

CONTENTS

APPENDIX I: APPARATUS FOR TESTS Lead Limit Test ........................................ 1270

AND ASSAYS ................................................. 1217 Manganese Limit Test ............................. 1275
Oxygen Flask Combustion ...................... 1217 Mercury Limit Test .................................. 1275
Thermometers ......................................... 1217 Nickel Limit Test...................................... 1276
Volumetric Apparatus .............................. 1218 Phosphorus Limit Test ............................. 1277
Weights And Balances ............................. 1219 Selenium Limit Test ................................. 1278
C. Others.................................................... 1279
APPENDIX II: PHYSICAL TESTS AND Alginates Assay ........................................ 1279
DETERMINATIONS ........................................ 1221 α-Amino Nitrogen (AN)
A. Chromatography................................. 1221 Determination...................................... 1280
Column Chromatography ....................... 1222 Ammonia Nitrogen (NH 3-N)
Paper Chromatography ........................... 1222 Determination...................................... 1280
Thin-Layer Chromatography ................... 1223 Benzene .................................................. 1280
Gas Chromatography .............................. 1224 Color Determination ............................... 1283
High-Performance Liquid Elemental Impurities by ICP .................... 1289
Chromatography ................................. 1226 Glutamic Acid ......................................... 1291
B. Physicochemical Properties ............... 1230 Hydroxypropoxyl Determination ............. 1291
Distillation Range .................................... 1230 Methoxyl Determination ......................... 1292
Melting Range or T emperature Nitrogen Determination .......................... 1293
Determination...................................... 1231 Spectrophotometric Identification
Optical (Specific) Rotation ....................... 1232 Tests .................................................... 1294
pH Determination ................................... 1233 Sulfur ...................................................... 1295
Readily Carbonizable Substances ............. 1233

General Tests and Assays

Refractive Index ....................................... 1234 APPENDIX IV: CHEWING GUM BASE
Solidification Point .................................. 1234 POLYMERS .................................................... 1298
Viscosity Determination ........................... 1236 Bound Styrene......................................... 1298
Water Determination ............................... 1239 Molecular Weight .................................... 1299
C. Others.................................................... 1242 Quinones................................................. 1299
Ash (Acid-Insoluble) ................................ 1242 Residual Styrene ...................................... 1300
Ash (Total)............................................... 1242 Sample Solution for Arsenic
Hydrochloric Acid Table .......................... 1242 Limit Test ............................................. 1301
Loss on Dr ying ........................................ 1243 Sample Solution for Lead
Nuclear Magnetic Resonance .................. 1244 Limit Test ............................................. 1301
Oil Content of Synthetic Paraffin ............. 1251 Total Unsaturation ................................... 1301
Plasma Spectrochemistry......................... 1253
Residue on Ignition (Sulfated Ash) .......... 1259 APPENDIX V: ENZYME ASSAYS...................... 1303
Sieve Analysis of Granular Enzyme Preparations Used in Food
Metal Powders ..................................... 1259 Processing............................................ 1303
Sulfuric Acid Table................................... 1260 Acid Phosphatase Activity ........................ 1306
Water-Insoluble Matter ............................ 1261 Aminopeptidase (Leucine) Activity .......... 1307
α-Amylase Activity (Nonbacterial) ........... 1308
APPENDIX III: CHEMICAL TESTS AND α-Amylase Activity (Bacterial) .................. 1309
DETERMINATIONS ........................................ 1262 Catalase Activity ...................................... 1309
A. Identification Tests ............................. 1262 Cellulase Activity ..................................... 1310
B. Limit Tests ............................................ 1264 Chymotrypsin Activity ............................. 1311
Aluminum Limit Test ............................... 1264 Diastase Activity (Diastatic Power) ........... 1312
Arsenic Limit Test .................................... 1264 α-Galactosidase Activity........................... 1313
Cadmium Limit Test ................................ 1266 β-Glucanase Activity ................................ 1313
Chloride and Sulfate Limit T ests .............. 1266 Glucoamylase Activity (Amyloglucosidase
1,4-Dioxane Limit Test ............................ 1267 Activity) ............................................... 1314
Fluoride Limit Test ................................... 1268 Glucose Isomerase Activity ...................... 1315
 1214 / Contents / General Tests and Assays FCC 8

Glucose Oxidase Activity ......................... 1316 Lovibond Color ....................................... 1342

Hemicellulase Activity .............................. 1317 Fatty Acid Composition ........................... 1343
Invertase Sumner Unit Activity ................ 1318 Fatty Acid Composition (Saturated,
Lactase (Neutral) ( β-Galactosidase) cis-Monounsaturated, and
Activity................................................. 1319 cis-Polyunsaturated) in
Lactase (Acid) ( β-Galactosidase) Oils Containing Long Chain
Activity................................................. 1320 Polyunsaturated Fatty Acids ................. 1344
Lipase Activity ......................................... 1321 Free Fatty Acids ....................................... 1346
Lipase (Microbial) Activity for Medium- Free Glycerin or Propylene Glycol ........... 1347
and Long-Chain Fatty Acids ................. 1322 Hexane-Insoluble Matter ......................... 1347
Lysozyme Activity .................................... 1323 Hydroxyl Value ........................................ 1347
Maltogenic Amylase Activity .................... 1324 Iodine Value ............................................ 1348
Milk-Clotting Activity............................... 1324 Melting Range (Fats and Related
Pancreatin Activity ................................... 1325 Substances).......................................... 1348
Pepsin Activity ......................................... 1327 1-Monoglycerides.................................... 1348
Phospholipase A 2 Activity ........................ 1328 Total Monoglycerides .............................. 1349
Phytase Activity ....................................... 1328 Oxyethylene Determination .................... 1350
Plant Proteolytic Activity .......................... 1330 Peroxide Value......................................... 1351
Proteolytic Activity, Bacterial (PC) ........... 1331 Reichert-Meissl Value ............................... 1352
Proteolytic Activity, Fungal (HUT) ........... 1331 Saponification Value ................................ 1352
Proteolytic Activity, Fungal (SAP) ............ 1332 Soap........................................................ 1353
Pullulanase Activity .................................. 1333 Specific Gravity ....................................... 1353
Transglutaminase Activity ........................ 1334 Stability (Fats and Related Substances) .... 1353
Trypsin Activity ........................................ 1335 Tocopherols............................................. 1354
Unsaponifiable Matter ............................. 1355
APPENDIX VI: ESSENTIAL OILS AND Volatile Acidity......................................... 1356
FLAVORS ....................................................... 1336
Acetals..................................................... 1336 APPENDIX VIII: OLEORESINS.......................... 1357
General Tests and Assays

Acid Value (Essential Oils and Flavors) ..... 1336 Color Value (Oleoresins) .......................... 1357
Aldehydes................................................ 1336 Curcumin Content .................................. 1357
Aldehydes and Ketones ........................... 1336 Piperine Content ..................................... 1357
Chlorinated Compounds ......................... 1337 Residual Solvent (Oleoresins) ................... 1358
Esters....................................................... 1337 Total Capsaicinoids Content .................... 1359
Linalool Determination ............................ 1338 Volatile Oil Content (Oleoresins) ............. 1359
Percentage of Cineole ............................. 1338
Phenols ................................................... 1338 APPENDIX IX: ROSINS AND RELATED
Phenols, Free ........................................... 1339 SUBSTANCES ................................................. 1360
Residue on Evaporation ........................... 1339 Acid Number (Rosins and Related
Solubility in Alcohol ................................ 1339 Substances).......................................... 1360
Total Alcohols .......................................... 1339 Softening Point ....................................... 1360
Ultraviolet Absorbance of Citrus Viscosity (Rosins and Related
Oils ...................................................... 1339 Substances).......................................... 1363
Volatile Oil Content (Essential Oils
and Flavors) ......................................... 1340 APPENDIX X: CARBOHYDRATES
(STARCHES, SUGARS, AND RELATED
APPENDIX VII: FATS AND RELATED SUBSTANCES ................................................. 1364
SUBSTANCES ................................................. 1341 Acetyl Groups.......................................... 1364
Acetyl Value............................................. 1341 Crude Fat ................................................ 1364
Acid Value (Fats and Related Invert Sugar Determination ..................... 1364
Substances).......................................... 1341 Lactose Determination ............................ 1366
Anisidine Value ........................................ 1341 Propylene Chlorohydrin Determination ... 1366
Chlorophyll ............................................. 1342 Reducing Sugars Assay ............................ 1367
Cold Test................................................. 1342 Sulfur Dioxide Determination .................. 1368
Color (Fats and Related Substances) ....... 1342 Total Solids .............................................. 1370
FCC 8
APPENDIX XI: FLAVOR CHEMICALS
(OTHER THAN ESSENTIAL OILS) ................ 1375
M-1 Assay by Gas Chromatography............... 1375
GC Conditions for Analysis...................... 1375
Calculations and Methods....................... 1375
GC System Suitability Test Sample .......... 1376
M-2 Assays for Certain Aldehydes and
Ketones....................................................... 1376
M-3 Assay by Titrimetric Procedures .............. 1377
M-4 Alcohol Content of Ethyl
Oxyhydrate................................................. 1378
M-5 Acidity Determination by
Iodometric Method..................................... 1378
M-6 Limit Test for Antioxidants in
Ethyl Acrylate.............................................. 1378
M-7 Limit Test for Hydrocarbons in
Eugenol ...................................................... 1379
M-8 Limit Test for Hydrocyanic Acid in
Benzaldehyde ............................................. 1379
M-9 Limit Test for Lead .................................. 1379
M-10 Limit Test for Methyl Compounds
in Ethyl Acetate........................................... 1379
M-11 Limit Test for Peroxide Value ................ 1379
M-12 Limit Test for Readily Carbonizable
Substances in Ethyl Acetate ........................ 1379
M-13 Limit Test for Readily Oxidizable
Substances in dl-Menthol............................ 1380
M-14 Limit Test for Reducing
Substances.................................................. 1380
M-15 Acid Value, Flavor Chemicals (Other than
Essential Oils).............................................. 1380
M-16 Residue On Evaporation ....................... 1380
M-17 Qualitative Test for Phenols Using
Ferric Chloride ............................................ 1380
General Tests and Assays / Contents / 1215
APPENDIX XII: MICROBIOLIGICAL TESTS ..... 1381
Media and Reagents................................ 1381
Microbiological Enumeration Tests .......... 1382
Total Aerobic Microbial Count................. 1382
Total Yeasts and Molds Count ................. 1382
Tests for Absence of Specific
Microorganisms ................................... 1382
Bile-Tolerant Gram-Negative Bacteria ...... 1383
Enterobacter sakazakii
(Cronobacter Spp.)................................ 1383
Salmonella Spp......................................... 1383
APPENDIX XIII: ADULTERANTS AND CONTAMI-
NANTS IN FOOD INGREDIENTS................... 1384
Diethylene Glycol and Ethylene Glycol in
Glycerin................................................... 1384
Pesticide Residues.................................... 1384
APPENDIX XIV: MARKERS FOR AUTHENTICITY
TESTING ........................................................ 1388
Biobased Content of 1,3-Propanediol...... 1388
SOLUTIONS AND INDICATORS ....................... 1393
Colormetric Solutions .............................. 1393
Standard Buffer Solutions ........................ 1393
Standard Solutions for the Preparation
of Controls and Standards ................... 1394
Test Solutions (TS) and Other
General Tests and Assays
Reagents .............................................. 1394
Volumetric Solutions................................ 1400
Indicators ................................................ 1404
Indicator Papers and Test Papers ............. 1406
Detector Tubes ........................................ 1406
General Tests and Assays
FCC 8
General Tests and Assays
APPENDIX I: APPARATUS FOR TESTS AND ASSAYS
OXYGEN FLASK COMBUSTION
Apparatus The apparatus consists of a heavy-walled,
deeply lipped or cupped, conical flask of a volume suitable
for the complete combustion of the sample in which the
particular element is being determined (e.g., see Selenium
Limit Test, Appendix IIIB). The flask is fitted with a ground-
glass stopper to which is fused a sample carrier consisting of
heavy-gauge platinum wire and a piece of welded platinum
gauze measuring about 1.5 × 2 cm. A suitable apparatus
may be obtained as Catalog Nos. 6513-C20 (500-mL capac-
ity) and 6513-C30 (1000-mL capacity) from the Arthur H.
Thomas Co., P.O. Box 779, Philadelphia, PA 19105. Equiva-
lent apparatus available from other sources, or other suitable
apparatus embodying the principles described herein, may
also be used.
[CAUTION—Analysts should wear safety
Procedure
glasses and should use a suitable safety shield between
themselves and the apparatus. Further safety measures
should be observed as necessary to ensure maximum pro-
tection of the analysts. Furthermore, the flask must be scru-
pulously clean and free from even traces of organic solvents.
Samples containing water of hydration or more than 1% of
moisture should be dried at 140° for 2 h before combus-
tion, unless otherwise directed.] Accurately weigh the
amount of sample specified in the monograph or general
test. Solids should be weighed on a 4-cm square piece of
halide-free paper, which should be folded around the sam-
ple. Liquid samples not exceeding 0.2 mL in volume should
be weighed in tared cellulose acetate capsules [available as
Catalog Nos. 6513-C80 (100 capsules) and 6513-C82 (1000
capsules) from the Arthur H. Thomas Co.]; gelatin capsules
are satisfactory for liquid samples exceeding 0.2 mL in
volume.
[NOTE—Gelatin capsules may contain significant
amounts of combined halide or sulfur, in which case a
blank determination should be made as necessary.]
Place the sample, together with a filter paper fuse-strip, in
the platinum gauze sample holder. Place the absorbing liq-
uid, as specified in the individual monograph or general
test, into the flask, moisten the joint of the stopper with
water, and flush the air from the flask with a stream of
rapidly flowing oxygen, swirling the liquid to facilitate its
taking up oxygen.
[NOTE—Saturation of the liquid with oxygen is essen-
tial for successful performance of this procedure.]
General Tests and Assays / Appendix I / 1217
Ignite the fuse-strip by suitable means. If the strip is ig-
nited outside the flask, immediately plunge the sample
holder into the flask, invert the flask so that the absorption
solution makes a seal around the stopper, and hold the
stopper firmly in place. If the ignition is carried out in a
closed system, the inversion of the flask may be omitted.
After combustion is complete, shake the flask vigorously,
and allow it to stand for not less than 10 min with intermit-
tent shaking. Continue as directed in the individual mono-
graph or general test chapter.
THERMOMETERS
Thermometers suitable for Food Chemicals Codex use con-
form to the specifications of the American Society for Test-
ing and Materials, ASTM Standards E 1, and are standard-
ized in accordance with ASTM Method E 77.
General Tests and Assays
The thermometers are of the mercury in glass type, and
the column above the liquid is filled with nitrogen. They
may be standardized for “total immersion” or for “partial im-
mersion” and should be used as near as practicable under
the same condition of immersion.
Total immersion means standardization with the ther-
mometer immersed to the top of the mercury column, with
the remainder of the stem and the upper expansion cham-
ber exposed to the ambient temperature. Partial immersion
means standardization with the thermometer immersed to
the indicated immersion line etched on the front of the
thermometer, with the remainder of the stem exposed to
the ambient temperature. If used under any other condition
of immersion, an emergent-stem correction is necessary to
obtain correct temperature readings.
Thermometer Specifications
Subdivi-
Immer-
Range sion
ASTM sion
No. E1 °C °F °C °F (mm)
For General Use
1C –20 to +150 — 1 — 76
1F — 0 to 302 — 2 76
a For determination of melting range of Class III solids.
b For determination of the titer of fatty acids.
c For determination of Saybolt viscosity.
d For determination of Engler viscosity.
e For determination of congealing point.
f For determination of oil in wax.
 1218 / Appendix I / General Tests and Assays FCC 8

Thermometer Specifications (continued) Thermometer Specifications (continued)

Subdivi- Subdivi-
Immer- Immer-
Range sion Range sion
ASTM sion ASTM sion
No. E1 °C °F °C °F (mm) No. E1 °C °F °C °F (mm)
2C –5 to +300 — 1 — 76 95 C 100 to 130 — 0.1 — 76
2F — 20 to 580 — 2 76 96 C 120 to 150 — 0.1 — 76
3C –5 to +400 — 1 — 76 100 C 145 to 205 — 0.2 — 76
3F — 20 to 760 — 2 76 101 C 195 to 305 — 0.5 — 76
For Determination of Softening Point For Special Use
a
15 C –2 to +80 — 0.2 — total 14 C 38 to 82 — 0.1 — 79
b
15 F — 30 to 180 — 0.5 total 38 C –2 to +68 — 0.2 — 45
c
16 C 30 to 200 — 0.5 — total 18 C 34 to 42 — 0.1 — total
c
16 F — 85 to 392 — 1 total 18 F — 94 to 108 — 0.2 total
c
For Determination of Kinematic Viscosity 22 C 95 to 103 — 0.1 — total
c
44 F — 66.5 to — 0.1 total 22 F — 204 to — 0.2 total
71.5 218
d
45 F — 74.5 to — 0.1 total 23 C 18 to 28 — 0.2 — 90
79.5 d
24 C 30 to 54 — 0.2 — 90
28 F — 97.5 to — 0.1 total e
54 F — 68 to 213 — 0.5 total
102.5
f
71 F — –35 to — 1 76
46 F — 119.5 to — 0.1 total
+70
124.5
a For determination of melting range of Class III solids.
29 F — 127.5 to — 0.1 total b For determination of the titer of fatty acids.
132.5 c For determination of Saybolt viscosity.
47 F — 137.5 to — 0.1 total d For determination of Engler viscosity.
142.5 e For determination of congealing point.
f For determination of oil in wax.
48 F — 177.5 to — 0.1 total
182.5
In selecting a thermometer, careful consideration should
30 F — 207.5 to — 0.1 total
General Tests and Assays

be given to the conditions under which it is to be used. The

212.5
preceding table lists several ASTM thermometers, together
For Determination of Distillation Range
with their usual conditions of use, which may be required in
37 C –2 to +52 — 0.2 — 100 Food Chemicals Codex tests. Complete specifications for these
38 C 24 to 78 — 0.2 — 100 thermometers are given in “ASTM Standards on
39 C 48 to 102 — 0.2 — 100 Thermometers.”
40 C 72 to 126 — 0.2 — 100
41 C 98 to 152 — 0.2 — 100 VOLUMETRIC APPARATUS
102 C 123 to 177 — 0.2 — 100
Most of the volumetric apparatus available in the United
103 C 148 to 202 — 0.2 — 100
States is calibrated at 20°, although the temperatures gener-
104 C 173 to 227 — 0.2 — 100 ally prevailing in laboratories more nearly approach 25°,
105 C 198 to 252 — 0.2 — 100 which is the temperature specified generally for tests and
106 C 223 to 277 — 0.2 — 100 assays. This discrepancy is inconsequential provided the
107 C 248 to 302 — 0.2 — 100 room temperature is reasonably constant and the apparatus
has been calibrated accurately prior to and under the condi-
For Determination of Solidification Point
tions of its intended use.
89 C –20 to +10 — 0.1 — 76
Before use, all volumetric ware must be cleaned in such a
90 C 0 to 30 — 0.1 — 76 manner that when rinsed with water, no droplet of water
91 C 20 to 50 — 0.1 — 76 can be seen on the inside walls. Many kinds of “degreasing”
92 C 40 to 70 — 0.1 — 76 solutions are available, and the user should consult the man-
93 C 60 to 90 — 0.1 — 76
ufacturer’s literature for the system of choice.
94 C 80 to 110 — 0.1 — 76 Use To attain the degree of precision required in many
assays involving volumetric measurements and directing that
a For determination of melting range of Class III solids. a quantity be “accurately measured” (see Tests and Assays
b For determination of the titer of fatty acids.
under General Provisions), the apparatus must be chosen
c For determination of Saybolt viscosity.
d For determination of Engler viscosity. and used with exceptional care. Where less than 10 mL of
e For determination of congealing point. titrant is to be measured, a 10-mL buret or microburet gen-
f For determination of oil in wax. erally is required.
FCC 8 General Tests and Assays / Appendix I / 1219

The design of volumetric apparatus is an important factor Volume readings on burets should be estimated to the near-
in ensuring accuracy. For example, the length of the gradu- est 0.01 mL for 25- and 50-mL burets, and to the nearest
ated portions of graduated cylinders should be not less than 0.005 mL for 5- and 10-mL burets. Pipets calibrated “to
five times the inside diameter, and the tips of burets should contain” may be called for in special cases, generally for
permit an outflow of not more than measuring viscous fluids. In such cases, the pipet should be
0.5 mL/s. washed clean, after draining, and the washings added to
Pipets and burets must be allowed to drain properly in the measured portion.
use. Usually, transfer pipets for dilute aqueous solutions
should drain for the time specified by the manufacturer WEIGHTS AND BALANCES
before the tip is touched to the wall of the vessel. Buret
volumes should not be read immediately upon delivery of Food Chemicals Codex tests and assays are designed for use
the titrant. A suitable length of time should elapse to allow with three types of analytical balances, known as micro-,
the titrant retained on the walls to drain down. A time inter- semimicro-, and macro-.
val of 5 to 10 s is usually sufficient. By custom, microbalances weigh objects with a sensitivity
Standards of Accuracy The capacity tolerances for vol- down to the microgram range (or lower);
umetric flasks, transfer pipets, and burets are those accepted semimicrobalances down to the 0.01-mg range; and analyti-
by the National Institute of Standards and Technology (Class cal macrobalances down to the 0.1-mg range.
A),1 as indicated in the accompanying tables. Use Class A Tolerances The analytical weights meet the tolerances of
volumetric apparatus unless otherwise specified in the indi- the American National Standard ANSI/ASTM E617, “Labora-
vidual monograph. For plastic volumetric apparatus, the ac- tory Weights and Precision Mass Standards.” This standard is
cepted capacity tolerances are Class B.2 incorporated by reference and should be consulted for full
descriptions and information on the tolerances and con-
Volumetric Flasks
struction of weights.3 Where quantities of 25 mg or less are
Designated Volume (mL) to be “accurately weighed” (see Tests and Assays under Gen-
10 25 50 100 250 500 1000 eral Provisions), any applicable corrections for weights should
Limit of error 0.02 0.03 0.05 0.08 0.12 0.15 0.30 be used.
(mL) Class 1.1 weights are used for calibration of low-capacity,
Limit of error 0.20 0.12 0.10 0.08 0.05 0.03 0.03 high-sensitivity balances. They are available in various de-
(%) nominations from 1 to 500 mg. The tolerance for any de-
nomination in this class is 5 µg. They are recommended for

General Tests and Assays

calibration of balances using optical or electrical methods
Transfer Pipets for accurately weighing quantities below 20 mg.
Designated Volume (mL) Class 1 weights are designated as high-precision standards
1 2 5 10 25 50 100 for calibration. They may be used for accurately weighing
Limit of error 0.006 0.006 0.01 0.02 0.03 0.05 0.08 quantities below 20 mg.
(mL) Class 2 weights are used as working standards for calibra-
Limit of error 0.6 0.30 0.20 0.20 0.12 0.10 0.08 tion, built-in weights for analytical balances, and laboratory
(%) weights for routine analytical work.
Class 3 and Class 4 weights are used with moderate-preci-
sion laboratory balances.
Burets
Use Where substances are to be “accurately weighed” (see
Designated Volume (mL) Tests and Assays under General Provisions), in an assay or a
10 (“micro” type) 25 50 test, the weighing is to be performed in such manner as to
Subdivisions (mL) 0.02 0.10 0.10 limit the error to 0.1% or less. For example, a quantity of
Limit of error (mL) 0.02 0.03 0.05
50 mg is to be weighed to the nearest 0.05 mg; a quantity
of 0.1 g is to be weighed to the nearest 0.1 mg; and a
The capacity tolerances for measuring (i.e., “graduated”) quantity of 10 g is to be weighed to the nearest 10 mg.
pipets of up to and including 10-mL capacity are somewhat Calibration All precision balances and weights should be
larger than those for the corresponding sizes of transfer calibrated periodically (preferably at least once a year) and a
pipets, namely, 0.01, 0.02, and 0.03 mL for the 2-, 5-, and record kept of the calibration date and results. The user may
10-mL sizes, respectively. have a set of weights calibrated by the nearest Department
Transfer and measuring pipets calibrated “to deliver” of Weights and Measurements (or its equivalent). This is
should be drained in a vertical position and then touched usually done for little or no charge. Alternatively, an inde-
against the wall of the receiving vessel to drain the tips. pendent, outside company may be retained for the purpose
of performing such calibrations.
Buoyancy Effect When a weighing is to be performed
1 See “Testing of Glass Volumetric Apparatus,” NBS Circ. 602, April 1, 1959. with an accuracy of 0.1% or better, the buoyancy effect
Apparatus meeting the specifications of NB SIR 74–461 (“The Calibration of
Small Volumetric Laboratory Glassware”), as well as of ANSI/ASTM E 694–79 3 Copies of ASTM Standard E 617-81 (Reapproved 1985) may be obtained

(“Specifications for Volumetric Ware”), is also acceptable. from the American Society for Testing and Materials, 1916 Race Street, Phila-
2 See ASTM E 288, Fed. Spec. NNN-F-289, and ISO Standard 284. delphia, PA 19103.
 1220 / Appendix I / General Tests and Assays FCC 8

should not be neglected. The equation to be used in cor- weighed object; and DW is the density of the calibrated
recting for this effect is: weights.

MV = MA[1 + 0.0012(1/DO + DW)]

in which MV is the mass in vacuum; MA is the mass in air;

0.0012 is the density of air; DO is the density of the
General Tests and Assays
FCC 8
APPENDIX II: PHYSICAL TESTS AND DETERMINATIONS
A. CHROMATOGRAPHY
[NOTE—Chromatographic separations may also be
characterized according to the type of instrumenta-
tions or apparatus used. The types of chromatography
that may be used in the Food Chemicals Codex (FCC)
are column, thin-layer, gas, and high-pressure or high-
performance liquid chromatography.
The Committee on Food Chemicals Codex recognizes
that the field of chromatography continues to ad-
vance. Accordingly, the use of equivalent or improved
systems is acceptable with appropriate validation.]
For the purposes of the FCC, chromatography is defined
as an analytical technique whereby a mixture of chemicals
may be separated by virtue of their differential affinities for
two immiscible phases. One of these, the stationary phase,
consists of a fixed bed of small particles with a large surface
area, while the other, the mobile phase, is a gas or liquid
that moves constantly through, or over the surface of, the
fixed phase. Chromatographic systems achieve their ability
to separate mixtures by selectively retarding the passage of
some compounds through the stationary phase while per-
mitting others to move more freely. Therefore, the chromat-
ogram may be evaluated qualitatively by determining the
RF, or retardation factor, for each of the eluted substances.
The RF is a measure of that fraction of its total elution time
that any compound spends in the mobile phase. Because
this fraction is directly related to the fraction of the total
amount of the solute that is in the mobile phase, the RF can
be expressed as:
RF = VmCm/(VmCm + VsCs)
in which Vm and Vs are the volumes of the mobile and sta-
tionary phase, respectively, and Cm and Cs are the concen-
trations of the solute in either phase at any time. This can
be simplified to:
RF = Vm/(Vm + KVs)
in which K = Cs/Cm and is an equilibrium constant that indi-
cates this differential affinity of the solute for the phases.
Alternatively, a new constant, k, the capacity factor, may be
introduced, giving another form of the expression:
RF = 1/(1 + k)
in which k = KVs/Vm. The capacity factor, k, which is nor-
mally constant for small samples, is a parameter that ex-
presses the ability of a particular chromatographic system to
interact with a solute. The larger the k value, the more the
sample is retarded.
General Tests and Assays / Appendix II / 1221
Both the retardation factor and the capacity factor may
be used for qualitative identification of a solute or for devel-
oping strategies for improving separation. In terms of pa-
rameters easily obtainable from the chromatogram, the RF is
defined as the ratio of the distance traveled by the solute
band to the distance traveled by the mobile solvent in a
particular time. The capacity factor, k, can be evaluated by
the expression:
k = (tr − to)/to
in which tr, the retention time, is the elapsed time from the
start of the chromatogram to the elution maximum of the
solute, and to is the retention time of a solute that is not
retained by the chromatographic system.
Retardation of the solutes by the stationary phase may be
achieved by one or a combination of mechanisms. Certain
substances, such as alumina or silica gel, interact with the
solutes primarily by adsorption, either physical adsorption, in
which the binding forces are weak and easily reversible, or
chemisorption, in which strong bonding to the surface can
occur. Another important mechanism of retardation is parti-
tion, which occurs when the solute dissolves in the station-
ary phase, usually a liquid coated as a thin layer on the
General Tests and Assays
surface of an inert particle or chemically bonded to it. If the
liquid phase is a polar substance (e.g., polyethylene glycol)
and the mobile phase is nonpolar, the process is termed
normal-phase chromatography. When the stationary phase is
nonpolar (e.g., octadecylsilane) and the mobile phase is po-
lar, the process is reversed-phase chromatography. For the
separation of mixtures of ionic species, insoluble polymers
called ion exchangers are used as the stationary phase. Ions
of the solutes contained in the mobile phase are adsorbed
onto the surface of the ion exchanger while at the same
time displacing an electrically equivalent amount of less
strongly bound ions to maintain the electroneutrality of
both phases. The chromatographic separation of mixtures of
large molecules such as proteins may be accomplished by a
mechanism called size exclusion chromatography. The sta-
tionary phases used are highly cross-linked polymers that
have imbibed a sufficient amount of solvent to form a gel.
The separation is based on the physical size of the solvated
solutes; those that are too large to fit within the interstices
of the gel are eluted rapidly, while the smaller molecules
permeate into the pores of the gel and are eluted later. In
any chromatographic separation, more than one of the
above mechanisms may be occurring simultaneously.
Chromatographic separations may also be characterized
according to the type of instrumentation or apparatus used.
The types of chromatography that may be used in the FCC
are column, thin-layer, gas, and high-performance liquid
chromatography.
 1222 / Appendix II / General Tests and Assays
COLUMN CHROMATOGRAPHY
Apparatus The equipment needed for column chroma-
tography is not elaborate, consisting only of a cylindrical
glass or Teflon tube that has a restricted outflow orifice. The
dimensions of the tube are not critical and may vary from
10 to 40 mm in inside diameter and from 100 to 600 mm
in length. For a given separation, greater efficiency may be
obtained with a long narrow column, but the resultant flow
rate will be lower. A fritted-glass disk may be seated in the
end of the tube to act as a support for the packing material.
The column is fitted at the end with a stopcock or other
flow-restriction device to control the rate of delivery of the
eluant.
Procedure The stationary phase is introduced into the
column either as a dry powder or as a slurry in the mobile
phase. Because a homogeneous bed free of void spaces is
necessary to achieve maximum separation efficiency, the
packing material is introduced in small portions and allowed
to settle before further additions are made. Settling may be
accomplished by allowing the mobile phase to flow through
the bed, by tapping or vibrating the column if a dry powder
is used, or by compressing each added portion using a
tamping rod. The rod can be a solid glass, plastic, or metal
cylinder whose diameter is slightly smaller than that of the
column, or it can be a thinner rod onto the end of which
has been attached a disk of suitable diameter. Ion-exchange
resins and exclusion polymers are never packed as dry
powders because after introduction of the mobile phase,
they will swell and create sufficient pressure to shatter the
General Tests and Assays
column. When the packing has been completed, the sample
is introduced onto the top of the column. If the sample is
soluble, it is dissolved in a minimum amount of the mobile
phase, pipetted onto the column, and allowed to percolate
into the top of the bed. If it is not soluble or if the volume
of solution is too large, it may be mixed with a small
amount of the column packing. This material is then trans-
ferred to the chromatographic tube to form the top of the
bed.
The chromatogram is then developed by adding the mo-
bile phase to the column in small portions and allowing it
to percolate through the packed bed either by gravity or
under the influence of pressure or vacuum. Development of
the chromatogram takes place by selective retardation of
the components of the mixture as a result of their interac-
tion with the stationary phase. In column chromatography,
the stationary phase may act by adsorption, partition, ion
exchange, exclusion of the solutes, or a combination of
these effects.
When the development is complete, the components of
the sample mixture may be detected and isolated by either
of two procedures. The entire column may be extruded
carefully from the tube, and if the compounds are colored
or fluorescent under ultraviolet light, the appropriate seg-
ments may be cut from the column using a razor blade. If
the components are colorless, they may be visualized by
painting or spraying a thin longitudinal section of the sur-
face of the chromatogram with color-developing reagents.
The chemical may then be separated from the stationary
phase by extraction with a strong solvent such as methanol
and subsequently quantitated by suitable methods.
FCC 8
In the second procedure, the mobile phase may be al-
lowed to flow through the column until the components of
the mixture successively appear in the effluent. This eluate
may be collected in fractions and the mobile phase evapo-
rated if desired. The chemicals present in each fraction may
then be determined by suitable analytical techniques.
PAPER CHROMATOGRAPHY
In this type of chromatography, the stationary phase ordi-
narily consists of a sheet of paper of suitable texture and
thickness. The paper used is made from highly purified cel-
lulose, which has a great affinity for water and other polar
solvents since it has many hydroxyl functional groups. The
tightly bound water acts as the stationary phase, and there-
fore the mechanism that predominates is liquid–liquid or
partition chromatography. Adsorption of solutes to the cel-
lulose surface may also occur, but this is of lesser impor-
tance. Papers especially impregnated to permit ion-ex-
change or reverse-phase chromatography are also available.
Apparatus The essential equipment for paper chroma-
tography consists of the following:
Vapor-Tight Chamber: The chamber is constructed prefera-
bly of glass, stainless steel, or porcelain. It is provided with
inlets for the addition of solvent or for releasing internal
pressure, and it is designed to permit observation of the
progress of the chromatographic run without being opened.
Tall glass cylinders are convenient if they are made vapor-
tight with suitable covers and a sealing compound.
Supporting Rack: The rack serves as a support for the sol-
vent troughs and antisiphoning rods. It is constructed of a
corrosion-resistant material about 5 cm shorter than the in-
side height of the chamber.
Solvent Troughs: The troughs, made of glass, are designed
to be longer than the width of the chromatographic sheets
and to contain a volume of solvent greater than that re-
quired for one chromatographic run.
Antisiphoning Rods: Constructed of heavy glass, the rods are
placed on the rack and arranged to run outside of, parallel
to, and slightly above the edge of the glass trough.
Chromatographic Sheets: Special chromatographic filter pa-
per is cut to length approximately equal to the height of
the chamber. The sheet is a least 2.5 cm wide but not wider
than the length of the trough. A fine pencil line is drawn
horizontally across the filter paper at a distance from one
end such that when the sheet is suspended from the an-
tisiphoning rods with the upper end of the paper resting in
the trough and the lower portion hanging free into the
chamber, the line is located a few cm below the rods. Care
is necessary to avoid contaminating the paper by excessive
handling or by contact with dirty surfaces.
Procedure for Descending Chromatography Separa-
tion of substances by descending chromatography is accom-
plished by allowing the mobile phase to flow downward on
the chromatographic sheet.
The substance or substances to be analyzed are dissolved
in a suitable solvent. Convenient volumes of the resulting
solution, normally containing 1–20 µg of the compound,
are placed in 6–10-mm spots along the pencil line not less
FCC 8
than 3 cm apart. If the total volume to be applied would
produce spots of a diameter greater than 6–10 mm, it is
applied in separate portions to the same spot, each portion
being allowed to dry before the next is added.
The spotted chromatographic sheet is suspended in the
chamber by use of the antisiphoning rod and an additional
heavy glass rod that holds the upper end of the sheet in the
solvent trough. The bottom of the chamber is covered with
a mixture containing both phases of the prescribed solvent
system. It is important to ensure that the portion of the
sheet hanging below the rods is freely suspended in the
chamber without touching the rack or the chamber walls.
The chamber is sealed to allow equilibration (saturation) of
the chamber and the paper with solvent vapor. Any excess
pressure is released as necessary. For large chambers, equili-
bration overnight may be necessary.
A volume of the mobile phase in excess of the volume
required for complete development of the chromatogram is
saturated with the immobile phase. After equilibration of the
chamber, the prepared mobile solvent is introduced into the
trough through the inlet. The inlet is closed, and the mobile
phase is allowed to travel down the paper the desired dis-
tance. Precautions must be taken against allowing the sol-
vent to run down the sheet when opening the chamber and
removing the chromatogram. The location of the solvent
front is quickly marked, and the sheets are dried.
The chromatogram is observed and measured directly or
after suitable development to reveal the location of the
spots of the isolated components of the mixture.
Procedure for Ascending Chromatography In as-
cending chromatography, the lower edge of the sheet (or
strip) is dipped into the mobile phase to permit the mobile
phase to rise on the chromatographic sheet.
The test materials are applied to the chromatographic
sheet as directed under Procedure for Descending Chromatog-
raphy. Enough of both phases of the solvent mixture to
cover the bottom of the chamber is added. Empty solvent
troughs are placed on the bottom of the chamber, and the
chromatographic sheet is suspended so that the end near
which the spots have been added hangs free inside the
empty trough.
The chamber is sealed, and equilibration is allowed to
proceed as described under Procedure for Descending Chro-
matography. Then the solvent is added through the inlet to
the trough in excess of the quantity of solvent required for
complete moistening of the chromatographic sheet. The
chamber is resealed. When the solvent front has reached the
desired height, the chamber is opened and the sheet is re-
moved, the location of the solvent front is quickly marked,
and the sheet is dried.
Small cylinders may be used without troughs so that only
the mobile phase is placed on the bottom. The chromato-
graphic sheet is suspended during equilibration with the
lower end just above the solvent, and chromatography is
started by lowering the sheet so that it touches the solvent.
Detection of Chromatographic Bands After the chro-
matogram has been fully developed, the bands correspond-
ing to the various solutes may be detected by means similar
to those described in Column Chromatography. If the com-
pounds are colored or fluorescent under ultraviolet light,
General Tests and Assays / Appendix II / 1223
they may be visualized directly. Colorless compounds may
be detected by spraying the paper with color-developing
reagents. The bands corresponding to the individual compo-
nents can be cut from the paper, and the chemical sub-
stances eluted from the cellulose by the use of a strong
solvent such as methanol.
Identification of Solutes Since the chromatographic
mobilities of the solutes may change from run to run due to
varying experimental conditions, presumptive identification
of a substance should be based on comparison with a refer-
ence standard. The RF values of the unknown substance and
the standard on the same chromatogram must be identical.
Alternatively, the ratio between the distances traveled by a
given compound and a reference substance, the Rr value,
must be 1.0. Identification may also be made by mixing a
small amount of the reference substance with the unknown
and chromatographing. The resulting chromatogram should
contain only one spot. Definitive identification of solutes
may be achieved by eluting them from the paper and sub-
jecting them to IR, NMR, or mass spectrometry.
THIN-LAYER CHROMATOGRAPHY
In thin-layer chromatography (TLC), the stationary phase is
a uniform layer of a finely divided powder that has been
coated on the surface of a glass or plastic sheet and that is
held in place by a binder. The capacity of the system is
dependent on the thickness of the layer, which may range
from 0.1–2.0 mm. The thinner layers are used primarily for
analytical separations, while the thicker layers, because of
General Tests and Assays
their greater sample-handling ability, are useful for prepara-
tive work.
Substances that are used as coatings in TLC include silica
gel, alumina, cellulose, and reversed-phase packings. Separa-
tions occur because of adsorption of the solutes from the
mobile phase onto the surface of the thin layer. However,
adsorption of water from the air or solvent components
from the mobile phase can give rise to partition or liq-
uid–liquid chromatography. Specially coated plates are avail-
able that permit ion-exchange or reversed-phase
separations.
Apparatus Acceptable apparatus and materials for thin-
layer chromatography consist of the following:
Glass Plates: Flat glass plates of uniform thickness through-
out their areas. The most common sizes are 20 cm, 10 cm,
and 5 cm × 20 cm. (Aluminum plates also are commonly
used.)
Aligning Tray: An aligning tray or other suitable flat surface
is used to align and hold plates during application of the
adsorbent.
Adsorbent: The adsorbent may consist of finely divided ad-
sorbent materials for chromatography. It can be applied di-
rectly to the glass plate, or it can be bonded to the plate by
means of plaster of Paris or with starch paste. Pretreated
chromatographic plates are available commercially.
Spreader: A suitable spreading device that, when moved
over the glass plate, applies a uniform layer of adsorbent of
the desired thickness over the entire surface of the plate.
 1224 / Appendix II / General Tests and Assays
Storage Rack: A rack of convenient size to hold the pre-
pared plates during drying and transportation.
Developing Chamber: A glass chamber that can accommo-
date one or more plates and can be properly closed and
sealed. It is fitted with a plate-support rack that can support
the plates when the lid of the chamber is in place.
[NOTE—Preformed TLC plates available commercially
may also be used.]
Procedure Clean the plates scrupulously, as by immer-
sion in a chromic acid cleansing mixture, rinse them with
copious quantities of water until the water runs off the
plates without leaving any visible water or oily spots, and
dry.
Arrange the plate or plates on the aligning tray, and se-
cure them so that they will not slip during the application
of the adsorbent. Mix an appropriate quantity of adsorbent
and liquid, usually water, which when shaken for 30 s gives
a smooth slurry that will spread evenly with the aid of a
spreader. Transfer the slurry to the spreader, and apply the
coating at once before the binder begins to harden. Move
the spreader smoothly over the plates from one end of the
tray to the other. Remove the spreader, and wipe away ex-
cess slurry. Allow the plates to set for 10 min, and then
place them in the storage rack and dry at 105° for 30 min
or as directed in the individual monograph. Store the fin-
ished plates in a desiccator.
Equilibrate the atmosphere in the Developing Chamber by
placing in it a volume of the mobile phase in excess of that
required for complete development of the chromatogram,
General Tests and Assays
cover the chamber with its lid, and allow it to stand for at
least 30 min.
Apply the Sample Solution and the Standard Solution at
points about 1.5 cm apart and about 2 cm from the lower
edge of the plate (the lower edge is the first part over
which the spreader moves in the application of the adsor-
bent layer), and allow to dry. A template will aid in deter-
mining the spot points and the 10-cm to 15-cm distance
through which the solvent front should move.
Arrange the plate on the supporting rack (sample spots
on the bottom), and introduce the rack into the developing
chamber. The solvent in the chamber must be deep enough
to reach the lower edge of the adsorbent, but must not
touch the spot points. Seal the cover in place, and maintain
the system until the solvent ascends to a point 10–15 cm
above the initial spots; this usually requires 15 min to 1 h.
Remove the plates, and dry them in air. Measure and record
the distance of each spot from the point of origin. If so
directed, spray the spots with the reagent specified, ob-
serve, and compare the sample with the standard
chromatogram.
Detection and Identification Detection and identifica-
tion of solute bands is done by methods essentially the
same as those described in Column Chromatography. How-
ever, in TLC an additional method called fluorescence
quenching is also used. In this procedure, an inorganic
phosphor is mixed with the adsorbent before it is coated on
the plate. When the developed chromatogram is irradiated
with ultraviolet light, the surface of the plate fluoresces with
FCC 8
a characteristic color, except in those places where ultra-
violet-absorbing solutes are situated. These quench the fluo-
rescence and are detectable as dark spots.
Detection with an ultraviolet light source suitable for ob-
servations with short (254-nm) and long (360-nm) ultra-
violet wavelengths may be called for in some cases.
Quantitative Analysis Two methods are available if
quantitation of the solute is necessary. In the first, the bands
are detected and their positions marked. Those areas of ad-
sorbent containing the compounds of interest are scraped
from the surface of the plate into a centrifuge tube. The
chemicals are extracted from the adsorbent with the aid of
a suitable strong solvent, the suspension is centrifuged, and
the supernatant layer is subjected to appropriate methods of
quantitative analysis.
The second method involves the use of a scanning densi-
tometer. This is a spectrophotometric device that directs a
beam of monochromatic radiation across the surface of the
plate. After interaction with the solutes in the adsorbent
layer, the radiation is detected as transmitted or reflected
light and a recording of light intensity versus distance trav-
eled is produced. The concentration of a particular species is
proportional to the area under its peak and can be deter-
mined accurately by comparison with standards.
GAS CHROMATOGRAPHY
The distinguishing features of gas chromatography are a
gaseous mobile phase and a solid or immobilized liquid sta-
tionary phase. Liquid stationary phases are available in
packed or capillary columns. In the packed columns, the
liquid phase is deposited on a finely divided, inert solid sup-
port, such as diatomaceous earth or porous polymer, which
is packed into a column that typically has a 2-mm to 4-mm
id and is 1–3 m long. In capillary columns, which contain
no particles, the liquid phase is deposited on the inner sur-
face of the fused silica column and may be chemically
bonded to it. In gas–solid chromatography, the solid phase
is an active adsorbent, such as alumina, silica, or carbon,
packed into a column. Polyaromatic porous resins, which
are sometimes used in packed columns, are not coated with
a liquid phase.
When a volatile compound is introduced into the carrier
gas and carried into the column, it is partitioned between
the gas and stationary phases by a dynamic countercurrent
distribution process. The compound is carried down the col-
umn by the carrier gas, retarded to a greater or lesser ex-
tent by sorption and desorption in the stationary phase. The
elution of the compound is characterized by the partition
ratio, k, a dimensionless quantity also called the capacity
factor. It is equivalent to the ratio of the time required for
the compound to flow through the column (the retention
time) to the retention time of a nonretarded compound.
The value of the capacity factor depends on the chemical
nature of the compound; the nature, amount, and surface
area of the liquid phase; and the column temperature.
Under a specified set of experimental conditions, a charac-
teristic capacity factor exists for every compound. Separa-
tion by gas chromatography occurs only if the compounds
concerned have different capacity factors.
FCC 8
Apparatus A gas chromatograph consists of a carrier gas
source, an injection port, column, detector, and recording
device. The injection port, column, and detector are care-
fully temperature controlled. The typical carrier gas is helium
or nitrogen, depending on the column and detector in use.
The gas is supplied from a high-pressure cylinder and passes
through suitable pressure-reducing valves to the injection
port and column. Compounds to be chromatographed, ei-
ther in solution or as gases, are injected into the gas stream
at the injection port. Depending on the configuration of the
apparatus, the test mixture may be injected directly into the
column or be vaporized in the injection port and mixed into
the flowing carrier gas before entering the column.
Once in the column, compounds in the test mixture are
separated by virtue of differences in their capacity factors,
which in turn depend on their vapor pressure and degree of
interaction with the stationary phase. The capacity factor,
which governs resolution and retention times of compo-
nents of the test mixture, is also temperature dependent.
The use of temperature-programmable column ovens takes
advantage of this dependence to achieve efficient separation
of compounds differing widely in vapor pressure.
As resolved compounds emerge from the column, they
pass through a detector, which responds to the amount of
each compound present. The type of detector to be used
depends on the nature of the compounds to be analyzed,
and is specified in the individual monograph. Detectors are
heated above the maximum column operating temperature
to prevent condensation of the eluting compounds.
Detector output is recorded as a function of time, produc-
ing a chromatogram, which consists of a series of peaks on
a time axis. Each peak represents a compound in the
vaporized test mixture, although some peaks may overlap.
The elution time is characteristic of the individual com-
pounds (qualitative analysis), and the peak area is a function
of the amount present (quantitative analysis).
Injectors: Sample injection devices range from simple sy-
ringes to fully programmable automatic injectors. The
amount of sample that can be injected into a capillary col-
umn without overloading is small compared with the
amount that can be injected into a packed column, and
may be less than the smallest amount that can be manipu-
lated satisfactorily by syringe. Capillary columns are there-
fore used with injectors able to split samples into two frac-
tions, a small one that enters the column and a large one
that goes to waste (split injector). Such injectors may also
be used in a splitless mode for analyses of trace or minor
components.
Purge and trap injectors are equipped with a sparging
device by which volatile compounds in solution are carried
into a low-temperature trap. When sparging is complete,
trapped compounds are thermally desorbed into the carrier
gas by rapid heating of the temperature-programmable
trap.
Headspace injectors are equipped with a thermostatically
controlled sample-heating chamber. Solid or liquid samples
in tightly closed containers are heated in the chamber for a
fixed period of time, allowing the volatile components in
the sample to reach an equilibrium between the non-
gaseous phase and the gaseous or headspace phase.
General Tests and Assays / Appendix II / 1225
After this equilibrium has been established, the injector
automatically introduces a fixed amount of the headspace in
the sample container into the gas chromatograph.
Columns: Capillary columns, which are usually made of
fused silica, have a 0.2-mm to 0.53-mm id and are 5–30 m
long. The liquid or stationary phase is 0.1–1.0 µm thick,
although nonpolar stationary phases may be up to 5 µm
thick.
Packed columns, made of glass or metal, are 1–3 m long,
with a 2-mm to 4-mm id. Those used for analysis typically
have liquid phase loadings of about 5% (w/w) on a solid
support.
Supports for analysis of polar compounds on low-capacity,
low-polarity liquid phase columns must be inert to avoid
peak tailing. The reactivity of support materials can be re-
duced by silanizing before coating with liquid phase. Acid-
washed, flux-calcined diatomaceous earth is often used for
drug analysis. Support materials are available in various
mesh sizes, with 80- to 100-mesh and 100- to 120-mesh
being more commonly used with 2-mm to 4-mm columns.
Because of the absence of a solid support, capillary com-
pounds are much more inert than packed columns.
Retention time and the peak efficiency depend on the
carrier gas flow rate; retention time is also directly propor-
tional to column length, while resolution is proportional to
the square root of the column length. For packed columns,
the carrier gas flow rate is usually expressed in mL/min at
atmospheric pressure and room temperature. It is measured
at the detector outlet with a soap film flow meter while the
column is at operating temperature. Unless otherwise speci-
General Tests and Assays
fied in the individual monograph, flow rates for packed col-
umns are 60–75 mL/min for 4-mm id columns and ~30 mL/
min for 2-mm id columns.
For capillary columns, linear flow velocity is often used
instead of flow rate. This is conveniently determined from
the length of the column and the retention time of a dilute
methane sample, provided a flame-ionization detector is in
use. Typical linear velocities are 20–60 cm/s for helium. At
high operating temperatures there is sufficient vapor pres-
sure to result in a gradual loss of liquid phase, a process
called “bleeding.”
Detectors: Flame-ionization detectors are used for most
analyses, with lesser use made of thermal conductivity, elec-
tron-capture, nitrogen–phosphorus, and mass spectrometric
detectors. For quantitative analyses, detectors must have a
wide linear dynamic range: the response must be directly
proportional to the amount of compound present in the
detector over a wide range of concentrations. Flame-ioniza-
tion detectors have a wide linear range (~106) and are sensi-
tive to organic compounds. Unless otherwise specified in in-
dividual monographs, flame-ionization detectors with either
helium or nitrogen carrier gas are to be used for packed
columns, and helium is used for capillary columns.
The thermal conductivity detector detects changes in the
thermal conductivity of the gas stream as solutes are eluted.
Although its linear dynamic range is smaller than that of the
flame-ionization detector, it is quite rugged and occasionally
used with packed columns, especially for compounds that
do not respond to flame-ionization detectors.
 1226 / Appendix II / General Tests and Assays
The alkali flame-ionization detector, sometimes called an
NP or nitrogen−phosphorus detector, contains a thermionic
source, such as an alkali-metal salt or a glass element con-
taining rubidium or other metal, that results in the efficient
ionization of organic nitrogen and phosphorus compounds.
It is a selective detector that shows little response to
hydrocarbons.
The electron-capture detector contains a radioactive
source (usually 63Ni) of ionizing radiation. It exhibits an ex-
tremely high response to compounds containing halogens
and nitro groups but little response to hydrocarbons. The
sensitivity increases with the number and atomic weight of
the halogen atoms.
Data Collection Devices: Modern data stations receive the
detector output, calculate peak areas, and print chromato-
grams, complete with run parameters and peak data. Chro-
matographic data may be stored and reprocessed, with inte-
gration and other calculation variables being changed as
required. Data stations are used also to program the chro-
matograph, controlling most operational variables and pro-
viding for long periods of unattended operation.
Data can also be collected for manual measurement on
simple recorders or on integrators whose capabilities range
from those providing a printout of peak areas to those pro-
viding chromatograms with peak areas and peak heights
calculated and data stored for possible reprocessing.
Procedure Capillary columns must be tested to ensure
that they comply with the manufacturers’ specifications
before they are used. These tests consist of the following
injections: a dilute methane sample to determine the linear
General Tests and Assays
flow velocity; a mixture of alkanes (e.g., C14, C15, and C16) to
determine resolution; and a polarity test mixture to check
for active sites on the column. The latter mixture may in-
clude a methyl ester, an unsaturated compound, a phenol,
an aromatic amine, a diol, a free carboxylic acid, and a
polycyclic aromatic compound, depending on the samples
to be analyzed.
Packed columns must be conditioned before use until the
baseline and other characteristics are stable. This may be
done by operation at a temperature above that called for by
the method or by repeated injections of the compound or
mixture to be chromatographed. A suitable test for support
inertness should be done. Very polar molecules (like free
fatty acids) may require a derivatization step.
Before any column is used for assay purposes, a calibra-
tion curve should be constructed to verify that the instru-
mental response is linear over the required range and that
the curve passes through the origin. If the compound to be
analyzed is adsorbed within the system, the calibration
curve will intersect the abscissa at a nonzero value. This may
result in error, particularly for compounds at low concentra-
tions determined by a procedure based on a single refer-
ence point. At high concentrations, the liquid phase may be
overloaded, leading to loss of peak height and symmetry.
Assays require quantitative comparison of one chromato-
gram with another. A major source of error is irreproducibil-
ity in the amount of sample injected, notably when manual
injections are made with a syringe. The effects of variability
can be minimized by addition of an internal standard, a
noninterfering compound present at the same concentration
FCC 8
as in the sample and standard solutions. The ratio of peak
response of the analyte to that of the internal standard is
compared from one chromatogram to another. Where the
internal standard is chemically similar to the substance be-
ing determined, there is also compensation for minor varia-
tions in column and detector characteristics. In some cases,
the internal standard may be carried through the sample
preparation procedure before gas chromatography to con-
trol other quantitative aspects of the assay. Automatic injec-
tors greatly improve the reproducibility of sample injections
and reduce the need for internal standards.
Many monographs require that system suitability require-
ments be met before samples are analyzed, see System Suit-
ability below.
HIGH-PERFORMANCE LIQUID
CHROMATOGRAPHY
High-performance liquid chromatography (HPLC) is a sepa-
ration technique based on a solid stationary phase and a
liquid mobile phase. Separations are achieved by partition,
adsorption, exclusion, or ion-exchange processes, depend-
ing on the type of stationary phase used. HPLC has distinct
advantages over gas chromatography for the analysis of
nonvolatile organic compounds. Compounds to be analyzed
are dissolved in a liquid, and most separations take place at
room temperature.
As in gas chromatography, the elution time of a com-
pound can be described by the capacity factor, k, which
depends on the chemical nature of the composition and
flow rate of the mobile phase, and the composition and
surface area of the stationary phase. Column length is an
important determinant of resolution. Only compounds hav-
ing different capacity factors can be separated by HPLC.
Apparatus A liquid chromatograph consists of one, two,
or more reservoirs containing the mobile phase, a pump to
force the mobile phase through the system at high pressure,
an injector to introduce the sample into the mobile phase, a
chromatographic column, a detector, and a data collection
device such as a computer, integrator, or recorder. Short,
3-cm, 5-cm, 10-cm, and 25-cm small-bore columns contain-
ing densely packed particles of stationary phase provide for
the rapid exchange of compounds between the mobile and
stationary phases. In addition to receiving and reporting de-
tector output, computers are used to control chromato-
graphic settings and operations, thus providing for long pe-
riods of unattended operation.
Pumping Systems: HPLC pumping systems deliver metered
amounts of mobile phase from the solvent reservoirs to the
column through high-pressure tubing and fittings. Modern
systems consist of one or more computer-controlled meter-
ing pumps that can be programmed to vary the ratio of
mobile phase components, as is required for gradient chro-
matography, or to mix isocratic mobile phases (i.e., mobile
phases having a fixed ratio of solvents). However, the pro-
portion of ingredients in premixed isocratic mobile phases
can be more accurately controlled than in those delivered
by most pumping systems. Operating pressures up to 5000
psi with delivery rates up to about 10 mL/min are typical.
Pumps used for quantitative analysis should be constructed
FCC 8
of materials inert to corrosive mobile phase components
and be capable of delivering the mobile phase at a constant
rate with minimal fluctuations over extended periods of
time.
Injectors: After dissolution in mobile phase or other suitable
solution, compounds to be chromatographed are injected
into the mobile phase, either manually by syringe or loop
injectors, or automatically by autosamplers. The latter con-
sist of a carousel or rack to hold sample vials with tops that
have a pierceable septum or stopper and an injection device
to transfer sample from the vials to a calibrated, fixed-vol-
ume loop from which it is loaded into the chromatograph.
Some autosamplers can be programmed to control sample
volume, the number of injections and loop rinse cycles, the
interval between injections, and other operating variables.
Some valve systems incorporate a calibrated sample loop
that is filled with test solution for transfer to the column in
the mobile phase. In other systems, test solution is trans-
ferred to a cavity by syringe and then switched into the
mobile phase.
Columns: For most analyses, separation is achieved by parti-
tion of compounds in the test solution between the mobile
and stationary phases. Systems consisting of polar stationary
phases and nonpolar mobile phases are described as normal
phase, while the opposite arrangement, polar mobile phases
and nonpolar stationary phases, is called reversed-phase
chromatography. Partition chromatography is almost always
used for hydrocarbon-soluble compounds of a molecular
weight that is less than 1000. The affinity of a compound
for the stationary phase, and thus its retention time on the
column, is controlled by making the mobile phase more or
less polar. Mobile phase polarity can be varied by the addi-
tion of a second, and sometimes a third or even a fourth,
component.
Stationary phases for modern, reversed-phase liquid chro-
matography typically consist of an organic phase chemically
bound to silica or other materials. Particles are usually 3 µm,
5 µm, or 10 µm in diameter, but sizes may range up to 50
µm for preparative columns. Small particles thinly coated
with organic phase allow fast mass transfer and, hence,
rapid transfer of compounds between the stationary and
mobile phases. Column polarity depends on the polarity of
the bound functional groups, which range from relatively
nonpolar octadecyl silane to very polar nitrile groups.
Columns used for analytical separations usually have inter-
nal diameters of 2–4.6 mm; larger diameter columns are
used for preparative chromatography. Columns may be
heated to give more efficient separations, but only rarely are
they used at temperatures above 60° because of potential
stationary phase degradation or mobile phase volatility. Un-
less otherwise specified in the individual monograph, col-
umns are used at an ambient temperature.
Ion-exchange chromatography is used to separate water-
soluble, ionizable compounds of molecular weights that are
less than 2000. The stationary phases are usually synthetic
organic resins; cation-exchange resins contain negatively
charged active sites and are used to separate basic sub-
stances such as amines; while anion-exchange resins have
positively charged active sites for separation of compounds
General Tests and Assays / Appendix II / 1227
with negatively charged groups such as phosphate, sulfo-
nate, or carboxylate groups. Water-soluble ionic or ionizable
compounds are attracted to the resins, and differences in
affinity bring about the chromatographic separation. The pH
of the mobile phase, temperature, ion type, ionic concentra-
tion, and organic modifiers affect the equilibrium, and these
variables can be adjusted to obtain the desired degree of
separation.
In size-exclusion chromatography, columns are packed
with a porous stationary phase. Molecules of the com-
pounds being chromatographed are filtered according to
size. Those too large to enter the pores pass unretained
through the column (total exclusion). Smaller molecules
enter the pores and are increasingly retained as molecular
size decreases. These columns are typically used to remove
high molecular weight matrices or to characterize the mo-
lecular weight distribution of a polymer.
Detectors: Many compendial HPLC methods require the use
of spectrophotometric detectors. Such a detector consists of
a flow-through cell mounted at the end of the column. A
beam of ultraviolet radiation passes through the flow cell
and into the detector. As compounds elute from the col-
umn, they pass through the cell and absorb the radiation,
resulting in measurable energy level changes.
Fixed, variable, and photodiode array (PDA) detectors are
widely available. Fixed wavelength detectors operate at a
single wavelength, typically 254 nm, emitted by a low-pres-
sure mercury lamp. Variable wavelength detectors contain a
continuous source, such as a deuterium or high-pressure xe-
non lamp, and a monochromator or an interference filter to
General Tests and Assays
generate monochromatic radiation at a wavelength selected
by the operator. Modern variable wavelength detectors can
be programmed to change wavelength while an analysis is
in progress. Multi-wavelength detectors measure absorbance
at two or more wavelengths simultaneously. In diode array
multi-wavelength detectors, continuous radiation is passed
through the sample cell, then resolved into its constituent
wavelengths, which are individually detected by the
photodiode array. These detectors acquire absorbance data
over the entire UV-visible range, thus providing the analyst
with chromatograms at multiple, selectable wavelengths
and spectra of the eluting peaks. Diode array detectors usu-
ally have lower signal-to-noise ratios than fixed or variable
wavelength detectors, and thus are less suitable for analysis
of compounds present at low concentrations.
Differential refractometer detectors measure the difference
between the refractive index of the mobile phase alone and
that of the mobile phase containing chromatographed com-
pounds as it emerges from the column. Refractive index de-
tectors are used to detect non-UV absorbing compounds,
but they are less sensitive than UV detectors. They are sensi-
tive to small changes in solvent composition, flow rate, and
temperature, so that a reference column may be required to
obtain a satisfactory baseline.
Fluorometric detectors are sensitive to compounds that
are inherently fluorescent or that can be converted to fluo-
rescent derivatives either by chemical transformation of the
compound or by coupling with fluorescent reagents at spe-
cific functional groups. If derivatization is required, it can be
done before chromatographic separation or, alternatively,
 1228 / Appendix II / General Tests and Assays
the reagent can be introduced into the mobile phase just
before its entering the detector.
Potentiometric, voltammetric, or polarographic electro-
chemical detectors are useful for the quantitation of species
that can be oxidized or reduced at a working electrode.
These detectors are selective, sensitive, and reliable, but re-
quire conducting mobile phases free of dissolved oxygen
and reducible metal ions. A pulseless pump must be used,
and care must be taken to ensure that the pH, ionic
strength, and temperature of the mobile phase remain con-
stant. Working electrodes are prone to contamination by re-
action products with consequent variable responses.
Electrochemical detectors with carbon-paste electrodes
may be used advantageously to measure nanogram quanti-
ties of easily oxidized compounds, notably phenols and
catechols.
Data Collection Devices: Modern data stations receive and
store detector output and print out chromatograms com-
plete with peak heights, peak areas, sample identification,
and method variables. They are also used to program the
liquid chromatograph, controlling most variables and pro-
viding for long periods of unattended operation.
Data also may be collected on simple recorders for man-
ual measurement or on stand-alone integrators, which range
in complexity, from those providing a printout of peak areas
to those providing a printout of peak areas and peak
heights calculated and data stored for possible subsequent
reprocessing.
Procedure The mobile phase composition significantly
influences chromatographic performance and the resolution
General Tests and Assays
of compounds in the mixture being chromatographed.
Composition has a much greater effect than temperature on
the capacity factor, k.
In partition chromatography, the partition coefficient, and
hence the separation, can be changed by addition of an-
other component to the mobile phase. In ion-exchange
chromatography, pH and ionic strength as well as changes
in the composition of the mobile phase affect capacity fac-
tors. The technique of continuously increasing mobile phase
strength during the chromatographic run is called gradient
elution or solvent programming. It is sometimes used to
chromatograph complex mixtures of components differing
greatly in their capacity factors. Detectors that are sensitive
to change in solvent composition, such as the differential
refractometer, are more difficult to use with the gradient
elution technique.
For accurate quantitative work, high-purity, “HPLC-grade”
solvents and reagents must be used. The detector must
have a broad linear dynamic range, and compounds to be
measured must be resolved from any interfering substances.
The linear dynamic range of a compound is the range over
which the detector signal response is directly proportional
to the amount of the compound. For maximum flexibility in
quantitative work, this range should be about three orders
of magnitude. HPLC systems are calibrated by plotting peak
responses in comparison with known concentrations of a
reference standard, using either an external or an internal
standardization procedure.
Reliable quantitative results are obtained by external cali-
bration if automatic injectors or autosamplers are used. This
FCC 8
method involves direct comparison of the peak responses
obtained by separately chromatographing the test and refer-
ence standard solutions. If syringe injection, which is irrepro-
ducible at the high pressures involved, must be used, better
quantitative results are obtained by the internal calibration
procedure where a known amount of a noninterfering com-
pound, the internal standard, is added to the test and refer-
ence standard solutions, and the ratios of peak responses of
the analyte and internal standard are compared.
Because of normal variations in equipment, supplies, and
techniques, a system suitability test is required to ensure
that a given operating system may be generally applicable.
The main features of System Suitability tests are described
below.
For information on the interpretation of results, see the
section Interpretation of Chromatograms.
Interpretation of Chromatograms Figure 1 repre-
sents a typical chromatographic separation of two sub-
stances, 1 and 2, in which tR(1) and tR(2) are the respective
retention times; h, h/2, and Wh/2 are the height, the half-
height, and the width at half-height, respectively, for peak
1; and W1 and W2 are the respective widths of peaks 1 and
2 at the baseline. Air peaks are a feature of gas chromato-
grams and correspond to the solvent front in liquid
chromatography.
Figure 1. Chromatographic Separation of Two Substances
Chromatographic retention times are characteristic of the
compounds they represent but are not unique. Coincidence
of retention times of a test and a reference substance can
be used as a feature in construction of an identity profile
but is insufficient on its own to establish identity. Absolute
retention times of a given compound vary from one chro-
matogram to the next. Comparisons are normally made in
terms of relative retention, which is calculated by the
equation:
α = (tR(2) − tR(0))/(tR(1) − tO)
in which tR(2) and tR(1) are the retention times, measured
from the point of injection, of the test and reference sub-
stances, respectively, determined under identical experimen-
tal conditions on the same column, and tO is the retention
time of a nonretained substance, such as methane in this
case, of gas chromatography.
In this and the following expressions, the corresponding
retention volumes or linear separations on the chromato-
gram, both of which are directly proportional to retention
time, may be substituted in the equations. Where the value
of tO is small, Rr may be estimated from the retention times
measured from the point of injection (tR(2)/tR(1)).
FCC 8
The number of theoretical plates, N, is a measure of col-
umn efficiency. For Gaussian peaks, it is calculated by the
equations:
N = 16(tR/W)2 or N = 5.54(tR/W / )2 1
2
in which tR is the retention time of the substance and W is
the width of the peak at its base, obtained by extrapolating
the relatively straight sides of the peak to the baseline. W /
1
2
is the peak width at half-height, obtained directly by elec-
tronic integrators. The value of N depends on the substance
being chromatographed as well as the operating conditions
such as mobile phase or carrier gas flow rates and tempera-
ture, the quality of the packing, the uniformity of the pack-
ing within the column, and for capillary columns, the thick-
ness of the stationary phase film and the internal diameter
and length of the column.
The separation of two components in a mixture, the reso-
lution, R, is determined by the equation:
R = 2(tR(2) − tR(1))/(W2 + W1)
in which tR(2) and tR(1) are the retention times of the two
components, and W2 and W1 are the corresponding widths
at the bases of the peaks obtained by extrapolating the rela-
tively straight sides of the peaks to the baseline.
Peak areas and peak heights are usually proportional to
the quantity of compound eluting. These are commonly
measured by electronic integrators but may be determined
by more classical approaches. Peak areas are generally used
but may be less accurate if peak interference occurs. For
manual measurements, the chart should be run faster than
usual, or a comparator should be used to measure the
width at half-height and the width at the base of the peak,
to minimize error in these measurements. For accurate
quantitative work, the components to be measured should
be separated from any interfering components. Peak tailing
and fronting and the measurement of peaks on solvent tails
are to be avoided (see Figure 2). The relative standard devia-
tion is expressed by the equation:
in which SR is the relative standard deviation in percent, X
is the mean of the set of N measurements, and Xi is an
individual measurement. When an internal standard is used,
the measurement Xi usually refers to the measurement of
relative area, As:
Xi = As = ar/ai
in which ar is the area of the peak corresponding to the
standard substance and ai is the area of the peak corre-
sponding to the internal standard. When peak heights are
used, the measurement Xi refers to the measurement of rel-
ative heights, Hs:
Xi = Hs = hr/hi
in which hr is the height of the peak corresponding to the
standard substance and hi is the height of the peak corre-
sponding to the internal standard.
General Tests and Assays / Appendix II / 1229
Figure 2. Asymmetrical Chromatographic Peak
System Suitability Such tests are an integral part of gas
and liquid chromatographic methods. They are used to ver-
ify that the resolution and reproducibility of the chromato-
graphic system are adequate for the analysis to be done.
The tests are based on the concept that the equipment,
electronics, analytical operations, and samples to be ana-
lyzed constitute an integral system that can be evaluated as
such.
The resolution, R, is a function of column efficiency, N,
and is specified to ensure that closely eluting compounds
are resolved from each other, to establish the general resolv-
ing power of the system, and to ensure that internal stan-
dards are resolved from the analyte. Column efficiency may
be specified also as a system suitability requirement, espe-
cially if there is only one peak of interest in the chromato-
gram; however, it is a less reliable means to ensure resolu-
tion than direct measurement. Column efficiency is a
measure of peak sharpness, which is important for the de-
General Tests and Assays
tection of trace components.
Replicate injections of a standard preparation used in the
assay or other standard solution are compared to ascertain
whether requirements for precision are met. Unless other-
wise specified in the individual monograph, data from five
replicate injections of the analyte are used to calculate the
relative standard deviation if the requirement is 2.0% or
less; data from six replicate injections are used if the relative
standard deviation requirement is more than 2.0%.
The tailing factor, T, a measure of peak symmetry, is unity
for perfectly symmetrical peaks, and its value increases as
tailing becomes more pronounced. In some cases, values
less than unity may be observed. As peak asymmetry in-
creases, integration, and hence precision, becomes less relia-
ble. The calculation is expressed by the equation:
tailing factor = T = W0.05/2f
These tests are performed by collecting data from repli-
cate injections of standard or other solutions as specified in
the individual monograph. The specification of definitive pa-
rameters in a monograph does not preclude the use of
other suitable operating conditions (see Procedures under
Tests and Assays in General Provisions). Adjustments of oper-
ating conditions to meet system suitability requirements
may be necessary.
Unless otherwise directed in the monograph, system suit-
ability parameters are determined from the analyte peak.
To ascertain the effectiveness of the final operating sys-
tem, it should be subjected to a suitability test before use
and during testing whenever there is a significant change in
 1230 / Appendix II / General Tests and Assays
equipment or in a critical reagent or when a malfunction is
suspected.
B. PHYSICOCHEMICAL PROPERTIES
DISTILLATION RANGE
Scope This method is to be used for determining the
distillation range of pure or nearly pure compounds or mix-
tures having a relatively narrow distillation range of about
40° or less. The result so determined is an indication of
purity, not necessarily of identity. Products having a distilla-
tion range of greater than 40° may be determined by this
method if a wide-range thermometer, such as ASTM E1, 1C,
2C, or 3C, is specified in the individual monograph.
Definitions
Distillation Range The difference between the temperature
observed at the start of a distillation and that observed at
which a specified volume has distilled, or at which the dry
point is reached.
Initial Boiling Point The temperature indicated by the distil-
lation thermometer at the instant the first drop of conden-
sate leaves the end of the condenser tube.
Dry Point The temperature indicated at the instant the last
drop of liquid evaporates from the lowest point in the distil-
lation flask, disregarding any liquid on the side of the flask.
General Tests and Assays
Apparatus
Distillation Flask: A 200-mL round-bottom distilling flask of
heat-resistant glass is preferred when sufficient sample (in
excess of 100 mL) is available for the test. If a sample of less
than 100 mL must be used, a smaller flask having a capacity
of at least double the volume of the liquid taken may be
employed. The 200-mL flask has a total length of 17–19 cm,
and the inside diameter of the neck is 20–22 mm. Attached
about midway on the neck, approximately 12 cm from the
bottom of the flask, is a side arm 10–12.7 cm long and 5
mm in internal diameter, which forms an angle of 70°–75°
with the lower portion of the neck.
Condenser: Use a straight glass condenser of heat-resistant
tubing, 56–60 cm long and equipped with a water jacket so
that about 40 cm of the tubing is in contact with the cool-
ing medium. The lower end of the condenser may be bent
to provide a delivery tube or it may be connected to a bent
adapter that serves as the delivery tube.
[NOTE—All-glass apparatus with standard-taper ground
joints may be used alternatively if the assembly em-
ployed provides results equal to those obtained with
the flask and condenser described above.]
Receiver: The receiver is a 100-mL cylinder that is gradu-
ated in 1-mL subdivisions and calibrated “to contain.” It is
used for measuring the sample as well as for receiving the
distillate.
Thermometer: An accurately standardized partial-immersion
thermometer having the smallest practical subdivisions (not
FCC 8
greater than 0.2°) is recommended to avoid the necessity
for an emergent stem correction. Suitable thermometers are
available as the ASTM E1 Series 37C through 41C, and
102C through 107C, or as the MCA types R-1 through R-4
(see Thermometers, Appendix I).
Source of Heat: A Bunsen burner is the preferred source of
heat. An electric heater may be used, however, if it is shown
to give results comparable to those obtained with the gas
burner.
Shield: The entire burner and flask assembly should be pro-
tected from external air currents. Any efficient shield may be
employed for this purpose.
Flask Support: A heat-resistant board, 5–7 mm in thickness
and having a 10-cm circular hole, is placed on a suitable
ring or platform support and fitted loosely inside the shield
to ensure that hot gases from the source of heat do not
come in contact with the sides or neck of the flask. A sec-
ond 5–7-mm thick heat-resistant board, 14–16-cm square
and provided with a 30–40-mm circular hole, is placed on
top of the first board. This board is used to hold the 200-
mL distillation flask, which should be fitted firmly on the
board so that direct heat is applied to the flask only through
the opening in the board.
Procedure [NOTE—For materials boiling below 50°, cool
the liquid to below 10° before sampling, receive the distil-
late in a water bath cooled to below 10°, and use water
cooled to below 10° in the condenser.]
Measure 100 ± 0.5 mL of the liquid in the 100-mL gradu-
ate, and transfer the sample, together with an efficient an-
tibumping device, into the distilling flask. Do not use a fun-
nel in the transfer or allow any of the sample to enter the
side arm of the flask. Place the flask on the heat-resistant
boards, which are supported on a ring or platform, and po-
sition the shield for the flask and burner. Connect the flask
and condenser, place the graduate under the outlet of the
condenser tube, and insert the thermometer. The thermom-
eter should be located in the center of the neck so that the
top of the contraction chamber (or bulb, if 37C or 38C is
used) is level with the bottom of the outlet to the side arm.
Regulate the heating so that the first drop of liquid is col-
lected within 5–10 min. Read the thermometer at the in-
stant the first drop of distillate falls from the end of the
condenser tube, and record as the initial boiling point. Con-
tinue the distillation at the rate of 4 or 5 mL/min of distil-
late, noting the temperature as soon as the last drop of
liquid evaporates from the bottom of the flask (dry point) or
when the specified percentage has distilled over. Correct the
observed temperature readings for any variation in the baro-
metric pressure from the normal (760 mm) by allowing 0.1°
for each 2.7 mm of variation, adding the correction if the
pressure is lower, or subtracting if higher, than 760 mm.
When a total-immersion thermometer is used, correct for
the temperature of the emergent stem:
Result = 0.00015 × N(T − t)
in which N represents the number of degrees of emergent
stem from the bottom of the stopper, T represents the ob-
served temperatures of the distillation, and t represents the
temperature registered by an auxiliary thermometer, the
FCC 8
bulb of which is placed midway of the emergent stem, add-
ing the correction to the observed readings of the main
thermometer.
MELTING RANGE OR TEMPERATURE
DETERMINATION
For purposes of the FCC, the melting range or temperature
of a solid is defined as those points of temperature within
which or the point at which the solid coalesces and is com-
pletely melted when determined as directed below. Any ap-
paratus or method capable of equal accuracy may be used.
The accuracy should be checked frequently by the use of
one or more of the six USP Melting Point Reference Stan-
dards, preferably the one that melts nearest the melting
temperature of the compound to be tested.
Five procedures for the determination of melting range or
temperature are given herein, varying in accordance with
the nature of the substance. When no class is designated in
the monograph, use the procedure for Class I.
The procedure known as the mixed melting point deter-
mination, whereby the melting range of a solid under test is
compared with that of an intimate mixture of equal parts of
the solid and an authentic specimen of it, may be used as a
confirmatory identification test. Agreement of the observa-
tions on the original and the mixture usually constitutes reli-
able evidence of chemical identity.
Apparatus The melting range apparatus consists of a
glass container for a bath of colorless fluid, a suitable stirring
device, an accurate thermometer (see Appendix I), and a
controlled source of heat. The bath fluid is selected consis-
tent with the temperature required, but light paraffin is
used generally, and certain liquid silicones are well adapted
to the higher temperature ranges. The fluid is deep enough
to permit immersion of the thermometer to its specified im-
mersion depth so that the bulb is still about 2 cm above the
bottom of the bath. The heat may be supplied electrically or
by an open flame. The capillary tube is about 10 cm long,
with an internal diameter of 0.8–1.2 mm, and with walls
0.2–0.3 mm thick.
The thermometer is preferably one that conforms to the
specifications provided under Thermometers, Appendix I, se-
lected for the desired accuracy and range of temperature.
Procedure for Class I Reduce the sample to a very fine
powder, and unless otherwise directed, render it anhydrous
when it contains water of hydration by drying it at the tem-
perature specified in the monograph, or when the sub-
stance contains no water of hydration, dry it over a suitable
desiccant for 16–24 h.
Charge a capillary glass tube, one end of which is sealed,
with a sufficient amount of the dry powder to form a col-
umn in the bottom of the tube 2.5–3.5 mm high when
packed down as closely as possible by moderate tapping on
a solid surface.
Heat the bath until a temperature approximately 30° be-
low the expected melting point is reached, attach the capil-
lary tube to the thermometer, and adjust its height so that
the material in the capillary is level with the thermometer
bulb. Return the thermometer to the bath, continue the
General Tests and Assays / Appendix II / 1231
heating, with constant stirring, at a rate of rise of approxi-
mately 3°/min until a temperature 3° below the expected
melting point is attained, then carefully regulate the rate to
about 1°–2°/min until melting is complete.
The temperature at which the column of the sample is
observed to collapse definitely against the side of the tube
at any point is defined as the beginning of melting, and the
temperature at which the sample becomes liquid through-
out is defined as the end of melting. The two temperatures
fall within the limits of the melting range.
Procedure for Class Ia Prepare the sample and charge
the capillary glass tube as directed for Class I. Heat the bath
until a temperature 10 ± 1° below the expected melting
range is reached, then introduce the charged tube, and
heat at a rate of rise of 3 ± 0.5°/min until melting is com-
plete. Record the melting range as for Class I.
Procedure for Class Ib Place the sample in a closed
container, and cool to 10° or lower for at least 2 h. Without
previous powdering, charge the cooled material into the
capillary tube as directed for Class I, immediately place the
charged tube in a vacuum desiccator, and dry at a pressure
not exceeding 20 mm Hg for 3 h. Immediately upon re-
moval from the desiccator, fire-seal the open end of the
tube. As soon as is practicable, proceed with the determina-
tion of the melting range as follows: Heat the bath until a
temperature of 10 ± 1° below the expected melting range is
reached, then introduce the charged tube, and heat at a
rate of rise of 3 ± 0.5°/min until melting is complete. Record
the melting range as directed in Class I.
If the particle size of the material is too large for the capil-
General Tests and Assays
lary, precool the sample as directed above, then with as
little pressure as possible, gently crush the particles to fit the
capillary, and immediately charge the tube.
Procedure for Class II Carefully melt the material to be
tested at as low a temperature as possible, and draw it into
a capillary tube that is left open at both ends to a depth of
about 10 mm. Cool the charged tube at 10°, or lower, for
24 h, or in contact with ice for at least 2 h. Then attach the
tube to the thermometer by means of a rubber band, adjust
it in a water bath so that the upper edge of the material is
10 mm below the water level, and heat as directed for Class
I, except within 5° of the expected melting temperature,
regulate the rate of rise of temperature to 0.5°–1.0°/min.
The temperature at which the material is observed to rise in
the capillary tube is the melting temperature.
Procedure for Class III Melt a quantity of the substance
slowly, while stirring, until it reaches a temperature of
90°–92°. Remove the source of heat, and allow the molten
substance to cool to a temperature of 8°–10° above the
expected melting point. Chill the bulb of an ASTM 14C
thermometer (see Appendix I) to 5°, wipe it dry, and while
it is still cold, dip it into the molten substance so that ap-
proximately the lower half of the bulb is submerged. With-
draw it immediately, and hold it vertically away from the
heat until the wax surface dulls, then dip it for 5 min into a
water bath having a temperature not higher than 16°.
Fix the thermometer securely in a test tube so that the
lower point is 15 mm above the bottom of the test tube.
Suspend the test tube in a water bath adjusted to about
16°, and raise the temperature of the bath at the rate of
 1232 / Appendix II / General Tests and Assays
2°/min to 30°, then change to a rate of 1°/min, and note
the temperature at which the first drop of melted substance
leaves the thermometer. Repeat the determination twice on
a freshly melted portion of the sample. If the variation of
three determinations is less than 1°, take the average of the
three as the melting point. If the variation of three determi-
nations is greater than 1°, make two additional determina-
tions and take the average of the five.
OPTICAL (SPECIFIC) ROTATION
Many chemicals in a pure state or in solution are optically
active in the sense that they cause incident polarized light
to emerge in a plane forming a measurable angle with the
plane of the incident light. When this effect is large enough
for precise measurement, it may serve as the basis for an
assay or an identity test. In this connection, the optical rota-
tion is expressed in degrees, as either angular rotation (ob-
served) or specific rotation (calculated with reference to the
specific concentration of 1 g of solute in 1 mL of solution,
measured under stated conditions).
Specific rotation of a liquid substance usually is expressed
by the equation [α]xt = a/ld, and for solutions of solid sub-
stances, expressed by the equation [α]xt = 100a/lpd = 100a/
lc, in which a is the corrected observed rotation, in degrees,
at temperature t; x is the wavelength of the light used; l is
the length of the polarimeter cell, in dm; d is the specific
gravity of the liquid or solution at the temperature of obser-
vation; p is the concentration of the solution expressed as
the number of grams of substance in 100 g of solution; and
General Tests and Assays
c is the concentration of the solution expressed as the num-
ber of grams of substance in 100 mL of solution. The con-
centrations p and c should be calculated on the dried or
anhydrous basis, unless otherwise specified. Spectral lines
most frequently employed are the D line of sodium (doublet
at 589.0 nm and 589.6 nm) and the yellow-green line of
mercury at 546.1 nm. The specific gravity and the rotatory
power vary appreciably with the temperature.
The accuracy and precision of optical rotatory measure-
ments will be increased if they are carried out with due
regard for the following general considerations.
Supplement the source of illumination with a filtering sys-
tem capable of transmitting light of a sufficiently monochro-
matic nature. Precision polarimeters generally are designed
to accommodate interchangeable disks to isolate the D line
from sodium light or the 546.1-nm line from the mercury
spectrum. With polarimeters not thus designed, cells con-
taining suitably colored liquids may be employed as filters
(see also A. Weissberger and B. W. Rossiter, Techniques of
Chemistry, Vol. I: Physical Methods of Chemistry, Part 3, Wi-
ley-Interscience, New York, 1972).
Pay special attention to temperature control of the solu-
tion and of the polarimeter. Make accurate and reproducible
observations to the extent that differences between repli-
cates, or between observed and true values of rotation (the
latter value having been established by calibration of the
polarimeter scale with suitable standards), calculated in
terms of either specific rotation or angular rotation, which-
ever is appropriate, do not exceed one-fourth of the range
given in the individual monograph for the rotation of the
article being tested. Generally, a polarimeter accurate to
FCC 8
0.05° of angular rotation, and capable of being read with
the same precision, suffices for FCC purposes; in some cases,
a polarimeter accurate to 0.01°, or less, of angular rotation,
and read with comparable precision, may be required.
Fill polarimeter tubes in such a way as to avoid creating
or leaving air bubbles, which interfere with the passage of
the beam of light. Interference from bubbles is minimized
with tubes in which the bore is expanded at one end. How-
ever, tubes of uniform bore, such as semimicro- or micro-
tubes, require care for proper filling. At the time of filling,
the tubes and the liquid or solution should be at a tempera-
ture not higher than that specified for the determination to
guard against the formation of a bubble upon cooling and
contraction of the contents.
In closing tubes having removable end plates fitted with
gaskets and caps, the latter should be tightened only
enough to ensure a leak-proof seal between the end plate
and the body of the tube. Excessive pressure on the end
plate may set up strains that result in interference with the
measurements. In determining the specific rotation of a sub-
stance of low rotatory power, loosen the caps and tighten
them again between successive readings in the measure-
ment of both the rotation and the zero point. Differences
arising from end plate strain thus generally will be revealed
and appropriate adjustments to eliminate the cause may be
made.
Procedure In the case of a solid, dissolve the substance
in a suitable solvent, reserving a separate portion of the lat-
ter for a blank determination. Make at least five readings of
the rotation of the solution, or of the substance itself if liq-
uid, at 25° or the temperature specified in the individual
monograph. Replace the solution with the reserved portion
of the solvent (or, in the case of a liquid, use the empty
tube), make the same number of readings, and use the av-
erage as the zero point value. Subtract the zero point value
from the average observed rotation if the two figures are of
the same sign, or add if opposite in sign, to obtain the
corrected observed rotation.
Calculation Calculate the specific rotation of a liquid
substance, or of a solid in solution, by application of one of
the following formulas:
1. for liquid substances,
2. for solutions of solids,
in which a is the corrected observed rotation, in degrees, at
temperature t; x is the wavelength of the light used; l is the
length, in dm, of the polarimeter cell; d is the specific grav-
ity of the liquid or solution at the temperature of observa-
tion; p is the concentration of the solution expressed as the
number of grams of substance in 100 g of solution; and c is
the concentration of the solution expressed as the number
of grams of substance in 100 mL of solution. The concentra-
tions p and c should be calculated on the dried or anhy-
drous basis, unless otherwise specified.
FCC 8
pH DETERMINATION
Principle The definition of pH is the negative log of the
hydrogen ion concentration in moles per liter of aqueous
solutions. Measure pH potentiometrically by using a pH
meter or colorimetrically by using pH indicator paper.
Scope This method is suitable to determine the pH of
aqueous solutions. While pH meters, calibrated with aque-
ous solutions, are sometimes used to make measurements in
semiaqueous solutions or in nonaqueous polar solutions, the
value obtained is the apparent pH value only and should
not be compared with the pH of aqueous solutions. For
nonpolar solutions, pH has no meaning, and pH electrodes
may be damaged by direct contact with these solutions.
References to the pH of nonpolar solutions or liquids usually
indicate the pH of a water extract of the nonpolar liquid or
the apparent pH of a mixture of the nonpolar liquid in a
polar liquid such as alcohol or alcohol–water mixtures.
Procedure [Potentiometric Method (pH Meter)]
Calibration: Select two standard buffers to bracket, if possi-
ble, the anticipated pH of the unknown substances. These
commercially available standards and the sample should be
at the same temperature, within 2°. Set the temperature
compensator of the pH meter to the temperature of the
samples and standards. Follow the manufacturer’s instruc-
tions for setting temperature compensation and for adjust-
ing the output during calibration. Rinse the electrodes with
distilled or deionized water, and blot them dry with clean,
absorbent laboratory tissue. Place the electrode(s) in the first
standard buffer solution, and adjust the standardization con-
trol so that the pH reading matches the stated pH of the
standard buffer. Repeat this procedure with fresh portions of
the first buffer solution until two successive readings are
within ±0.02 pH units with no further adjustment. Rinse the
electrodes, blot them dry, and place them in a portion of
the second standard buffer solution. Following the manufac-
turer’s instructions, adjust the slope control (not the stan-
dardization control) until the output displays the pH of the
second standard buffer.
Repeat the sequence of standardization with both buffers
until pH readings are within ± 0.02 pH units for both buffers
without adjustments to either the slope or standardization
controls. The pH of the unknown may then be measured,
using either a pH electrode in combination with a reference
electrode or a single combination electrode. Select elec-
trodes made of chemically resistant glass when measuring
samples of either low or high pH.
pH Indicator Paper: Test papers impregnated with acid–base
indicators, although less accurate than pH meters, offer a
convenient way to determine the pH of an aqueous solu-
tion. They may be purchased in rolls or strips covering all or
part of the pH range; papers covering a narrow part of the
pH range can be sensitive to differences of 0.2 pH units.
Some test papers comprise a plastic strip with small squares
of test paper attached. The different squares are sensitive to
different pH ranges. When using this type of test paper, wet
all of the squares with the test sample to ensure a correct
pH reading.
Test paper can contaminate the sample being tested;
therefore, do not dip it into the sample. Either use a clean
General Tests and Assays / Appendix II / 1233
glass rod to remove a drop of the test solution and place it
on the test paper, or transfer a small amount of the sample
to a small container, dip the test paper into this portion,
and compare the developed color with the color compari-
son chart provided with the test paper to determine the pH
of the sample.
READILY CARBONIZABLE SUBSTANCES
Reagents
Sulfuric Acid, 95%: Add a quantity of sulfuric acid of known
concentration to sufficient water to adjust the final concen-
tration to 94.5%–95.5% of H2SO4. Because the acid concen-
tration may change upon standing or upon intermittent use,
check the concentration frequently and either adjust solu-
tions assaying more than 95.5% or less than 94.5% by add-
ing either diluted or fuming sulfuric acid, as required, or
discard them.
Cobaltous Chloride CS: Dissolve about 65 g of cobaltous
chloride (CoCl2·6H2O) in enough of a mixture of 25 mL of
hydrochloric acid and 975 mL of water to make 1000 mL.
Pipet 5 mL of this solution into a 250-mL iodine flask, add 5
mL of hydrogen peroxide TS (3%) and 15 mL of a solution
of sodium hydroxide (1:5), boil for 10 min, cool, and add 2
g of potassium iodide and 20 mL of sulfuric acid (1:4).
When the precipitate has dissolved, titrate the liberated io-
dine with 0.1 N sodium thiosulfate. The titration is sensitive
to air oxidation and should be blanketed with carbon diox-
ide. Each mL of 0.1 N sodium thiosulfate is equivalent to
23.79 mg of CoCl2·6H2O. Adjust the final volume of the
General Tests and Assays
solution by adding enough of the mixture of hydrochloric
acid and water so that each mL contains 59.5 mg of
CoCl2·6H2O.
Cupric Sulfate CS: Dissolve about 65 g of cupric sulfate
(CuSO4·5H2O) in enough of a mixture of 25 mL of hydro-
chloric acid and 975 mL of water to make 1000 mL. Pipet
10 mL of this solution into a 250-mL iodine flask; add 40
mL of water, 4 mL of acetic acid, and 3 g of potassium
iodide; and titrate the liberated iodine with 0.1 N sodium
thiosulfate, adding starch TS as the indicator. Each mL of
0.1 N sodium thiosulfate is equivalent to 24.97 mg of
CuSO4·5H2O. Adjust the final volume of the solution by add-
ing enough of the mixture of hydrochloric acid and water
so that each mL contains 62.4 mg of CuSO4·5H2O.
Ferric Chloride CS: Dissolve about 55 g of ferric chloride
(FeCl3·6H2O) in enough of a mixture of 25 mL of hydrochlo-
ric acid and 975 mL of water to make 1000 mL. Pipet 10
mL of this solution into a 250-mL iodine flask; add 15 mL of
water, 5 mL of hydrochloric acid, and 3 g of potassium io-
dide; and allow the mixture to stand for 15 min. Dilute with
100 mL of water, and titrate the liberated iodine with 0.1 N
sodium thiosulfate, adding starch TS as the indicator. Per-
form a blank determination with the same quantities of the
same reagents and in the same manner, and make any nec-
essary correction. Each mL of 0.1 N sodium thiosulfate is
equivalent to 27.03 mg of FeCl3·6H2O. Adjust the final vol-
ume of the solution by adding the mixture of hydrochloric
acid and water so that each mL contains 45.0 mg of
FeCl3·6H2O.
 1234 / Appendix II / General Tests and Assays FCC 8

a
Platinum–Cobalt CS: Transfer 1.246 g of potassium Matching Fluids (continued)
chloroplatinate (K2PtCl6) and 1.00 g of crystallized cobaltous Parts of Parts of
chloride (CoCl2·6H2O) into a 1000-mL volumetric flask, dis- Cobaltous Ferric Parts of
Matching Chloride Chloride Cupric Parts of
solve in about 200 mL of water and 100 mL of hydrochloric
Fluid CS CS Sulfate CS Water
acid, dilute with water to volume, and mix. This solution
O 0.1 4.8 0.1 0.0
has a color of 500 APHA units.
P 0.2 0.4 0.1 4.3
[NOTE—Use this solution only when specified in an in-
Q 0.2 0.3 0.1 4.4
dividual monograph.]
R 0.3 0.4 0.2 4.1
Procedure Unless otherwise directed, add the specified S 0.2 0.1 0.0 4.7
quantity of the substance, finely powdered if in solid form,
T 0.5 0.5 0.4 3.6
in small portions to the comparison container, which is
made of colorless glass resistant to the action of sulfuric acid
a Solutions A–D, very light brown-yellow. Solutions E–L, yellow through red-
yellow. Solutions M–O, green-yellow. Solutions P–T, light pink.
and contains the specified volume of 95% Sulfuric Acid.
Stir the mixture with a glass rod until solution is com- REFRACTIVE INDEX
plete, allow the solution to stand for 15 min, unless other-
wise directed, and compare the color of the solution with The refractive index of a transparent substance is the ratio
that of the specified matching fluid in a comparison con- of the velocity of light in air to its velocity in that material
tainer that also is of colorless glass and has the same inter- under like conditions. It is equal to the ratio of the sine of
nal and cross-section dimensions, viewing the fluids trans- the angle of incidence made by a ray in air to the sine of
versely against a background of white porcelain or white the angle of refraction made by the ray in the material be-
glass. ing tested. The refractive index values specified in this Co-
When heat is directed to effect solution of the substance dex are for the D line of sodium (589 nm) unless otherwise
in the 95% Sulfuric Acid, mix the sample and the acid in a specified. The determination should be made at the temper-
test tube, heat as directed, cool, and transfer the solution to ature specified in the individual monograph, or at 25° if no
the comparison container for matching. temperature is specified. This physical constant is used as a
Matching Fluids For purposes of comparison, a series of means for identification of, and detection of impurities in,
20 matching fluids, each designated by a letter of the al- volatile oils and other liquid substances. The Abbé refrac-
phabet, is provided, the composition of each being as indi- tometer, or other refractometers of equal or greater accu-
cated in the accompanying table. To prepare the matching racy, may be employed at the discretion of the operator.
General Tests and Assays

fluid specified, pipet the prescribed volumes of the colori-

metric test solutions (CS) and water into one of the match- SOLIDIFICATION POINT
ing containers, and mix the solutions in the container.
a Scope This method is designed to determine the solidifi-
Matching Fluids
cation point of food-grade chemicals having appreciable
Parts of Parts of
heats of fusion. It is applicable to chemicals having solidifi-
Cobaltous Ferric Parts of
Matching Chloride Chloride Cupric Parts of cation points between −20° and +150°. Necessary modifica-
Fluid CS CS Sulfate CS Water tions will be noted in individual monographs.
A 0.1 0.4 0.1 4.4 Definition Solidification Point is an empirical constant
B 0.3 0.9 0.3 8.5 defined as the temperature at which the liquid phase of a
C 0.1 0.6 0.1 4.2
substance is in approximate equilibrium with a relatively
small portion of the solid phase. It is measured by noting
D 0.3 0.6 0.4 3.7
the maximum temperature reached during a controlled
E 0.4 1.2 0.3 3.1 cooling cycle after the appearance of a solid phase.
F 0.3 1.2 0.0 3.5 The solidification point is distinguished from the freezing
G 0.5 1.2 0.2 3.1 point in that the latter term applies to the temperature of
H 0.2 1.5 0.0 3.3 equilibrium between the solid and liquid state of pure
compounds.
I 0.4 2.2 0.1 2.3
Some chemical compounds have more than one tempera-
J 0.4 3.5 0.1 1.0
ture at which there may be an equilibrium between the
K 0.5 4.5 0.0 0.0 solid and liquid state depending on the crystal form of the
L 0.8 3.8 0.1 0.3 solid that is present.
M 0.1 2.0 0.1 2.8 Apparatus The apparatus illustrated in Figures 3 and 4
N 0.0 4.9 0.1 0.0 consists of the components described in the following
paragraphs.
a Solutions A–D, very light brown-yellow. Solutions E–L, yellow through red-

yellow. Solutions M–O, green-yellow. Solutions P–T, light pink.

FCC 8
Figure 3. Apparatus for Determination of Solidification Point
Figure 4. Stirrer for Solidification Point Determination
Thermometer: A thermometer having a range not exceed-
ing 30°, graduated in 0.1° divisions, and calibrated for 76-
mm immersion should be employed. A satisfactory series of
thermometers, covering a range from −20° to +150°, is
available as ASTM-E1 89C through 96C (see Thermometers,
Appendix I). A thermometer should be chosen such that the
solidification point is not obscured by the cork stopper of
the sample container.
Sample Container: Use a standard glass 25-mm × 150-mm
test tube with a lip, fitted with a two-hole cork stopper to
hold the thermometer in place and to allow adequate stir-
ring with a stirrer.
Air Jacket: For the air jacket, use a standard glass 38-mm ×
200-mm test tube with a lip and fitted with a cork or rub-
ber stopper bored with a hole into which the sample con-
tainer can easily be inserted up to the lip.
Cooling Bath: Use a 2000-mL beaker or a similar, suitable
container as a cooling bath. Fill it with an appropriate cool-
ing medium such as glycerin, mineral oil, water, water and
ice, or alcohol–dry ice.
Stirrer: The stirrer (Figure 4) consists of a 1-mm in diameter
(B & S gauge 18), corrosion-resistant wire bent into a series
of three loops about 25 mm apart. It should be made so
General Tests and Assays / Appendix II / 1235
that it will move freely in the space between the thermome-
ter and the inner wall of the sample container. The shaft of
the stirrer should be of a convenient length designed to
pass loosely through a hole in the cork holding the ther-
mometer. Stirring may be hand operated or mechanically
activated at 20–30 strokes/min.
Assembly: Assemble the apparatus in such a way that the
cooling bath can be heated or cooled to control the desired
temperature ranges. Clamp the air jacket so that it is held
rigidly just below the lip, and immerse it in the cooling bath
to a depth of 160 mm.
Sample Preparation The solidification point of chemi-
cals is usually determined as they are received. Some may
be hygroscopic, however, and will require special drying. If
this is necessary, it will be noted in the individual
monographs.
Products that are normally solid at room temperature
must be carefully melted at a temperature about 10° above
the expected solidification point. Care should be observed
to avoid heating in such a way as to decompose or distill
any portion of a sample.
Procedure Adjust the temperature of the cooling bath to
about 5° below the expected solidification point. Fit the
thermometer and stirrer with a cork stopper so that the
thermometer is centered and the bulb is about 20 mm from
the bottom of the sample container. Transfer a sufficient
amount of the sample, previously melted if necessary, into
the sample container to fill it to a depth of about 90 mm
when in the molten state. Place the thermometer and stirrer
in the sample container, and adjust the thermometer so that
General Tests and Assays
the immersion line will be at the surface of the liquid and so
that the end of the bulb is 20 ± 4 mm from the bottom of
the sample container. When the temperature of the sample
is about 5° above the expected solidification point, place
the assembled sample tube in the air jacket.
Allow the sample to cool while stirring, at the rate of
20–30 strokes/min, in such a manner that the stirrer does
not touch the thermometer. Stir the sample continuously
during the remainder of the test.
The temperature at first will gradually fall, then will be-
come constant as crystallization starts and continues under
equilibrium conditions, and finally will start to drop again.
Some chemicals may supercool slightly below (0.5°) the so-
lidification point; as crystallization begins, the temperature
will rise and remain constant as equilibrium conditions are
established. Other products may cool more than 0.5° and
cause deviation from the normal pattern of temperature
change. If the temperature rise exceeds 0.5° after the initial
crystallization begins, repeat the test, and seed the melted
compound with small crystals of the sample at 0.5° intervals
as the temperature approaches the expected solidification
point. Crystals for seeding may be obtained by freezing a
small sample in a test tube directly in the cooling bath. It is
preferable that seed of the stable phase be used from a
previous determination.
Observe and record the temperature readings at regular
intervals until the temperature rises from a minimum, due
to supercooling, to a maximum and then finally drops. The
maximum temperature reading is the solidification point.
Readings 10 s apart should be taken to establish that the
 1236 / Appendix II / General Tests and Assays
temperature is at the maximum level and should continue
until the drop in temperature is established.
VISCOSITY DETERMINATION
Viscosity is a fluid’s measured internal resistance to flow.
Thick, slow-moving fluids have higher viscosities than thin,
free-flowing fluids. The basic unit of measure for viscosity is
the poise or Pascal second, Pa·s, in SI units. The relationship
between poise and Pa·s is 1 poise = 0.1 Pa·s. Since com-
monly encountered viscosities are often fractions of 1 poise,
viscosities are commonly expressed as centipoises (one cen-
tipoise = 0.01 poise). Poise or centipoise is the unit of meas-
ure for absolute viscosity. Kinematic viscosity also is com-
monly used and is determined by dividing the absolute
viscosity of the test liquid by the density of the test liquid at
the same temperature as the viscosity measurement and is
expressed as stokes or centistokes (poise/density = stokes).
The specified temperature is important: viscosity varies
greatly with temperature, generally decreasing with increas-
ing temperature.
Absolute viscosity can be determined directly if accurate
dimensions of the measuring instruments are known. It is
common practice to calibrate an instrument with a fluid of
known viscosity and to determine the unknown viscosity of
another fluid by comparison with that of the known
viscosity.
Many substances, such as gums, have a variable viscosity,
and most of them are less resistant to flow at higher flow
(more correctly, shear) rates. In such cases, select a given
General Tests and Assays
set of conditions for measurement, and consider the meas-
urement obtained to be an apparent viscosity. Since a
change in the conditions of measurement would yield a dif-
ferent value for the apparent viscosity of such substances,
the operator must closely adhere to the instrument dimen-
sions and conditions for measurement.
Measuring Viscosity Several common methods are
available for measuring viscosity. Two very common ones
are the use of capillary tubes such as Ubbelohde, Ostwald,
or Cannon-Fenske viscometer tubes and the use of a rotat-
ing spindle such as the Brookfield viscometer.
Determine the viscosity in capillary tubes by measuring
the amount of time it takes for a given volume of liquid to
flow through a calibrated capillary tube. Calibrate the capil-
lary tube by using liquids of known viscosity. The calibration
may be supplied with the viscometer tube when purchased
along with specific instructions for its use. Many types of
capillary viscometer tubes are available, and exact proce-
dures will vary with the type of tube chosen. Examples of
procedures are in the following sections: Viscosity of Dimeth-
ylpolysiloxane and Viscosity of Methylcellulose. In general, cali-
brate capillary viscometers by filling the viscometers per the
manufacturer’s instructions and allowing the filled tube to
equilibrate to the given temperature in a constant-tempera-
ture bath. Draw the liquid to the top graduation line, and
measure the time, in seconds, it takes for the liquid to flow
FCC 8
from the upper mark to the lower mark in the capillary
tube. Calculate the viscometer constant, k:
k = v/dt
in which v is the known viscosity, in centipoises, of the stan-
dard liquid; d is the density, at the specified temperature, of
the liquid; and t is the time, in seconds, for the liquid to
pass from the upper mark to the lower mark. It is not neces-
sary to recalibrate the tube unless changes or repairs are
made to it. To measure viscosity, introduce the unknown
liquid into the viscometer tube in the same way as the cali-
bration standard was introduced, and measure the time, in
seconds, it takes for the liquid to flow from the upper mark
to the lower mark. Calculate viscosity:
v = kdt
in which v is the viscosity to be determined, k is the viscom-
eter constant, and d is the density of the liquid being
measured.
Using rotational viscometers provides a particularly rapid
and convenient method for determining viscosity. They em-
ploy a rotating spindle or cup immersed in the liquid, and
they measure the resistance of the liquid to the rotation of
the spindle or cup. A wide range of viscosities can be meas-
ured with one instrument by using spindles or cups of dif-
ferent sizes and by rotating them at different speeds. The
manufacturer supplies the calibration of viscosity versus the
spindle size and speed, which can be checked by using
fluids of known viscosity. Take a measurement by allowing
the sample to come to the desired temperature in a
constant-temperature bath and immersing the spindle or
cup to the depth specified by the manufacturer. Allow the
spindle or cup to rotate until a constant reading is obtained.
Multiply the reading by a factor supplied by the manufac-
turer for a given spindle or cup and given rotational speed
to obtain the viscosity. The exact procedures will vary with
the particular instrument. An example is given in the section
on Viscosity of Cellulose Gum.
Another method to determine viscosity uses the falling-
ball viscometer. Determine viscosity by noting the time it
takes for a ball to fall through the distance between two
marks on a tube filled with the unknown liquid (the tube is
generally in a constant-temperature bath). Use balls of dif-
ferent weights to measure a wide range of viscosities. Calcu-
late the viscosity by using manufacturer-supplied constants
for the ball used. These instruments can be quite precise for
Newtonian liquids, that is, liquids that do not have viscosi-
ties that vary with flow (more correctly, shear) rate.
Three specific methods are described below:
Viscosity of Dimethylpolysiloxane
Apparatus The Ubbelohde suspended level viscometer,
shown in Figure 5 is preferred to determine the viscosity of
dimethylpolysiloxane. Alternatively, a Cannon-Ubbelohde
viscometer may be used.
FCC 8 General Tests and Assays / Appendix II / 1237

in which cs is the viscosity, in centistokes, and t1 is the ef-

flux time, in seconds, for the standard liquid.
Determination of the Viscosity of Dimethylpolysilox-
ane Charge the viscometer with the sample in the same
manner as described for the calibration procedure; deter-
mine the efflux time, t2; and calculate the viscosity of the
dimethylpolysiloxane:

V = C × t2

Viscosity of Methylcellulose
Apparatus Viscometers used to determine the viscosity of
methylcellulose and some related compounds are illustrated
in Figure 6 and consist of three parts: a large filling tube, A;
an orifice tube, B; and an air vent to the reservoir, C.

Figure 5. Ubbelohde Viscometer for Dimethylpolysiloxane

(all dimensions are in mm)
Select a viscometer having a minimum flow time of at
least 200 s. Use a No. 3 size Ubbelohde, or a No. 400 size
Cannon-Ubbelohde, viscometer for the range of 300–600
centistokes. The viscometer should be fitted with holders
that satisfy the dimensional positions of the separate tubes
as shown in the diagram and that hold the viscometer verti-
cally. Filling lines in bulb A indicate the minimum and maxi-
mum volumes of liquid to be used for convenient operation.

General Tests and Assays

The volume of bulb B is approximately 5 mL.
Calibration of the Viscometer Determine the viscosity
constant, C, for each viscometer by using an oil of known
viscosity.1 Charge the viscometer by tilting the instrument
about 30 degrees from the vertical, with bulb A below the
capillary, and then introduce enough of the sample into
tube l to bring the level up to the lower filling line. The
level should not be above the upper filling line when the
viscometer is returned to the vertical position and the sam-
ple has drained from tube l. Charge the viscometer in such Figure 6. Methylcellulose Viscometers
a manner that the U-tube at the bottom fills completely
There are two basic types of methylcellulose
without trapping air.
viscometers—one for cellulose derivatives of a range be-
After the viscometer has been in a constant-temperature
tween 1500 and 4000 centipoises, and the other for less
bath (25 ± 0.2°) long enough for the sample to reach tem-
viscous ones. Each type of viscometer is modified slightly for
perature equilibrium, place a finger over tube 3, and apply
the different viscosities.
suction to tube 2 until the liquid reaches the center of bulb
C. Remove suction from tube 2, then remove the finger Calibration of the Viscometer Determine the viscome-
from tube 3, and place it over tube 2 until the sample drops ter constant, K, for each viscometer by using an oil of
away from the lower end of the capillary. Remove the finger known viscosity.2 Place an excess of the liquid that is to be
from tube 2, and measure the time, to the nearest 0.1 s, tested (adjusted to 20 ± 0.1°) in the filling tube, A, and
required for the meniscus to pass from the first timing mark transfer it to the orifice tube, B, by gentle suction, taking
(T1) to the second (T2). care to keep the liquid free from air bubbles by closing the
Calculate the viscometer constant, C: air vent tube, C. Adjust the column of liquid in tube B so it
is even with the top graduation line. Open both tubes B
C = cs/t1 and C to permit the liquid to flow into the reservoir against
atmospheric pressure.

1 Oils of known viscosities may be obtained from the Cannon Instrument 2 Oils of known viscosities may be obtained from the Cannon Instrument

Co., P.O. Box 812, State College, PA 16801. For determining the viscosity of Co., P.O. Box 812, State College, PA 16801. For determining the viscosity of
dimethylpolysiloxane, choose an oil with a viscosity as close as possible to methylcellulose, choose an oil that has a viscosity as close as possible to that
that of the type of sample to be tested. of the type of sample to be tested.
 1238 / Appendix II / General Tests and Assays FCC 8

[NOTE—Failure to open air vent tube C before deter-

mining the viscosity will yield false values.]
Record the time, in seconds, for the liquid to flow from the
upper mark to the lower mark in tube B.
Calculate the viscometer constant, K:

K = V/dt

in which V is the viscosity, in centipoises, of the liquid; K is

the viscometer constant; d is the specific gravity of the liq-
uid tested at 20°/20°; and t is the time, in seconds, for the
liquid to pass from the upper to the lower mark.
For the calibration, all values in the equation are known
or can be determined except K, which must be solved. If a
tube is repaired, it must be recalibrated to avoid obtaining
significant changes in the value of K.
Determination of the Viscosity of Methylcellulose
Prepare a 2% solution of methylcellulose or other cellulose
derivative, by weight, as directed in the monograph. Place
the solution in the proper viscometer and determine the
time, t, required for the solution to flow from the upper
mark to the lower mark in orifice tube B. Separately deter-
mine the specific gravity, d, at 20°/20°. Viscosity, V = Kdt.
Viscosity of Cellulose Gum
Figure 7. Agitator for Viscosity of Cellulose Gum
Apparatus Use a Brookfield Model LV series viscometer,
analog or digital, or equivalent type viscometer for the de- [NOTE—The agitator may be fabricated from stainless
termination of viscosity of aqueous solutions of cellulose steel (Hercules, Inc., Wilmington, Delaware, or equiva-
gum within the range of 25–10,000 centipoises at 25°. Ro- lent.) or glass as shown in Figure 7. Where this proce-
tational viscometers of this type have spindles for use in dure is specified for viscosity measurements by refer-
determining the viscosity of different viscosity types of cellu- ence in other monographs, equivalent three-blade
General Tests and Assays

lose gum. The spindles and speeds for determining viscosity agitators may be used.]
within different ranges are tabulated below.
Sample Container: Use a glass jar about 152 mm deep hav-
Viscometer Spindles Required for Given Speeds ing an od of approximately 64 mm and a capacity of about
Viscosity 340 g.
Range Water Bath: Use a water bath capable of maintaining a
(centi- Spindle Speed
constant temperature. Set the temperature to 25°, and
poises) No. (rpm) Scale Factor
maintain it within ±0.2°.
10–100 1 60 100 1
Thermometer: Use an ASTM Saybolt Viscosity Thermometer
100–200 1 30 100 2
having a range from 19° to 27° and conforming to the
200–1000 2 30 100 10 requirements for Thermometer 17C as described in ASTM
1000–4000 3 30 100 40 Specification E1.
4000–10, Sample Preparation Accurately weigh an amount of
4 30 100 200
000
sample equivalent to 4.8 g of cellulose gum on the dried
basis, and record the actual quantity required, in grams, as
Mechanical Stirrer: Use an agitator, essentially as shown S. Transfer an accurately measured volume of water equiva-
in Figure 7, that can be attached to a variable-speed motor lent to 240 − S g into the sample container. Position the
capable of operating at 900 ± 100 rpm under varying load stirrer in the sample container, allowing minimal clearance
conditions. between the stirrer and the bottom of the container. Begin
stirring, and slowly add the sample. Adjust the stirring speed
to approximately 900 ± 100 rpm. Mix for exactly 2 h. Do
not allow the stirring speed to exceed 1200 rpm. Remove
the stirrer, cap the sample container, and transfer the sam-
ple container into a constant-temperature water bath, main-
tained at 25 ± 0.2°, for 1 h. Check the sample temperature
with a thermometer at the end of 1 h to ensure that the
test temperature has been reached.
Procedure Remove the sample container from the water
bath, shake vigorously for 10 s, and measure the viscosity
with the Brookfield viscometer, using the proper spindle and
FCC 8
speed indicated in the accompanying table. Be sure to use
the viscometer guard, and allow the spindle to rotate for 3
min before taking the reading. Calculate the viscosity, in
centipoises, by multiplying the reading observed by the ap-
propriate factor from the table.
WATER DETERMINATION
Method I (Karl Fischer Titrimetric Method) Deter-
mine the water by Method Ia, unless otherwise specified in
the individual monograph.
Method Ia (Direct Titration)
Principle The titrimetric determination of water is based
on the quantitative reaction of water with an anhydrous so-
lution of sulfur dioxide and iodine in the presence of a
buffer that reacts with hydrogen ions.
In the original titrimetric solution, known as Karl Fischer
Reagent, the sulfur dioxide and iodine are dissolved in pyri-
dine and methanol. Pyridine-free reagents are more com-
monly used now. The test specimen may be titrated with
the Karl Fischer Reagent directly, or the analysis may be car-
ried out by a residual titration procedure. The stoichiometry
of the reaction is not exact, and the reproducibility of the
determination depends on such factors as the relative con-
centrations of the Karl Fischer Reagent ingredients, the na-
ture of the inert solvent used to dissolve the test specimen,
the apparent pH of the final mixture, and the technique
used in the particular determination. Therefore, an empiri-
cally standardized technique is used to achieve the desired
accuracy. Precision in the method is governed largely by the
extent to which atmospheric moisture is excluded from the
system. The titration of water is usually carried out with the
use of anhydrous methanol as the solvent for the test speci-
men; however, other suitable solvents may be used for spe-
cial or unusual test specimens.
Substances that may interfere with the test results are fer-
ric ion, chlorine, and similar oxidizing agents, as well as sig-
nificant amounts of strong acids or bases, phosgene, or any-
thing that will reduce iodide to iodine, poison the reagent,
and show the sample to be bone dry when water may be
present (false negative). 8-Hydroxyquinoline may be added
to the vessel to eliminate interference from ferric ion. Chlo-
rine interference can be eliminated with sulfur dioxide or
unsaturated hydrocarbon. Excess pyridine or other amines
may be added to the vessel to eliminate the interference of
strong acids. Excess acetic acid or other carboxylic acid can
be added to reduce the interference of strong bases. Al-
dehydes and ketones may react with the solution, showing
the sample to be wet while the detector never reaches an
endpoint (false positive).
Apparatus Any apparatus may be used that provides for
adequate exclusion of atmospheric moisture and for deter-
mination of the endpoint. In the case of a colorless solution
that is titrated directly, the endpoint may be observed visu-
ally as a change in color from canary yellow to amber. The
reverse is observed in the case of a test specimen that is
titrated residually. More commonly, however, the endpoint
is determined electrometrically with an apparatus employing
a simple electrical circuit that serves to impress about 200
General Tests and Assays / Appendix II / 1239
mV of applied potential between a pair of platinum elec-
trodes (about 5 mm2 in area and about 2.5 cm apart) im-
mersed in the solution to be titrated. At the endpoint of the
titration, a slight excess of the reagent increases the flow of
current to 50–150 microamperes for 30 s to 30 min, de-
pending on the solution being titrated. The time is shortest
for substances that dissolve in the reagent. The longer times
are required for solid materials that do not readily go into
solution in the Karl Fischer Reagent. With some automatic
titrators, the abrupt change in current or potential at the
endpoint serves to close a solenoid-operated valve that con-
trols the buret delivering the titrant. A commercially availa-
ble apparatus generally comprises a closed system consisting
of one or two automatic burets and a tightly covered titra-
tion vessel fitted with the necessary electrodes and a mag-
netic stirrer. The air in the system is kept dry with a suitable
desiccant such as phosphorus pentoxide, and the titration
vessel may be purged by means of a stream of dry nitrogen
or a current of dry air.
Reagent The Karl Fischer Reagent may be prepared as fol-
lows: Add 125 g of iodine to a solution containing 670 mL
of methanol and 170 mL of pyridine, and cool. Place 100
mL of pyridine in a 250-mL graduated cylinder, and keeping
the pyridine cold in an ice bath, pass in dry sulfur dioxide
until the volume reaches 200 mL. Slowly add this solution,
with shaking, to the cooled iodine mixture. Shake to dis-
solve the iodine, transfer the solution to the apparatus, and
allow the solution to stand overnight before standardizing.
One mL of this solution, when freshly prepared, is equiva-
lent to approximately 5 mg of water, but it deteriorates
General Tests and Assays
gradually; therefore, standardize it within 1 h before use, or
daily in continual use. Protect the solution from light while
in use. Store any bulk stock of the solution in a suitably
sealed, glass-stoppered container, fully protected from light
and under refrigeration.
A commercially available, stabilized solution of a Karl
Fischer-type reagent may be used. Commercially available
reagents containing solvents or bases other than pyridine
and/or alcohols other than methanol also may be used.
These may be single solutions or reagents formed in situ by
combining the components of the reagents present in two
discrete solutions. The diluted Karl Fischer Reagent called for
in some monographs should be diluted as directed by the
manufacturer. Either methanol, or another suitable solvent
such as ethylene glycol monomethyl ether, may be used as
the diluent.
Test Preparation Unless otherwise specified in the indi-
vidual monograph, use an accurately weighed or measured
amount of the specimen under test estimated to contain
10–250 mg of water.
Where the monograph specifies that the specimen under
test is hygroscopic, accurately weigh a sample of the speci-
men into a suitable container. Use a dry syringe to inject an
appropriate volume of methanol, or other suitable solvent,
accurately measured, into the container and shake to dis-
solve the specimen. Dry the syringe, and use it to remove
the solution from the container and transfer it to a titration
vessel prepared as directed under Procedure. Repeat the pro-
cedure with a second portion of methanol, or other suitable
 1240 / Appendix II / General Tests and Assays
solvent, accurately measured; add this washing to the titra-
tion vessel; and immediately titrate. Determine the water
content, in milligrams, of a portion of solvent of the same
total volume as that used to dissolve the specimen and to
wash the container and syringe, as directed under Standardi-
zation of Water Solution for Residual Titration, and subtract
this value from the water content, in mg, obtained in the
titration of the specimen under test.
Standardization of the Reagent Place enough metha-
nol or other suitable solvent in the titration vessel to cover
the electrodes, and add sufficient Karl Fischer Reagent to give
the characteristic color or 100 ± 50 microamperes of direct
current at about 200 mV of applied potential. Pure metha-
nol can make the detector overly sensitive, particularly at
low ppm levels of water, causing it to deflect to dryness and
slowly recover with each addition of reagent. This slows
down the titration and may allow the system to actually
pick up ambient moisture during the resulting long titration.
Adding chloroform or a similar nonconducting solvent will
retard this sensitivity and can improve the analysis.
For determination of trace amounts of water (less than
1%), quickly add 25 µL (25 mg) of pure water, using a 25-
or 50-µL syringe, and titrate to the endpoint. The water
equivalence factor F, in mg of water per mL of reagent, is
given below:
Result = 25/V
in which V is the volume, in mL, of the Karl Fischer Reagent
consumed in the second titration.
For the precise determination of significant amounts of
General Tests and Assays
water (more than 1%), quickly add 25–250 mg (25–250
µL) of pure water, accurately weighed by difference from a
weighing pipet or from a precalibrated syringe or
micropipet, the amount of water used being governed by
the reagent strength and the buret size, as referred to under
Volumetric Apparatus. Titrate to the endpoint. Calculate the
water equivalence factor, F, in mg of water per mL of
reagent:
Result = W/V
in which W is the weight, in mg, of the water, and V is the
volume, in mL, of the Karl Fischer Reagent required.
Procedure Unless otherwise specified, transfer 35–40 mL
of methanol or other suitable solvent to the titration vessel,
and titrate with the Karl Fischer Reagent to the electrometric
or visual endpoint to consume any moisture that may be
present. (Disregard the volume consumed because it does
not enter into the calculations.) Quickly add the Test Prepa-
ration, mix, and again titrate with the Karl Fischer Reagent to
the electrometric or visual endpoint. Calculate the water
content of the specimen, in mg:
Result = SF
in which S is the volume, in mL, of the Karl Fischer Reagent
consumed in the second titration, and F is the water equiva-
lence factor of the Karl Fischer Reagent.
Method Ib (Residual Titration)
Principle See the information in the section entitled Princi-
ple under Method Ia. In the residual titration, add excess Karl
FCC 8
Fischer Reagent to the test specimen, allow sufficient time for
the reaction to reach completion, and titrate the uncon-
sumed Karl Fischer Reagent with a standard solution of water
in a solvent such as methanol. The residual titration proce-
dure is generally applicable and avoids the difficulties that
may be encountered in the direct titration of substances
from which the bound water is released slowly.
Apparatus, Reagent, and Test Preparation Use those
in Method Ia.
Standardization of Water Solution for Residual Titra-
tion Prepare a Water Solution by diluting 2 mL of pure
water to 1000 mL with methanol or another suitable sol-
vent. Standardize this solution by titrating 25.0 mL with the
Karl Fischer Reagent, previously standardized as directed
under Standardization of the Reagent. Calculate the water
content, in mg/mL, of the Water Solution:
Result = VF/25
in which V is the volume of the Karl Fischer Reagent con-
sumed, and F is the water equivalence factor of the Karl
Fischer Reagent. Determine the water content of the Water
Solution weekly, and standardize the Karl Fischer Reagent
against it periodically as needed. Store the Water Solution in
a tightly capped container.
Procedure Where the individual monograph specifies the
water content is to be determined by Method Ib, transfer
35–40 mL of methanol or other suitable solvent into the
titration vessel, and titrate with the Karl Fischer Reagent to
the electrometric or visual endpoint. Quickly add the Test
Preparation, mix, and add an accurately measured excess of
the Karl Fischer Reagent. Allow sufficient time for the reac-
tion to reach completion, and titrate the unconsumed Karl
Fischer Reagent with standardized Water Solution to the elec-
trometric or visual endpoint. Calculate the water content of
the specimen, in mg:
Result = F(X′ − XR)
in which F is the water equivalence factor of the Karl Fischer
Reagent; X′ is the volume, in mL, of the Karl Fischer Reagent
added after introduction of the specimen; X is the volume,
in mL, of standardized Water Solution required to neutralize
the unconsumed Karl Fischer Reagent; and R is the ratio V/25
(mL of Karl Fischer Reagent/mL of Water Solution), deter-
mined from the Standardization of Water Solution for Residual
Titration.
Method Ic (Coulometric Titration)
Principle Use the Karl Fischer reaction in the coulometric
determination of water. In this determination, iodine is not
added in the form of a volumetric solution, but is produced
in an iodide-containing solution by anodic oxidation. The
reaction cell usually consists of a large anode compartment
and a small cathode compartment that are separated by a
diaphragm. Other suitable types of reaction cells (e.g., with-
out diaphragms) may be used. Each compartment has a
platinum electrode that conducts current through the cell.
Iodine, which is produced at the anode electrode, immedi-
ately reacts with the water present in the compartment.
When all the water has been consumed, an excess of iodine
FCC 8 General Tests and Assays / Appendix II / 1241

occurs, which can be detected potentiometrically, thus indi- Procedure Quickly inject the Test Preparation, or transfer
cating the endpoint. Pre-electrolysis, which can take several the solid sample, into the anolyte, mix, and perform the
hours, eliminates moisture from the system. Therefore, coulometric titration to the electrometric endpoint. Read the
changing the Karl Fischer Reagent after each determination is water content of the Test Preparation directly from the in-
not practical. Individual determinations may be carried out strument’s display, and calculate the percent that is present
in succession in the same reagent solution. A requirement in the substance.
for this method is that each component of the test speci- Method II (Toluene Distillation Method)
men be compatible with the other components and that no
Principle This method determines water by distillation of
side reactions take place. Samples may be transferred into
a sample with an immiscible solvent, usually toluene.
the vessel as solids or as solutions by means of injection
through a septum. Gases can be introduced into the cell by Apparatus Use a glass distillation apparatus (see Figure 8)
means of a suitable gas inlet tube. For the water determina- provided with 24/40 ground-glass connections. The compo-
tion of solids, another common technique is to dissolve the nents consist of a 500-mL short-neck, round-bottom flask
solid in a suitable solvent and then inject a portion of this connected by means of a trap to a 400-mm water-cooled
solution into the cell. In the case of insoluble solids, water condenser. The lower tip of the condenser should be about
may be extracted using suitable solvents, and then the ex- 7 mm above the surface of the liquid in the trap after distil-
tracts injected into the coulometric cell. Alternatively, an lation conditions have been established (see Procedure).
evaporation technique may be used in which the sample is
heated in a tube and the water is evaporated and carried
into the cell by means of a stream of dry, inert gas. Preci-
sion in the method is predominantly governed by the ex-
tent to which atmospheric moisture is excluded from the
system. Control of the system may be monitored by meas-
uring the amount of baseline drift. The titration of water in
solid test specimens is usually carried out with the use of
anhydrous methanol as the solvent. Other suitable solvents
may be used for special or unusual test specimens. This
method is particularly suited to chemically inert substances
such as hydrocarbons, alcohols, and ethers. In comparison
with the volumetric Karl Fischer titration, coulometry is a
micro-method. The method uses extremely small amounts

General Tests and Assays

of current. It is predominantly used for substances with a
very low water content (0.1%–0.0001%).
Apparatus Any commercially available apparatus consist-
ing of an absolutely tight system fitted with the necessary
electrodes and a magnetic stirrer is appropriate. The instru-
ment’s microprocessor controls the analytical procedure and
displays the results. Calibration of the instrument is not nec-
essary as the current consumed can be measured absolutely.
Proper operation of the instrument can be confirmed by
injecting 1 µL of water into the vessel. The instrument
should read 1000 µg of water on reaching the endpoint.
Reagent See Reagent under Method Ia.
Test Preparation Using a dry syringe, inject an appropri-
ate volume of test specimen estimated to contain 0.5–5 mg
of water, accurately measured, into the anolyte solution.
The sample may also be introduced as a solid, accurately Figure 8. Moisture Distillation Apparatus
weighed, into the anolyte solution. Perform coulometric ti- The trap should be constructed of well-annealed glass, the
tration, and determine the water content of the specimen receiving end of which is graduated to contain 5 mL and
under test. subdivided into 0.1-mL divisions, with each 1-mL line num-
Alternatively, when the specimen is a suitable solid, dis- bered from 5 mL beginning at the top. Calibrate the re-
solve an appropriate quantity, accurately weighed, in anhy- ceiver by adding 1 mL of water, accurately measured, to
drous methanol or another suitable solvent, and inject a 100 mL of toluene contained in the distillation flask. Con-
suitable portion into the anolyte solution. duct the distillation, and calculate the volume of water ob-
When the specimen is an insoluble solid, extract the water tained as directed in the Procedure. Add another mL of
by using a suitable anhydrous solvent from which an appro- water to the cooled apparatus, and repeat the distillation.
priate quantity, accurately weighed, may be injected into Continue in this manner until five 1-mL portions of water
the anolyte solution. Alternatively use an evaporation have been added. The error at any indicated capacity
technique. should not exceed 0.05 mL. The source of heat is either an
oil bath or an electric heater provided with a suitable means
 1242 / Appendix II / General Tests and Assays FCC 8

of temperature control. The distillation may be better con- free ash is still not obtained, cool the crucible, add 15 mL of
trolled by insulating the tube leading from the flask to the ethanol, break up the ash with a glass rod, then burn off
receiver. It is also advantageous to protect the flask from the ethanol, again heat the whole to a dull redness, cool it
drafts. Clean the entire apparatus with potassium dichro- in a desiccator, and weigh.
mate-sulfuric acid cleaning solution, rinse thoroughly, and
dry completely before using. HYDROCHLORIC ACID TABLE
Procedure Place in the previously cleaned and dried flask
a quantity of the substance, weighed accurately to the near-
est 0.01 g, that is expected to yield 1.5–4 mL of water. If °Bé Sp. Gr. Percent HCl
the substance is of a pastelike consistency, weigh it in a 1.00 1.0069 1.40
boat of metal foil that will pass through the neck of the 2.00 1.0140 2.82
flask. If the substance is likely to cause bumping, take suita-
3.00 1.0211 4.25
ble precautions to prevent it. Transfer about 200 mL of ACS
reagent-grade toluene into the flask, and swirl to mix it with 4.00 1.0284 5.69
the sample. Assemble the apparatus, fill the receiver with 5.00 1.0357 7.15
toluene by pouring it through the condenser until it begins 5.25 1.0375 7.52
to overflow into the flask, and insert a loose cotton plug in 5.50 1.0394 7.89
the top of the condenser. Heat the flask so that the distilla-
5.75 1.0413 8.26
tion rate will be about 200 drops/min, and continue distil-
6.00 1.0432 8.64
ling until the volume of water in the trap remains constant
for 5 min. Discontinue the heating, use a copper or 6.25 1.0450 9.02
nichrome wire spiral to dislodge any drops of water that 6.50 1.0469 9.40
may be adhering to the inside of the condenser tube or 6.75 1.0488 9.78
receiver, and wash down with about 5 mL of toluene. Dis- 7.00 1.0507 10.17
connect the receiver, immerse it in water at 25° for at least
7.25 1.0526 10.55
15 min or until the toluene layer is clear, and then read the
volume of water. Conduct a blank determination using the 7.50 1.0545 10.94
same volume of toluene as used when distilling the sample 7.75 1.0564 11.32
mixture, and make any necessary correction (see General 8.00 1.0584 11.71
Provisions). 8.25 1.0603 12.09
General Tests and Assays

8.50 1.0623 12.48

8.75 1.0642 12.87
9.00 1.0662 13.26
C. OTHERS 9.25 1.0681 13.65
9.50 1.0701 14.04
9.75 1.0721 14.43
ASH (Acid-Insoluble) 10.00 1.0741 14.83
10.25 1.0761 15.22
Boil the ash obtained as directed under Ash (Total), below,
with 25 mL of 2.7 N hydrochloric acid for 5 min, collect the 10.50 1.0781 15.62
insoluble matter on a tared, porous-bottom porcelain filter 10.75 1.0801 16.01
crucible or ashless filter, wash it with hot water, ignite to 11.00 1.0821 16.41
constant weight at 675 ± 25°, and weigh. Calculate the per- 11.25 1.0841 16.81
cent acid-insoluble ash from the weight of the sample
11.50 1.0861 17.21
taken.
11.75 1.0881 17.61
[NOTE—Avoid exposing the crucible to sudden tem-
12.00 1.0902 18.01
perature changes.]
12.25 1.0922 18.41
12.50 1.0943 18.82
ASH (Total) 12.75 1.0964 19.22
13.00 1.0985 19.63
Unless otherwise directed, accurately weigh about 3 g of the
sample in a tared crucible, ignite it at a low temperature 13.25 1.1006 20.04
(about 550°), not to exceed a very dull redness, until it is 13.50 1.1027 20.44
free from carbon, cool it in a desiccator, and weigh. If a 13.75 1.1048 20.86
carbon-free ash is not obtained, wet the charred mass with 19.2 1.1526 30.00
hot water, collect the insoluble residue on an ashless filter
19.3 1.1535 30.18
paper, and ignite the residue and filter paper until the ash is
19.4 1.1544 30.35
white or nearly so. Finally, add the filtrate, evaporate it to
dryness, and heat the whole to a dull redness. If a carbon-
FCC 8 General Tests and Assays / Appendix II / 1243

°Bé Sp. Gr. Percent HCl °Bé Sp. Gr. Percent HCl
19.5 1.1554 30.53 24.6 1.2043 40.78
19.6 1.1563 30.71 24.7 1.2053 41.01
19.7 1.1572 30.90 24.8 1.2063 41.24
19.8 1.1581 31.08 24.9 1.2073 41.48
19.9 1.1590 31.27 25.0 1.2083 41.72
20.0 1.1600 31.45 25.1 1.2093 41.99
20.1 1.1609 31.64 25.2 1.2103 42.30
20.2 1.1619 31.82 25.3 1.2114 42.64
20.3 1.1628 32.01 25.4 1.2124 43.01
20.4 1.1637 32.19 25.5 1.2134 43.40
20.5 1.1647 32.38
Specific gravity determinations were made at 60°F, com-
20.6 1.1656 32.56
pared with water at 60°F.
20.7 1.1666 32.75
From the specific gravities, the corresponding degrees
20.8 1.1675 32.93 Baumé were calculated by the following formula:
20.9 1.1684 33.12
degrees Baumé = 145 − (145/sp. gr.)
21.0 1.1694 33.31
21.1 1.1703 33.50 Baumé hydrometers for use with this table must be grad-
21.2 1.1713 33.69 uated by the above formula, which should always be
21.3 1.1722 33.88 printed on the scale.
21.4 1.1732 34.07 Allowance for Temperature
10°–15°Bé: 1/40 °Bé or 0.0002 sp. gr. for 1°F
21.5 1.1741 34.26
15°–22°Bé: 1/30 °Bé or 0.0003 sp. gr. for 1°F
21.6 1.1751 34.45
22°–25°Bé: 1/28 °Bé or 0.00035 sp. gr. for 1°F
21.7 1.1760 34.64
21.8 1.1770 34.83 LOSS ON DRYING
21.9 1.1779 35.02

General Tests and Assays

22.0 1.1789 35.21 This procedure is used to determine the amount of volatile
22.1 1.1798 35.40 matter expelled under the conditions specified in the mono-
22.2 1.1808 35.59
graph. Because the volatile matter may include material
other than adsorbed moisture, this test is designed for com-
22.3 1.1817 35.78
pounds in which the loss on drying may not definitely be
22.4 1.1827 35.97 attributable to water alone. For substances appearing to
22.5 1.1836 36.16 contain water as the only volatile constituent, the Direct
22.6 1.1846 36.35 (Karl Fischer) Titration Method, provided under Water, Ap-
22.7 1.1856 36.54 pendix IIB, is usually appropriate.
22.8 1.1866 36.73 Procedure Unless otherwise directed in the monograph,
22.9 1.1875 36.93
conduct the determination on 1–2 g of the substance, previ-
ously mixed and accurately weighed. If the sample is in the
23.0 1.1885 37.14
form of large crystals, reduce the particle size to about 2
23.1 1.1895 37.36 mm, quickly crushing the sample to avoid absorption or loss
23.2 1.1904 37.58 of moisture. Tare a glass-stoppered, shallow weighing bottle
23.3 1.1914 37.80 that has been dried for 30 min under the same conditions
23.4 1.1924 38.03 to be used in the determination. Transfer the sample to the
bottle, replace the cover, and weigh the bottle and its con-
23.5 1.1934 38.26
tents. By gentle sideways shaking, distribute the sample as
23.6 1.1944 38.49
evenly as possible to a depth of about 5 mm for most sub-
23.7 1.1953 38.72 stances and not over 10 mm in the case of bulky materials.
23.8 1.1963 38.95 Place the loaded bottle in the drying chamber, removing
23.9 1.1973 39.18 the stopper and leaving it also in the chamber, and dry at
24.0 1.1983 39.41
the temperature and for the length of time specified in the
monograph. Upon opening the chamber, close the bottle
24.1 1.1993 39.64
promptly and allow it to come to room temperature, prefer-
24.2 1.2003 39.86 ably in a desiccator, before weighing.
24.3 1.2013 40.09 Where drying in vacuum is specified in the monograph,
24.4 1.2023 40.32 use a pressure as low as that obtainable by an aspirating
24.5 1.2033 40.55 water pump (NMT 20 mm Hg).
 1244 / Appendix II / General Tests and Assays
If the test substance melts at a temperature lower than
that specified for the determination, preheat the bottle and
its contents for 1–2 h at a temperature 5°–10° below the
melting range, then continue drying at the specified tem-
perature for the determination. When drying the sample in
a desiccator, ensure that the desiccant is kept fully effective
by replacing it frequently.
NUCLEAR MAGNETIC RESONANCE
Nuclear magnetic resonance (NMR) spectroscopy is an ana-
lytical procedure based on the magnetic properties of cer-
tain atomic nuclei. It is similar to other types of spectros-
copy in that absorption or emission of electromagnetic
energy at characteristic frequencies provides analytical infor-
mation. NMR differs in that the discrete energy levels be-
tween which the transitions take place are created artificially
by placing the nuclei in a magnetic field.
Atomic nuclei are charged and behave as if they were
spinning on the nuclear axis, thus creating a magnetic di-
pole of moment µ along this axis. The angular momentum
of the spinning nucleus is characterized by a spin quantum
number (I). If the mass number is odd, I is 1/2 or an integer
plus 1/2; otherwise, it has a value of 0 or a whole number.
Nuclei having a spin quantum number I ≠ 0, when placed
in an external uniform static magnetic field of strength, H0,
align with respect to the field in (2I + 1) possible orienta-
tions. Thus, for nuclei with I = 1/2, which include most iso-
topes of analytical significance, as shown in the table below,
there are two possible orientations, corresponding to two
General Tests and Assays
different energy states. A nuclear resonance is the transition
between these states, by absorption or emission of the cor-
responding amount of energy. In a static magnetic field the
nuclear magnetic axis precesses (Larmor precession) about
the external field axis. The precessional angular velocity, ω0,
Properties of Some Nuclei Amenable to NMR Study
Nucleus I Natural Abundance, %
1 H 1
/2 99.980
13 C 1
/2 1.108
19 F 1
/2 100.000
31 P 1
/2 100.000
11 B ( / 2)
3 80.420
* T = tesla, 1 T = 10,000 Gauss.
Apparatus The distinctive components of an NMR spec-
trometer are a magnet and a source of radio frequency. The
instruments are described by the approximate resonance
frequency of the analytical nucleus, e.g., 1H NMR. More re-
cently, instruments are being referred to by their field
strengths. Some spectrometers are dedicated to the analysis
of one type of nucleus; others are designed to obtain spec-
tra of different nuclei.
There are two types of commercial NMR spectrometers:
the classical continuous wave (CW) instruments and the
more modern pulse Fourier-transform (FT) instruments. The
CW spectrometers use a technique similar to that of classical
optical spectrometers: a slow scan of the radio frequency (at
FCC 8
is related to the external magnetic field strength through
the equation:
ω0 = γH0
in which γ is the magnetogyric ratio and is a constant for all
nuclei of a given isotope. If energy from an oscillating radio-
frequency field is introduced, the absorption of radiation
takes place according to the relationship:
∆E = hν = µHO/I
where h is Planck’s constant, and
ν = ω0/2π = γH0/2π
Thus, when the frequency (ν0) of the external energy field
(E = hν) is the same as the precessional angular velocity,
resonance is achieved.
The energy difference between the two levels corresponds
to electromagnetic radiation in the radio-frequency range. It
is a function of γ, which is a property of the nucleus, and
H0, the external field strength. As shown in the table below,
the resonance frequency of a nucleus increases with the in-
crease of the magnetic field strength.
NMR is a technique of high specificity but relatively low
sensitivity. The basic reason for the low sensitivity is the
comparatively small difference in energy between the ex-
cited and the ground states (0.02 calories at 15–20 kilogauss
field strength), which results in a population difference be-
tween the two levels of only a few ppm. Another important
aspect of the NMR phenomenon, with negative effects on
the sensitivity, is the long lifetime of most nuclei in the ex-
cited state, which affects the design of the NMR analytical
test, especially in pulsed repetitive experiments. Simultane-
ous acquisition of the entire spectrum instead of frequency-
swept spectra can give sensitivity enhancement.
Resonance Frequency (MHZ) at
*
Sensitivity 1.4093 T 2.3488 T 4.6975 T
1.000 60.000 100.000 200.000
0.0159 15.087 25.144 50.288
0.830 56.446 94.077 188.154
0.0663 24.289 40.481 80.961
0.170 19.250 32.084 64.167
fixed magnetic field) or the magnetic field (at fixed radio
frequency) over a domain corresponding to the resonance
of the nuclei being studied. The signal generated by the
absorption of energy is detected, amplified, and recorded.
Various instrument configurations are possible. The ar-
rangement of a typical double-coil spectrometer, as one
might see in the lower resolution 60-MHz and 100-MHz CW
instruments, is illustrated in Figure 9.
The limitations of the CW spectrometers are low sensitiv-
ity and long analysis time. In pulsed NMR spectrometers, a
single pulse of radio frequency energy is used to simultane-
ously activate all nuclei. The excited nuclei returning to the
FCC 8
Figure 9. Block Diagram of a Typical NMR Spectrometer
Figure 10. Block Diagram of a Typical Pulsed FT-NMR Spectrometer
lower energy level generate a free induction decay (FID) sig-
nal that contains in a time domain all the information ob-
tained in a frequency domain with a CW spectrometer. The
time domain and the frequency domain responses form a
pair of FTs; the mathematical operation is performed by a
computer after analog-to-digital conversion. After a delay al-
lowing for relaxation of the excited nuclei, the pulse experi-
ment (transient) may be repeated and the response coher-
ently added in the computer memory, with random noise
being averaged out. (A similar signal-to-noise increase can
be obtained by combining CW spectrometers with com-
puters that average transients.)
The block diagram of a typical high-resolution pulsed
spectrometer is shown in Figure 10.
It is a typical configuration of the high-resolution spectrom-
eter that uses a superconducting (cryogenic) solenoid as the
source of the magnetic field. Introduction of the pulsed
NMR spectrometer has made the acquisition of spectra of
many nuclei, other than protons, routine. It has also allowed
proton spectra to be obtained in much less time, and with
smaller amounts of specimen, as compared to CW
techniques.
NMR spectrometers have strict stability and homogeneity
requirements. Stability is often achieved by a field-frequency
locking system that “locks” the magnetic field to the
resonance frequency of a reference signal. The lock signal
can be homonuclear or heteronuclear. In the latter case, the
reference resonance is usually a deuterium signal from a
General Tests and Assays / Appendix II / 1245
General Tests and Assays
deuterated solvent. On older spectrometers, using deute-
rium as a locking nucleus permits noise decoupling of pro-
tons to be carried out while studying nuclei like 13C. While
internal homonuclear locks are still used in CW proton spec-
trometers (where tetramethylsilane at about 0.5% provides
a convenient lock), they are hardly ever used in pulsed FT
spectrometers.
No type of magnet is capable of producing a homogene-
ous field over the space occupied by the specimen. Two
techniques are usually employed to compensate for this lack
of homogeneity: specimen spinning and the use of addi-
tional (shim) coils. Because of design, particularly probe de-
sign, the spinning in the case of the electromagnet or per-
manent magnet is perpendicular to the basic field. In the
superconducting magnet, the axis of rotation can only be
parallel to the basic magnetic field. The spin rate should be
sufficient to produce averaging of the field, but not fast
enough to produce an extended vortex in the specimen
tube. A vortex extended near the region exposed to the
radio-frequency coils decreases resolution. The shim coils are
adjusted by the operator until instrumental contributions to
the observed line width are minimized.
An electronic integrator is a feature of most NMR spec-
trometers. On a CW instrument (1H and 19F) the integrator,
connected to the spectrometer output stage, determines the
relative areas of the resonance peaks and presents these ar-
eas as a series of stepped horizontal lines when a sweep is
made in the integration mode. On FT-NMR spectrometers,
 1246 / Appendix II / General Tests and Assays
Figure 11. NMR Spectrum of 2,3-Dimethyl-2-butenyl methyl ether (15% in CCl4) showing four nonequivalent, apparently
uncoupled protons with a normal integral trace (peak area ratio from low H0 to high H0 of 2:3:3:6). (Tetramethylsilane, the
NMR Reference, appears at 0 ppm.) The system of units represented by δ is defined under The Spectrum, in this section.
an integration algorithm is included in the spectrometer
software, and the resonance peak areas may be presented
graphically as stepped lines or tabulated as numeric values.
The use of computer-generated tabulated/numeric integra-
tion data should not be accepted without a specific demon-
stration of precision and accuracy on the spectrometer in
question.
The Spectrum The signals (peaks) in an NMR spectrum
are characterized by four attributes: resonance frequency,
General Tests and Assays
multiplicity, line width, and relative intensity. The analytical
usefulness of the NMR technique resides in the fact that the
same types of nuclei, when located in different molecular
environments, exhibit different resonance frequencies. The
reason for this difference is that the effective field exper-
ienced by a particular nucleus is a composite of the external
field provided by the instrument and the field generated by
the circulation of the surrounding electrons. (The latter is
generally opposed to the external field and the phenome-
non is called “shielding.”) In contrast with other spectro-
scopic methods, it is not possible to measure accurately the
absolute values of transition frequencies. However, it is pos-
sible to measure accurately the difference in frequencies be-
tween two resonance signals. The position of a signal in an
NMR spectrum is described by its separation from another
resonance signal arbitrarily taken as standard. This separa-
tion is called chemical shift.
The chemical shift, being the difference between two
resonance frequencies, is directly proportional to the mag-
netic field strength (or to the frequency of the oscillator).
However, the ratio between the chemical shift, in frequency
units, and the instrument frequency is constant. This allows
definition of a dimensionless chemical shift parameter (δ)
that is independent of the instrument frequency:
δ = (νs − νr)/νo + δr
in which νs is the test substance line frequency, νr is the
reference line frequency, νo is the instrument frequency, in
mHz, and δr is the chemical shift of the reference.
FCC 8
By employing the above equation, it is possible to use
(with appropriate caution) the chemical shift of any known
species (such as the residual 1H-containing species in deuter-
ated solvent) as a chemical shift reference. The above equa-
tion, now in common use, is applicable to nearly all meth-
ods except in the relatively rare cases where extremely
precise chemical shift values must be determined, and is
readily adaptable to nuclei where non-zero reference stan-
dards are the only practical method of chemical shift
determinations.
For CW instruments, tetramethylsilane (TMS) is the most
widely used chemical shift reference for proton and carbon
spectra. It is chemically inert, exhibits only one line, which is
at a higher field than most signals, and is volatile, thus al-
lowing for ready specimen recovery. Sodium 3-(trimethylsi-
lyl)propionate (TSP) or sodium 2,2-dimethyl-2-silapentane-5-
sulfonate (DSS) are used as NMR references for aqueous so-
lutions. The resonance frequency of the TSP or DSS methyl
groups closely approximate that of the TMS signal; how-
ever, DSS has the disadvantage of showing a number of
methylene multiplets that may interfere with signals from
the test substance. Where the use of an internal NMR refer-
ence material is not desirable, an external reference may be
used.
Conventional NMR spectra are shown with the magnetic
field strength increasing from left to right. Nuclei that reso-
nate at high magnetic field strengths (to the right) are said
to be more shielded (greater electron density) than those
that resonate at lower magnetic field strengths: these are
said to be de-shielded (lower electron density).
Figure 11 shows the proton NMR spectrum of 2,3-di-
methyl-2-butenyl methyl ether. This compound contains
protons in a methylene group (marked d in the graphic
formula) and in four methyl groups (a, a, b, and c). Methyl
groups b and c are situated in distinctly different molecular
environments than the two a methyl groups. Three different
methyl proton resonances are observed as spectral peaks in
addition to the peak corresponding to methylene proton
resonance. The two a methyl groups, being in very similar
FCC 8
environments, have the same chemical shift. Interaction be-
tween magnetically active nuclei situated within a few bond
lengths of each other leads to coupling, which results in a
mutual splitting of the respective signals into sets of peaks
or multiplets.
The coupling between two nuclei may be described in
terms of the spin-spin coupling constant, J, which is the
separation (in hertz) between the individual peaks of the
multiplet. Where two nuclei interact and cause reciprocal
splitting, the measured coupling constants in the two result-
ing mutiplets are equal. Furthermore, J is independent of
magnetic field strength.
In a first-order, comparatively noncomplex spin system,
the number of individual peaks that are expected to be
present in a multiplet and the relative peak intensities are
predictable. The number of peaks is determined by 2 nI + 1,
where n is the number of nuclei on adjacent groups that are
active in splitting. For protons this becomes (n + 1) peaks.
In general, the relative intensity of each peak in the multi-
plet follows the coefficient of the binomial expansion (a +
b)n. These coefficients may conveniently be found by use of
Pascal’s triangle, which produces the following relative areas
for the specified multiplets: doublet, 1:1; triplet, 1:2:1; quar-
tet, 1:3:3:1; quintet, 1:4:6:4:1; sextet, 1:5:10:10:5:1; and
septet, 1:6:15:20:15:6:1. This orderly arrangement, gener-
ally referred to as first-order behavior, may be expected
when the ratio of Dν to J is greater than about 10; Dν is the
chemical shift difference between two nuclei or two groups
of equivalent nuclei. Two examples of idealized spectra aris-
ing from first-order coupling are shown in Figure 12.
Figure 12. Diagrammatic Representation of Simple First-Or-
der Coupling of Adjacent Protons
General Tests and Assays / Appendix II / 1247
Figure 13 shows a spectrum displaying triplet signals result-
ing from the mutual splitting of two adjacent methylene
groups.
Coupling may occur between 1H and other nuclei, such as
19F, 13C, and 31P. In some cases, e.g., in the CW mode, the
coupling constants may be large enough so that part of the
multiplet is off scale at either the upfield or downfield end.
This type of coupling may occur over the normal “three-
bond distance,” as for 1H-1H coupling.
Magnetically active nuclei with I ≥ 1, such as 14N, possess
an electrical quadrupole moment, which produces line-
broadening of the signal due to neighboring nuclei.
Another characteristic of the signal, its relative intensity,
has wide analytical applications. In carefully designed experi-
ments (see General Method, below), the area or intensity of
a signal is directly proportional to the number of protons
giving rise to the signal. As a result, it is possible to deter-
mine the relative ratio of the different kinds of protons or
other nuclei in a specimen or to perform NMR assays with
the aid of an internal standard.
The NMR spectra may contain extraneous signals due to
the inhomogeneity of the magnetic field throughout the
specimen. These artifacts, called spinning side bands, appear
as minor lines symmetrically located around each signal. The
presence of large spinning side bands indicates that the
non-spinning shims require adjustment. The separation is
equal to the frequency of the specimen tube spin rate or
some integral multiple of that frequency. Thus, spinning
side bands are readily identifiable.
General Method Inadequate specimen preparation or
General Tests and Assays
incorrect instrumental adjustments and parameters may lead
to poor resolution, decreased sensitivity, spectral artifacts,
and erroneous data. It is preferable that the operator be
familiar with the basic theory of NMR, the properties of the
specimen, and the operating principles of the instruments.
Strict adherence to the instruction manuals provided by the
manufacturer and frequent checks of the performance of
the instrument are essential.
The method and procedures discussed here refer specifi-
cally to 1H (proton) and 19F NMR. They are applicable, with
modification, to other nuclei. The discussion presumes that
the NMR spectra are obtained from liquid test substances or
solutions in suitable solvents.
Selection of Solvent In addition to having good solubil-
ity properties, suitable solvents do not exhibit resonance
peaks that obscure resonance peaks of the specimen being
analyzed. The most commonly used solvents for proton and
carbon NMR are listed in the table below. Deuterated sol-
vents also provide the signal for the heteronuclear system
lock. If solvent peaks might interfere with any signals from
the specimen, then the isotopic purity of the solvent should
be as high as possible. Deuterium (I = 1) does not exhibit
resonance under 1H conditions but may cause J-coupling to
 1248 / Appendix II / General Tests and Assays FCC 8

Figure 13. NMR Spectrum of 3-Keto-tetrahydrofuran (10% in CCl4) showing three nonequivalent protons, with a normal
integral trace (peak area ratio from low H0 to high H0 of 1:1:1). Note two sets of methylene groups coupled to each other at
4.2 and 2.4 ppm. (Tetramethylsilane, the NMR Reference, appears at 0 ppm.)

be observed. The residual protons generate solvent peaks However, unless they are decoupled, protonated functional
whose chemical shifts are shown in the table below. groups on the 19F-containing specimen will provide J-
coupling.
Solvents Commonly Used for Proton NMR
Specimen Preparation Directions are usually given in in-
a
Solvent Residual Proton Signal, δ dividual monographs. The solute concentration depends on
b
CCl4 — the objective of the experiment and on the type of instru-
CS2
b
— ment. Detection of minor contaminants may require higher
SO2 (liquid) —
concentrations. The solutions are prepared in separate vials
and transferred to the NMR specimen tube. The volume re-
General Tests and Assays

(CF3)2CO —
quired depends on the size of the specimen tube and on
CDCl3 7.27 the geometry of the instrument. The level of the solution in
c
CD3OD 3.35, 4.8 the tube must be high enough to extend beyond the coils
(CD3)2CO 2.05 when the tube is inserted in the instrument probe and
D2O 4.7
c spun.
d The NMR specimen tubes must meet narrow tolerance
DMSO-d6 2.50
specifications in diameter, wall thickness, concentricity, and
C6D6 7.20
camber. The most widely used tubes have a 5-mm or 10-
p-Dioxane-d8 3.55 mm outside diameter and a length of 15–20 cm. Micro-
c
CD3CO2D 2.05, 8.5 tubes are available for the analysis of small amounts of
DMF-d7
e
2.77, 2.93, 8.05 specimen.
a δ in ppm relative to tetramethylsilane arbitrarily taken as 0δ or 0 ppm. Procedure The specimen tube is placed in a probe lo-
b Spectrophotometric grade. cated in the magnetic field. The probe contains electronic
c Highly variable; depends on solute and temperature. circuitry including the radio-frequency coil(s), and is pro-
d Dimethyl sulfoxide-d6.
e N,N-Dimethylformamide-d7 per Aldrich, Alfa, Fluka, and Sigma catalogs.
vided with attachments for the air supply that spins the
specimen tubes.
Some solvents (e.g., D2O or CD3OD) enter into fast ex- Instrument adjustments are made before each experi-
change reactions with protons and may eliminate resonance ment. The spinning rate of the specimen tube is adjusted so
signals from –COOH, –OH, and –NH2 structural groups. The that spinning side bands do not interfere with the peaks of
protons in alcohols and amines do not take part in rapid interest and the vortex does not extend beyond the coils in
exchange unless catalyzed by small concentrations of acid the probe. To optimize the instrument performance, the
or base, except in the presence of D2O and some other magnetic shim gradients on FT-NMR spectrometers are ad-
solvents (e.g., CD3OD). justed. In adjusting resolution on CW spectrometers, a good
For 19F NMR, most solvents used in proton NMR may be indicator is the definite “ringing” of the TMS peak. The phe-
employed, the most common ones being CHCl3, CCl4, H2O, nomenon of ringing is the oscillation of the recorder trace
CS2, aqueous acids and bases, and dimethylacetamide. In after the magnetic field has passed through a resonance fre-
general, any nonfluorinated solvent may be used, provided quency. Ringing, evident on a number of the peaks in
that it is of spectral quality. Obviously, there is no interfer- Figures 13 and 14, arises during rapid scans and decays ex-
ence from the protonated functional groups of the solvent. ponentially to the baseline value.
FCC 8
Figure 14. Continuous Wave Proton Spectrum of Ethyl Ether
Figure 15 clearly indicates the absence, in an FT experiment, of
the ringing phenomenon. Ringing will not appear because the
spectrum obtained is the result of analysis of the FID by
Fourier transformation and not a magnetic field or frequency
sweep through the individual resonance positions.
Figure 15. Proton NMR Spectrum of Ethyl Ether in Deuter-
ated Chloroform
With proton CW instruments the spectrum is scanned
from 0 ppm to about 10 ppm with a scan time of about
1–5 min. The amplification is adjusted so that all peaks re-
main on scale. If the response is low at reasonable ampli-
tude, the radio-frequency power is increased to obtain the
highest possible peak response without peak broadening.
After the initial scan, the presence of peaks downfield of 10
ppm is quickly checked by off-setting the instrument re-
sponse by about 5 ppm. With CW instrumentation, it is
common for the TMS peak to shift slightly during an ex-
tended scan. The extent of the shift is usually obtained by
comparing the relative positions of another peak in the ini-
tial scan with the same peak in the offset scan.
The operation of an FT-NMR spectrometer is a much
more elaborate experiment. The computer serves to control
the spectrometer, to program the experiment, and to store
and process the data. Programming the experiment involves
setting values for a large number of variables including the
General Tests and Assays / Appendix II / 1249
spectral width to be examined, the duration (“width”) of the
excitation pulse, the time interval over which data will be
acquired, the number of transients to be accumulated, and
the delay between one acquisition and the next. The analy-
sis time for one transient is in the order of seconds. The
number of transients is a function of the specimen concen-
tration, the type of nucleus, and the objective of the experi-
ment. At the end of the experiment, the FID signal is stored
in digitized form in the computer memory and is displayed
on the video screen. The signal can be processed mathe-
matically to enhance either the resolution or the sensitivity,
and it can be Fourier-transformed into a frequency-domain
spectrum. The instrument provides a plot of the spectrum.
The integration routine, accessed through keyboard com-
mands, results in a stepped-line plot. Considerably more ac-
curate integrals are obtained if the signals or regions of in-
terest are separately integrated.
FT-NMR spectrometers may yield qualitative and quantita-
tive data from the same experiment, but this is seldom
done in practice. In quantitative FT experiments, special pre-
cautions must be taken for the signal areas to be propor-
tional to the number of protons. The delays between pulses
must be long enough to allow complete relaxation of all
excited nuclei. This results in a considerable increase in anal-
ysis time and in some loss of resolution. Qualitative analysis
is usually performed in nonquantitative conditions, with the
design of the experiment directed to fast analysis with maxi-
mum resolution or sensitivity.
Qualitative and Quantitative Analysis NMR spectros-
copy has been used for a wide range of applications such as
General Tests and Assays
structure elucidation; thermodynamic, kinetic, and mechan-
istic studies; and quantitative analysis. Some of these appli-
cations are beyond the scope of compendial methods.
All five characteristics of the signal (chemical shift, multi-
plicity, line width, coupling constants, and relative intensity)
contribute analytical information.
Qualitative Applications Comparison of a spectrum
from the literature or from an authentic specimen with that
of a test specimen may be used to confirm the identity of a
compound and to detect the presence of impurities that
generate extraneous signals. The NMR spectra of simple
structures can be adequately described by the numeric value
of the chemical shifts and coupling constants, and by the
number of protons under each signal. (The software of
modern instruments includes programs that generate simu-
lated spectra using these data.) Experimental details, such as
the solvent used, the specimen concentration, and the
chemical shift reference, must also be provided.
For unknown specimens, NMR analysis, usually coupled
with other analytical techniques, is a powerful tool for struc-
ture elucidation. Chemical shifts provide information on the
chemical environment of the nuclei. Extensive literature is
available with correlation charts and rules for predicting
chemical shifts. The multiplicity of the signals provides im-
portant stereochemical information. Mutual signal splitting
of functional groups indicates close proximity. The magni-
tude of the coupling constant, J, between residual protons
on substituted aromatic, olefinic, or cycloalkyl structures is
used to identify the relative position of the substituents.
 1250 / Appendix II / General Tests and Assays
Several special techniques (double resonance, chemical
exchange, use of shift reagents, two-dimensional analysis,
etc.) are available to simplify some of the more complex
spectra, to identify certain functional groups, and to deter-
mine coupling correlations.
Double resonance, or spin decoupling, is a technique that
removes the coupling between nuclei and thus simplifies the
spectrum and identifies the components in a coupling rela-
tionship. For example, in a simple two-proton system, gen-
erally designated an AX system (see Figure 12), each proton
appears as a doublet. If a strong radio-frequency field is in-
troduced at the frequency of X, while the normal radio-
frequency field is maintained at the frequency that causes A
to resonate, the coupling between A and X is removed
(homonuclear decoupling). A is no longer split, but instead
appears as a singlet. Routine 13C spectra are obtained under
proton decoupling conditions that remove all heteronuclear
13C-1H couplings. As a result of this decoupling, the carbon
signals appear as singlets, unless other nuclei that are not
decoupled are present (e.g., 19F, 31P).
Functional groups containing exchangeable protons
bound to hetero-atoms such as –OH, –NH2, or –COOH
groups may be identified by taking advantage of the rapid
exchange of these protons with D2O. To determine the
presence and position of these groups, scan the test sub-
stance in CDCl3 or DMSO-d6, then add a few drops of D2O
to the specimen tube, shake, and scan again. The resonance
peaks from these groups collapse in the second scan and
are replaced by the HDO singlet between 4.7 and 5.0 ppm.
This chemical exchange is an example of the effect of
intermolecular and intramolecular rate processes on NMR
General Tests and Assays
spectra. If a proton can experience different environments
by virtue of such a process (tautomerism, rotation about a
bond, exchange equilibria, ring inversion, etc.), the appear-
ance of the spectrum will be a function of the rate of the
process. Slow processes (on an NMR time scale) result in
more than one signal, fast processes average these signals to
one line, and intermediate processes produce broad signals.
The software of modern FT-NMR spectrometers allows for
sequences of pulses much more complex than the repetitive
accumulation of transients described above. Such experi-
ments include homonuclear or heteronuclear two-dimen-
sional analysis, which determines the correlation of cou-
plings and may simplify the interpretation of otherwise
complex spectra.
Quantitative Applications If appropriate instrument set-
tings for quantitative analysis have been made, the areas (or
intensities) of two signals are proportional to the total num-
ber of protons generating the signals.
A1/A2 = N1/N2 (1)
If the two signals originate from two functional groups of
the same molecule, the equation can be simplified to:
A1/A2 = n1/n2 (2)
in which n1 and n2 are the number of protons in the respec-
tive functional groups.
FCC 8
If the two signals originate from different molecular spe-
cies,
A1/A2 = n1m1/n2m2 = (n1W1/M1)/(n2W2/M2) (3)
in which m1 and m2 are the numbers of moles; W1 and W2
are the masses; and M1 and M2 are the molecular weights
of compounds 1 and 2, respectively.
Examination of Equations 2 and 3 shows that NMR quan-
titative analysis can be performed in an absolute or relative
manner. In the absolute method, an internal standard is
added to the specimen and a resonance peak area arising
from the test substance is compared with a resonance peak
area from the internal standard. If both test substance and
internal standard are accurately weighed, the absolute purity
of the substance may be calculated. A good internal stan-
dard has the following properties: it presents a reference
resonance peak, preferably a singlet, at a field position re-
moved from all specimen peaks; it is soluble in the analytical
solvent; its proton equivalent weight, i.e., the molecular
weight divided by the number of protons giving rise to the
reference peak, is low; and it does not interact with the
compound being tested. Typical examples of useful stan-
dards are 1,2,4,5-tetrachlorobenzene, 1,4-dinitrobenzene,
benzyl benzoate, and maleic acid. The choice of a standard
will be dictated by the spectrum of the specimen.
The relative method may be used to determine the molar
fraction of an impurity in a test substance (or of the compo-
nents in a mixture) as calculated by Equation 3.
Quantitative analysis, as well as detection of trace impuri-
ties, is markedly improved with modern instrumentation.
Stronger magnetic fields and the ability to accumulate and/
or average signals over long periods of time greatly enhance
the sensitivity of the method.
Absolute Method of Quantitation Where the individual
monograph directs that the Absolute Method of Quantitation
be employed, proceed as follows.
Solvent, Internal Standard, and NMR Reference: Use as di-
rected in the individual monograph.
Test Preparation: Transfer an accurately weighed quantity of
the test substance, containing about 4.5 proton mEq, to a
glass-stoppered, graduated centrifuge tube. Add about 4.5
proton mEq of Internal Standard, accurately weighed, and
3.0 mL of Solvent, insert the stopper, and shake. When dis-
solution is complete, add about 30 µL (30 mg if a solid) of
NMR Reference, provided that it does not interfere with sub-
sequent measurements, and shake.
Procedure: Transfer an appropriate amount (0.4–0.8 mL) of
Test Preparation to a standard 5-mm NMR spinning tube,
and record the spectrum, adjusting the spin rate so that no
spinning side bands interfere with the peaks of interest.
Measure the area under each of the peaks specified in the
individual monograph by integrating not fewer than five
times. Record the average area of the Internal Standard peak
as AS and that of the Test Preparation peak as AU.
Calculate the quantity, in mg, of the analyte in the Test
Preparation:
WS(AU/AS)(EU/ES)
FCC 8 General Tests and Assays / Appendix II / 1251

in which WS is the weight, in mg, of Internal Standard taken; pressure slowly from the air source, and immerse the filter
and EU and ES are the proton equivalent weights (i.e., the just below the surface of water contained in a beaker.
molecular weights divided by the number of protons giving
rise to the reference peak) of the analyte and the Internal
Standard, respectively.
Relative Method of Quantitation Where the individual
monograph directs that the Relative Method of Quantitation
be employed, proceed as follows.
Solvent, NMR Reference, and Test Preparation: Use as di-
rected under Absolute Method of Quantitation.
Procedure: Transfer an appropriate amount (0.4–0.8 mL) of
Test Preparation to a standard 5-mm NMR spinning tube,
and record the spectrum, adjusting the spin rate so that no
spinning side bands interfere with the peaks of interest.
Measure the area or intensity under each of the peaks speci-
fied in the individual monograph by integrating not fewer
than five times. Record the average area or intensity result-
ing from the resonances of the groups designated in the
individual monograph as A1 and A2. Figure 16. Assembly for Checking Pore Diameter of Filter
Calculate the quantity, in mole percent, of the analyte in Sticks
the Test Preparation:
[NOTE—If a head of liquid is noted above the surface
(100 × (A1/n1)/[(A1/n1) + (A2/n2)] of the filter after it is inserted into the water, the back
pressure thus produced should be subtracted from the
in which n1 and n2 are, respectively, the numbers of protons observed pressure when the pore diameter is calcu-
in the designated groups. lated as directed below.]
Increase the air pressure to 10 mm below the acceptable
OIL CONTENT OF SYNTHETIC PARAFFIN pressure limit, and then increase the pressure at a slow, uni-
form rate of about 3 mm Hg per minute until the first bub-
Apparatus
ble passes through the filter. This can be conveniently ob-

General Tests and Assays

Filter Stick: Use either a 10-mm diameter sintered-glass filter
stick of 10–15-µm maximum pore diameter, or a filter stick
made of stainless steel and having a 0.5-in. disk of 10–15-
µm maximum pore diameter. Determine conformance with
the pore diameter specified as follows: Clean sintered-glass
filter sticks by soaking in hydrochloric acid, or stainless steel
sticks by soaking in nitric acid, wash with water, rinse with
acetone, and dry in air followed by drying in an oven at
105° for 30 min.
Thoroughly wet the clean filter stick by soaking in water,
and then connect it with an apparatus (see Figure 16) con-
sisting of a mercury-filled manometer, readable to 0.5 mm;
a clean and filtered air supply; a drying bulb filled with silica
gel; and a needle-valve type air pressure regulator. Apply
served by placing the beaker over a mirror. Read the
manometer when the first bubble passes off the underside
of the filter. Calculate the pore diameter, in µm:
Result = 2180/p
in which p is the observed pressure, in mm, corrected for
any back pressure as mentioned above.
Filtration Assembly: Connect the Filter Stick with an air pres-
sure inlet tube and delivery nozzle and ground-glass joint to
fit a 25-mm × 170-mm test tube as shown in Figure 17. If a
stainless steel Filter Stick is used, make the connection to the
test tube by means of a cork.
 1252 / Appendix II / General Tests and Assays
Figure 17. Filtration Assembly for Determination of Oil
Content
Cooling Bath: Use a suitable insulated box having 1-in.
holes in the center to accommodate any desired number of
test tubes. The bath may be filled with a suitable medium
such as kerosene and may be cooled by circulating a refrig-
erant through coils, or by using solid carbon dioxide, to
produce a temperature of 30 ± 2°F.
General Tests and Assays
Air Pressure Regulator: Use a suitable pressure-reduction
valve, or other suitable regulator, that will supply air to the
Filtration Assembly at the volume and pressure required to
give an even flow of filtrate (see Procedure). Connect the
regulator with rubber tubing to the end of the Filter Stick in
the Filtration Assembly.
Thermometer: Use an ASTM Oil in Wax Thermometer hav-
ing the range of −35° to +70°F and conforming to the re-
quirements for an ASTM 71F thermometer (see Thermome-
ters, Appendix I).
Weighing Bottles: Use glass-stoppered conical bottles having
a capacity of 15 mL. The bottles are used as evaporating
flasks in the Procedure.
Evaporation Assembly: The assembly consists of an evaporat-
ing cabinet capable of maintaining a temperature of 95 ±
2°F around the evaporation flasks, and air jets (4 ± 0.2 mm
id) for delivering a stream of clean, dry air vertically down-
ward into the flasks. In the Procedure below, support each
jet so that the tip is 15 ± 5 mm above the surface of the
liquid at the start of the evaporation. Supply the air (purified
by passage through a tube of 1-cm bore packed loosely to a
height of 20 cm with absorbent cotton) at the rate of 2 to
3 L/min per jet. The cleanliness of the air should be checked
periodically to ensure that NMT 0.1 mg of residue is ob-
tained when 4 mL of methyl ethyl ketone is evaporated as
directed in the Procedure.
Wire Stirrer Use a 250-mm length of stiff iron or nichrome
wire of about No. 20 B & S gauge. Form a 10-mm diameter
FCC 8
loop at each end, and bend the loop at the bottom end so
that the plane of the loop is perpendicular to the length of
the wire.
Sample Selection If the sample weighs about 1 kg or
less, obtain a representative portion by melting the entire
sample and stirring thoroughly. For samples heavier than
about 1 kg, exercise special care to ensure that a truly repre-
sentative portion is obtained, noting that the oil may not be
distributed uniformly throughout the sample and that me-
chanical operations may have expressed some of the oil.
Procedure Melt a representative portion of the sample in
a beaker, using a water bath or oven maintained at
160°–210°F. As soon as the sample is completely melted,
thoroughly mix it by stirring. Preheat a dropper pipet, pro-
vided with a rubber bulb and calibrated to deliver 1 ± 0.05 g
of molten sample, and withdraw a 1-g portion of the sam-
ple as soon as possible after it has melted. Hold the pipet in
a vertical position, and carefully transfer its contents into a
clean, dry test tube previously weighed to the nearest milli-
gram. Evenly coat the bottom of the tube by swirling, allow
the tube to cool, and weigh to the nearest milligram. Calcu-
late the sample weight, in grams, and record it as B (see
Calculation). Pipet 15 mL of methyl ethyl ketone (ASTM
Specification D 740, or equivalent) into the tube, and im-
merse the tube up to the top of the liquid in a hot water or
steam bath. Stir with an up-and-down motion with the wire
stirrer, and continue heating and stirring until a homogene-
ous solution is obtained, exercising care to avoid loss of sol-
vent by prolonged boiling.
[NOTE—If it appears that a clear solution will not be
obtained, stir until any undissolved material is well dis-
persed so as to produce a slightly cloudy solution.]
After the sample solution is prepared, plunge the test
tube into an 800-mL beaker of ice water, and continue to
stir until the contents are cold. Remove the stirrer, then re-
move the test tube from the bath, dry the outside of the
tube with a cloth, and weigh to the nearest 100 mg. Calcu-
late the weight, in grams, of solvent in the test tube, and
record it as C (see Calculation). Place the tube in the cooling
bath, maintained at −30 ± 2°F, and stir continuously with
the thermometer until the temperature reaches −25 ± 0.5°F,
maintaining the slurry at a uniform consistency and taking
precautions to prevent the sample from setting up on the
walls of the tube or forming crystals.
Place the filter stick in a test tube and cool at −30 ± 2°F in
the cooling bath for a minimum of 10 min. Immerse the
cooled filter stick in the sample, then connect the filtration
assembly, seating the ground-glass joint of the filter so as to
make an airtight seal. Place an unstoppered weighing bot-
tle, previously weighed together with the glass stopper to
the nearest 0.1 mg, under the delivery nozzle of the filtra-
tion assembly.
[NOTE—Suitable precautions and proper analytical
technique should be applied to ensure the accuracy of
the weight of the bottle. Before determining its
weight, the bottle and its stopper should have been
FCC 8
cleaned and dried, then rinsed with methyl ethyl ke-
tone, wiped dry on the outside, dried in the evapora-
tion assembly for about 5 min, and cooled. Then al-
low it to stand for about 10 min near the balance
before weighing.]
Apply air pressure to the filtration assembly, immediately
collect about 4 mL of filtrate in the weighing bottle, and
release the air pressure to permit the liquid to drain back
slowly from the delivery nozzle. Stopper the bottle, and
weigh it to the nearest 10 mg without waiting for it to
come to room temperature. Remove the stopper, transfer
the bottle to the evaporation assembly maintained at 95 ±
2°F, and place it under an air jet centered inside the neck,
with the tip 15 ± 5 mm above the surface of the liquid.
After the solvent has evaporated (usually less than 30 min),
stopper the bottle, and allow it to stand near the balance
for about 10 min before it is weighed to the nearest 0.1
mg. Repeat the evaporation procedure for 5-min periods
until the loss between successive weighings is NMT 0.2 mg.
Determine the weight of the oil residue, in grams, by sub-
tracting the weight of the empty stoppered bottle from the
weight of the stoppered bottle plus the oil residue after the
evaporation procedure, and record the results as A (see Cal-
culation). Determine the weight of solvent evaporated, in
grams, by subtracting the weight of the bottle plus oil resi-
due from the weight of the bottle plus filtrate, and record
the result as D (see Calculation).
Calculation Calculate the percent, by weight, of oil in
the sample:
Result = (100 AC/BD) − 0.15
in which 0.15 is a factor to correct for solubility of the sam-
ple in the solvent at −25°F.
PLASMA SPECTROCHEMISTRY
Plasma-based instrumental techniques that are useful for
food ingredient analyses fall into two major categories:
those based on the inductively coupled plasma, and those
where a plasma is generated at or near the surface of the
sample. An inductively coupled plasma (ICP) is a high-tem-
perature excitation source that desolvates, vaporizes, and at-
omizes aerosol samples and ionizes the resulting atoms. The
excited analyte ions and atoms can then subsequently be
detected by observing their emission lines, a method
termed inductively coupled plasma–atomic emission spec-
troscopy (ICP–AES), also known as inductively coupled
plasma–optical emission spectroscopy (ICP–OES); or the ex-
cited or ground state ions can be determined by a tech-
nique known as inductively coupled plasma–mass spectrom-
etry (ICP–MS). ICP–AES and ICP–MS may be used for either
single- or multi-element analysis, and they provide good
general-purpose procedures for either sequential or simulta-
neous analyses over an extended linear range with good
sensitivity.
An emerging technique in plasma spectrochemistry is la-
ser-induced breakdown spectroscopy (LIBS). In LIBS, a solid,
liquid, or gaseous sample is heated directly by a pulsed la-
ser, or indirectly by a plasma generated by the laser. As a
General Tests and Assays / Appendix II / 1253
result, the sample is volatilized at the laser beam contact
point, and the volatilized constituents are reduced to atoms,
molecular fragments, and larger clusters in the plasma that
forms at or just above the surface of the sample. Emission
from the atoms and ions in the sample is collected, typically
using fiber optics or a remote viewing system, and is meas-
ured using an array detector such as a charge-coupled de-
vice (CCD). LIBS can be used for qualitative analysis or
against a working standard curve for quantitative analysis.
Although LIBS is not currently in wide use, it might be
suited for at-line or on-line measurements in a production
setting, as well as in the laboratory. Because of its potential,
it should be considered a viable technique for plasma spec-
trochemistry in the laboratory. However, because LIBS is still
an emerging technique, details will not be further discussed
here.3
Sample Preparation Sample preparation is critical to
the success of plasma-based analysis and is the first step in
performing any analysis via ICP–AES or ICP–MS. Plasma-
based techniques are heavily dependent on sample trans-
port into the plasma, and because ICP–AES and ICP–MS
share the same sample introduction system, the means by
which samples are prepared may be applicable to either
technique. The most conventional means by which samples
are introduced into the plasma is via solution nebulization. If
solution nebulization is employed, solid samples must be
dissolved in order to be presented into the plasma for analy-
sis. Samples may be dissolved in any appropriate solvent.
There is a strong preference for the use of aqueous or dilute
nitric acid solutions, because there are minimal interferences
General Tests and Assays
with these solvents compared to other solvent choices. Hy-
drogen peroxide, hydrochloric acid, sulfuric acid, perchloric
acid, combinations of acids, or various concentrations of
acids can all be used to dissolve the sample for analysis.
Dilute hydrofluoric acid may also be used, but great care
must be taken to ensure the safety of the analyst, as well as
to protect the quartz sample introduction equipment when
using this acid; specifically, the nebulizer, spray chamber,
and inner torch tube should be manufactured from hydro-
fluoric acid-tolerant materials. Additionally, alternative
means of dissolving the sample can be employed. These
include, but are not limited to, the use of dilute bases,
straight or diluted organic solvents, combinations of acids or
bases, and combinations of organic solvents.
When samples are introduced into the plasma via solution
nebulization, it is important to consider the potential matrix
effects and interferences that might arise from the solvent.
The use of an appropriate internal standard and/or match-
ing the standard matrix with samples should be applied for
ICP–AES and ICP–MS analyses in cases where accuracy and
precision are not adequate. In either event, the selection of
an appropriate internal standard should consider the analyte
in question, ionization energy, wavelengths or masses, and
the nature of the sample matrix.
Where a sample is found not to be soluble in any accept-
able solvent, a variety of digestion techniques can be em-
ployed. These include hot-plate digestion and microwave-
3 Yueh F-Y, Singh JP, Zhang H. Laser-induced breakdown spectroscopy, ele-
mental analysis. In: Encyclopedia of Analytical Chemistry: Instrumentation and
Applications. New York: Wiley; 2000:2066–2087.
 1254 / Appendix II / General Tests and Assays
assisted digestions, including open- and closed-vessel ap-
proaches. The decision regarding the type of digestion tech-
nique to use depends on the nature of the sample being
digested, as well as on the analytes of interest.
Open-vessel digestion is generally not recommended for
the analysis of volatile metals, e.g., selenium and mercury.
The suitability of a digestion technique, whether open- or
closed-vessel, should be supported by spike recovery experi-
ments in order to verify that, within an acceptable toler-
ance, volatile metals have not been lost during sample prep-
aration. Use acids, bases, and hydrogen peroxide of ultra-
high purity, especially when ICP–MS is employed. Deionized
water must be at least 18 megaohm. Check diluents for
interferences before they are used in an analysis. Because it
is not always possible to obtain organic solvents that are
free of metals, use organic solvents of the highest quality
possible with regard to metal contaminants.
It is important to consider the selection of the type, mate-
rial of construction, pretreatment, and cleaning of analytical
labware used in ICP–AES and ICP–MS analyses. The material
must be inert and, depending on the specific application,
resistant to caustics, acids, and/or organic solvents. For
some analyses, diligence must be exercised to prevent the
adsorption of analytes onto the surface of a vessel, particu-
larly in ultra-trace analyses. Contamination of the sample so-
lutions from metal and ions present in the container can
also lead to inaccurate results.
The use of labware that is not certified to meet Class A
tolerances for volumetric flasks is acceptable if the linearity,
accuracy, and precision of the method have been experi-
mentally demonstrated to be suitable for the purpose at
General Tests and Assays
hand.
Sample Introduction There are two ways to introduce
the sample into the nebulizer: by a peristaltic pump and by
self-aspiration. The peristaltic pump is preferred, and serves
to ensure that the flow rate of sample and standard solution
to the nebulizer is the same, irrespective of sample viscosity.
In some cases, where a peristaltic pump is not required, self-
aspiration can be used.
A wide variety of nebulizer types is available, including
pneumatic (concentric and cross-flow), grid, and ultrasonic
nebulizers. Micronebulizers, high-efficiency nebulizers, di-
rect-injection high-efficiency nebulizers, and flow-injection
nebulizers are also available. The selection of the nebulizer
for a given analysis should consider the sample matrix,
analyte, and desired sensitivity. Some nebulizers are better
suited for use with viscous solutions or those containing a
high concentration of dissolved solids, whereas others are
better suited for use with organic solutions.
Note that the self-aspiration of a fluid is due to the Ber-
noulli or Venturi effect. Not all types of nebulizers will sup-
port self-aspiration. The use of a concentric nebulizer, for
example, is required for self-aspiration of a solution.
Once a sample leaves the nebulizer as an aerosol, it enters
the spray chamber, which is designed to permit only the
smallest droplets of sample solution into the plasma; as a
result, typically only 1%–2% of the sample aerosol reaches
the ICP, although some special-purpose nebulizers have
been designed that permit virtually all of the sample aerosol
to enter the ICP. As with nebulizers, there is more than one
type of spray chamber available for use with ICP–AES or
FCC 8
ICP–MS. Examples include the Scott double-pass spray
chamber, as well as cyclonic spray chambers of various con-
figurations. The spray chamber must be compatible with the
sample and solvent, and must equilibrate and wash out in
as short a time as possible. When a spray chamber is se-
lected, the nature of the sample matrix, the nebulizer, the
desired sensitivity, and the analyte should all be considered.
Gas and liquid chromatography systems can be interfaced
with ICP–AES and ICP–MS for molecular speciation, ionic
speciation, or other modes of separation chemistry, based
on elemental emission or mass spectrometry.
Ultimately, the selection of sample introduction hardware
should be demonstrated experimentally to provide sufficient
specificity, sensitivity, linearity, accuracy, and precision of
the analysis at hand.
In addition to solution nebulization, it is possible to ana-
lyze solid samples directly via laser ablation (LA). In such
instances, the sample enters the torch as a solid aerosol.
LA–ICP–AES and LA–ICP–MS are better suited for qualitative
analyses of compounds, because of the difficulty in ob-
taining appropriate standards. Nonetheless, quantitative
analyses can be performed if it can be demonstrated
through appropriate method validation that the available
standards are adequate.4
Standard Preparation Single- or multi-element stan-
dard solutions, which have concentrations traceable to pri-
mary reference standards, such as those of the National In-
stitute of Standards and Technology (NIST), can be
purchased for use in the preparation of working standard
solutions. Alternatively, standard solutions of elements can
be accurately prepared from standard materials and their
concentrations, determined independently, as appropriate.
Working standard solutions, especially those used for ultra-
trace analyses, may have limited shelf life. As a general rule,
working standard solutions should be retained for no more
than 24 h, unless stability is demonstrated experimentally.
The selection of the standard matrix is of fundamental im-
portance in the preparation of element standard solutions.
Spike recovery experiments should be conducted with spe-
cific sample matrices in order to determine the accuracy of
the method. If sample matrix effects cause excessive inaccu-
racies, standards, blanks, and sample solutions should be
matrix matched, if possible, in order to minimize matrix
interferences.
In cases where matrix matching is not possible, an appro-
priate internal standard or the method of standard additions
should be used for ICP–AES or ICP–MS. Internal standards
can also be introduced through a T connector into the sam-
ple uptake tubing. In any event, the selection of an appro-
priate internal standard should consider the analytes in
question, their ionization and excitation energies, their
chemical behavior, their wavelengths or masses, and the na-
ture of the sample matrix. Ultimately, the selection of an
internal standard should be demonstrated experimentally to
provide sufficient specificity, sensitivity, linearity, accuracy,
and precision of the analysis at hand.
The method of standard additions involves adding a
known concentration of the analyte element to the sample
4 For additional information on laser ablation, see Russo R, Mao X, Borisov
O, Liu H. Laser ablation in atomic spectrometry. In: Encyclopedia of Analytical
Chemistry: Instrumentation and Applications. New York: Wiley; 2000.
FCC 8
at no fewer than two concentration levels plus an unspiked
sample preparation. The instrument response is plotted
against the concentration of the added analyte element,
and a linear regression line is drawn through the data
points. The absolute value of the x-intercept multiplied by
any dilution factor is the concentration of the analyte in the
sample.
The presence of dissolved carbon at concentrations of a
small percentage in aqueous solutions enhances ionization
of selenium and arsenic in an inductively coupled argon
plasma. This phenomenon frequently results in a positive
bias for ICP–AES and ICP–MS selenium and arsenic quantifi-
cation measurements, which can be remedied by using the
method of standard additions or by adding a small percent-
age of carbon, such as analytically pure glacial acetic acid,
to the linearity standards.
ICP The components that make up the ICP excitation
source include the argon gas supply, torch, radio frequency
(RF) induction coil, impedance-matching unit, and RF gener-
ator. Argon gas is almost universally used in an ICP. The
plasma torch consists of three concentric tubes designated
as the inner, the intermediate, and the outer tube. The in-
termediate and outer tubes are almost universally made of
quartz. The inner tube can be made of quartz or alumina if
the analysis is conducted with solutions containing hydroflu-
oric acid. The nebulizer gas flow carries the aerosol of the
sample solution into and through the inner tube of the
torch and into the plasma. The intermediate tube carries the
intermediate (sometimes referred to as the auxiliary) gas.
The intermediate gas flow helps to lift the plasma off the
inner and intermediate tubes to prevent their melting and
the deposition of carbon and salts on the inner tube. The
outer tube carries the outer (sometimes referred to as the
plasma or coolant) gas, which is used to form and sustain
the toroidal plasma. The tangential flow of the coolant gas
through the torch constricts the plasma and prevents the
ICP from expanding to fill the outer tube, keeping the torch
from melting. An RF induction coil, also called the load coil,
surrounds the torch and produces an oscillating magnetic
field, which in turn sets up an oscillating current in the ions
and electrons produced from the argon. The impedance-
matching unit serves to efficiently couple the RF energy
from the generator to the load coil. The unit can be of
either the active or passive type. An active matching unit
adjusts the impedance of the RF power by means of a ca-
pacitive network, whereas the passive type adjusts the impe-
dance directly through the generator circuitry. Within the
load coil of the RF generator, the energy transfer between
the coil and the argon creates a self-sustaining plasma. Colli-
sions of the ions and electrons liberated from the argon
ionize and excite the analyte atoms in the high-temperature
plasma. The plasma operates at temperatures of 6,000 to
10,000 K, so most covalent bonds and analyte-to-analyte
interactions have been eliminated.
ICP–AES An inductively coupled plasma can use either an
optical or a mass spectral detection system. In the former
case, ICP–AES, analyte detection is achieved at an emission
wavelength of the analyte in question. Because of differ-
ences in technology, a wide variety of ICP–AES systems are
available, each with different capabilities, as well as different
General Tests and Assays / Appendix II / 1255
advantages and disadvantages. Simultaneous-detection sys-
tems are capable of analyzing multiple elements at the same
time, thereby shortening analysis time and improving back-
ground detection and correction. Sequential systems move
from one wavelength to the next to perform analyses, and
often provide a larger number of analytical lines from which
to choose. Array detectors, including charge-coupled de-
vices and charge-injection devices, with detectors on a chip,
make it possible to combine the advantages of both simul-
taneous and sequential systems. These types of detection
devices are used in the most powerful spectrometers, pro-
viding rapid analysis and a wide selection of analytical lines.
The ICP can be viewed in either axial or radial (also called
lateral) mode. The torch is usually positioned horizontally in
axially viewed plasmas and is viewed end on, whereas it is
positioned vertically in radially viewed plasmas and is
viewed from the side. Axial viewing of the plasma can pro-
vide higher signal-to-noise ratios (better detection limits and
precision); however, it also incurs greater matrix and spec-
tral interferences. Methods validated on an instrument with
a radial configuration will probably not be completely trans-
ferable to an instrument with an axial configuration, and
vice versa.
Additionally, dual-view instrument systems are available,
making it possible for the analyst to take advantage of ei-
ther torch configuration. The selection of the optimal torch
configuration will depend on the sample matrix, analyte in
question, analytical wavelength(s) used, cost of instrumenta-
tion, required sensitivity, and type of instrumentation availa-
ble in a given laboratory.
Regardless of torch configuration or detector technology,
General Tests and Assays
ICP–AES is a technique that provides a qualitative and/or
quantitative measurement of the optical emission from ex-
cited atoms or ions at specific wavelengths. These measure-
ments are then used to determine the analyte concentration
in a given sample. Upon excitation, an atom or atomic ion
emits an array of different frequencies of light that are char-
acteristic of the distinct energy transition allowed for that
element. The intensity of the light is generally proportional
to the analyte concentration. It is necessary to correct for
the background emission from the plasma. Sample concen-
tration measurements are usually determined from a work-
ing curve of known standards over the concentration range
of interest. It is, however, also possible to perform a single-
point calibration under certain circumstances, such as with
limit tests, if the methodology has been validated for suffi-
cient specificity, sensitivity, linearity, accuracy, precision,
ruggedness, and robustness.
Because there are distinct transitions between atomic en-
ergy levels, and because the atoms in an ICP are rather
dilute, emission lines have narrow bandwidths. However,
because the emission spectra from the ICP contain many
lines, and because “wings” of these lines overlap to produce
a nearly continuous background on top of the continuum
that arises from the recombination of argon ions with elec-
trons, a high-resolution spectrometer is required in ICP–AES.
The decision regarding which spectral line to measure
should include an evaluation of potential spectral interfer-
ences. All atoms in a sample are excited simultaneously;
however, the presence of multiple elements in some sam-
ples can lead to spectral overlap. Spectral interference can
 1256 / Appendix II / General Tests and Assays FCC 8

also be caused by background emission from the sample or
plasma. Modern ICPs usually have background correction
available, and a number of background correction tech-
niques can be applied. Simple background correction typi-
cally involves measuring the background emission intensity
at some point away from the main peak and subtracting
this value from the total signal being measured. Mathemati-
cal modeling to subtract the interfering signal as a back-
ground correction can also be performed with certain types
of ICP–AES spectrometers.
The selection of the analytical spectral line is critical to the
success of an ICP–AES analysis, regardless of torch configura-
tion or detector type. Though some wavelengths are pre-
ferred, the final choice must be made in the context of the
sample matrix, the type of instrument being used, and the
sensitivity required. Analysts might choose to start with the
wavelengths recommended by the manufacturer of their
particular instrument, and select alternative wavelengths
based on manufacturer recommendations or published
wavelength tables.5,6,7,8,9 Ultimately, the selection of analyti-
cal wavelengths should be demonstrated experimentally to
provide sufficient specificity, sensitivity, linearity, accuracy,
and precision of the analysis at hand.
Forward power, gas flow rates, viewing height, and torch
position can all be optimized to provide the best signal.
However, it must also be kept in mind that these same vari-
ables can influence matrix and spectral interferences.
In general, it is desirable to operate the ICP under robust
conditions, which can be gauged on the basis of the MgII/
MgI line pair at (280.270 nm/285.213 nm). If that ratio of
intensities is above 6.0 in an aqueous solution, the ICP is
General Tests and Assays
be employed for aqueous solutions, as well as a reduction in
the nebulizer gas flow. When using organic solvents, it may
also be necessary to bleed small amounts of oxygen into the
torch to prevent carbon buildup in the torch.
Calibration The wavelength accuracy for ICP–AES detec-
tion must comply with the manufacturer’s applicable oper-
ating procedures. Because of the inherent differences
among the types of instruments available, there is no gen-
eral system suitability procedure that can be employed. Cali-
bration routines recommended by the instrument manufac-
turer for a given ICP–AES instrument should be followed.
These might include, but are not limited to, use of a multi-
element wavelength calibration with a reference solution, in-
ternal mercury (Hg) wavelength calibration, and peak
search. The analyst should perform system checks in accor-
dance with the manufacturer’s recommendations.
Standardization The instrument must be standardized
for quantification at time of use. However, because ICP–AES
is a technique generally considered to be linear over a range
of 6–8 orders of magnitude, it is not always necessary to
continually demonstrate linearity by the use of a standard
curve composed of multiple standards. Once a method has
been developed and is in routine use, it is possible to cali-
brate with a blank and a single standard. One-point stan-
dardizations are suitable for conducting limit tests on pro-
duction materials and final products if the methodology has
been rigorously validated for sufficient specificity, sensitivity,
linearity, accuracy, precision, ruggedness, and robustness.
The use of a single-point standardization is also acceptable
for qualitative ICP–AES analyses, where the purpose of the

experiment is to confirm the presence or absence of ele-

said to be robust, and is less susceptible to matrix interfer-
ments without the requirement of an accurate
ences. A ratio of about 10.0 is generally what is sought.
quantification.
Note that the term robust conditions is unrelated to robust-
An appropriate blank solution and standards that bracket
ness as applied to analytical method validation. Operation of
the expected range of the sample concentrations should be
an instrument with an MgII/MgI ratio greater than 6.0 is
assayed and the detector response plotted as a function of
not mandated, but is being suggested as a means of opti-
analyte concentration, as in the case where the concentra-
mizing instrument parameters in many circumstances.
tion of a known component is being determined within a
The analysis of the Group I elements can be an exception
specified tolerance. However, it is not always possible to
to this strategy. When atomic ions are formed from ele-
employ a bracketing standard when an analysis is performed
ments in this group, they assume a noble gas electron con-
at or near the detection limit. This lack of use of a bracket-
figuration, with correspondingly high excitation energy. Be-
ing standard is acceptable for analyses conducted to
cause the first excited state of these ions is extremely high,
demonstrate the absence or removal of elements below a
few are excited, so emission intensity is correspondingly
specified limit. The number and concentrations of standard
low. This situation can be improved by reducing the frac-
solutions used should be based on the purpose of the quan-
tional ionization, which can in turn be achieved by using
tification, the analyte in question, the desired sensitivity, and
lower forward power settings in combination with adjusted
the sample matrix. Regression analysis of the standard plot
viewing height or nebulizer gas flow, or by adding an ioni-
should be employed to evaluate the linearity of detector
zation suppression agent to the samples and standards.
response, and individual monographs may set criteria for
When organic solvents are used, it is often necessary to
the residual error of the regression line. Optimally, a correla-
use a higher forward power setting, higher intermediate and
tion coefficient of NLT 0.99, or as indicated in the individual
outer gas flows, and a lower nebulizer gas flow than would
monograph, should be demonstrated for the working curve.
Here, too, however, the nature of the sample matrix, the
5 Payling R, Larkins P. Optical Emission Lines of the Elements. New York: Wiley;
2000.
analyte(s), the desired sensitivity, and the type of instrumen-
6 Harrison GR. Massachusetts Institute of Technology Wavelength Tables [also tation available may dictate a correlation coefficient lower
referred to as MIT Wavelength Tables]. Cambridge, MA: MIT Press; 1969. than 0.99. The analyst should use caution when proceeding
7 Winge RK, Fassel VA, Peterson VJ, Floyd MA. Inductively Coupled Plasma
with such an analysis, and should employ additional work-
Atomic Emission Spectroscopy: An Atlas of Spectral Information. New York: El-
ing standards.
sevier; 1985.
8 Boumans PWJM. Spectrochim Acta A. 1981;36B:169. To demonstrate the stability of the system’s initial stan-
9 Boumans PWJM. Line Coincidence Tables for Inductively Coupled Plasma dardization, a solution used in the initial standard curve
Atomic Emission Spectrometry. 2nd ed.; Oxford, UK: Pergamon; 1984.
FCC 8
must be reassayed as a check standard at appropriate inter-
vals throughout the analysis of the sample set. The reas-
sayed standard should agree with its expected value to
within ±10%, or as specified in an individual monograph,
for single-element analyses when analytical wavelengths are
200–500 nm, or concentrations are >1 µg/mL. The reas-
sayed standard should agree with its theoretical value to
within ±20%, or as specified in an individual monograph,
for multi-element analyses, when analytical wavelengths are
<200 nm or >500 nm, or at concentrations of <1 µg/mL. In
cases where an individual monograph provides different
guidance regarding the reassayed check standard, the re-
quirements of the monograph take precedence.
Procedure Follow the procedure as directed in the indi-
vidual monograph for the instrumental parameters. Because
of differences in manufacturers’ equipment configurations,
the manufacturer’s suggested default conditions may be
used and modified as needed. The specification of definitive
parameters in a monograph does not preclude the use of
other suitable operating conditions, and adjustments of op-
erating conditions may be necessary. Alternative conditions
must be supported by suitable validation data, and the con-
ditions in the monograph will take precedence for official
purposes. Data collected from a single sample introduction
are treated as a single result. This result might be the aver-
age of data collected from replicate sequential readings
from a single solution introduction of the appropriate stan-
dard or sample solution. Sample concentrations are calcu-
lated versus the working curve generated by plotting the
detector response versus the concentration of the analyte in
the standard solutions. This calculation is often performed
directly by the instrument.
ICP–MS When an inductively coupled plasma uses a mass
spectral detection system, the technique is referred to as
inductively coupled plasma–mass spectrometry (ICP–MS). In
this technique, analytes are detected directly at their atomic
masses. Because these masses must be charged to be de-
tected in ICP–MS, the method relies on the ability of the
plasma source to both atomize and ionize sample constitu-
ents. As is the case with ICP–AES, a wide variety of ICP–MS
instrumentation systems are available.
The systems most commonly in use are quadrupole-based
systems. Gaining in interest is time-of-flight ICP–MS. Al-
though still not in widespread use, this approach may see
greater use in the future. Additionally, high-resolution sector
field instruments are available.
Regardless of instrument design or configuration, ICP–MS
provides both a qualitative and a quantitative measurement
of the components of the sample. Ions are generated from
the analyte atoms by the plasma. The analyte ions are then
extracted from the atmospheric-pressure plasma through a
sampling cone into a lower-pressure zone, ordinarily held at
a pressure near 1 Torr. In this extraction process, the sam-
pled plasma gases, including the analyte species, form a su-
personic beam, which dictates many of the properties of the
resulting analyte ions. A skimmer cone, located behind the
sampling cone, “skims” the supersonic beam of ions as they
emerge from the sampling cone. Behind the skimmer cone
is a lower-pressure zone, often held near a milliTorr. Lastly,
the skimmed ions pass a third-stage orifice to enter a zone
General Tests and Assays / Appendix II / 1257
held near a microTorr, where they encounter ion optics and
are passed into the mass spectrometer. The mass spectrom-
eter separates the ions according to their mass-to-charge (m
/z) ratios. The ICP–MS has a mass range up to 240 atomic
mass units (amu). Depending on the equipment configura-
tion, analyte adducts can form with diluents, with argon, or
with their decomposition products. Also formed are oxides
and multiply-charged analyte ions, which can increase the
complexity of the resulting mass spectra. Interferences can
be minimized by appropriate optimization of operational pa-
rameters, including gas flows (central, intermediate, and
outer gas flow rates), sample-solution flow, RF power, ex-
traction-lens voltage, etc., or by the use of collision or reac-
tion cells, or cool plasma operation, if available on a given
instrument. Unless a laboratory is generating or examining
isotopes that do not naturally occur, a list of naturally occur-
ring isotopes will provide the analyst with acceptable iso-
topes for analytical purposes. Isotopic patterns also serve as
an aid to element identification and confirmation. Addition-
ally, tables of commonly found interferences and polyatomic
isobaric interferences and correction factors can be used.
ICP–MS generally offers considerably lower (better) de-
tection limits than ICP–AES, largely because of the extremely
low background that it generates. This ability is a major ad-
vantage of ICP–MS for determination of very low analyte
concentrations or when elimination of matrix interferences is
required. In the latter case, some interferences can be
avoided simply by additional dilution of the sample solution.
In some applications, analytes can be detected below the
parts per trillion (ppt) level using ICP–MS. As a general rule,
ICP–MS as a technique requires that samples contain signifi-
General Tests and Assays
cantly less total dissolved solids than does ICP–AES.
The selection of the analytical mass to use is critical to
the success of an ICP–MS analysis, regardless of instrument
design. Though some masses are often considered to be the
primary ones, because of their high natural abundance, an
alternative mass for a given element is often used to avoid
spectral overlaps (isobaric interferences). Selection of an an-
alytical mass must always be considered in the context of
the sample matrix, the type of instrument being used, and
the concentrations to be measured. Analysts might choose
to start with masses recommended by the manufacturer of
their particular instrument, and select alternate masses
based on manufacturer’s recommendations or published ta-
bles of naturally occurring isotopes.10
Optimization of an ICP–MS method is also highly depen-
dent on the plasma parameters and means of sample intro-
duction. Forward power, gas flow rates, and torch position
may all be optimized to provide the best signal. When or-
ganic solvents are used, it is often necessary to use a higher
forward power setting and a lower nebulizer flow rate than
would be used for aqueous solutions. Additionally, when or-
ganic solvents are used, it might be necessary to introduce
small amounts of oxygen into the central or intermediate
gas to prevent carbon buildup in the torch or on the sam-
pler cone orifice. The use of a platinum-tipped sampling or
skimmer cone may also be required in order to reduce cone
degradation with some organic solvents.
10 Horlick G, Montaser A. Analytical characteristics of ICPMS. In: Montaser A,
Editor. Inductively Coupled Plasma Mass Spectrometry. New York: Wiley-VCH;
1998:516–518.
 1258 / Appendix II / General Tests and Assays
Calibration The mass spectral accuracy for ICP–MS detec-
tion must be in accordance with the applicable operating
procedures. Because of the inherent differences between the
types of instruments available, there is no general system
suitability procedure that can be employed. Analysts should
refer to the tests recommended by the instrument manufac-
turer for a given ICP–MS instrument. These may include,
but are not limited to, tuning on a reference mass or
masses, peak search, and mass calibration. The analyst
should perform system checks recommended by the instru-
ment manufacturer.
Standardization The instrument must be standardized
for quantification at the time of use. Because the response
(signal vs. concentration) of ICP–MS is generally considered
to be linear over a range of 6–8 orders of magnitude, it is
not always necessary to continually demonstrate linearity by
the use of a working curve. Once a method has been devel-
oped and is in routine use, it is common practice to cali-
brate with a blank and a single standard. One-point stan-
dardizations are suitable for conducting limit tests on
production materials and final products, provided that the
methodology has been rigorously validated for sufficient
specificity, sensitivity, linearity, accuracy, precision, rugged-
ness, and robustness. An appropriate blank solution and
standards that bracket the expected range of the sample
concentrations should be assayed and the detector response
plotted as a function of analyte concentration. The number
and concentration of standard solutions used should be
based on the analyte in question, the expected concentra-
tions, and the sample matrix, and should be left to the dis-
General Tests and Assays
cretion of the analyst. Optimally, a correlation coefficient of
NLT 0.99, or as indicated in the individual monograph,
should be demonstrated for the working standard curve.
Here, too, however, the nature of the sample matrix, the
analyte, the desired sensitivity, and the type of instrumenta-
tion available might dictate a correlation coefficient lower
than 0.99. The analyst should use caution when proceeding
with such an analysis and should employ additional working
standards.
To demonstrate the stability of the system since initial
standardization, a solution used in the initial standard curve
must be reassayed as a check standard at appropriate inter-
vals throughout the analysis of the sample set. Appropriate
intervals may be established as occurring after every fifth or
tenth sample, or as deemed adequate by the analyst, on the
basis of the analysis being performed. The reassayed stan-
dard should agree with its expected value to within ±10%
for single-element analyses when analytical masses are free
of interferences and when concentrations are >1 ng/mL.
The reassayed standard should agree with its expected value
to within ±20% for multi-element analyses, or when con-
centrations are <1 ng/mL. In cases where an individual
monograph provides different guidance regarding the reas-
sayed check standard, the requirements of the monograph
take precedence.
The method of standard additions should be employed in
situations where matrix interferences are expected or sus-
pected. This method involves adding a known concentration
of the analyte element to the sample solution at no fewer
than two concentration levels. The instrument response is
FCC 8
plotted against the concentration of the added analyte ele-
ment, and a linear regression line is drawn through the data
points. The absolute value of the x-intercept multiplied by
any dilution factor is the concentration of the analyte in the
sample.
Procedure Follow the procedure as directed in the indi-
vidual monograph for the detection mode and instrument
parameters. The specification of definitive parameters in a
monograph does not preclude the use of other suitable op-
erating conditions, and adjustments of operating conditions
may be necessary. Alternative conditions must be supported
by suitable validation data, and the conditions in the mono-
graph will take precedence for official purposes. Because of
differences in manufacturers’ equipment configurations, the
analyst may wish to begin with the manufacturer’s sug-
gested default conditions and modify them as needed. Data
collected from a single sample introduction are treated as a
single result. Data collected from replicate sequential read-
ings from a single introduction of the appropriate standard
or sample solutions are averaged as a single result. Sample
concentrations are calculated versus the working curve gen-
erated by plotting the detector response versus the concen-
tration of the analyte in the standard solutions. With mod-
ern instruments, this calculation is often performed by the
instrument.
Glossary
AUXILIARY GAS See Intermediate (or Auxiliary) Gas.
AXIAL VIEWING A configuration of the plasma for AES in
which the plasma is directed toward the spectrometer opti-
cal path, also called “end-on viewing.”
CENTRAL (OR NEBULIZER) GAS One of three argon gas flows in
an ICP torch. The central gas is used to help create a fine
mist of the sample solution when solution nebulization is
employed. This fine mist is then directed through the cen-
tral tube of the torch and into the plasma.
COLLISION CELL A design feature of some ICP–MS instru-
ments. Collision cells are used to reduce interferences from
argon species or polyatomic ions and to facilitate the analy-
sis of elements that might be affected by those
interferences.
COOL PLASMA Plasma conditions used for ICP–MS that result
in a plasma that is cooler than that normally used for an
analysis. This condition is achieved by using a lower forward
power setting and higher central-gas flow rate, and is used
to help reduce isotopic interferences caused by argon and
some polyatomic ions.
COOLANT GAS See Outer (or Coolant or Plasma) Gas.
FORWARD POWER The number of watts used to ignite and
sustain the plasma during an analysis. Forward power re-
quirements may vary, depending on sample matrix and
analyte.
INTERMEDIATE (OR AUXILIARY) GAS Gas used to “lift” the plasma
off the surface of the torch, thereby preventing melting of
the intermediate tube and the formation of carbon and salt
deposits on the inner tube.
INTERNAL STANDARD An element added to or present in the
same concentration in blanks, standards, and samples to act
FCC 8
as an intensity reference for the analysis. An internal stan-
dard should be used for ICP–AES work and must always be
used for quantitative ICP–MS analyses.
LATERAL VIEWING See Radial Viewing.
m The ion mass of interest.
MULTIPLY-CHARGED IONS Atoms that, when subjected to the
high-ionization temperature of the ICP, can form doubly or
triply charged ions (X++, X+++, etc.). When detected by MS,
the apparent mass of these ions will be half or one-third
that of the atomic mass.
NEBULIZER Used to form a consistent sample aerosol that
mixes with the argon gas, which is subsequently sent into
the ICP.
OUTER (OR COOLANT OR PLASMA) GAS The main gas supply for
the plasma.
PLASMA GAS See Outer (or Coolant or Plasma) Gas.
RADIAL VIEWING A configuration of the plasma for AES in
which the plasma is viewed orthogonal to the spectrometer
optic path. Also called “side-on viewing.” See also Lateral
Viewing.
REACTION CELL Similar to Collision Cell, but operating on a
different principle. Designed to reduce or eliminate spectral
interferences.
SAMPLING CONE A metal cone (usually nickel-, aluminum-, or
platinum-tipped) with a small opening, through which ion-
ized sample material flows after leaving the plasma.
SEQUENTIAL A type of detector configuration for AES or MS
in which discrete emission lines or isotopic peaks are ob-
served by scanning or hopping across the spectral range by
means of a monochromator or scanning mass spectrometer.
SIMULTANEOUS A type of detector configuration for AES or
MS in which all selected emission lines or isotopic peaks are
observed at the same time by using a polychromator or
simultaneous mass spectrometer, offering increased analysis
speed for analyses of multi-element samples.
SKIMMER CONE A metal cone through which ionized sample
flows after leaving the sampling cone and before entering
the high-vacuum region of an ICP–MS.
STANDARD ADDITIONS A method used to determine the actual
analyte concentration in a sample when viscosity or matrix
effects might cause erroneous results.
TORCH A series of three concentric tubes, usually manufac-
tured from quartz, in which the ICP is formed.
RESIDUE ON IGNITION (Sulfated Ash)
Method I (for Solids)
Transfer the quantity of the sample directed in the individual
monograph onto a tared 50-mL to 100-mL platinum dish or
other suitable container, and add sufficient 2 N sulfuric acid
to moisten the entire sample. Heat gently, using a hot plate,
an Argand burner, or an infrared heat lamp, until the sam-
ple is dry and thoroughly charred, then continue heating
until all of the sample has been volatilized or nearly all of
the carbon has been oxidized, and cool. Moisten the residue
General Tests and Assays / Appendix II / 1259
with 0.1 mL of sulfuric acid, and heat in the same manner
until the remainder of the sample and any excess sulfuric
acid have been volatilized. To promote volitilization of sulfu-
ric acid, add a few pieces of ammonium carbonate just
before completing ignition. Finally, ignite to constant
weight in a muffle furnace at 800 ± 25° for 15 min, or
longer if necessary to complete ignition, cool in a desicca-
tor, and weigh.
Method II (for Liquids)
Unless otherwise directed, transfer the required weight of
the sample onto a tared 75-mL to 100-mL platinum dish.
Heat gently, using an Argand or Meker burner, until the
sample ignites, then allow the sample to burn until it self-
extinguishes. Cool, then wet the residue with 2 mL of con-
centrated sulfuric acid, and heat the sample over a low
flame until dry. Ignite to constant weight in a muffle fur-
nace at 800 ± 25° for 30 min, or longer if necessary for
complete ignition, cool in a desiccator, and weigh.
SIEVE ANALYSIS OF GRANULAR METAL
POWDERS (Based on ASTM Designation: B
214)11
Apparatus
Sieves: Use a set of standard sieves, ranging from 80-mesh
to 325-mesh, conforming to the specifications in ASTM Des-
ignation: E 11 (Sieves for Testing Purposes).
Sieve Shaker: Use a mechanically operated sieve shaker that
imparts to the set of sieves a horizontal rotary motion of
270–300 rotations/min and a tapping action of140–160
General Tests and Assays
taps/min. The sieve shaker is fitted with a plug to receive
the impact of the tapping device. The entire apparatus is
rigidly mounted—bolted to a solid foundation, preferably of
concrete. Preferably a time switch is provided to ensure the
accuracy of test duration.
Procedure Assemble the sieves in consecutive order by
opening size, with the coarsest sieve (80-mesh) at the top,
and place a solid-collecting pan below the bottom sieve
(325-mesh). Place 100.0 g of the test sample, W, on the
top sieve, and close the sieve with a solid cover. Securely
fasten the assembly to the sieve shaker, and operate the
shaker for 15 min. Remove the most coarse sieve from the
nest, gently tap its contents to one side, and pour the con-
tents onto a tared, glazed paper. Using a soft brush, transfer
onto the next finer sieve any material adhering to the bot-
tom of the sieve and frame. Place the sieve just removed
upside down on the paper containing the retained portion,
and tap the sieve. Accurately weigh the paper and its con-
tents, and record the net weight of the fraction, F, ob-
tained. Repeat this process for each sieve in the nest and for
the portion of the sample that has been collected in the
bottom pan. Record the total of the fractions retained on
the sieves as T and that portion collected in the pan as t.
11 Adapted from ASTM B214 Standard Test Method for Sieve Analysis of
Metal Powders. The original ASTM method is available in its entirety from
ASTM International, 100 Barr Harbor Drive, West Conshohocken, PA 19428.
Phone: 610-832-9585, Fax: 610-832-9555, Email: service@astm.org, Website:
www.astm.org.
 1260 / Appendix II / General Tests and Assays FCC 8

The combined total, S, of T + t is the amount of the sam- °Bé Sp. Gr. Percent H2SO4
ple, W, recovered in the test. Calculate the percent 33 1.2946 38.58
recovery: 34 1.3063 39.92
35 1.3182 41.27
Result = S/W × 100
36 1.3303 42.63
If the percent recovery is less than 99.0%, check the condi- 37 1.3426 43.99
tion of the sieves and for possible errors in weighing, and
38 1.3551 45.35
repeat the test. If the percent recovery is NLT 99.0%, calcu-
39 1.3679 46.72
late the percent retained on each sieve:
40 1.3810 48.10
Result = F/W × 100 41 1.3942 49.47

Calculate the percent through the smallest mesh sieve from 42 1.4078 50.87
the portion collected in the pan: 43 1.4216 52.26
44 1.4356 53.66
Result = [(100 − t)/W] × 100
45 1.4500 55.07
46 1.4646 56.48
SULFURIC ACID TABLE 47 1.4796 57.90
48 1.4948 59.32
49 1.5104 60.75
°Bé Sp. Gr. Percent H2SO4
50 1.5263 62.18
0 1.0000 0.00
51 1.5426 63.66
1 1.0069 1.02
52 1.5591 65.13
2 1.0140 2.08
53 1.5761 66.63
3 1.0211 3.13
54 1.5934 68.13
4 1.0284 4.21
55 1.6111 69.65
5 1.0357 5.28
56 1.6292 71.17
6 1.0432 6.37
57 1.6477 72.75
General Tests and Assays

7 1.0507 7.45
58 1.6667 74.36
8 1.0584 8.55
59 1.6860 75.99
9 1.0662 9.66
60 1.7059 77.67
10 1.0741 10.77
61 1.7262 79.43
11 1.0821 11.89
62 1.7470 81.30
12 1.0902 13.01
63 1.7683 83.34
13 1.0985 14.13
64 1.7901 85.66
14 1.1069 15.25
64.25 1.7957 86.33
15 1.1154 16.38
64.50 1.8012 87.04
16 1.1240 17.53
64.75 1.8068 87.81
17 1.1328 18.71
65 1.8125 88.65
18 1.1417 19.89
65.25 1.8182 89.55
19 1.1508 21.07
65.50 1.8239 90.60
20 1.1600 22.25
66 1.8354 93.19
21 1.1694 23.43
22 1.1789 24.61 Specific gravity determinations were made at 60°F, com-
23 1.1885 25.81 pared with water at 60°F. The values given above for aque-
24 1.1983 27.03
ous sulfuric acid solutions were adopted as standard in 1904
by the Manufacturing Chemists’ Association of the United
25 1.2083 28.28
States.
26 1.2185 29.53 From the specific gravities, the corresponding degrees
27 1.2288 30.79 Baumé were calculated by the following equation:
28 1.2393 32.05
°Baumé = 145 − (145/sp. gr.)
29 1.2500 33.33
30 1.2609 34.63 Baumé hydrometers for use with this table must be grad-
31 1.2719 35.93 uated by the above formula, which should always be
32 1.2832 37.26
printed on the scale. Acids stronger than 66°Bé should have
FCC 8
their percentage compositions determined by chemical
analysis.
WATER-INSOLUBLE MATTER
Sample Preparation Add 5 g of sample (if a different
amount of sample is specified in the individual monograph,
use that amount) to 100 mL of water, and stir until the
sample is dissolved.
General Tests and Assays / Appendix II / 1261
Procedure Dry a membrane filter (cellulose nitrate, 0.45-
µm porosity) at 110°C for 1 h, allow to cool in a desiccator,
and weigh to the nearest 0.1 mg. Pass the Sample Prepara-
tion through the dried membrane filter and wash with three
successive 10-mL portions of water. Dry the membrane filter
at 110°C for 1 h. Cool in a desiccator, and weigh the mem-
brane filter to the nearest 0.1 mg. Calculate the insoluble
matter as percentage.
General Tests and Assays
 1262 / Appendix III / General Tests and Assays
APPENDIX III: CHEMICAL TESTS AND DETERMINATIONS
A. IDENTIFICATION TESTS
The identification tests described in section A of this Appen-
dix are frequently referred to in the Food Chemicals Codex
for the presumptive identification of FCC-grade chemicals
taken from labeled containers. These tests are not intended
to be applied to mixtures unless so specified.
Acetate
Acetic acid or acetates, when warmed with sulfuric acid and
alcohol, form ethyl acetate, recognizable by its characteristic
odor. With neutral solutions of acetates, ferric chloride TS
produces a deep red color that is destroyed by the addition
of a mineral acid.
Aluminum
Solutions of aluminum salts yield with 6 N ammonia a
white, gelatinous precipitate that is insoluble in an excess of
the 6 N ammonia. The same precipitate is produced by 1 N
sodium hydroxide, but it dissolves in an excess of this
reagent.
Ammonium
Ammonium salts are decomposed by 1 N sodium hydroxide
with the evolution of ammonia, recognizable by its alkaline
effect on moistened red litmus paper. The decomposition is
General Tests and Assays
accelerated by warming.
Benzoate
Neutral solutions of benzoates yield a salmon colored pre-
cipitate with ferric chloride TS. From moderately concen-
trated solutions of benzoate, 2 N sulfuric acid precipitates
free benzoic acid, which is readily soluble in ether.
Bicarbonate
See Carbonate.
Bisulfite
See Sulfite.
Bromide
Free bromine is liberated from solutions of bromides upon
the dropwise addition of chlorine TS. When shaken with
chloroform, the bromine dissolves, coloring the chloroform
red to red-brown. A yellow-white precipitate, which is insol-
uble in nitric acid and slightly soluble in 6 N ammonia, is
produced when solutions of bromides are treated with silver
nitrate TS.
Calcium
Insoluble oxalate salts are formed when solutions of calcium
salts are treated in the following manner: using 2 drops of
methyl red TS as the indicator, neutralize a 1:20 solution of
a calcium salt with 6 N ammonia, then add 2.7 N hydro-
chloric acid, dropwise, until the solution is acid. A white
precipitate of calcium oxalate forms upon the addition of
ammonium oxalate TS. This precipitate is insoluble in acetic
acid but dissolves in hydrochloric acid.
Calcium salts moistened with hydrochloric acid impart a
transient yellow-red color to a nonluminous flame.
FCC 8
Carbonate
Carbonates and bicarbonates effervesce with acids, yielding
a colorless gas that produces a white precipitate immedi-
ately when passed into calcium hydroxide TS. Cold solutions
of soluble carbonates are colored red by phenolphthalein
TS, whereas solutions of bicarbonates remain unchanged or
are slightly changed.
Chloride
Solutions of chlorides yield with silver nitrate TS a white,
curdy precipitate that is insoluble in nitric acid but soluble
in a slight excess of 6 N ammonia.
Citrate
To 15 mL of pyridine add a few mg of a citrate salt, dis-
solved or suspended in 1 mL of water, and shake. Add 5 mL
of acetic anhydride to this mixture, and shake. A light red
color appears.
Cobalt
Solutions of cobalt salts (1:20) in 2.7 N hydrochloric acid
yield a red precipitate when heated on a steam bath with
an equal volume of a hot, freshly prepared 1:10 solution of
1-nitroso-2-naphthol in 9 N acetic acid. Solutions of cobalt
salts yield a yellow precipitate when saturated with potas-
sium chloride and treated with potassium nitrite and acetic
acid.
Copper
When solutions of cupric compounds are acidified with hy-
drochloric acid, a red film of metallic copper is deposited on
a bright untarnished surface of metallic iron. An excess of 6
N ammonia, added to a solution of a cupric salt, produces
first a blue precipitate and then a deep blue colored solu-
tion. Solutions of cupric salts yield with potassium ferrocya-
nide TS a red-brown precipitate, insoluble in diluted acids.
Hypophosphite
Hypophosphites evolve spontaneously flammable phosphine
when strongly heated. Solutions of hypophosphites yield a
white precipitate with mercuric chloride TS. This precipitate
becomes gray when an excess of hypophosphite is present.
Hypophosphite solutions, acidified with sulfuric acid and
warmed with copper sulfate TS, yield a red precipitate.
Iodide
Solutions of iodides, upon the dropwise addition of chlorine
TS, liberate iodine, which colors the solution yellow to red.
Chloroform is colored violet when shaken with this solution.
The iodine thus liberated gives a blue color with starch TS.
In solutions of iodides, silver nitrate TS produces a yellow,
curdy precipitate that is insoluble in nitric acid and in 6 N
ammonia.
Iron
Solutions of ferrous and ferric compounds yield a black pre-
cipitate with ammonium sulfide TS. This precipitate is dis-
solved by cold 2.7 N hydrochloric acid with the evolution of
hydrogen sulfide.
FCC 8
Ferric Salts: Potassium ferrocyanide TS (10%) produces a
dark blue precipitate in acid solutions of ferric salts. With an
excess of 1 N sodium hydroxide, a red-brown precipitate is
formed. Solutions of ferric salts produce with ammonium
thiocyanate TS (1.0 N) a deep red color that is not de-
stroyed by diluted mineral acids.
Ferrous Salts: Potassium ferricyanide TS (10%) produces a
dark blue precipitate in solutions of ferrous salts. This precip-
itate, which is insoluble in dilute hydrochloric acid, is de-
composed by 1 N sodium hydroxide. Solutions of ferrous
salts yield with 1 N sodium hydroxide a green-white precipi-
tate, the color rapidly changing to green and then to brown
when shaken.
Lactate
When solutions of lactates are acidified with sulfuric acid,
potassium permanganate TS (0.1 N) is added, and the mix-
ture is heated, acetaldehyde is evolved. This can be de-
tected by allowing the vapor to come into contact with a
filter paper that has been moistened with a freshly prepared
mixture of equal volumes of 20% aqueous morpholine and
sodium nitroferricyanide TS. A blue color is produced.
Magnesium
Solutions of magnesium salts in the presence of ammonium
chloride yield no precipitate with ammonium carbonate TS,
but a white crystalline precipitate, which is insoluble in 6 N
ammonium hydroxide, is formed on the subsequent addi-
tion of sodium phosphate TS (6%).
Manganese
Solutions of manganous salts yield with ammonium sulfide
TS a salmon colored precipitate that dissolves in acetic acid.
Nitrate
When a solution of a nitrate is mixed with an equal volume
of sulfuric acid, the mixture cooled, and a solution of ferrous
sulfate superimposed, a brown color is produced at the
junction of the two liquids. Brown-red fumes are evolved
when a nitrate is heated with sulfuric acid and metallic cop-
per. Nitrates do not decolorize acidified potassium perman-
ganate TS (0.1 N) (distinction from nitrites).
Nitrite
Nitrites yield brown-red fumes when treated with diluted
mineral acids or acetic acid. A few drops of potassium io-
dide TS (15%) and a few drops of 2 N sulfuric acid added
to a solution of nitrite liberate iodine, which colors starch TS
blue.
Peroxide
Solutions of peroxides slightly acidified with sulfuric acid
yield a deep blue color on the addition of potassium dichro-
mate TS. On shaking the mixture with an equal volume of
diethyl ether and allowing the liquids to separate, the blue
color is transferred to the ether layer.
Phosphate
Neutral solutions of orthophosphates yield with silver nitrate
TS (0.1 N) a yellow precipitate, which is soluble in 1.7 N
General Tests and Assays / Appendix III / 1263
nitric acid or in 6 N ammonium hydroxide. With ammonium
molybdate TS, a yellow precipitate, which is soluble in 6 N
ammonium hydroxide, is formed.
Potassium
Potassium compounds impart a violet color to a nonlumi-
nous flame if not masked by the presence of small quanti-
ties of sodium. In neutral, concentrated or moderately con-
centrated solutions of potassium salts, sodium bitartrate TS
(10%) slowly produces a white, crystalline precipitate that is
soluble in 6 N ammonium hydroxide and in solutions of al-
kali hydroxides or carbonates. The precipitation may be ac-
celerated by stirring or rubbing the inside of the test tube
with a glass rod or by the addition of a small amount of
glacial acetic acid or alcohol.
Sodium
Dissolve 0.1 g of the sodium compound in 2 mL of water.
Add 2 mL of 15% potassium carbonate, and heat to boiling.
No precipitate is formed. Add 4 mL of potassium pyroan-
timonate TS, and heat to boiling. Allow to cool in ice water,
and if necessary, rub the inside of the test tube with a glass
rod. A dense precipitate is formed. Sodium compounds im-
part an intense yellow color to a nonluminous flame.
Sulfate
Solutions of sulfates yield with barium chloride TS a white
precipitate that is insoluble in hydrochloric and nitric acids.
Sulfates yield with lead acetate TS a white precipitate that is
soluble in ammonium acetate solution. Hydrochloric acid
produces no precipitate when added to solutions of sulfates
(distinction from thiosulfates).
Sulfite
General Tests and Assays
When treated with 2.7 N hydrochloric acid, sulfites and bi-
sulfites yield sulfur dioxide, recognizable by its characteristic
odor. This gas blackens filter paper moistened with mercu-
rous nitrate TS.
Tartrate
When a few mg of a tartrate are added to a mixture of 15
mL of pyridine and 5 mL of acetic anhydride, an emerald
green color is produced.
Thiosulfate
With hydrochloric acid, solutions of thiosulfates yield a white
precipitate that soon turns yellow, liberating sulfur dioxide,
recognizable by its odor. The addition of ferric chloride TS
to solutions of thiosulfates produces a dark violet color that
quickly disappears.
Zinc
Zinc salts, in the presence of sodium acetate, yield a white
precipitate with hydrogen sulfide. This precipitate, which is
insoluble in acetic acid, is dissolved by 2.7 N hydrochloric
acid. A similar precipitate is produced by ammonium sulfide
TS in neutral or alkaline solutions. Solutions of zinc salts
yield with potassium ferrocyanide TS (10%) a white precipi-
tate that is insoluble in 2.7 N hydrochloric acid.
 1264 / Appendix III / General Tests and Assays FCC 8

ARSENIC LIMIT TEST

B. LIMIT TESTS
Silver Diethyldithiocarbamate Colorimetric Method

ALUMINUM LIMIT TEST [NOTE—All reagents used in this test should be very
low in arsenic content.]
[NOTE—The Standard Solutions and Sample Solution may be Apparatus Use the general apparatus shown in Figure 1
modified, if necessary, to obtain solutions of suitable con- unless otherwise specified in an individual monograph. It
centrations adaptable to the linear or working range of the consists of a 125-mL arsine generator flask (a) fitted with a
instrument.] scrubber unit (c) and an absorber tube (e), with a 24/40
Nitric Acid Diluent Dilute 40 mL of nitric acid with standard-taper joint (b) and a ball-and-socket joint (d), se-
water to 1000 mL. cured with a No. 12 clamp, connecting the units. The tub-
Standard Aluminum Solutions Treat a quantity of alu- ing between d and e and between d and c is a capillary
minum wire with 6 N hydrochloric acid at 80° for a few having an id of 2 mm and an od of 8 mm. Alternatively, an
min. Dissolve 100 mg of the treated wire in a mixture con- apparatus embodying the principle of the general assembly
sisting of 10 mL of hydrochloric acid and 2 mL of nitric described and illustrated may be used.
acid, by heating at 80° for about 30 min. Continue heating
until the volume is reduced to about 4 mL. Cool to room
temperature, and add 4 mL of water. Evaporate to about 2
mL by heating. Cool, and transfer this solution, with the aid
of water, to a 100-mL volumetric flask, and dilute with
water to volume (1 mg/mL aluminum). Transfer 10.0 mL of
this solution to a second 100-mL volumetric flask, and dilute
with water to volume (100 µg/mL aluminum). Transfer 1.0
mL of this solution to a third 100-mL volumetric flask, and
dilute with water to volume (1 µg/mL aluminum). [NOTE—
If more diluted Standard Aluminum Solutions are required,
transfer 1.0-mL, 2.0-mL, and 4.0-mL portions of the 1 µg/
mL Standard Aluminum Solution to separate 100-mL volu-
metric flasks, dilute with Nitric Acid Diluent to volume, and
General Tests and Assays

mix. These solutions contain 0.01 µg/mL, 0.02 µg/mL, and

0.04 µg/mL of aluminum, respectively.]
Sample Solution Transfer the amount of sample speci-
fied in the monograph to a plastic 100-mL volumetric flask. Figure 1. General Apparatus for Arsenic Limit Test (Courtesy
Add 50 mL of water, and sonicate for 30 min. Add 4 mL of of the Fisher Scientific Co., Pittsburgh, PA.)
nitric acid, and dilute with water to volume.
Procedure Determine the absorbances of the Standard [NOTE—The special assemblies shown in Figures 2, 3,
Aluminum Solutions and the Sample Solution at the aluminum and 4 are to be used only when specified in certain
emission line at 309.3 nm with a suitable atomic absorption monographs.]
spectrophotometer equipped with an aluminum hollow-
cathode lamp and a flameless electrically heated furnace,
using the Nitric Acid Diluent as the blank. Plot the ab-
sorbances of the Standard Solutions versus the concentration
of aluminum, in µg/mL, drawing a straight line best fitting
the three points. From the graph so obtained, determine
the concentration, in µg/mL, of aluminum in the Sample
Solution.
Calculate the amount of aluminum in the sample taken,
in µg/g:

Result = CA/CS

in which CA is the concentration of aluminum in the Sample

Solution, in µg/mL, obtained from the standard curve; and
CS is the concentration of the Sample Solution, in g/mL.

Figure 2. Modified Bethge Apparatus for the Distillation of

Arsenic Tribromide
FCC 8
Figure 3. Special Apparatus for the Distillation of Arsenic
Trichloride (Flask A contains 150 mL of hydrochloric acid;
flasks D and F contain 20 mL of water. Flask D is placed in
an ice water bath, E.)
Figure 4. Special Apparatus for the Determination of Inor-
ganic Arsenic (A, 250-mL distillation flask; B, receiver cham-
ber, approximately 50-mL capacity; C, reflux condenser; D,
splash head.)
Standard Arsenic Solution Accurately weigh 132.0 mg
of arsenic trioxide that has been previously dried at 105° for
1 h, and dissolve it in 5 mL of a 1:5 sodium hydroxide
solution. Neutralize the solution with 2 N sulfuric acid, add
10 mL in excess, and dilute with recently boiled water to
1000.0 mL. Transfer 10.0 mL of this solution into a 1000-mL
volumetric flask, add 10 mL of 2 N sulfuric acid, dilute with
recently boiled water to volume, and mix. Use this final so-
lution, which contains 1 µg of arsenic in each mL, within 3
days.
Silver Diethyldithiocarbamate Solution Dissolve 1 g of
ACS reagent-grade silver diethyldithiocarbamate in 200 mL
of recently distilled pyridine. Store this solution in a light-
resistant container and use within 1 month.
Stannous Chloride Solution Dissolve 40 g of stannous
chloride dihydrate (SnCl2·2H2O) in 100 mL of hydrochloric
acid. Store the solution in glass containers and use within 3
months.
General Tests and Assays / Appendix III / 1265
Lead Acetate-Impregnated Cotton Soak cotton in a
saturated solution of lead acetate trihydrate, squeeze out
the excess solution, and dry in a vacuum at room
temperature.
Sample Solution Use directly as the Sample Solution in
the Procedure the solution obtained by treating the sample
as directed in an individual monograph. Prepare sample so-
lutions of organic compounds in the generator flask (a), un-
less otherwise directed, according to the following general
procedure:
[CAUTION—Some substances may react unexpectedly
with explosive violence when digested with hydrogen
peroxide. Use appropriate safety precautions at all
times.]
[NOTE—If halogen-containing compounds are present,
use a lower temperature while heating the sample
with sulfuric acid; do not boil the mixture; and add
the peroxide, with caution, before charring begins to
prevent loss of trivalent arsenic.]
Transfer 1.0 g of the sample into the generator flask, add 5
mL of sulfuric acid and a few glass beads, and digest at a
temperature not exceeding 120° until charring begins, pref-
erably using a hot plate in a fume hood. (Additional sulfuric
acid may be necessary to completely wet some samples, but
the total volume added should not exceed about 10 mL.)
After the acid has initially decomposed the sample, cau-
tiously add, dropwise, hydrogen peroxide (30%), allowing
the reaction to subside and reheating the sample between
General Tests and Assays
drops. Add the first few drops very slowly with sufficient
mixing to prevent a rapid reaction, and discontinue heating
if foaming becomes excessive. Swirl the solution in the flask
to prevent unreacted substance from caking on the walls or
bottom of the flask during digestion.
[NOTE—Maintain oxidizing conditions at all times dur-
ing the digestion by adding small quantities of the
peroxide whenever the mixture turns brown or
darkens.]
Continue the digestion until the organic matter is destroyed,
gradually raising the temperature of the hot plate to 250° to
300° until fumes of sulfur trioxide are copiously evolved and
the solution becomes colorless or retains only a light straw
color. Cool, cautiously add 10 mL of water, heat again to
strong fuming, and cool. Cautiously add 10 mL of water,
mix, wash the sides of the flask with a few mL of water, and
dilute to 35 mL.
Procedure If the Sample Solution was not prepared in the
generator flask, transfer to the flask a volume of the solu-
tion, prepared as directed, equivalent to 1.0 g of the sub-
stance being tested, and add water to make 35 mL. Add 20
mL of 1:5 sulfuric acid, 2 mL of potassium iodide TS, 0.5
mL of Stannous Chloride Solution, and 1 mL of isopropyl al-
cohol, and mix. Allow the mixture to stand for 30 min at
room temperature. Pack the scrubber unit (c) with two
plugs of Lead Acetate-Impregnated Cotton, leaving a small air
space between the two plugs, lubricate joints b and d with
stopcock grease, if necessary, and connect the scrubber unit
with the absorber tube (e). Transfer 3.0 mL of Silver
Diethyldithiocarbamate Solution to the absorber tube, add
 1266 / Appendix III / General Tests and Assays
3.0 g of granular zinc (20-mesh) to the mixture in the flask,
and immediately insert the standard-taper joint (b) into the
flask. Allow the evolution of hydrogen and color develop-
ment to proceed at room temperature (25 ± 3°) for 45 min,
swirling the flask gently at 10-min intervals. Disconnect the
absorber tube from the generator and scrubber units, and
transfer the Silver Diethyldithiocarbamate Solution to a 1-cm
absorption cell. Determine the absorbance at the wave-
length of maximum absorption between 535 nm and 540
nm, with a suitable spectrophotometer or colorimeter, using
Silver Diethyldithiocarbamate Solution as the blank. The ab-
sorbance due to any red color from the solution of the sam-
ple does not exceed that produced by 3.0 mL of Standard
Arsenic Solution (3 µg As) when treated in the same manner
and under the same conditions as the sample. The room
temperature during the generation of arsine from the stan-
dard should be held to within ±2° of that observed during
the determination of the sample.
Interferences Metals or salts of metals such as chromium,
cobalt, copper, mercury, molybdenum, nickel, palladium,
and silver may interfere with the evolution of arsine. Anti-
mony, which forms stibine, is the only metal likely to pro-
duce a positive interference in the color development with
the silver diethyldithiocarbamate. Stibine forms a red color
with silver diethyldithiocarbamate that has a maximum ab-
sorbance at 510 nm, but at 535–540 nm, the absorbance of
the antimony complex is so diminished that the results of
the determination would not be altered significantly.
CADMIUM LIMIT TEST
General Tests and Assays
Spectrophotometer Use any suitable atomic absorption
spectrophotometer equipped with a Boling-type burner, an
air–acetylene flame, and a hollow-cathode cadmium lamp.
The instrument should be capable of operating within the
sensitivity necessary for the determination.
Standard Solution Transfer 100 mg of cadmium chlo-
ride crystals (CdCl2·21/2H2O), accurately weighed, into a
1000-mL volumetric flask, dissolve in and dilute with water
to volume, and mix. Pipet 25 mL of this solution into a 100-
mL volumetric flask, add 1 mL of hydrochloric acid, dilute
with water to volume, and mix. Each mL contains 12.5 µg
of cadmium.
Sample Solution Transfer 10 g of sample, accurately
weighed, into a 50-mL volumetric flask, dissolve in and di-
lute with water to volume, and mix.
Test Solutions Transfer 5.0 mL of the Sample Solution
into each of five separate 25-mL volumetric flasks. Dilute the
contents of Flask 1 with water to volume, and mix. Add
1.00 mL, 2.00 mL, 3.00 mL, and 4.00 mL of Standard Solu-
tion to Flasks 2, 3, 4, and 5, respectively; then dilute each
flask with water to volume; and mix. The Test Solutions con-
tain, respectively, 0 µg/mL, 0.5 µg/mL, 1.0 µg/mL, 1.5 µg/
mL, and 2.0 µg/mL of cadmium.
Procedure Determine the absorbance of each Test Solu-
tion at 228.8 nm, setting the instrument to previously estab-
lished optimum conditions, using water as a blank. Plot the
absorbance of the Test Solutions versus their contents of cad-
mium, in µg/mL. Draw the straight line best fitting the five
FCC 8
points, and extrapolate the line until it intercepts the con-
centration axis. From the intercept, determine the amount,
in µg, of cadmium in each mL of the Test Solution contain-
ing 0 mL of the Standard Preparation. Calculate the quan-
tity, in mg/kg, of cadmium in the sample by multiplying
this value by 25.
CHLORIDE AND SULFATE LIMIT TESTS
Where limits for chloride and sulfate are specified in the
individual monograph, compare the Sample Solution and
control in appropriate glass cylinders of the same dimen-
sions and matched as closely as practicable with respect to
their optical characteristics.
If the solution is not perfectly clear after acidification, pass
it through filter paper that has been washed free of chloride
and sulfate. Add identical quantities of the precipitant (silver
nitrate TS or barium chloride TS) in rapid succession to both
the Sample Solution and the control solution.
Experience has shown that visual turbidimetric compari-
sons are best made between solutions containing from 10
to 20 µg of chloride (Cl) ion or from 200 to 400 µg of
sulfate (SO4) ion in 50 mL. Weights of samples are specified
on this basis in the individual monographs in which these
limits are included.
Chloride Limit Test
Standard Chloride Solution Dissolve 165 mg of sodium
chloride in water and dilute to 100.0 mL. Transfer 10.0 mL
of this solution into a 1000-mL volumetric flask, dilute with
water to volume, and mix. Each mL of the final solution
contains 10 µg of chloride (Cl) ion.
Procedure Unless otherwise directed, dissolve the speci-
fied amount of the test substance in 30–40 mL of water;
neutralize to litmus external indicator with nitric acid, if nec-
essary; and add 1 mL in excess. Add 1 mL of silver nitrate
TS to the clear solution or filtrate, dilute with water to 50
mL, mix, and allow to stand for 5 min protected from direct
sunlight. Compare the turbidity, if any, with that produced
similarly in a control solution containing the required vol-
ume of Standard Chloride Solution and the quantities of the
reagents used for the sample.
Sulfate Limit Test
Standard Sulfate Solution Dissolve 148 mg of anhy-
drous sodium sulfate in water, and dilute to 100.0 mL.
Transfer 10.0 mL of this solution to a 1000-mL volumetric
flask, dilute with water to volume, and mix. Each mL of the
final solution contains 10 µg of sulfate (SO4).
Procedure Unless otherwise directed, dissolve the speci-
fied amount of the test substance in 30–40 mL of water;
neutralize to litmus external indicator with hydrochloric
acid, if necessary; then add 1 mL of 2.7 N hydrochloric
acid. Add 3 mL of barium chloride TS to the clear solution
or filtrate, dilute with water to 50 mL, and mix. After 10
min compare the turbidity, if any, with that produced in a
solution containing the required volume of Standard Sulfate
Solution and the quantities of the reagents used for the
sample.
FCC 8
1,4-DIOXANE LIMIT TEST
Vacuum Distillation Apparatus Assemble a closed-sys-
tem vacuum distillation apparatus employing glass vacuum
stopcocks (A, B, and C), as shown in Figure 5.
Figure 5. Closed-System Vacuum Distillation Apparatus for 1,
4-Dioxane
The concentrator tube (D) is made of borosilicate or quartz
(not flint) glass, graduated precisely enough to measure the
0.9 mL or more of distillate and marked so that the analyst
can accurately dilute to 2.0 mL (available as Chromaflex
concentrator tube, Kontes Glass Co., Vineland, NJ, Catalog
No. K42560-0000).
Standard Preparation Prepare a solution of 1,4-diox-
ane in water containing 100 µg/mL. Keep the solution re-
frigerated, and prepare fresh weekly.
Sample Preparation Transfer 20 g of the sample, accu-
rately weighed, into a 50-mL round-bottom flask (E) having
a 24/40 ground-glass neck. Semisolid or waxy samples
should be liquefied by heating on a steam bath before mak-
ing the transfer. Add 2.0 mL of water to the flask for crystal-
line samples, and 1.0 mL for liquid, semisolid, or waxy sam-
ples. Place a small Teflon-covered stirring bar in the flask,
stopper, and stir to mix. Immerse the flask in an ice bath,
and chill for about 1 min.
Wrap heating tape around the tube connecting the
Chromaflex tube (D) and the round-bottom flask (E), and
apply about 10 V to the tape. Apply a light coating of high-
vacuum silicone grease to the ground-glass joints, and con-
nect the Chromaflex tube to the 10/30 joint and the round-
bottom flask to the 24/40 joint. Immerse the vacuum trap
in a Dewar flask filled with liquid nitrogen, close stopcocks A
and B, open stopcock C, and begin evacuating the system
with a vacuum pump. Prepare a slush bath from powdered
dry ice and methanol, and raise the bath to the neck of the
round-bottom flask. After freezing the contents of the flask
for about 10 min, and when the vacuum system is operat-
ing at 0.05 mm pressure or lower, open stopcock A for 20
s, and then close it. Remove the slush bath, and allow the
flask to warm in air for about 1 min. Immerse the flask in a
water bath at 20°–25°, and after about 5 min warm the
water in the bath to 35°–40° (sufficient to liquefy most sam-
ples) while stirring slowly but constantly with the magnetic
bar. Cool the water in the bath by adding ice, and chill for
about 2 min. Replace the water bath with the slush bath,
freeze the contents of the flask for about 10 min, then open
stopcock A for 20 s, and close it. Remove the slush bath,
General Tests and Assays / Appendix III / 1267
and repeat the heating steps as before, this time reaching a
final temperature of 45°–50° or a temperature necessary to
melt the sample completely. If there is any condensation in
the tube connecting the round-bottom flask to the
Chromaflex tube, slowly increase the voltage to the heating
tape and heat until condensation disappears.
Stir with the magnetic stirrer throughout the following
steps: very slowly immerse the Chromaflex tube in the
Dewar flask containing liquid nitrogen. [CAUTION—When
there is liquid distillate in the Chromaflex tube, the tube
must be immersed in the nitrogen very slowly, or the tube
will break.]
Water will begin to distill into the tube. As ice forms in
the tube, raise the Dewar flask to keep the liquid nitrogen
level only slightly below the level of ice in the tube. When
water begins to freeze in the neck of the 10/30 joint, or
when liquid nitrogen reaches the 2.0-mL graduation mark
on the Chromaflex tube, remove the Dewar flask and let the
ice melt without heating. After the ice has melted, check
the volume of water that has distilled, and repeat the se-
quence of chilling and thawing until at least 0.9 mL of
water has been collected. Freeze the tube once again for
about 2 min, and release the vacuum first by opening stop-
cock B, followed by stopcock A. Remove the Chromaflex
tube from the apparatus, close it with a greased stopper,
and let the ice melt without heating. Mix the contents of
the tube by swirling, note the volume of distillate, and di-
lute with water to 2.0 mL, if necessary. Use this Sample
Preparation as directed under Chromatography.
Chromatography (See Chromatography, Appendix IIA.)
General Tests and Assays
Use a gas chromatograph equipped with a flame-ionization
detector. Under typical conditions, the instrument contains
a 4-mm (id) × 6-ft glass column, or equivalent, packed with
80-/100- or 100-/120-mesh Chromosorb 104, or equivalent.
The column is maintained isothermally at about 140°, the
injection port at 200°, and the detector at 250°. Nitrogen is
the carrier gas, flowing at a rate of about 35 mL/min. Install
an oxygen scrubber between the carrier gas line and the
column. The column should be conditioned for about 72 h
at 250° with 30–40 mL/min carrier flow.
[NOTE—Chromosorb 104 is oxygen sensitive. Both
new and used columns should be flushed with carrier
gas for 30–60 min before heating each time they are
installed in the gas chromatograph.]
Inject a volume of the Standard Preparation, accurately
measured, to give about 20% of maximum recorder re-
sponse. Where possible, keep the injection volume in the
range of 2–4 µL, and use the solvent-flush technique to
minimize errors associated with injection volumes. In the
same manner, inject an identical volume of the Sample Prep-
aration. The height of the peak produced by the Sample
Preparation does not exceed that produced by the Standard
Preparation.1
1 If the sample fails the test because of known or suspected interference,
another aliquot may be run on a 6-ft × 2-mm (id) column, or equivalent, of
0.2% Carbowax 1500 on Carbopak C, operating at 100° isothermal, with 20
mL/min of helium carrier flow. Under these conditions, the 1,4-dioxane elutes
in about 4 min.
 1268 / Appendix III / General Tests and Assays
FLUORIDE LIMIT TEST
Method I (Thorium Nitrate Colorimetric Method)
Use this method unless otherwise directed in the individual
monograph.
[CAUTION—When applying this test to organic com-
pounds, rigidly control at all times the temperature at
which the distillation is conducted to the recom-
mended range of 135°–140° to avoid the possibility of
explosion.]
[NOTE—To minimize the distillation blank resulting
from fluoride leached from the glassware, treat the
distillation apparatus as follows: treat the glassware
with hot 10% sodium hydroxide solution, followed by
flushing with tap water and rinsing with distilled
water. At least once daily, treat in addition by boiling
down 15–20 mL of 1:2 sulfuric acid until the still is
filled with fumes; cool, pour off the acid, treat again
with 10% sodium hydroxide solution, and rinse thor-
oughly. For further details, see AOAC method
944.08.]
Unless otherwise directed, place a 5.0-g sample and 30
mL of water in a 125-mL Pyrex distillation flask having a
side arm and trap. The flask is connected with a condenser
and carries a thermometer and a capillary tube, both of
which must extend into the liquid. Slowly add, with contin-
uous stirring, 10 mL of 70% perchloric acid, and then add 2
or 3 drops of a 1:2 solution of silver nitrate and a few glass
General Tests and Assays
beads. Connect a small dropping funnel or a steam genera-
tor to the capillary tube. Support the flask on a flame-resis-
tant mat or shielding board, with a hole that exposes about
one-third of the flask to the low, “clean” flame of a Bunsen
burner.
[NOTE—The shielding is essential to prevent the walls
of the flask from overheating above the level of its
liquid contents.]
Distill until the temperature reaches 135°. Add water from
the funnel or introduce steam through the capillary, main-
taining the temperature between 135° and 140° at all
times. Continue the distillation until 100 mL of distillate has
been collected. After the 100-mL portion (Distillate A) is col-
lected, collect an additional 50-mL portion of distillate (Dis-
tillate B) to ensure that all of the fluorine has been
volatilized.
Place 50 mL of Distillate A in a 50-mL Nessler tube. In
another, similar Nessler tube, place 50 mL of water distilled
through the apparatus as a control. Add to each tube 0.1
mL of a filtered 1:1000 solution of sodium alizarinsulfonate
and 1 mL of a freshly prepared 1:4000 solution of hydroxyl-
amine hydrochloride, and mix well. Add, dropwise and with
stirring, either 1 N or 0.05 N sodium hydroxide, depending
on the expected volume of volatile acid distilling over, to
the tube containing the distillate until its color just matches
that of the control, which is faintly pink. Then add to each
tube 1.0 mL of 0.1 N hydrochloric acid, and mix well. From
a buret, graduated in 0.05 mL, add slowly to the tube con-
taining the distillate enough of a 1:4000 solution of thorium
nitrate so that, after mixing, the color of the liquid just
FCC 8
changes to a faint pink. Note the volume of the solution
added, then add exactly the same volume to the control,
and mix. Now add to the control solution sodium fluoride
TS (10 µg F/mL) from a buret to make the colors of the two
tubes match after dilution to the same volume. Mix well,
and allow all air bubbles to escape before making the final
color comparison. Check the endpoint by adding 1 or 2
drops of sodium fluoride TS to the control. A distinct
change in color should take place. Note the volume of so-
dium fluoride TS added.
Dilute Distillate B to 100 mL, and mix well. Place 50 mL
of this solution in a 50-mL Nessler tube, and follow the
procedure used for Distillate A. The total volume of sodium
fluoride TS required for the solutions from both Distillate A
and Distillate B should not exceed 2.5 mL.
Method II (Ion-Selective Electrode Method A)
Buffer Solution Dissolve 36 g of cyclohexylenedinitrilote-
traacetic acid (CDTA) in sufficient 1 N sodium hydroxide to
make 200 mL. Transfer 20 mL of this solution (equivalent to
4 g of disodium CDTA) into a 1000-mL beaker containing
500 mL of water, 57 mL of glacial acetic acid, and 58 g of
sodium chloride, and stir to dissolve. Adjust the pH of the
solution to 5.0–5.5 by the addition of 5 N sodium hydrox-
ide, then cool to room temperature, dilute with water to
1000 mL, and mix.
Procedure Unless otherwise directed in the individual
monograph, transfer 8.0 g of sample and 20 mL of water
into a 250-mL distilling flask, cautiously add 20 mL of per-
chloric acid, and then add 2 or 3 drops of a 1:2 solution of
silver nitrate and a few glass beads.
[CAUTION—Handle perchloric acid in an appropriate
fume hood.]
Following the directions, and observing the Caution and
Notes, as given under Method I, distill the solution until 200
mL of distillate has been collected.
Transfer a 25.0-mL aliquot of the distillate into a 250-mL
plastic beaker, and dilute with the Buffer Solution to 100 mL.
Place the fluoride ion and reference electrodes (or a combi-
nation fluoride electrode) of a suitable ion-selective elec-
trode apparatus in the solution. Adjust the calibration con-
trol until the indicator needle points to the center on the
logarithmic concentration scale, allowing sufficient time for
equilibration (about 20 min), and stirring constantly during
the equilibration period and throughout the remainder of
the procedure. Pipet 1.0 mL of a solution containing 100 µg
of fluoride (F) ion per mL (prepared by dissolving 22.2 mg
of sodium fluoride, previously dried at 200° for 4 h, in suffi-
cient water to make 100.0 mL) into the beaker, allow the
electrode to come to equilibrium, and record the final read-
ing on the logarithmic concentration scale.
[NOTE—Follow the instrument manufacturer’s instruc-
tions regarding precautions and interferences, elec-
trode filling and check, temperature compensation,
and calibration.]
Calculations Calculate the fluoride content, in mg/kg, of
the sample taken:
Result = [IA/(R – I)] × 100 × (200/25W)
FCC 8
in which I is the initial scale reading before the addition of
the sodium fluoride solution; A is the concentration, in µg/
mL, of fluoride in the sodium fluoride solution added to the
sample solution; R is the final scale reading after addition of
the sodium fluoride solution; and W is the original weight,
in grams, of the sample.
Method III (Ion-Selective Electrode Method B)
Sodium Fluoride Solution (5 µg F/mL) Transfer 2.210 g
of sodium fluoride, previously dried at 200° for 4 h and
accurately weighed, into a 400-mL plastic beaker, add 200
mL of water, and stir until dissolved. Quantitatively transfer
this solution into a 1000-mL volumetric flask with the aid of
water, dilute with water to volume, and mix. Store this
stock solution in a plastic bottle. On the day of use, transfer
5.0 mL of the stock solution into a 1000-mL volumetric
flask, dilute with water to volume, and mix.
Calibration Curve Transfer 1.0 mL, 2.0 mL, 3.0 mL, 5.0
mL, 10.0 mL, and 15.0 mL of the Sodium Fluoride Solution
into separate 250-mL plastic beakers; add 50 mL of water, 5
mL of 1 N hydrochloric acid, 10 mL of 1 M sodium citrate,
and 10 mL of 0.2 M disodium EDTA to each beaker; and
mix. Transfer each solution into separate 100-mL volumetric
flasks, dilute with water to volume, and mix. Transfer a 50-
mL portion of each solution into separate 125-mL plastic
beakers, and measure the potential of each solution with a
suitable ion-selective electrode apparatus (such as the Orion
Model No. 94-09, with solid-state membrane), using a suita-
ble reference electrode (such as the Orion Model No. 90-01,
with single junction). Plot the calibration curve on two-cycle
semilogarithmic paper (such as K & E No. 465130) or with
the use of a suitable graphing calculator or spreadsheet pro-
gram, with µg of F per 100 mL solution on the logarithmic
scale.
Procedure Transfer 1.00 g of sample into a 150-mL glass
beaker, add 10 mL of water, and, while stirring continu-
ously, slowly add 20 mL of 1 N hydrochloric acid to dissolve
the sample. Boil rapidly for 1 min, then transfer into a 250-
mL plastic beaker, and cool rapidly in ice water. Add 15 mL
of 1 M sodium citrate and 10 mL of 0.2 M disodium EDTA,
and mix. Adjust the pH to 5.5 ± 0.1 with 1 N hydrochloric
acid or 1 N sodium hydroxide, if necessary; transfer into a
100-mL volumetric flask; dilute with water to volume; and
mix. Transfer a 50-mL portion of this solution into a 125-mL
plastic beaker, and measure the potential of the solution
with the apparatus described under Calibration Curve. Deter-
mine the fluoride content, in µg, of the sample from the
Calibration Curve. Determine the percentage of fluoride in
the sample taken:
Result = (C/WS) × 0.000001 × 100%
in which C is the content of fluoride, in µg, in the sample,
determined from the Calibration Curve; WS is the sample
weight, in g; and 0.000001 is a factor converting µg to
grams.
Method IV (Ion-Selective Electrode Method C)
[NOTE—Unless directed otherwise by the individual
monograph, use Buffer Solution A for samples with a
neutral to higher pH, and use Buffer Solution B for
samples with a neutral to lower pH.]
General Tests and Assays / Appendix III / 1269
Buffer Solution A Add 2 volumes of 6 N acetic acid to 1
volume of water, and adjust the pH to 5.0 with 50% potas-
sium hydroxide solution.
Buffer Solution B Dissolve 150 g of sodium citrate dihy-
drate and 10.3 g of disodium EDTA dihydrate in 800 mL of
water, adjust the pH to 8.0 with 50% sodium hydroxide
solution, and dilute with water to 1000 mL.
Fluoride Standard Solutions
1000 mg/kg Fluoride Standard: Transfer 2.2108 g of so-
dium fluoride, previously dried at 200° for 4 h, into a 1000-
mL volumetric flask, and dissolve in and dilute with water to
volume. The resulting solution contains 1000 µg of fluoride
per mL.
50 mg/kg Fluoride Standard: Pipet 50 mL of the 1000 mg
/kg Fluoride Standard into a 1000-mL volumetric flask. Dilute
with water to volume.
10 mg/kg Fluoride Standard: Pipet 100 mL of the 50 mg/
kg Fluoride Standard into a 500-mL volumetric flask. Dilute
with water to volume.
Fluoride Limit Solutions (for a 1-g sample)
50 mg/kg Fluoride Limit Solution (1 mg/kg fluoride stan-
dard): Pipet 50 mL of the 10 mg/kg Fluoride Standard into
a 500-mL volumetric flask, and dilute with water to volume.
10 mg/kg Fluoride Limit Solution (0.2 mg/kg fluoride stan-
dard): Pipet 10 mL of the 10 mg/kg Fluoride Standard into
a 500-mL volumetric flask, and dilute with water to volume.
Fluoride Limit Solutions (for a 2-g sample)
50 mg/kg Fluoride Limit Solution (2 mg/kg fluoride stan-
General Tests and Assays
dard): Pipet 100 mL of the 10 mg/kg Fluoride Standard into
a 500-mL volumetric flask, and dilute with water to volume.
10 mg/kg Fluoride Limit Solution (0.4 mg/kg fluoride stan-
dard): Pipet 20 mL of the 10 mg/kg Fluoride Standard into
a 500-mL volumetric flask, and dilute with water to volume.
[NOTE—Store all standard and limit solutions in plastic
containers.]
Sample Preparation Accurately weigh the amount of
sample specified in the monograph, transfer it into a 100-
mL volumetric flask, and dissolve it in a minimal amount of
water. Add 50.0 mL of the appropriate Buffer Solution, dilute
with water to volume, and mix.
Electrode Calibration Pipet 50 mL of the appropriate
Buffer Solution into a plastic beaker. Place the fluoride ion
and reference electrodes (or a combination fluoride elec-
trode) into the plastic beaker and stir. At 5-min intervals,
add 100 µL and 1000 µL of the 1000 mg/kg Fluoride Stan-
dard and read the potential, in mV, after each addition. The
difference between the two readings is the slope of the fluo-
ride electrode and should typically be in the range of 63–70
mV at 25° for Buffer Solution A and in the range of 54–60
mV at 25° for Buffer Solution B. If the difference in potential
is not within this range, check, and, if necessary, replace the
electrode, instrument, or solutions.
Alternatively, the electrode calibration should be per-
formed according to the manufacturer’s instructions and
should comply with the manufacturer’s calibration range at
25°. If the difference in potential is not within this range,
evaluate the system and equipment as necessary.
 1270 / Appendix III / General Tests and Assays
Procedure Transfer the entire sample into a plastic beaker.
Place the electrode into the beaker, allow the solution to
equilibrate for 5 min with stirring, and read the potential, in
mV. Remove and rinse the electrode(s) with water. In an-
other beaker, using a pipet, add 50 mL of the appropriate
Buffer Solution followed by 50 mL of the Fluoride Limit Solu-
tion that best reflects the fluoride limit of the sample. Place
the electrode in the beaker, equilibrate for 3 min, and read
the potential in mV. If the potential of the Fluoride Limit
Solution is less than that of the sample, the sample passes
the test criteria for maximum acceptable fluoride level limit.
Method V
Lime Suspension Carefully shake about 56 g of low-fluor-
ine calcium oxide (about 2 mg/kg of F) with 250 mL of
water, and while stirring, slowly add 250 mL of 60% per-
chloric acid. Add a few glass beads, and boil until copious
fumes of perchloric acid evolve, then cool, add 200 mL of
water, and boil again.
[CAUTION—Handle perchloric acid in an appropriate
fume hood.]
Repeat the dilution and boiling once more, cool, dilute
considerably, and if precipitated silicon dioxide forms, pass
through a fritted-glass filter. While stirring, pour the clear
solution into 1000 mL of a 1:10 solution of sodium hydrox-
ide, allow the precipitate to settle, and siphon off the super-
natant liquid. Remove the sodium salts from the precipitate
by washing five times with water in large centrifuge bottles,
shaking the mass thoroughly each time. Finally, shake the
precipitate into a suspension, and dilute with water to 2000
General Tests and Assays
mL. Store in paraffin-lined bottles, and shake well before
use.
[NOTE—100 mL of this suspension should give no ap-
preciable fluoride blank when evaporated, distilled,
and titrated as directed under Method I.]
Procedure Assemble the distilling apparatus as described
under Method I, and add 1.67 g of sample, accurately
weighed, and 25 mL of 1:2 sulfuric acid to the distilling
flask. Distill until the temperature reaches 160°, then main-
tain at 160°–165° by adding water from the funnel, collect-
ing 300 mL of distillate. Oxidize the distillate by cautiously
adding 2 or 3 mL of fluorine-free 30% hydrogen peroxide
(to remove sulfates), allow to stand for a few minutes, and
evaporate in a platinum dish with an excess of Lime Suspen-
sion. Ignite briefly at 600°, then cool and wet the ash with
about 10 mL of water. Cover the dish with a watch glass,
and cautiously introduce under the watch glass just suffi-
cient 60% perchloric acid to dissolve the ash. Add the con-
tents of the dish through the dropping funnel of a freshly
prepared distilling apparatus (the distilling flask should con-
tain a few glass beads), using a total of 20 mL of the 60%
perchloric acid to dissolve the ash and transfer the solution.
Add 10 mL of water and a few drops of a 1:2 solution of
silver perchlorate through the dropping funnel, and con-
tinue as directed under Method I, beginning with “Distill un-
til the temperature reaches 135°…”.
FCC 8
LEAD LIMIT TEST
[NOTE—Unless otherwise specified in the monograph,
use the Dithizone Method to determine lead levels.]
Dithizone Method
Special Reagents Select reagents having as low a lead
content as practicable, and store all solutions in containers
of borosilicate glass. Rinse all glassware thoroughly with
warm, 1:2 nitric acid followed by water.
Ammonia–Cyanide Solution Dissolve 2 g of potassium
cyanide in 15 mL of ammonium hydroxide, and dilute with
water to 100 mL.
Ammonium Citrate Solution Dissolve 40 g of citric acid
in 90 mL of water, add 2 or 3 drops of phenol red TS, then
cautiously add ammonium hydroxide until the solution ac-
quires a red color. Extract it with 20-mL portions of
Dithizone Extraction Solution until the dithizone solution re-
tains its green color or remains unchanged.
Diluted Standard Lead Solution (1 µg Pb in 1 mL)
Lead Nitrate Stock Solution: Dissolve 159.8 mg of ACS
Reagent-Grade Lead Nitrate [Pb(NO3)2] in 100 mL of water
containing 1 mL of nitric acid, dilute with water to 1000.0
mL, and mix. Prepare and store this solution in glass con-
tainers that are free from lead salts.
Standard Lead Solution: On the day of use, dilute 10.0
mL of Lead Nitrate Stock Solution with water to 100.0 mL.
Each mL of Standard Lead Solution contains the equivalent of
10 µg of lead (Pb) ion.
Diluted Standard Lead Solution: Immediately before use,
transfer 10.0 mL of Standard Lead Solution into a 100-mL
volumetric flask, dilute with 1:100 nitric acid to volume, and
mix.
Dithizone Extraction Solution Dissolve 30 mg of
dithizone in 1000 mL of chloroform, add 5 mL of alcohol,
and mix. Store in a refrigerator. Before use, shake a suitable
volume of the solution with about half its volume of 1:100
nitric acid, discarding the nitric acid.
Hydroxylamine Hydrochloride Solution Dissolve 20 g
of hydroxylamine hydrochloride in sufficient water to make
about 65 mL, transfer the solution into a separator, add a
few drops of thymol blue TS, then add ammonium hydrox-
ide until the solution assumes a yellow color. Add 10 mL of
a 1:25 solution of sodium diethyldithiocarbamate, mix, and
allow to stand for 5 min. Extract the solution with successive
10- to 15-mL portions of chloroform until a 5-mL test por-
tion of the chloroform extract does not assume a yellow
color when shaken with cupric sulfate TS. Add 2.7 N hydro-
chloric acid until the extracted solution is pink, adding 1 or
2 drops more of thymol blue TS if necessary, then dilute
with water to 100 mL, and mix.
Potassium Cyanide Solution Dissolve 50 g of potassium
cyanide in sufficient water to make 100 mL. Remove the
lead from the solution by extraction with successive portions
of Dithizone Extraction Solution as described under Ammo-
nium Citrate Solution, then extract any dithizone remaining
in the cyanide solution by shaking with chloroform. Finally,
FCC 8
dilute the cyanide solution with sufficient water so that each
100 mL contains 10 g of potassium cyanide.
Standard Dithizone Solution Dissolve 10 mg of
dithizone in 1000 mL of chloroform, keeping the solution in
a glass-stoppered, lead-free bottle suitably wrapped to pro-
tect it from light and stored in a refrigerator.
Sample Solution Use the solution obtained by treating
the sample as directed in an individual monograph as the
Sample Solution in the Procedure. Sample solutions of organic
compounds are prepared, unless otherwise directed, accord-
ing to the following general method: [CAUTION—Some sub-
stances may react unexpectedly with explosive violence
when digested with hydrogen peroxide. Use appropriate
safety precautions at all times.]
Transfer 1.0 g of sample into a suitable flask, add 5 mL of
sulfuric acid and a few glass beads, and digest at a tempera-
ture not exceeding 120° until charring begins, using prefer-
ably a hot plate in a fume hood. (Additional sulfuric acid
may be necessary to completely wet some samples, but the
total volume added should not exceed about 10 mL.) After
the sample has initially been decomposed by the acid, add
with caution, dropwise, hydrogen peroxide (30%), allowing
the reaction to subside and reheating between drops. The
first few drops must be added very slowly with sufficient
mixing to prevent a rapid reaction, and heating should be
discontinued if foaming becomes excessive. Swirl the solu-
tion in the flask to prevent unreacted substance from caking
on the walls or bottom of the flask during the digestion.
[NOTE—Add small quantities of the peroxide when the
solution begins to darken.]
Continue the digestion until the organic matter is destroyed,
gradually raising the temperature of the hot plate to
250°–300° until fumes of sulfur trioxide are copiously
evolved and the solution becomes colorless or retains only a
light straw color. Cool, cautiously add 10 mL of water,
again evaporate to strong fuming, and cool. Quantitatively
transfer the solution into a separator with the aid of small
quantities of water.
Procedure Transfer the Sample Solution, prepared as di-
rected in the individual monograph, into a separator, and
unless otherwise directed, add 6 mL of Ammonium Citrate
Solution and 2 mL of Hydroxylamine Hydrochloride Solution.
(Use 10 mL of the citrate solution when determining lead in
iron salts.) Add 2 drops of phenol red TS to the separator,
and make the solution just alkaline (red in color) by the
addition of ammonium hydroxide. Cool the solution, if nec-
essary, under a stream of tap water, then add 2 mL of Po-
tassium Cyanide Solution. Immediately extract the solution
with 5-mL portions of Dithizone Extraction Solution, draining
each extract into another separator, until the dithizone solu-
tion retains its green color. Shake the combined dithizone
solutions for 30 s with 20 mL of 1:100 nitric acid, discard
the chloroform layer, add 5.0 mL of Standard Dithizone Solu-
tion and 4 mL of Ammonia–Cyanide Solution to the acid solu-
tion, and shake for 30 s. The purple hue in the chloroform
solution of the sample caused by any lead dithizonate pres-
ent does not exceed that in a control, containing the vol-
ume of Diluted Standard Lead Solution equivalent to the
General Tests and Assays / Appendix III / 1271
amount of lead specified in the monograph, when treated
in the same manner as the sample.
Flame Atomic Absorption Spectrophotometric
Method
Select reagents having as low a lead content as practicable,
and store all solutions in high-density polyethylene contain-
ers. Rinse all plastic and glassware thoroughly with warm,
1:2 nitric acid followed by water.
Lead Nitrate Stock Solution (100 µg/mL) Dissolve
159.8 mg of reagent-grade lead nitrate [Pb(NO3)2] in 100
mL of water containing 1 mL of nitric acid in a 1000-mL
volumetric flask, and dilute with water to volume.
Standard Lead Solution (10 µg/mL) On the day of use,
transfer 10 mL of Lead Nitrate Stock Solution into a 100-mL
volumetric flask, and dilute with water to volume.
Diluted Standard Lead Solutions On the day of use,
prepare a set of standard lead solutions that corresponds to
the lead limit specified in the monograph:
1 mg/kg Lead Limit (0.5 µg/mL, 1.0 µg/mL, and 1.5 µg/
mL standards): On the day of use, transfer 5.0 mL, 10.0
mL, and 15.0 mL of Standard Lead Solution into three sepa-
rate 100-mL volumetric flasks, add 10 mL of 3 N hydrochlo-
ric acid to each, and dilute with water to volume.
5 mg/kg Lead Limit (1.0 µg/mL, 5.0 µg/mL, and 10.0 µg/
mL standards): On the day of use, transfer 10.0 mL and
50.0 mL of Standard Lead Solution into two separate 100-mL
volumetric flasks, add 10 mL of 3 N hydrochloric acid to
each, and dilute with water to volume. The final standard,
10.0 µg/mL, is taken directly from the Standard Lead
General Tests and Assays
Solution.
10 mg/kg Lead Limit (5.0 µg/mL, 10.0 µg/mL, and 15.0
µg/mL standards): On the day of use, transfer 5.0 mL, 10.0
mL, and 15.0 mL of Lead Nitrate Stock Solution into three
separate 100-mL volumetric flasks, add 10 mL of 3 N hydro-
chloric acid to each, and dilute with water to volume.
25% Sulfuric Acid Solution (by volume) Cautiously
add 100 mL of sulfuric acid to 300 mL of water with con-
stant stirring while cooling in an ice bath.
Sample Preparation Transfer the sample weight as
specified in the monograph, weighed to the nearest 0.1
mg, into an evaporating dish. Add a sufficient amount of
25% Sulfuric Acid Solution, and distribute the sulfuric acid
uniformly through the sample. Within a hood, place the
dish on a steam bath to evaporate most of the water. Place
the dish on a burner, and slowly pre-ash the sample by
expelling most of the sulfuric acid. Place the dish in a muffle
furnace that has been set at 525°, and ash the sample until
the residue appears free from carbon. Prepare a Sample
Blank by ashing 5 mL of 25% sulfuric acid. Cool and cau-
tiously wash down the inside of each evaporation dish with
water.
Add 5 mL of 1 N hydrochloric acid. Place the dish on a
steam bath, and evaporate to dryness. Add 1.0 mL of 3 N
hydrochloric acid and approximately 5 mL of water, and
heat briefly on a steam bath to dissolve any residue. Transfer
each solution quantitatively to a 10-mL volumetric flask, di-
lute to volume, and mix.
Procedure Concomitantly determine the absorbances of
the Sample Blank, the Diluted Standard Lead Solutions, and
 1272 / Appendix III / General Tests and Assays
the Sample Preparation at the lead emission line of 283.3
nm, using a slit-width of 0.7 nm. Use a suitable atomic ab-
sorption spectrophotometer equipped with a lead electrode-
less discharge lamp (EDL), an air–acetylene flame, and a 4-in
burner head. Use water as the blank.
Calculations Determine the corrected absorbance values
by subtracting the Sample Blank absorbance from each of
the Diluted Standard Lead Solutions and from the Sample
Preparation absorbances. Prepare a standard curve by plot-
ting the corrected Diluted Standard Lead Solutions absorb-
ance values versus their corresponding concentrations ex-
pressed as µg/mL. Determine the lead concentration in the
Sample Preparation by reference to the calibration curve.
Calculate the quantity of lead, in mg/kg, in the sample
taken:
Result = 10C/WS
in which C is the concentration, in µg/mL, of lead from the
standard curve; and WS is the weight, in grams, of the sam-
ple taken.
Change to read:
Atomic Absorption Spectrophotometric Graphite
Furnace Method
The following methods are primarily intended for the analy-
sis of applicable substances containing less than 1 mg/kg of
lead.
Method I
This method is intended for the quantitation of lead in
substances that are soluble in water, such as sugars and
General Tests and Assays
sugar syrups, at levels as low as 0.03 mg/kg. The method
detection limit is approximately 5 ng/kg.
Apparatus Use a suitable graphite furnace atomic absorp-
tion spectrophotometer set at 283.3 nm and equipped with
an autosampler, pyrolytically coated graphite tubes, solid
pyrolytic graphite platforms, and an adequate means of
background correction. Zeeman effect or Smith-Hieftje back-
ground correction is preferred, but deuterium arc back-
ground correction should be acceptable. (This method was
developed on a Perkin-Elmer Model Z5100, 0.7-nm slit,
HGA-600 furnace, AS-60 autosampler with Zeeman back-
ground correction.) If the instrument does not have a well-
defined calibration function, a separate calculator or com-
puter is required for linear least squares, nonlinear, or quad-
ratic calibrations. Use either a hollow-cathode lamp or an
electrode-less discharge lamp as the source, and use argon
as the purge gas and breathing-quality air (for oxygen ash-
ing to avoid residue build up during the char step) as the
alternate gas. Set up the instrument according to the manu-
facturer’s specifications with consideration of current good
GFAAS practices—addressing such factors as line voltage,
cooling water temperature, graphite part specifications, and
furnace temperature. If an optical pyrometer or thermocou-
ple is not available to check the furnace controller tempera-
ture calibration, dim the room lights, and observe the fur-
nace emission through the sample introduction port while
increasing the furnace temperature. A characteristic cherry
red glow should begin to appear at 800°. If it glows at a
lower temperature, then the furnace is hotter, and tempera-
tures must be adjusted downward accordingly.
FCC 8
Use acid-cleaned [in a mixture of 5% sub-boiling, distilled
nitric acid and 5% sub-boiling, distilled hydrochloric acid
made up in deionized, distilled water (18 megohm), and
thoroughly rinsed with deionized, distilled water (18 meg-
ohm)] autosampler cups (PE B008-7600 Teflon, or equiva-
lent) to avoid contamination. Use micropipets with disposa-
ble tips free of lead contamination for dilution. Ensure
accuracy and precision of micropipets and tips by dispens-
ing and weighing 5–10 replicate portions of water onto a
microbalance. Use acid-cleaned volumetric glassware to pre-
pare standards and dilute samples to a final volume. For
digestion, use acid-cleaned, high-density polyethylene tubes,
polypropylene tubes, Teflon tubes, or quartz tubes. Store
final diluted samples in plastic tubes.
Standard Solutions Prepare all lead solutions in 5% sub-
boiling distilled nitric acid. Use a single-element 1000- or
10,000-µg/mL lead stock to prepare (weekly) an intermedi-
ate 10-µg/mL standard in 5% nitric acid. Prepare (daily) a
Lead Standard Solution (1 µg/mL) by diluting the intermedi-
ate 10-µg/mL stock solution 1:10. Prepare Working Calibra-
tion Standards of 100.0 ng/mL, 50.0 ng/mL, 25.0 ng/mL,
and 10.0 ng/mL from this, using appropriate dilutions. Store
standards in acid-cleaned polyethylene test tubes or bottles.
If the GFAAS autosampler is used to automatically dilute
standards, ensure calibration accuracy by pipetting volumes
of 3 µL or greater.
Modifier Stock Solution Weigh 20 g of ultrapure mag-
nesium nitrate hexahydrate, and dilute to 100 mL. Just
before use, prepare a Modifier Working Solution by diluting
stock solution 1:10. A volume of 5 µL will provide 0.06 mg
of magnesium nitrate.
Sample Digestion [CAUTION—Perform the procedure in
a fume hood, and wear safety glasses.] Obtain a representa-
tive subsample to be analyzed. For liquid samples such as
sugar syrups, ultrasonicate and/or vortex mix before weigh-
ing. For solid samples such as crystalline sucrose, make a
sugar solution using equal weights of sample (5-g mini-
mum) and deionized, distilled (18 megohm) water. Mix
samples until completely dissolved. Transfer approximately
1.5 g (record to nearest mg) of sample (or 3.0 g of sugar
solution), accurately weighed, into a digestion tube. Run a
Sample Preparation Blank of 1.5 g of deionized, distilled (18
megohm) water through the entire procedure with each
batch of samples. Add 0.75 mL of sub-boiling, distilled nitric
acid. Heat plastic tubes in a water bath, quartz tubes in a
water bath or heating block, warming slowly to 90°–95° to
avoid spattering. Monitor the temperature by using a
“dummy” sample. Heat until all brown vapors have dissi-
pated and any rust-colored tint is gone (20–30 min). Cool.
Add 0.5 mL of 50% hydrogen peroxide dropwise, heat at
90°–95° for 5 min, and cool. Add a second 0.5-mL portion
of 50% hydrogen peroxide, dropwise, and heat at 90°–100°
for 5–10 min until clear. Cool, and dilute  with water FCC8 to
a final volume of 10 mL.
Procedure The furnace program is as follows: (1) Dry at
200°, using a 20-s ramp and a 30-s hold and a 300-mL/min
argon flow; (2) char the sample at 750°, using a 40-s ramp
and a 40-s hold and a 300-mL/min air flow; (3) cool down,
and purge the air from the furnace for 60 s, using a 20° set
temperature and a 300-mL/min argon flow; (4) atomize at
FCC 8
1800°, using a 0-s ramp and a 10-s hold with the argon
flow stopped; (5) clean out at 2600°, with a 1-s ramp and a
7-s hold; (6) cool down the furnace (if necessary) at 20°,
with a 1-s ramp and a 5-s hold with a 300-mL/min argon
flow.
Use the autosampler to inject 20 µL each of blanks, cali-
bration standards, and sample solutions and 5 µL of Modifier
Working Solution. Inject each respective solution in triplicate,
and average results. Use peak area measurements for all
quantitation. After ensuring that the furnace is clean by run-
ning a 5% nitric acid blank, check the instrument sensitivity
 according to manufacturer’s specifications
 FCC8 by running
the 25-ng/mL calibration standard.   FCC8 Calculate the char-
acteristic mass (mo) (mass of Pb pg necessary to produce an
integrated absorbance of 0.0044 abs-sec) as follows:
mo = (0.0044 abs-sec)(25 pg/µL)(20 µL)/
(measured 25 pg/µL abs-sec)
Record and track the integrated absorbance and mo for ref-
erence and quality assurance.
Standard Curve: Inject each calibration standard in triplicate
 and determine the instrument linearity according to manu-
facturer’s instructions. FCC8 Use the calibration algorithms
provided in the instrument software. Recheck calibration pe-
riodically (≤15 samples) by running a 25- or 50-ng/mL cali-
bration standard interspersed with samples. If recheck differs
from calibration by >10%, recalibrate the instrument. The
instrumental detection limit (DL) and quantitation limit
(QL), in picograms, may be based on 7–10 replicates of the
Sample Preparation Blank and calculated as follows:
DL = (3)(s.d. blank abs-sec)(10 pg/µL)(20µL)/
(abs-sec 10 ng/mL std)
QL = (10)(s.d. blank abs-sec)(10 pg/µL)(20 µL)/
(abs-sec 10 ng/mL std)
During method development, detection limits were typically
10–14 pg, corresponding to 0.5–0.7 ng/mL for 20 µL. This
corresponds to a method detection limit of 3.3–4.7 ng/g of
sugar.
Sample Analyses: Inject each sample digest in triplicate, and
record the integrated absorbance. If instrument response ex-
ceeds that of the calibration curve, dilute with 5% nitric
acid to bring the sample response into working range, and
note the dilution factor (DF). Sample solutions having a final
concentration  beyond the linearity range FCC8 should be di-
luted 1:10 to facilitate analysis in the linear range for sys-
tems not equipped with nonlinear calibration. All sample
analyses should be blank corrected using the sample prepa-
ration blank. This can typically be done automatically by the
software after identifying and running a representative sam-
ple preparation blank. Use the calibration algorithm pro-
vided in the instrument software to calculate a blank-
corrected, digest lead concentration (in ng/mL).
Calculation of Lead Content: Calculate the lead level in the
original sample as follows:
Pb (ng/g) = (blank-corrected Pb ng/mL)(DF)[sample vol (10
mL)]/[sample wt (approx. 1.5 g)]2
General Tests and Assays / Appendix III / 1273
Quality Assurance To ensure analytical accuracy, Na-
tional Institute of Standards and Technology (NIST) SRM
1643c acidified water or a similar material should be ana-
lyzed before the unknown samples are. The certified content
of SRM 1643c is 35.3 ± 0.9 ng/mL. If the concentration
determined is not within 10% of the mean reference value
(31.8–38.8 ng/mL), the reason for inaccuracy should be
evaluated, and unknown samples should not be analyzed
until acceptable accuracy is achieved. Also prepare an in-
house control solution made from uncontaminated table
sugar or reagent-grade sucrose (or other appropriate sub-
stance with a Pb content <5 ng/g as received) mixed with
an equal volume of water. Spike this solution with Pb to
produce a concentration of 100 ng/g. Analyze with each
batch of samples. Recoveries should be 100 ± 20%, and the
precision for complete replicate digestions should be <5%
RSD. Periodically, a sample digest should be checked using
the method of standard additions to ensure that there are
no multiplicative or chemical interferences. Spiking samples
and checking recoveries is always a good practice.
Method II
This method is primarily intended for the determination of
lead at levels of less than 1 mg/kg in substances immiscible
with water, such as edible oils.
Apparatus Use a suitable atomic absorption spectropho-
tometer (Perkin-Elmer Model 3100 or equivalent) fitted with
a graphite furnace (Perkin-Elmer HGA 600 or equivalent).
Use a lead hollow-cathode lamp (Perkin-Elmer or equivalent)
with argon as the carrier gas. Follow the manufacturers’ di-
rections for setting the appropriate instrument parameters
General Tests and Assays
for lead determination.
[NOTE—For this test, use reagent-grade chemicals with
as low a lead content as is practicable, as well as high-
purity water and gases. Before use in this analysis,
rinse all glassware and plasticware twice with 10% ni-
tric acid and twice with 10% hydrochloric acid, and
then rinse them thoroughly with high-purity water,
preferably obtained from a mixed-bed strong-acid,
strong-base ion-exchange cartridge capable of produc-
ing water with an electrical resistivity of 12–15
megohms.]
Hydrogen Peroxide–Nitric Acid Solution Dissolve
equal volumes of 10% hydrogen peroxide and 10% nitric
acid.
[NOTE—Use caution.]
Lead Nitrate Stock Solution Dissolve 159.8 mg of ACS
Reagent-Grade Lead Nitrate (alternatively, use NIST Stan-
dard Reference Material, containing 10 mg of lead per kg,
or equivalent) in 100 mL of Hydrogen Peroxide–Nitric Acid
Solution. Dilute with Hydrogen Peroxide–Nitric Acid Solution to
1000.0 mL, and mix. Prepare and store this solution in glass
containers that are free from lead salts. Each mL of this solu-
tion contains the equivalent of 100 µg of lead (Pb) ion.
2 If a sample solution was prepared initially to ensure sample homogeneity,
this is the weight of the original sugar digested (not the weight of the
solution).
 1274 / Appendix III / General Tests and Assays
Standard Lead Solution On the day of use, dilute 10.0
mL of Lead Nitrate Stock Solution with Hydrogen Perox-
ide–Nitric Acid Solution to 100.0 mL, and mix. Each mL of
Standard Lead Solution contains the equivalent of 10 µg of
lead (Pb) ion.
Butanol–Nitric Acid Solution Slowly add 50 mL of nitric
acid to approximately 500 mL of butanol contained in a
1000-mL volumetric flask. Dilute with butanol to volume,
and mix.
Standard Solutions Prepare a series of lead standard so-
lutions serially diluted from the Standard Lead Solution in
Butanol–Nitric Acid Solution. Pipet into separate 100-mL volu-
metric flasks 0.2 mL, 0.5 mL, 1 mL, and 2 mL, respectively,
of Standard Lead Solution, dilute with Butanol–Nitric Acid So-
lution to volume, and mix. The Standard Solutions contain,
respectively, 0.02 µg, 0.05 µg, 0.1 µg, and 0.2 µg of lead
per mL. (For lead limits greater than 1 mg/kg, prepare a
series of standard solutions in a range encompassing the
expected lead concentration in the sample.)
Sample Solution [CAUTION—Perform this procedure in a
fume hood, and wear safety glasses.] Transfer 1 g of sample,
accurately weighed, into a large test tube. Add 1 mL of
nitric acid. Place the test tube in a rack in a boiling water
bath. As soon as the rusty tint is gone, add 1 mL of 30%
hydrogen peroxide dropwise to avoid a vigorous reaction,
and wait for bubbles to form. Stir with an acid-washed
plastic spatula if necessary. Remove the test tube from the
water bath, and let it cool. Transfer the solution into a 10-
mL volumetric flask, and dilute with Butanol–Nitric Acid Solu-
tion to volume, and mix. Use this solution for analysis.
General Tests and Assays
Procedure
Tungsten Solution: Transfer 0.1 g of tungstic acid (H2WO4)
and 5 g of sodium hydroxide pellets into a 50-mL plastic
bottle. Add 5.0 mL of high-purity water, and mix. Heat the
mixture in a hot water bath until complete solution is
achieved. Cool, and store at room temperature.
Procedure: Place the graphite tube in the furnace. Inject a
20-µL aliquot of the Tungsten Solution into the graphite
tube, using a 300-mL/min argon flow and the following se-
quence of conditions: dry at 110° for 20 s, char at
700°–900° for 20 s, and with the argon flow stopped, at-
omize at 2700° for 10 s; repeat this procedure once more
using a second 20-µL aliquot of the Tungsten Solution. Clean
the quartz windows.
Standard Curve: [NOTE—The sample injection technique is
the most crucial step in controlling the precision of the anal-
ysis; the volume of the sample must remain constant. Rinse
the µL pipet tip (Eppendorf or equivalent) three times with
either the Standard Solutions or Sample Solution before injec-
tion. Use a fresh pipet tip for each injection, and start the
atomization process immediately after injecting the sample.
Between injections, flush the graphite tube of any residual
lead by purging at a high temperature as recommended by
the manufacturer.]
With the hollow-cathode lamp properly aligned for maxi-
mum absorbance and the wavelength set at 283.3 nm, at-
omize 20-µL aliquots of the four Standard Solutions, using a
300-mL/min argon flow and the following sequence of con-
ditions: dry at 110° for 30 s, with a 20-s ramp period and a
FCC 8
10-s hold time; then char at 700° for 42 s, with a 20-s ramp
period and a 22-s hold time; and then, with the argon flow
stopped, atomize at 2300° for 7 s.
Plot a standard curve using the concentration, in µg/mL,
of each Standard Solution versus its maximum absorbance
value compensated for background correction as directed
for the particular instrument, and draw the best straight
line.
Atomize 20 µL of the Sample Solution under identical con-
ditions, and measure its corrected maximum absorbance.
From the Standard Curve, determine the concentration, C, in
µg/mL, of the Sample Solution. Calculate the quantity, in
mg/kg, of lead in the sample:
Result = 10C/W
in which W is the weight, in grams, of the sample taken.
APDC Extraction Method
Select reagents having as low a lead content as practicable,
and store all solutions in high-density polyethylene contain-
ers. Rinse all plastic and glassware thoroughly with warm,
1:2 nitric acid followed by water.
2% APDC Solution Dissolve 2.0 g of ammonium pyr-
rolidinedithiocarbamate (APDC) in 100 mL of water. Filter
any slight residue of insoluble APDC from the solution
before use.
Lead Nitrate Stock Solution (100 µg/mL) Dissolve
159.8 mg of reagent-grade lead nitrate [Pb(NO3)2] in 100
mL of water containing 1 mL of nitric acid in a 1000-mL
volumetric flask, and dilute with water to volume.
Standard Lead Solutions
2 mg/kg Lead Standard: On the day of use, transfer 2.0
mL of Lead Nitrate Stock Solution into a 100-mL volumetric
flask, and dilute with water to volume. The resulting solu-
tion contains 2 µg of lead per mL.
3 mg/kg Lead Standard: On the day of use, transfer 3.0
mL of Lead Nitrate Stock Solution into a 100-mL volumetric
flask, and dilute with water to volume. The resulting solu-
tion contains 3 µg of lead per mL.
4 mg/kg Lead Standard: On the day of use, transfer 4.0
mL of Lead Nitrate Stock Solution into a 100-mL volumetric
flask, and dilute with water to volume. The resulting solu-
tion contains 4 µg of lead per mL.
10 mg/kg Lead Standard: On the day of use, transfer
10.0 mL of Lead Nitrate Stock Solution into a 100-mL volu-
metric flask, and dilute with water to volume. The resulting
solution contains 10 µg of lead per mL.
Sample Preparation Transfer a 10.0-g sample to a clean
150-mL beaker, and 10 mL of water to a second 150-mL
beaker to serve as the blank. Add to each 30 mL of water
and the minimum amount of hydrochloric acid needed to
dissolve the sample, plus an additional 1 mL of hydrochloric
acid to ensure the dissolution of any lead present. Heat to
boiling, and boil for several minutes. Allow to cool, and di-
lute with deionized water to about 100 mL. Adjust the pH
of the resulting solution to 1.0–1.5 with 25% NaOH. Quan-
titatively transfer the pH-adjusted solution to a clean 250-
mL separatory funnel, and dilute with water to about 200
mL. Add 2 mL of 2% APDC Solution, and mix. Extract with
FCC 8 General Tests and Assays / Appendix III / 1275

two 20-mL portions of chloroform, collecting the extracts in
a clean 50-mL beaker. Evaporate to dryness on a steam
bath. Add 3 mL of nitric acid to the residue, and heat to
near dryness. Then add 0.5 mL of nitric acid and 10 mL of
deionized water to the beaker, and heat until the volume is
reduced to about 3–5 mL. Transfer the digested extract to a
clean 10-mL volumetric flask, and dilute with water to
volume.
Procedure Concomitantly determine the absorbances of
the appropriate Standard Lead Solution and the Sample Prep-
aration against the blank at the lead emission line of 283.3
nm, using a slit-width of 0.7 nm. Use a suitable atomic ab-
sorption spectrophotometer equipped with a lead electrode-
less discharge lamp (EDL), or equivalent; an air–acetylene
flame; and a 4-in burner head. Use water as the blank. The
absorbance of the Sample Preparation is not greater than
that of the Standard Lead Solution.
MANGANESE LIMIT TEST
Manganese Detection Instrument Use any suitable
atomic absorption spectrophotometer equipped with a fast-
response recorder or other readout device and capable of
measuring the radiation absorbed by manganese atoms at
the manganese resonance line of 279.5 nm.
Standard Preparations Transfer 1000 mg, accurately
weighed, of manganese metal powder into a 1000-mL volu-
metric flask, dissolve by warming in a mixture of 10 mL of
water and 10 mL of 0.5 N hydrochloric acid, cool, dilute
with water to volume, and mix. Pipet 5.0 mL of this solu-
MERCURY LIMIT TEST
Method I
Mercury Detection Instrument Use any suitable atomic
absorption spectrophotometer equipped with a fast-re-
sponse recorder and capable of measuring the radiation ab-
sorbed by mercury vapors at the mercury resonance line of
253.6 nm. A simple mercury vapor meter or detector
equipped with a variable span recorder also is satisfactory.
[NOTE—Wash all glassware associated with the test
with nitric acid, and rinse thoroughly with water
before use.]
Aeration Apparatus The apparatus, shown in Figure 6,
consists of a flowmeter (a), capable of measuring flow rates
from 500 to 1000 mL/min, connected via a three-way stop-
cock (b), with a Teflon plug, to 125-mL gas washing bottles
(c and d), followed by a drying tube (e), and finally a suita-
ble quartz liquid absorption cell (f), terminating with a vent
(g) to a fume hood.
[NOTE—The absorption cell will vary in optical
pathlength depending on the type of mercury detec-
tion instrument used.]

General Tests and Assays

tion into a 50-mL volumetric flask, dilute with water to vol-
ume, and mix. Finally, pipet 5.0 mL, 10.0 mL, 15.0 mL, and
25.0 mL of this solution into separate 1000-mL volumetric
flasks, dilute each flask with water to volume, and mix. The Figure 6. Aeration Apparatus for Mercury Limit Test
final solutions contain 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg,
and 2.5 mg/kg of Mn, respectively. Bottle c is fitted with an extra-coarse fritted bubbler (Corn-
ing 31770 125 EC, or equivalent), and the bottle is marked
Sample Preparation Transfer 10.000 g of the sample
with a 60-mL calibration line. The drying tube e is lightly
into a 200-mL Kohlrausch volumetric flask, previously rinsed
packed with magnesium perchlorate. Bottle c is used for the
with 0.5 N hydrochloric acid, add 140 mL of 0.5 N hydro-
test solution, and bottle d, which remains empty through-
chloric acid, and shake vigorously for 15 min, preferably
out the procedure, is used to collect water droplets.
with a mechanical shaker. Dilute with 0.5 N hydrochloric
Alternatively, an apparatus embodying the principle of the
acid to volume, and shake. Centrifuge approximately 100
assembly described and illustrated may be used. The aerat-
mL of the sample mixture in a heavy-walled centrifuge tube
ing medium may be either compressed air or compressed
at 2000 rpm for 5 min, and use the clear supernatant liquid
nitrogen.
in the following Procedure.
Standard Preparation Transfer 1.71 g of mercuric nitrate
Procedure Aspirate 0.5 N hydrochloric acid through the
[Hg(NO3)·H2O] into a 1000-mL volumetric flask, dissolve in
air–acetylene burner for 5 min, and obtain a baseline read-
a mixture of 100 mL of water and 2 mL of nitric acid, dilute
ing at 279.5 nm, following the manufacturer’s instructions
with water to volume, and mix. Discard after 1 month.
for operating the atomic absorption spectrophotometer be-
Transfer 10.0 mL of this solution into a second 1000-mL
ing used for the analysis. Aspirate a portion of each Stan-
volumetric flask, acidify with 5 mL of a 1:5 sulfuric acid
dard Preparation in the same manner, note the readings,
solution, dilute with water to volume, and mix. Discard after
then aspirate a portion of the Sample Preparation, and note
1 week. On the day of use, transfer 10.0 mL of the second
the reading. Prepare a standard curve by plotting the mg/
solution into a 100-mL volumetric flask, acidify with 5 mL of
kg of Mn in each Standard Preparation against the respective
1:5 sulfuric acid, dilute with water to volume, and mix. Each
readings. From the graph determine the mg/kg of Mn in
mL of this solution contains 1 µg of mercury. Transfer 2.0
the Sample Preparation, and multiply this value by 20 to
mL of this solution (2 µg Hg) into a 50-mL beaker, and add
obtain the mg/kg of Mn in the original sample taken for
20 mL of water, 1 mL of a 1:5 sulfuric acid solution, and 1
analysis.
mL of a 1:25 solution of potassium permanganate. Cover
 1276 / Appendix III / General Tests and Assays
the beaker with a watch glass, boil for a few seconds, and
cool.
Sample Preparation Prepare as directed in the individual
monograph.
Procedure Assemble the aerating apparatus as shown in
Figure 16, with bottles c and d empty and stopcock b in the
bypass position. Connect the apparatus to the absorption
cell (f) in the instrument, and adjust the air or nitrogen flow
rate so that in the following procedure, maximum absorp-
tion and reproducibility are obtained without excessive
foaming in the test solution. Obtain a baseline reading at
253.6 nm, following the manufacturer’s instructions for op-
erating the instrument.
Treat the Standard Preparation as follows: destroy the ex-
cess permanganate by adding a 1:10 solution of hydroxyla-
mine hydrochloride, dropwise, until the solution is colorless.
Immediately wash the solution into bottle c with water, and
dilute with water to the 60-mL mark. Add 2 mL of 10%
stannous chloride solution (prepared fresh each week by dis-
solving 10 g of SnCl2·2H2O in 20 mL of warm hydrochloric
acid and diluting with 80 mL of water), and immediately
reconnect bottle c to the aerating apparatus. Turn stopcock
b from the bypass to the aerating position, and continue
the aeration until the absorption peak has been passed and
the recorder pen has returned to the baseline. Disconnect
bottle c from the aerating apparatus, discard the Standard
Preparation mixture, wash bottle c with water, and repeat
the foregoing procedure using the Sample Preparation; any
absorbance produced by the Sample Preparation does not
exceed that produced by the Standard Preparation.
General Tests and Assays
Method II
Dithizone Extraction Solution Dissolve 30 mg of
dithizone in 1000 mL of chloroform, add 5 mL of alcohol,
and mix. Store in a refrigerator. Before use, shake a suitable
volume of the solution with about half its volume of 1:100
nitric acid, discarding the nitric acid. Discard the solution
after 1 month.
Diluted Dithizone Extraction Solution Just before use,
dilute 5 mL of Dithizone Extraction Solution with 25 mL of
chloroform.
Hydroxylamine Hydrochloride Solution Dissolve 20 g
of hydroxylamine hydrochloride in sufficient water to make
about 65 mL, transfer the solution into a separator, add a
few drops of thymol blue TS, and then add ammonium hy-
droxide until a yellow color develops. Add 10 mL of a 1:25
solution of sodium diethyldithiocarbamate, mix, and allow
to stand for 5 min. Extract the solution with successive 10-
to 15-mL portions of chloroform until a 5-mL test portion of
the chloroform extract does not develop a yellow color
when shaken with a dilute solution of cupric sulfate. Add
2.7 N hydrochloric acid until the extracted solution is pink,
adding one or two more drops of thymol blue TS, if neces-
sary, then dilute with water to 100 mL, and mix.
Mercury Stock Solution Transfer 135.4 mg of mercuric
chloride, accurately weighed, into a 100-mL volumetric
flask, dissolve in and dilute with 1 N sulfuric acid to volume,
and mix. Dilute 5.0 mL of this solution with 1 N sulfuric
acid to 500.0 mL. Each mL contains the equivalent of 10 µg
of mercury.
FCC 8
Diluted Standard Mercury Solution On the day of use,
transfer 10.0 mL of Mercury Stock Solution into a 100-mL
volumetric flask, dilute with 1 N sulfuric acid to volume, and
mix. Each mL contains the equivalent of 1 µg of mercury.
Sodium Citrate Solution Dissolve 250 g of sodium cit-
rate dihydrate in 1000 mL of water.
Sample Solution Dissolve 1 g of sample in 30 mL of 1.7
N nitric acid by heating on a steam bath. Cool to room
temperature in an ice bath, stir, and pass through S and S
No. 589, or equivalent, filter paper that has been previously
washed with 1.7 N nitric acid, followed by water. Add 20
mL of Sodium Citrate Solution and 1 mL of Hydroxylamine
Hydrochloride Solution to the filtrate.
Procedure [NOTE—Because mercuric dithizonate is light
sensitive, perform this procedure in subdued light.] Pre-
pare a control containing 3.0 mL of Diluted Standard Mer-
cury Solution (3 µg Hg), 30 mL of 1.7 N nitric acid, 5 mL of
Sodium Citrate Solution, and 1 mL of Hydroxylamine Hydro-
chloride Solution. Treat the control and the Sample Solution
as follows: using a pH meter, adjust the pH of each solution
to 1.8 with ammonium hydroxide, and transfer the solutions
into different separators. Extract each with two 5-mL por-
tions of Dithizone Extraction Solution, and then extract again
with 5 mL of chloroform, discarding the aqueous solutions.
Transfer the combined extracts from each separator into dif-
ferent separators, add 10 mL of 1:2 hydrochloric acid to
each, shake well, and discard the chloroform layers. Extract
the acid solutions with about 3 mL of chloroform, shake
well, and discard the chloroform layers. Add 0.1 mL of 0.05
M disodium EDTA and 2 mL of 6 N acetic acid to each
separator, mix, and then slowly add 5 mL of ammonium
hydroxide. Stopper the separators, cool under a stream of
cold water, and dry the outside of the separators. To avoid
loss, carefully pour the solutions through the tops of the
separators into separate beakers, and using a pH meter, ad-
just the pH of both solutions to 1.8 with 6 N ammonium
hydroxide. Return the sample and control solutions to their
original separators, add 5.0 mL of Diluted Dithizone Extrac-
tion Solution, and shake vigorously. Any color developed in
the Sample Solution does not exceed that in the control.
NICKEL LIMIT TEST
[NOTE—Unless otherwise specified in the individual
monograph, use Method I.]
Method I
Atomic Absorption System Apparatus Use a suitable
atomic absorption spectrometer equipped with a nickel
hollow-cathode lamp and an air–acetylene flame to measure
the absorbance of the Blank Preparation, the Standard Prepa-
rations, and the Test Preparation as directed under Procedure.
Test Preparation Dissolve 20.0 g of sample in dilute ace-
tic acid TS, and dilute with the same solvent to 150.0 mL.
Add 2.0 mL of a saturated solution of ammonium pyr-
rolidinedithiocarbamate (about 10 g/L of water) and 10.0
mL of methyl isobutyl ketone, and shake for 30 s. Protect
from bright light. Allow the two layers to separate, and use
the methyl isobutyl ketone layer.
FCC 8
Blank Preparation Prepare in the same manner as in the
Test Preparation, but omit the sample.
Standard Preparations Prepare three Standard Prepara-
tions in the same manner as in the Test Preparation, but add
0.5 mL, 1.0 mL, and 1.5 mL, respectively, of 10 mg/kg
nickel standard solution TS in addition to 20.0 g of sample.
Procedure Zero the instrument with the Blank Preparation.
Concomitantly determine the absorbances of each of the
Standard Preparations and of the Test Preparation at least
three times each, and record the average of the steady
readings for each. Between each measurement, aspirate the
Blank Preparation, and ascertain that the reading returns to
its initial blank value.
Calculation Calculate the linear equation of the graph us-
ing a least-squares fit, and derive from it the concentration
of nickel in the Test Preparation. Alternatively, plot on a
graph the mean of the readings against the added quantity
of nickel. Extrapolate the line joining the points on the
graph until it meets the concentration axis. The distance
between this point and the intersection of the axes repre-
sents the concentration of nickel in the Test Preparation.
Method II
[NOTE—All glassware used must be soaked in 1% Nitric
Acid for at least 2 h, and then rinsed with water.]
1% Nitric Acid Cautiously add 10 mL of nitric acid to a
1000-mL volumetric flask containing about 500 mL of
water. Mix, and dilute with water to volume.
Blank Solution Use 1% Nitric Acid.
Nickel Stock Standard Solution Immediately before
use, dilute an appropriate amount of nickel standard3 with
1% Nitric Acid to prepare a solution containing the equiva-
lent of 10 µg of nickel per mL.
Standard Solutions Into three identical 100-mL volumet-
ric flasks, introduce respectively 2.0 mL, 5.0 mL, and 10.0
mL of Nickel Stock Standard solution. Dilute with 1% Nitric
Acid to volume, and mix. These standards contain 0.2 µg,
0.5 µg, and 1.0 µg of nickel per mL.
Test Solution Weigh accurately a quantity of test speci-
men containing about 5 g of solids into a 100-mL volumet-
ric flask. Dissolve in and dilute with 1% Nitric Acid to vol-
ume, and mix.
Procedure Concomitantly determine the absorbances of
the Standard Solutions and the Test Solution at least three
times each, at the wavelength of maximum absorbance at
232.0 nm, with a suitable atomic absorption spectropho-
tometer equipped with an air–acetylene flame and a nickel
hollow-cathode lamp using the Blank Solution to zero the
instrument. Record the average of the steady readings for
each of the Standard Solutions and the Test Solution. Clear
the nebulizer using the Blank Solution and aspirate each of
the Standard Solutions and the Test Solution in turn. The
standard chosen for reslope should be run every 4 to 5 sam-
ples. If there is a significant change in its response, reslope
and repeat the previous samples. The standard deviation for
3 Suitable nickel standards are available from e.g. Fisher Scientific, Fair Lawn,
NJ (nickel, reference standard solution, 1000 ppm ± 1%, certified, applica-
tion: for atomic absorption) or RICCA Chemical Company, Arlington, TX
(nickel standard, 1000 ppm Ni, for atomic absorption).
General Tests and Assays / Appendix III / 1277
the Standard Solution of 0.2 µg of nickel per mL must be
less than 20%. Plot the absorbances of the Standard Solu-
tions versus the concentration, in µg/mL, of nickel, and
draw the straight line best fitting the three plotted points.
From the graph so obtained, determine the concentration,
C, in µg/mL, of nickel in the Test Solution. Calculate the
quantity, in µg, of nickel in each g of test specimen taken:
Result = 100C/W
in which W is the weight, in g, of test specimen taken to
prepare the Test Solution.
PHOSPHORUS LIMIT TEST
Reagents
Ammonium Molybdate Solution (5%): Dissolve 50 g of
ammonium molybdate tetrahydrate, (NH4)6Mo7O24·4H2O, in
900 mL of warm water, cool to room temperature, dilute
with water to 1000 mL, and mix.
Ammonium Vanadate Solution (0.25%): Dissolve 2.5 g of
ammonium metavanadate, NH4VO3, in 600 mL of boiling
water, cool to 60°–70°, and add 20 mL of nitric acid. Cool
to room temperature, dilute with water to 1000 mL, and
mix.
Zinc Acetate Solution (10%): Dissolve 120 g of zinc ace-
tate dihydrate, Zn(C2H3O2)2·2H2O, in 880 mL of water, and
pass through Whatman No. 2V or equivalent filter paper
before use.
Nitric Acid Solution (29%): Add 300 mL of nitric acid (sp.
General Tests and Assays
gr. 1.42) to 600 mL of water, and mix.
Standard Phosphorus Solution (100 µg P in 1 mL): Dis-
solve 438.7 mg of monobasic potassium phosphate,
KH2PO4, in water in a 1000-mL volumetric flask, dilute with
water to volume, and mix.
Standard Curve Pipet 5.0 mL, 10.0 mL, and 15.0 mL of
the Standard Phosphorus Solution into separate 100-mL volu-
metric flasks. To each of these flasks, and to a fourth, blank
flask, add in the order stated 10 mL of Nitric Acid Solution,
10 mL of Ammonium Vanadate Solution, and 10 mL of Am-
monium Molybdate Solution, mixing thoroughly after each
addition. Dilute with water to volume, mix, and allow to
stand for 10 min. Determine the absorbance of each stan-
dard solution in a 1-cm cell at 460 nm, with a suitable
spectrophotometer, using the blank to set the instrument to
zero. Prepare a standard curve by plotting the absorbance
of each solution versus its concentration, in mg of phos-
phorus (P) per 100 mL.
Treated Sample Place 20–25 g of the starch sample in a
250-mL beaker, add 200 mL of a 7:3 mixture of methanol
and water, disperse the sample, and agitate mechanically for
15 min. Recover the starch by vacuum filtration in a 150-mL
medium-porosity fritted-glass or Büchner funnel, and wash
the wet cake with 200 mL of the methanol and water mix-
ture. Reslurry the wet cake in the solvent, and wash it a
second time in the same manner. Dry the filter cake in an
air oven at a temperature below 50°, then grind the sample
to 20-mesh or finer, and blend thoroughly. Determine the
 1278 / Appendix III / General Tests and Assays
amount of dry substance by drying a 5-g portion in a vac-
uum oven, not exceeding 100 mm Hg, at 120° for 5 h.
[NOTE—The treatment outlined above is satisfactory
for starch products that are insoluble in cold water.
For pregelatinized starch and other water-soluble
starches, prepare a 1%–2% aqueous paste, place it in
a cellophane tube, and dialyze against running dis-
tilled water for 30–40 h. Precipitate the starch by
pouring the solution into 4 volumes of acetone per
volume of paste while stirring. Recover the starch by
vacuum filtration in a medium-porosity fritted-glass or
Büchner funnel, and wash the filter cake with absolute
ethanol. Dry the filter cake, and determine the
amount of dry substance as directed for water-insolu-
ble starches.]
Sample Preparation Transfer about 10 g of the Treated
Sample, calculated on the dry-substance basis and accurately
weighed, into a Vycor dish, and add 10 mL of Zinc Acetate
Solution in a fine stream, distributing the solution uniformly
in the sample. Carefully evaporate to dryness on a hot plate,
then increase the heat, and carbonize the sample on the
hot plate or over a gas flame. Ignite in a muffle furnace at
550° until the ash is free from carbon (about 1–2 h), and
cool. Wet the ash with 15 mL of water, and slowly wash
down the sides of the dish with 5 mL of Nitric Acid Solution.
Heat to boiling, cool, and quantitatively transfer the mixture
into a 200-mL volumetric flask, rinsing the dish with three
20-mL portions of water and adding the rinsings to the
flask. Dilute with water to volume, and mix. Transfer an ac-
curately measured aliquot (V, in mL) of this solution, con-
General Tests and Assays
taining not more than 1.5 mg of phosphorus, into a 100-
mL volumetric flask, and add 50 mL of water to a second
flask to serve as a blank. To each flask add in the order
stated 10 mL of Nitric Acid Solution, 10 mL of Ammonium
Vanadate Solution, and 10 mL of Ammonium Molybdate Solu-
tion, mixing thoroughly after each addition. Dilute with
water to volume, mix, and allow to stand for 10 min.
Procedure Determine the absorbance of the Sample
Preparation in a 1-cm cell at 460 nm, with a suitable spec-
trophotometer, using the blank to set the instrument at
zero. From the Standard Curve, determine the mg of phos-
phorus in the aliquot taken, recording this value as a. Calcu-
late the amount, in mg/kg, of phosphorus (P) in the original
sample:
mg/kg P = (a × 200 × 1000)/(V × W)
in which W is the weight, in grams, of the sample taken.
SELENIUM LIMIT TEST
Change to read:
Reagents and Solutions
2,3-Diaminonaphthalene Solution: On the day of use, dis-
solve 100 mg of 2,3-diaminonaphthalene (C10H10N2) and
FCC 8
500 mg of hydroxylamine hydrochloride (NH2OH · HCl) in
sufficient 0.1 N hydrochloric acid to make 100 mL.
Selenium Stock Solution: Transfer 40.0 mg of powdered
metallic selenium into a 1000-mL volumetric flask, and dis-
solve in 100 mL of 1:2 nitric acid, warming gently on a
steam bath to effect solution. Cool, dilute with water to
volume, and mix.
Selenium Standard Solution: Pipet 5.0 mL of Selenium
Stock Solution into a 200-mL volumetric flask, dilute with
water to volume, and mix. Each mL of this solution contains
the equivalent of 1 µg of selenium (Se).  Alternatively, the
solution may be prepared using a commercially available
stock solution diluted to 1 µg/mL. FCC8
Method I
Standard Preparation Pipet 6.0 mL of Selenium Standard
Solution into a 150-mL beaker, add 50 mL of 0.25 N nitric
acid, and mix.
Sample Preparation Using a 1000-mL combustion flask
and 25 mL of 0.5 N nitric acid as the absorbing liquid,
proceed as directed under Oxygen Flask Combustion, Appen-
dix I, using the amount of sample specified in the individual
monograph (and the magnesium oxide or other reagent,
where specified).
[NOTE—If the sample contains water of hydration or
more than 1% of moisture, dry it at 140° for 2 h
before combustion, unless otherwise directed.]
Upon completion of combustion, place a few mL of water in
the cup or lip of the combustion flask, loosen the stopper of
the flask, and rinse the stopper, sample holder, and sides of
the flask with about 10 mL of water. Transfer the solution,
with the aid of about 20 mL of water, into a 150-mL
beaker, heat gently to boiling, boil for 10 min, and cool.
Procedure Treat the Sample Preparation, the Standard
Preparation, and 50 mL of 0.25 N nitric acid, to serve as the
blank, similarly and in parallel as follows: add a 1:2 solution
of ammonium hydroxide to adjust the pH of the solution to
2.0 ± 0.2. Dilute with water to 60.0 mL, and transfer to a
low-actinic separator with the aid of 10.0 mL of water, add-
ing the 10.0 mL of rinsings to the separator. Add 200 mg of
hydroxylamine hydrochloride, swirl to dissolve, immediately
add 5.0 mL of 2,3-Diaminonaphthalene Solution, insert the
stopper, and swirl to mix. Allow the solution to stand at
room temperature for 100 min. Add 5.0 mL of cyclohexane,
shake vigorously for 2 min, and allow the layers to separate.
Discard the aqueous phases, and centrifuge the cyclohexane
extracts to remove any traces of water. Determine the ab-
sorbance of each extract in a 1-cm cell at the maximum at
about 380 nm with a suitable spectrophotometer, using the
extract from the blank to set the instrument. The absorb-
ance of the extract from the Sample Preparation is not
greater than that from the Standard Preparation when a
200-mg sample is tested, or not greater than one-half the
absorbance of the extract from the Standard Preparation
when a 100-mg sample is tested.
FCC 8 General Tests and Assays / Appendix III / 1279

Method II
Standard Preparation Pipet 6.0 mL of Selenium Standard
Solution into a 150-mL beaker, add 50 mL of 2 N hydrochlo-
ric acid, and mix.
Sample Preparation Transfer the amount of the sample
specified in the individual monograph into a 150-mL
beaker, dissolve in 25 mL of 4 N hydrochloric acid, swirling
if necessary to effect solution, heat gently to boiling, and
digest on a steam bath for 15 min. Remove from heat, add
25 mL of water, and allow to cool to room temperature.
Procedure Place the beakers containing the Standard
Preparation and the Sample Preparation in a fume hood, and
to a third beaker add 50 mL of 2 N hydrochloric acid to
serve as the blank. Cautiously add 5 mL of ammonium hy-
droxide to each beaker, mix, and allow the solution to cool.
Treat each solution, similarly and in parallel, as directed
under Procedure in Method I, beginning with “Add a 1:2
solution of ammonium hydroxide...”.

C. OTHERS

ALGINATES ASSAY
Figure 7. Apparatus for Alginates Assay
In a suitable closed system, liberate the carbon dioxide from
The reaction flask is provided with a reflux condenser, F,
the uronic acid groups of about 250 mg of the test sample
to which is fitted a delivery tube, G, of 40-mL capacity,
by heating with hydrochloric acid, and sweep the carbon

General Tests and Assays

dioxide, by means of an inert gas, into a titration vessel
containing excess standardized sodium hydroxide. Any suita-
ble system may be used as long as it provides precautions
against leakage and overheating of the reaction mixture, ad-
equate sweeping time, avoidance of entrainment of hydro-
chloric acid, and meets the requirements of the System Suit-
ability Test. One suitable system, with accompanying
procedure, is given below.
Apparatus The apparatus is shown in Figure 7. It consists
essentially of a soda lime column, A, a mercury valve, B,
connected through a side arm, C, to a reaction flask, D, by
means of a rubber connection. Flask D is a 100-mL round-
bottom, long-neck boiling flask, resting in a suitable heating
mantle, E.
having a stopcock, H. The reflux condenser terminates in a
trap, I, containing 25 g of 20-mesh zinc or tin, which can
be connected with an absorption tower, J.
The absorption tower consists of a 45-cm tube fitted with
a medium-porosity fritted glass disk sealed to the inner part
above the side arm and having a delivery tube sealed to it
extending down to the end of the tube. A trap, consisting
of a bulb of approximately 100-mL capacity, is blown above
the fritted disk and the outer portion of a ground spherical
joint is sealed on above the bulb. A 250-mL Erlenmeyer
flask, K, is connected to the bottom of the absorption
tower. The top of the tower is connected to a soda lime
tower, L, which is connected to a suitable pump to provide
vacuum and air supply, the choice of which is made by a
three-way stopcock, M. The volume of air or vacuum is con-
trolled by a capillary-tube regulator or needle valve, N.
All joints are a size 35/25 ground spherical type.
Standard D-Glucurono-6,3-lactone This chemical
(C6H8O6) is available as a reference standard with an assay
of 100.0 ± 1.0% (24.99 ± 0.25% CO2) from Aldrich Chemi-
cal Co.
 1280 / Appendix III / General Tests and Assays FCC 8

System Suitability Test Transfer about 250.0 mg of 0.2 N barium hydroxide or 0.2 N sodium hydroxide in ex-
Standard D-Glucurono-6,3-lactone, accurately weighed, into cess, and back titrate to neutrality with 0.2 N hydrochloric
the reaction flask, D, and carry out the Procedure described acid. Conduct a blank titration using the same reagents,
below. The system is considered suitable when the net titra- with 20 mL of water in place of the test solution. Each mL
tion results in a calculation of %CO2 in a range of of 0.2 N barium hydroxide or 0.2 N sodium hydroxide is
24.73–25.26, which is equivalent to a range of equivalent to 2.8 mg of α-amino nitrogen.
98.95%–101.06% D-Glucurono-6,3-lactone.
Procedure Transfer about 250 mg of sample, accurately AMMONIA NITROGEN (NH3-N)
weighed, into the reaction flask, D, add 25 mL of 0.1 N DETERMINATION
hydrochloric acid, insert several boiling chips, and connect
the flask to the reflux condenser, F, using syrupy phosphoric
acid as a lubricant. [CAUTION—Provide adequate ventilation.]
[NOTE—Stopcock grease may be used for the other
[NOTE—Use nitrogen-free reagents, where available, or
connections.]
reagents very low in nitrogen content.]
Check the system for air leaks by forcing mercury up into
Transfer between 700 mg and 2.2 g of sample into a 500-
the inner tube of the mercury valve, B, to a height of about
to 800-mL Kjeldahl digestion flask of hard, moderately
5 cm. Turn off the pressure using the stopcock, M. If the
thick, well-annealed glass. If desired, wrap the sample, if
mercury level does not fall appreciably after 1–2 min, the
solid or semisolid, in nitrogen-free filter paper to facilitate
apparatus may be considered to be free from leaks. Draw
the transfer.
carbon dioxide-free air through the apparatus at a rate of
Add about 200 mL of water, and mix. Add a few granules
3000–6000 mL/h. Raise the heating mantle, E, to the flask,
of zinc to prevent bumping, tilt the flask, and cautiously
heat the sample to boiling, and boil gently for 2 min. Turn
pour sodium hydroxide pellets, or a 2:5 sodium hydroxide
off and lower the mantle, and allow the sample to cool for
solution, down the inside of the flask so that it forms a layer
15 min. Charge the delivery tube, G, with 23 mL of hydro-
under the solution, using a sufficient amount (usually about
chloric acid. Disconnect the absorption tower, J, rapidly
25 g of solid sodium hydroxide) to make the mixture
transfer 25.0 mL of 0.25 N sodium hydroxide into the
strongly alkaline. Immediately connect the flask to a distilla-
tower, add 5 drops of n-butanol, and reconnect the absorp-
tion apparatus consisting of a Kjeldahl connecting bulb and
tion tower. Draw carbon dioxide-free air through the appa-
a condenser that has a delivery tube extending well beneath
ratus at the rate of about 2000 mL/h, add the hydrochloric
the surface of a measured excess of 0.5 N hydrochloric or
acid to the reaction flask through the delivery tube, raise
General Tests and Assays

sulfuric acid contained in a 500-mL flask. Add 5 to 7 drops

the heating mantle, and heat the reaction mixture to boil-
ing. After 2 h, discontinue the current of air and heating.
Force the sodium hydroxide solution down into the flask, K,
using gentle air pressure, and then rinse down the absorp-
tion tower with three 15-mL portions of water, forcing each
washing into the flask with air pressure. Remove the flask,
and add to it 10 mL of a 10% solution of barium chloride
(BaCl2·2H2O). Stopper the flask, shake gently for about 2
min, add phenolphthalein TS, and titrate with 0.1 N hydro-
chloric acid. Perform a blank determination (see General Pro-
visions). Each mL of 0.25 N sodium hydroxide consumed is
equivalent to 5.5 mg of carbon dioxide (CO2). Calculate the
results on the dried basis.
α-AMINO NITROGEN (AN)
DETERMINATION
Transfer 7–25 g of sample, accurately weighed, into a 500-
mL volumetric flask with the aid of several 50-mL portions
of warm, ammonia-free water, dilute with water to volume,
and mix. Neutralize 20.0 mL of the solution with 0.2 N
barium hydroxide or 0.2 N sodium hydroxide, using phe-
nolphthalein TS as the indicator, and add 10 mL of freshly
prepared phenolphthalein–formol solution (50 mL of 40%
formaldehyde containing 1 mL of 0.05% phenolphthalein in
50% alcohol neutralized exactly to pH 7 with 0.2 N barium
hydroxide or 0.2 N sodium hydroxide). Titrate with 0.2 N
barium hydroxide or 0.2 N sodium hydroxide to a distinct
red color, add a small, but accurately measured, volume of
of methyl red indicator (1 g of methyl red in 200 mL of
alcohol) to the receiver flask. Rotate the Kjeldahl flask to mix
its contents thoroughly, and heat until all of the ammonia
has distilled, collecting at least 150 mL of distillate. Wash
the tip of the delivery tube, collecting the washings in the
receiving flask, and titrate the excess acid with 0.5 N so-
dium hydroxide. Perform a blank determination (see General
Provisions), substituting 2 g of sucrose for the sample, and
make any necessary correction. Each mL of 0.5 N acid con-
sumed is equivalent to 7.003 mg of ammonia nitrogen.
[NOTE—If it is known that the substance to be deter-
mined has a low nitrogen content, 0.1 N acid and
alkali may be used, in which case each mL of 0.1 N
acid consumed is equivalent to 1.401 mg of
nitrogen.]
Calculate the percent ammonia nitrogen:
Result = (NH3-N/S) × 100
in which NH3-N is the weight, in mg, of ammonia nitrogen,
and S is the weight, in mg, of the sample.
BENZENE (in Paraffinic Hydrocarbon
Solvents)
Apparatus (See Chromatography, Appendix IIA.) Use a
suitable gas chromatograph, equipped with a column, or
equivalent, that will elute n-decane before benzene under
FCC 8
the conditions of the System Suitability Test. Column materi-
als and conditions that have been found suitable for this
method are listed in the accompanying tables. See Figure 8
for a typical chromatogram obtained with column No. 5.
Figure 8. Typical Chromatogram for the Determination of
Benzene in Hexanes Using Column No. 5
Reagents
Isooctane: 99 mole percent minimum containing less
than 0.05 mole percent aromatic material
Benzene: 99.5 mole percent minimum
Internal Standard: n-Decane and either n-undecane or n-
dodecane according to the requirement of the System Suita-
bility Test
Reference Solution A: Prepare a standard solution contain-
ing 0.5% by weight each of the Internal Standard and of
benzene in isooctane.
Reference Solution B: Prepare a standard solution contain-
ing about 0.5% by weight each of n-decane, of Internal
Standard, and of benzene in isooctane.
Calibration Select the instrument conditions necessary
to give the desired sensitivity. Inject a known volume of
Reference Solution A, and change the attenuation, if neces-
sary, so that the benzene peak is measured with a chart
deflection of not less than 25% or more than 95% of full
scale. When choosing the attenuation, consider all un-
resolved peaks to represent a single compound. There may
be tailing of the nonaromatic peak, but do not use any
conditions that lead to a depth of the valley ahead of the
benzene peak (A) less than 50% of the weight of the ben-
zene peak (B) as depicted in Figure 9.
General Tests and Assays / Appendix III / 1281
Figure 9. lllustration of A/B Ratio
If there is tailing of the nonaromatic material, construct a
baseline by drawing a line from the bottom of the valley
ahead of the benzene peak to the point of tangency after
the peak (see Figure 10). Measure the areas of the benzene
peak and the internal standard peak by any of the following
means: triangulation, planimeter, paper cutout, or mechani-
cal or electronic integrator. Do not use integrators on peaks
without a constant baseline, unless the integrator has provi-
sion for making baseline corrections with accuracy at least
as good as that of manual methods.
General Tests and Assays
Figure 10. Illustration of A/B Ratio for a Small Component
Peak on the Tail of a Large Peak
Calculate a response factor for benzene (Rb) relative to the
Internal Standard:
Result = Ai/Wi × Wb/Ab
in which Ai is the area of the Internal Standard peak in arbi-
trary units corrected for attenuation; Wi is the weight per-
cent of Internal Standard in Reference Solution A; Wb is the
weight percent of benzene in Reference Solution A; and Ab is
the area of the benzene peak in arbitrary units corrected for
attenuation.
Procedure Place approximately 0.1 mL of Internal Stan-
dard into a tared 25-mL volumetric flask, weigh on an ana-
lytical balance, and dissolve in and dilute with the sample to
be analyzed to volume.
Using the exact instrumental conditions that were used in
the calibration, inject the same volume of sample containing
the Internal Standard. Before measuring the area of the In-
ternal Standard and benzene peaks, change the attenuation
to ensure at least 25% chart deflection.
 1282 / Appendix III / General Tests and Assays FCC 8

Measure the area of the Internal Standard and benzene
peaks in the same manner as was used for the calibration.
Calculate the weight percentage of benzene in the sample
(WB):
Result = (Ab × Rb × Wi × 100)/(Ai × S)
in which Ab is the area of the benzene peak corrected for
attenuation; Rb is the relative response factor for benzene;
Wi is the weight, in grams, of Internal Standard added; Ai is
the area of the Internal Standard peak corrected for attenua-
tion; and S is the weight, in grams, of the sample taken.
System Suitability Test Inject the same volume of Refer-
ence Solution B as in the Calibration and record the chromat-
ogram. n-Decane must be eluted before benzene, and the
ratio of A to B (Figure 19) must be at least 0.5 where A is
equal to the depth of the valley between the n-decane and
benzene peaks and B is equal to the height of the benzene
peak.

Column Materials and Conditions for the Determination of Benzene in Hexanes

Column No. 1 2 3 4 5 6 7
Liquid phase CEF PEF 200 CEF DEGS TCEPE TCEPE DEGS
Length, ft 15 6 16 10 15 100 12
m — 4.5 2 5 3.1 — 313.7
Diameter, in (mm)
Inside 0.07(1.8) — 0.07 0.18(4.5) 0.06(1.5) 0.01(.254)
Outside 1
/8(3.2) 1
/4(6.4) /8
1
— — — 1
/8
Weight, percent 17 30 20 20 10 — 20
Solid support Chromosorb P Chromosorb P Chromosorb P Chromosorb P Chromosorb P Capillary Chromosorb P
Mesh 60–80 60–80 60–80 80–100 60–80 — 80–100
Treatment AW AW AW none AW none AW Sil
Inlet, deg 200 210 250 260 250 275 260
Detector, deg 200 155 250 200 175 250 240
Column, deg 115 95 90 100 115 95 65
Carrier gas N2 He He He N2 N2 He
Flow rate,
cm3/min 30 60 60 60 1 3 52
General Tests and Assays

Detector FI TC FI FI FI FI FI
Recorder, mV 5 1 1 1 10 1 1
Sample, 1 5 10 1 2 5 0.8 5
Split 9+1 — — — 100 + 1 100 – 1 —
Area Tri EI DI Tri Plan EI EI Tri

Abbreviations Used in Table: AW—Acid washed; CEF—N,N-Bis(2-cyanoethyl)formamide; DEGS—Diethylene Glycol Suc-

cinate; DI—Disk integrator; EI—Electronic integrator; FI—Flame ionization; Sil—Silanized; TC—Thermal conductivity; TCEPE—
Tetracyanoethylated Pentaerythritrol; Tri—Triangulation.

Retention Times in Minutes for Selected Hydrocarbons under the Conditions for the Determination
of Benzene in Hexanes
Column No. 1 2 3 4 5 6 7
Benzene 3.4 2.0 6.5 6.7 5.4 6.1 6.7
Toluene 4.4 3.2 9.0 10.3 7.8 7.0 10.3
Ethylbenzene 5.4 5.2 11.5 14.8 10.8 8.0 14.8
p-m-Xylenes 5.8 — 12.5 — 11.4 8.5 —
o-Xylene 7.5 6.8 17.0 16.1 14.5 10.0 —
n-Undecane 3.0 2.8 3.5 — — — —
n-Dodecane — — — 12.8 8.5 6.5 —
FCC 8 General Tests and Assays / Appendix III / 1283

Color Determination4 bright copper wire through the condenser, and place a
small coil of copper wire (about 0.5 g) in the distillation
Chromium flask.
Standards
Standard Chromium Solution (1000 mg/kg): Transfer
2.829 g of K2Cr2O7, accurately weighed (NIST No. 136) into
a 1-L volumetric flask; dissolve in and dilute with water to
volume.
Standard Colorant Solution: Transfer 62.5 g of colorant
previously shown to be free of chromium to a 1-L volumet-
ric flask; dissolve in and dilute with water to volume.
Apparatus Use any suitable atomic absorption spectro-
photometer equipped with a fast response recorder and ca-
pable of measuring the radiation absorbed at 357.9 nm.
Instrument Parameters Wavelength setting: 357.9 nm;
optical passes: 5; lamp current: 8 mA; lamp voltage: 500 v;
fuel: hydrogen; oxidant: air; recorder: l mv with a scale ex-
pansion of 5 or 10. Alternatively, follow the instructions sup-
plied with the instrument.
Procedure Set the instrument at the optimum conditions
for measuring chromium as directed by the manufacturer’s
instructions. Prepare a series of seven standard chromium
solutions containing Cr at approximately 5 mg/kg, 10 mg/
kg, 15 mg/kg, 20 mg/kg, 40 mg/kg, 50 mg/kg, and 60 mg
/kg by appropriate dilutions of the Standard Chromium Solu-
tion into 100-mL volumetric flasks; add 80 mL of the Stan-
dard Colorant Solution, and dilute each flask with water to
volume.
Transfer 5 g of the colorant to be analyzed to a 100-mL

General Tests and Assays

volumetric flask; dissolve in and dilute with water to vol-
ume. Prepare a calibration curve using the series of stan-
dards, and using this curve, determine the chromium con-
tent of the colorant samples.
Ether Extracts
[CAUTION—Isopropyl ether forms explosive peroxides.
To ensure the absence of peroxides, perform the fol-
lowing test: prepare a colorless solution of ferrous thi-
ocyanate by mixing equal volumes of 0.1 N ferrous
sulfate and 0.1 N ammonium thiocyanate. Using titan-
ous chloride, carefully discharge any red coloration
due to ferric ions. Add 10 mL of ether to 50 mL of the
solution, and shake vigorously for 2–3 min. A red
color indicates the presence of peroxides. If redistilla-
tion is necessary, the usual precautions against perox-
ide detonation should be observed. Immediately
before use, pass the ether through a 30-cm column of
chromatography-grade aluminum oxide to remove
peroxides and inhibitors.]
Apparatus Use an upward displacement-type liquid–liquid
extractor, as shown in Figure 11, with a sintered-glass dif-
fuser and a working capacity of 200 mL. Suspend a piece of
4 To be used or sold for use to color food that is marketed in the United
States, color additives must be from batches that have been certified by the
U.S. Food and Drug Administration (FDA). If color additives are not from
FDA-certified batches, they are not permitted color additives for food use in
the United States, even if they are compositionally equivalent. The FD&C
names can be applied only to FDA-certified batches of these color additives.
Figure 11. Upward Displacement-Type Liquid–Liquid Extrac-
tor with Sintered-Glass Diffuser
Alkaline Ether Extract Transfer 5 g of the colorant to a
beaker, and dissolve in 150 mL of water. Add 2 mL of 2.5 N
NaOH solution, transfer the solution into the extractor, and
dilute with water to approximately 200 mL. Add 200 mL of
ether to the distillation flask, and extract for 2 h with a
reflux rate of about 15 mL/min. Set the extracted colorant
solution aside. Transfer the ether extract into a separatory
funnel, and wash with two 25-mL portions of 0.1 N NaOH
followed by two 25-mL portions of water. Reduce the vol-
ume of the ether extract to about 5 mL by distillation (in
portions) from a tared flask containing a small piece of clean
copper coil.
Acid Ether Extract Add 5 mL of 3 N hydrochloric acid to
the extracted colorant solution set aside in the alkaline ether
extract procedure above, mix, and extract with ether as di-
rected above. Wash the ether extract with two 25-mL por-
tions of 0.1 N hydrochloric acid and water. Transfer the
washed ether in portions to the flask containing the evapo-
rated alkaline extract, and carefully remove all the ether by
distillation. Dry the residue in an oven at 85° for 20 min.
Then allow the flask to cool in a desiccator for 30 min, and
weigh. Repeat drying and cooling until a constant weight is
 1284 / Appendix III / General Tests and Assays FCC 8

obtained. The increase in weight of the tared flask, ex- porcelain combustion boat (60 × 10 × 8 mm) and a small
pressed as a percentage of the sample weight, is the com- piece of copper oxide wire. The combustion tube is placed
bined ether extract. in a heavy-duty hinged combustion tube furnace (Lindburg
Leuco Base Type 70T, or equivalent), and it is connected by clamped
Reagents and Solutions ball-joints at one end to a source of nitrogen and connected
to a series of three traps at the other. The traps are con-
Cupric Chloride Solution: Transfer 10.0 g of CuCl2·2H2O to structed of a linear array of 18- × 2-mm Pyrex tubes con-
a 1-L volumetric flask; dissolve in and dilute with dimethyl- nected by clamped ball-joints and extend from the connec-
formamide (DMF) to volume. tion at the combustion tube. Trap I contains anhydrous
Sample Solution: Prepare as directed in the individual calcium sulfate packed between quartz-wool plugs, trap II
monograph. contains ascarite packed between cotton plugs, and trap III
Procedure contains aluminum oxide packed between cotton plugs. The
Solution 1: Pipet 50 mL of DMF into a 250-mL volumet- nitrogen flow forces the mercury through the combustion
ric flask, cover, and place in the dark. tube, the three traps, and a section of Tygon tube to a
mercury vapor meter (Beckman model K-23, or equivalent).
Solution 2: Pipet 10 mL of the Sample Solution into a The mercury released from a sample during combustion is
250-mL volumetric flask, add 50 mL of DMF, and place in quantitated by comparing the recorder response with that
the dark. given by a series of mercury standards.
Solution 3: Pipet 50 mL of Cupric Chloride Solution into a
250-mL volumetric flask, and gently bubble air through the
solution for 30 min.
Solutions 4a and 4b: Pipet 10 mL of the Sample Solution
into each of two 250-mL volumetric flasks, add 50 mL of
Cupric Chloride Solution to each, and bubble air gently
through the solutions for 30 min.
Dilute all of the solutions with water nearly to volume;
incubate for 5–10 min, but no longer, in a water bath
cooled with tap water; and dilute to volume. Record the
spectrum for each solution between 500 nm and 700 nm
using an absorbance range of 0 to 1 and a 1-cm pathlength
General Tests and Assays

cell; record all spectra on the same spectrogram.

Solution in Solution in
Curve No. Sample Cell Reference Cell
I 1 1
II 1 2
III 3 3
IVa 3 4a
IVb 3 4b

Calculation

Figure 12. (a) Schematic Diagram of Apparatus for Photo-

metric Mercury Vapor M Method:
in which the Roman numerals I through IV represent the
absorbance readings for solutions of the corresponding A. Tank of nitrogen G. Dehydrite trap
Arabic numerals (above) at the wavelength maximum; a is B. Two-stage pressure regulator H. Ascarite trap
the absorptivity (for Fast Green, a = 0.156 at 625 nm; for
C. Low-pressure regulator I. Aluminum oxide trap
Brillant Blue, a = 0.164 at 630 nm); W is the weight, in
D. Flowmeter J. Mercury vapor meter
grams, of the sample taken; and r is the ratio of the molec-
ular weights of colorant and leuco base (for Fast Green, r = E. Combustion tube K. Atenuator
0.9712; for Brillant Blue, r = 0.9706). F. Combustion-tube furnace L. Recorder
Mercury (b) Quartz Combustion Tube with Boat and Copper Oxide Packing;
Apparatus The apparatus used for the direct (c) Schematic Diagram of Trap Used to Contain Ascarite, Dehydrite,
microdetermination of mercury is shown in Figure 12. It and Aluminum Oxide.
consists of a quartz combustion tube designed to hold a
FCC 8
Reagents and Equipment
Absorbent Cotton
Aluminum Oxide: Anhydrous
Calcium Sulfate: Anhydrous, dehydrate, or equivalent
Asbestos Pads: (1 × 0.5 × 1 cm) Preheated at 800° for 1 h
Ascarite: 20- to 30-mesh
Copper Oxide Wire: Preheated at 850° for 2 h
Nitrogen: Purified grade
Quartz Wool
Sodium Carbonate: Anhydrous, fine granular
Standard Solution: Transfer approximately 1.35 g of rea-
gent-grade mercurous chloride, accurately weighed, into a
1-L volumetric flask. Dissolve in and dilute with water to
volume. When diluted 100-fold, the solution contains 0.01
µg Hg per microliter (Diluted Standard Solution).
Procedure Preheat the furnace to 650°, and adjust the
nitrogen flow to 1 L/min.
Blank Analysis: Place a square piece of preheated asbestos
pad in the combustion boat, and cover it with sodium car-
bonate. Stop the nitrogen flow, disconnect the ball-joint,
quickly insert the boat into the combustion tube with large
forceps, and reconnect the joint. Note the time, allow the
boat to sit in the tube with no nitrogen flow for exactly 1
min, and then restart the flow of nitrogen. Mercury elutes
almost immediately with the reinstated nitrogen flow; note
the recorder response. Allow about 30 s between runs.
Calibration: Determine the recorder response after the
application to the asbestos pad of 1 µL, 2 µL, and 3 µL of
the Diluted Standard Solution.
Sample Analysis: Transfer 25 mg of colorant, accurately
weighed, to the combustion boat, and cover the sample
completely with sodium carbonate. Follow the procedure
used for the Blank Analysis, and calculate the mercury con-
tent using the standard curve.
Trap Problems
1. Some colorants (e.g., Brillant Blue and Fast Green) may
give a response that is symmetrically dissimilar to the Hg
peak. If such a response “carries over” to the next sample,
then the aluminum oxide trap may need to be changed.
2. If the recorder response is of inadequate sensitivity (peak
height induced by 0.01 µg less than 0.5 cm), then the
traps are packed too tightly. Remove or redistribute
packing first in the aluminum oxide trap, then try the
other traps.
3. The traps will need changing periodically as indicated by
a change in the physical appearance of the trap material
or by chart responses of different retention times or dif-
ferent symmetry from that of mercury standards.
4. If two or more standards are run in succession, a later
sample might give an erroneous mercury response. Run
blanks and then repeat the sample analysis to confirm
the validity of the response.
Sodium Chloride
Dissolve approximately 2 g of colorant, accurately weighed,
in 100 mL of water, and add 10 g of activated carbon that
is free of chloride and sulfate. Boil gently for 2–3 min. Cool
to room temperature, add 1 mL of 6 N nitric acid, and stir.
General Tests and Assays / Appendix III / 1285
Dilute with water to volume in a 200-mL volumetric flask,
and then filter through dry paper. Repeat the treatment
with 2-g portions of carbon until no color is adsorbed onto
filter paper dipped into the filtrate.
Transfer 50 mL of filtrate to a 250-mL flask. Add 2 mL of
6 N nitric acid, 5 mL of nitrobenzene, and 10 mL of stan-
dardized 0.1 N silver nitrate solution. Shake the flask until
the silver chloride coagulates. Prepare a saturated solution of
ferric ammonium sulfate, and add just enough concentrated
nitric acid to discharge the red color; add 1 mL of this solu-
tion to the 250-mL flask to serve as the indicator. Titrate
with 0.1 N ammonium thiocyanate solution that has been
standardized against the silver nitrate solution until the color
persists after shaking for 1 min. Calculate the weight per-
cent of sodium chloride, P:
P = [(V × N)/W] × 22.79
in which V is the net volume, in mL, of silver nitrate solution
required; N is the normality of the silver nitrate solution;
and W is the weight, in grams, of the sample taken. The
factor 22.79 incorporates a total volume of 195 mL because
10 g of activated carbon occupies 5 mL.
Sodium Sulfate
Place 25 mL of the decolorized filtrate obtained from the
Sodium Chloride test into a 125-mL Erlenmeyer flask, and
add 1 drop of a 0.5% phenolphthalein solution in 50% eth-
anol. Add 0.05 N sodium hydroxide, dropwise, until the so-
lution is alkaline to pH paper, and then add 0.002 N hydro-
chloric acid until the indicator is decolorized. Add 25 mL of
ethanol and about 0.2 g of tetrahydroquinone sulfate indi-
General Tests and Assays
cator. Titrate with 0.03 N barium chloride solution to a red
endpoint. Make a blank determination.
Calculate the weight percent, P, of sodium sulfate:
P = [(V − B) × N/W] × 55.4
in which V is the volume, in mL, of barium chloride solution
required to titrate the sample; B is the volume, in mL, of
barium chloride solution required for the blank; N is the
normality of the barium chloride solution; and W is the
weight, in grams, of the sample taken. The factor 55.4 in-
corporates a total volume of 195 mL because 10 g of acti-
vated carbon occupies 5 mL.
Total Color
Method I (Spectrophotometric)
Pipet 10.0 mL of the dissolved colorant into a 250-mL
Erlenmeyer flask containing 90 mL of 0.04 N ammonium
acetate, and mix well. Determine the net absorbance of the
solution relative to water at the wavelength maximum given
for each color. Calculate the percentage of colorant present:
%total color = A/(a × C × b) × 100
in which A is the absorbance; a is the absorptivity specified
in the individual monograph (L/(mg·cm); C is the concen-
tration of sample in the final test solution (mg/L); and b is
the cell pathlength (cm).
Method II (Titration with Titanium Chloride)
Apparatus The apparatus for determining total color by
titration with titanium chloride (TiCl3) is shown in Figure 13.
It consists of a storage bottle, A, of 0.1 N titanium chloride
 1286 / Appendix III / General Tests and Assays
titrant maintained under hydrogen produced by a Kipp gen-
erator; an Erlenmeyer flask, B, equipped with a source of
CO2 or N2 to maintain an inert atmosphere in which the
reaction takes place; a stirrer; and the buret, C.
General Tests and Assays
Figure 13. Titanous Chloride Titration Apparatus
Reagents and Solutions
Titanium Chloride Solution (0.1 N): Transfer 73 mL of com-
mercially prepared 20% TiCl3 solution into a storage bottle,
and carefully add 82 mL of concentrated HCl per L of final
solution. Mix well, and bubble CO2 or N2 through the solu-
tion for 1 h. Before standardizing, maintain the solution
under a hydrogen atmosphere for at least 16 h using a Kipp
generator.
Potassium Dichromate Solution (0.1 N, primary standard):
Transfer 4.9032 g of K2Cr2O7 (NIST No. 136) to a 1-L volu-
metric flask; dissolve in and dilute with water to volume.
Ammonium Thiocyanate (50%): Transfer 500 g of NH4SCN,
ACS certified, to a 1-L volumetric flask; dissolve in about
600 mL of water, warming if necessary; and dilute to
volume.
Ferrous Ammonium Sulfate: Fe(NH4)2(SO4)2·6H2O, ACS
certified
FCC 8
Sodium Bitartrate
Standardization of the Titanium Chloride Solution
Drain any standing titanium chloride (TiCl3) from the feed
lines and buret, and refill with fresh solution. Add 3.0 g of
Ferrous Ammonium Sulfate to a wide-mouth Erlenmeyer flask
followed by 200 mL of water, 25 mL of 50% sulfuric acid,
25 mL of 0.1 N Potassium Dichromate Solution (by pipet),
and 2 or 3 boiling chips. Boil the solution vigorously on a
hot plate for 30 s to remove dissolved air, then quickly
transfer the flask to the titration apparatus, securely connect
the stopper assembly, and start the carbon dioxide flow and
stirrer. Pass carbon dioxide over the solution for 1 min
before beginning the titration.
Add the 0.1 N Titanium Chloride Solution at a fast, steady
drip to within 1 mL of the estimated endpoint (about 20
mL). Reduce the carbon dioxide flow, remove the solid-glass
rod from the stopper assembly, pipet 10 mL of Ammonium
Thiocyanate (50%) into the flask, insert the glass rod, and
increase the carbon dioxide flow. Continue titrating slowly
until the endpoint: a color change from brown-red to light
green is observed. Perform a blank determination using the
same reagents and quantities, and calculate the normality,
N, of the 0.1 N Titanium Chloride Solution on the basis of
three titrations:
N = (Vr × Nr/Vt − Vb)
in which Vr is the volume, in mL, of 0.1 N Potassium Dichro-
mate used; Nr is the normality of the 0.1 N Potassium
Dichromate; Vt is the volume, in mL, of 0.1 N Titanium Chlo-
ride Solution used; and Vb is the volume, in mL, of titanium
dichloride used in the blank titration.
Procedure Transfer the quantity of colorant prescribed in
the individual monograph into a 500-mL wide-mouth
Erlenmeyer flask and add 21–22 g of Sodium Bitartrate (so-
dium citrate for Sunset Yellow), 275 mL of water, and two
or three boiling chips. Boil the solution vigorously on a hot
plate for 30 s to remove dissolved air, then quickly transfer
the flask to the titration apparatus, securely connect the
stopper assembly, and start the carbon dioxide flow and
stirrer. Pass carbon dioxide over the solution for 1 min
before beginning the titration.
Titrate the sample until the color lightens, wait 20 s, and
then continue the addition with about 2 s between drops.
When the color is almost completely bleached, wait 20 s,
and then continue the addition with 5 s between drops. A
complete color change indicates the endpoint. Perform a
blank determination using the same reagents and quantities,
and calculate the total color, T, in percent and on the basis
of three titrations:
T = [(Vt − Vb)/(W × Fs)] × 100 × N
in which Vt is the volume of titrant used; Vb is the volume of
titrant required to produce the endpoint in a blank; W is the
weight, in grams, of the sample taken; Fs is a factor derived
from the stoichiometry of the reaction characteristics of
each colorant and is given in the individual monograph;
and N is the normality of the titrant.
Method III (Gravimetric)
Transfer approximately 0.5 g of colorant, accurately
weighed, to a 400-mL beaker, add 100 mL of water, and
FCC 8
heat to boiling. Add 25 mL of 1:50 hydrochloric acid, and
bring to a boil. Wash down the sides of the beaker with
water, cover, and keep on a steam bath for several hours or
overnight. Cool to room temperature, and quantitatively
transfer the precipitate into a tared filtering crucible with
1:100 hydrochloric acid. Wash the precipitate with two 15-
mL portions of water, and dry the crucible for 3 h at 135°.
Cool in a desiccator, and weigh. Calculate the total color, P,
in weight percent:
P = [(Wp × F)/Ws] × 100
in which Wp is the weight, in grams, of the precipitate; F is
the gravimetric conversion factor given in the individual
monograph; and Ws is the original weight, in grams, of the
sample taken.
Uncombined Intermediates and Products of Side
Reactions
Method I
Sample Solution Transfer approximately 2 g of colorant
to a 100-mL volumetric flask; dissolve in and dilute with
water to volume.
Apparatus Pack a 2.5-cm × 45-cm glass column with ap-
proximately 20 g of cellulose (Whatman CF-11 grade, or
equivalent) that has been slurried in the eluant and from
which the fines have been removed by decantation. Equili-
brate the column thoroughly with the eluant, 35% ammo-
nium sulfate.
Procedure Pipet 5 mL of the Sample Solution into a
beaker containing 5 g of cellulose that has been slurried in
eluant and from which the fines have been removed by
decantation. Stir the mixture thoroughly, add 10 g of am-
monium sulfate, and stir until uniformly mixed. Mix the
slurry with 15 mL of eluant, and apply it to the column.
Allow the fluid to enter the column, and wash the beaker
with eluant until the sample is quantitatively transferred.
Elute the column with approximately 500 mL of 35% am-
monium sulfate, and collect a total of eight 60-mL fractions.
Divide each collected fraction in half and add 0.5 mL of
NH4OH to one half and 0.5 mL of HCl to the other.
Calculation After identifying each intermediate and side
product by comparing spectra of the fractions with com-
mercial standards, calculate the concentration, C, of each:
C = A/(a × b)
in which A is the absorbance at the wavelength of maximal
absorption; a is the absorptivity given in the individual mon-
ograph; and b is the cell pathlength, in cm.
Method II
Apparatus Use a suitable high-performance liquid chro-
matography system (see Chromatography, Appendix IIA)
equipped with a dual wavelength detector system such that
the effluent can be monitored serially at 254 nm and
325–385 nm (wide-band pass). Use a 1-m × 2.1-mm (id)
column, or equivalent, packed with a strong anion-ex-
change resin (Dupont No. 830950405, or equivalent).
Operating Conditions The operating conditions required
may vary depending on the system used. The following
General Tests and Assays / Appendix III / 1287
conditions have been shown to give suitable results for Al-
lura Red, Tartrazine, and Sunset Yellow.
Allura Red
Primary Eluant: 0.01 M aqueous Na2B4O7
Secondary Eluant: 0.20 M NaClO4 in aqueous 0.01 M
Na2B4O7
Sample Size: 20 µL of a 0.25% solution
Flow Rate: 0.60 mL/min
Gradient: Linear, in two phases: 0%–18% in 40 min,
18%–62% in 8 min more, then hold for 18 min more at
62%
Temperature: 50°
Pressure: 1000 psi
Order of Elution: (1) Cresidinesulfonic acid (CSA); (2) un-
known; (3) Schaeffer’s salt (SS); (4) unknown; (5) 4,4′-
diazoaminobis(5-methoxy-2-methylbenzenesulfonic acid)
(DMMA); (6) unknown; (7) Allura Red; (8) 6,6′-oxybis(2-
naphthalenesulfonic acid) (DONS)
Tartrazine
Primary Eluant: 0.01 M aqueous Na2B4O7
Secondary Eluant: 0.10 M NaClO4 in aqueous 0.01 M
Na2B4O7
Sample Size: 50 µL of a 0.15% solution, prepared within
13 min of injection
Flow Rate: 1.00 mL/min
Gradient: Exponential at 4%/min: 0.95%
Temperature: 50°
Pressure: 1000 psi
Order of Elution: (1) Phenylhydrazine-p-sulfonic acid
(PHSA); (2) sulfanilic acid (SA); (3) 1-(4-sulfophenyl)-3-ethyl-
General Tests and Assays
carboxy-5-hydroxypyrazolone (PY-T); (4) 1-(4-sulfophenyl)-
3-carboxy-5-hydroxypyrazolone (EEPT); (5) 4,4′-
(diazoamino)-dibenzenesulfonic acid (DAADBSA)
Sunset Yellow
Primary Eluant: 0.01 M aqueous Na2B4O7
Secondary Eluant: 0.20 M NaClO4 in aqueous 0.01 M
Na2B4O7
Sample Size: 5 µL of a 1% solution
Flow Rate: 0.50 mL/min
Gradient: Linear in four phases: 0%–11% in 10 min; hold
25 min; 11%–38% in 10 min; 38%–42% in 10 min;
42%–98% in 20 min; hold 20 min
Temperature: 50°
Pressure: 1000 psi
Order of Elution: (1) Sulfanilic acid (SA); (2) Schaeffer’s salt
(SS); (3) 4,4′-(diazoamino)-dibenzenesulfonic acid
(DAADBSA); (4) R-salt dye; (5) Sunset Yellow; (6) 6,6′-ox-
ybis(2-naphthalenesulfonic acid) (DONS)
Standard Solutions
Allura Red: Prepare a solution containing 0.25 g of
colorant, 0.5 mg of CSA, 0.75 mg of SS, 0.25 mg of
DMMA, and 1.25 mg of DONS in a 100-mL volumetric
flask. Dissolve in and dilute with 0.1 M Na2B4O7 to volume.
Tartrazine: Prepare a solution containing 0.15 g of colorant
and 0.3 mg each of PHSA, SA, PY-T, EEPT, and DAADBSA in
a 100-mL volumetric flask. Dissolve in and dilute with 0.1 M
Na2B4O7 to volume.
Sunset Yellow: Prepare a solution containing 0.25 g of
colorant, 0.5 mg of SA, 0.75 mg of SS, 0.25 mg of
 1288 / Appendix III / General Tests and Assays FCC 8

DAADBSA, and 1.25 mg of DONS in a 100-mL volumetric

flask. Dissolve in and dilute with 0.1 M Na2B4O7 to volume.
Test Solutions Prepare at least four test solutions, each
containing the colorant, and one impurity, accurately
weighed, dissolved in 0.1 M Na2B4O7, and diluted to vol-
ume in a 100-mL volumetric flask. The solutions should en-
compass the range of concentrations, evenly spaced, given
below for each constituent:
Allura Red (250 mg): CSA (0.05–0.5 mg); SS (0.05–0.75
mg); DONS (0.5–2.5 mg); DMMA (0.025–0.25 mg). Inject
20 µL of each solution.
Tartrazine (150 mg): SA (7.5 to 300 µg); PY-T (7.5–300
µg); EEPT (7.5–300 µg); DAADBSA (7.5–300 µg). Inject 50
µL of each solution.
Sunset Yellow (250 mg): SA (0.05–0.5 mg); SS (0.05–0.75
mg); DONS (0.5–2.5 mg); DAADBSA (0.05–0.25 mg). Inject
20 µL of each solution.
System Suitability
Resolution: Elute the column, or equivalent, with the gradi-
ent specified under Operating Conditions until a smooth
baseline is obtained. Inject an aliquot of the Standard Solu-
tion. The resolution of the eluted components matches or
exceeds that shown for the corresponding colorant (see
Figures 14, 15, and 16). After determining that the column,
or equivalent, will give the required resolution, allow it to
rest for 2 weeks before use.
General Tests and Assays

Figure 15. Tartrazine–Top Trace: Eluant Monitored at 254

nm; Bottom Trace: Eluant Monitored at 375–385 nm

Figure 14. Allura Red–Top Trace: Eluant Monitored at 254

nm; Bottom Trace: Eluant Monitored at 375–385 nm
FCC 8
Figure 16. Sunset Yellow–Top Trace: Eluant Monitored at
254 nm; Bottom Trace: Eluant Monitored at 375–385 nm
Calibration Inject the designated volume of each Test Solu-
tion onto a conditioned column, and prepare a standard
curve corresponding to each unreacted intermediate and
side reaction product. Determine the area, A, for each peak
from the integrator if an automated system is used or by
multiplying the peak height by the width at one-half the
height. The peak height alone may be used for EEPT, PY-T,
and DAADBSA. Calculate the concentration, Ci, of each in-
termediate or side product:
Ci = mAi + b
in which Ai is the area of its corresponding chromatographic
peak. Calculate the slope, m, and intercept, b, using the
following linear regression equations:
m = [NΣCiAi − ΣCiΣAi]/[NΣAi2 − [(ΣAi)2]
b = [A]i − m[C]i
in which A and C are the calculated averages of the peak
areas and concentrations, respectively, used to construct the
standard curve for one intermediate or side reaction prod-
uct. Calculate the correlation coefficient, r:
r = [Σ(Ci − C)(Ai − A)]/[Σ(Ci − C2) × Σ(Ai − A)2]
Each time the system is calibrated, add the new data to
those accumulated from previous analyses. The correlation
coefficient must be 0.95–1.00 for any single experiment or
from accumulated data.
General Tests and Assays / Appendix III / 1289
Recalibrate the system after every 10 determinations or 2
days, whichever occurs first.
Sample Preparation Prepare as directed in the individual
monograph.
Procedure Inject the volume of Sample Preparation as des-
ignated in the monograph into the column. Determine the
concentration of intermediates and side reaction products
from the peak areas using the slope, m, and intercept, b,
calculated under Calibration:
Cs = mAs + b
in which Cs is the concentration of the unknown in the
Sample Preparation and As its corresponding peak area.
Loss on Drying (Volatile Matter)
Transfer 1.5–2.5 g of colorant, accurately weighed, to a
tared crucible. Heat in a vacuum oven at 135° for 12–15 h.
Lower the pressure in the oven to −125 mm Hg, and con-
tinue heating for an additional 2 h. Cover the crucible, and
allow to cool in a desiccator. Reweigh the crucible when
cool. The loss of weight is defined as the volatile matter.
Water-Insoluble Matter
Transfer about 1 g of colorant, accurately weighed, to a
250-mL beaker, and add 200 mL of boiling water. Stir to
facilitate dissolution of the color.
Tare a filtering crucible equipped with a glass fiber filter
(Reeve Angel, No. 5270, or equivalent). Filter the solution
with the aid of suction when it has cooled to ambient tem-
perature. Rinse the beaker three times, pouring the rinsings
through the crucible. Wash the filter with water until the
General Tests and Assays
filtrate is colorless.
Dry the crucible and filter in an oven at 135° for at least 3
h, cool them in a desiccator, and reweigh to the nearest 0.1
mg. Calculate the percent water-insoluble matter, I:
I = (Wc/Ws) × 100
in which Wc is the difference in crucible weight and Ws is
the sample weight.
ELEMENTAL IMPURITIES BY ICP
Before the initial use of either of the procedures below, the
analyst should ensure that the procedure is appropriate for
the instrument and sample used. Method I can be used for
elemental impurities generally amenable to detection by in-
ductively coupled plasma–atomic (optical) emission spec-
troscopy (ICP–OES). Method II can be used for elemental
impurities generally amenable to detection by inductively
coupled plasma–mass spectrometry (ICP–MS). If no method
is specified in the individual monograph, analysts are in-
structed to use Method II (ICP–MS).
Method I: ICP–OES
Reagents All reagents used for the preparation of the
sample and standard solutions should be free of elemental
impurities. Reagents should be commercial elemental stock
standards that are NIST-traceable, or equivalent, at a recom-
mended concentration of 100 µg/mL or greater; or appro-
priate USP Reference Standards, as either single element or
multielement.
 1290 / Appendix III / General Tests and Assays
Aqua Regia: Ultra pure nitric acid/hydrochloric acid (1:3)
prepared as needed. (A 1%–5% solution of aqua regia is
used as a rinsing solution between analyses and as calibra-
tion blanks.)
Sample Preparation Use this sample preparation proce-
dure unless otherwise specified in the individual mono-
graph. [NOTE—Weights and volumes provided may be ad-
justed to meet the requirements of the microwave digestion
apparatus used, if proportions remain constant.] Dehydrate
and predigest 0.5 g of sample in 5 mL of freshly prepared
Aqua Regia. Sulfuric acid may also be used as a last resort.
[NOTE—Sulfuric acid should be used only when absolutely
needed because addition of sulfuric acid may cause an ex-
treme exothermic reaction and result in elements being lost
and because the viscosity of sulfuric acid is higher than that
of other acids, which affects the overall flow of solu-
tion.] Allow the sample to sit loosely covered for 30 min in
a fume hood. Add an additional 10 mL of Aqua Regia and
digest, using a closed vessel microwave technique. Micro-
wave until digestion or extraction is complete. Repeat if
necessary by adding an additional 5 mL of Aqua Regia.
[NOTE—Follow the recommended procedures provided by
the manufacturer of the closed vessel microwave digestion
apparatus to ensure safe usage. In closed vessel microwave
digestion, the use of concentrated hydrofluoric acid (HF) is
not recommended; however, when its use is necessary,
practice the utmost caution in the preparation of test arti-
cles, and review or establish local procedures for safe han-
dling, safe disposal, and HF-tolerant instrumental
configurations.]
General Tests and Assays
Sample Solution Allow the digestion vessel containing
the Sample Preparation to cool (for mercury measurements,
add an appropriate stabilizer, such as gold at about 0.1
ppm), and dilute with water to 50.0 mL.
Calibration Solution 1 2J of the element of interest in a
matched matrix (acid concentrations similar to that of the
Sample Solution), where J is the limit for the specific elemen-
tal impurity. [NOTE—Multiple elements of interest may be
included in this solution at the same concentration ratio. For
mercury analysis, add an appropriate stabilizer, such as gold
at about 0.1 ppm.]
Calibration Solution 2 0.1J of the element of interest in
a matched matrix (acid concentrations similar to that of the
Sample Solution), where J is the limit for the specific elemen-
tal impurity. [NOTE—Multiple elements of interest may be
included in this solution at the same concentration ratio. For
mercury analysis, add an appropriate stabilizer, such as gold
at about 0.1 ppm.]
Check Standard Solution 1 ppm of the element of in-
terest in a matched matrix (acid concentrations similar to
that of the Sample Solution). [NOTE—Multiple elements of
interest may be included in this solution at the same con-
centration ratio. For mercury analysis, add an appropriate
stabilizer, such as gold at about 0.1 ppm.]
Blank Matched matrix (acid concentrations similar to that
of the Sample Solution)
Elemental Spectrometric System (see Plasma Spectro-
chemistry, Appendix IIC)
FCC 8
Mode: ICP
Detector: Optical emission spectroscopy
Rinse: 5% Aqua Regia
Calibration: Two-point, using Calibration Solution 1, Cali-
bration Solution 2, and Blank
System Suitability
Sample: Check Standard Solution
Suitability requirement: The concentration determined
from the resulting chromatogram differs from actual con-
centration by NMT 20%. [NOTE—If samples are high in
mineral content, to minimize sample carryover, rinse system
well (60 s) before introducing the Check Standard Solution.]
Analysis Analyze according to manufacturer’s suggestions
for program and wavelength. Calculate and report results
on the basis of the original sample size.
Calculation Upon completion of the analysis, calculate
the final concentration of a given element in the test article
(µg/g) from the solution element concentration (µg/mL) as
follows:
C = [(A × V1)/W] × (V2/V3)
where C is the concentration of the analyte, µg/g; A is the
instrument reading, µg/mL; V1 is the volume of the initial
test article preparation, mL; W is the weight of the test arti-
cle preparation, g; V2 is the total volume of any dilution
performed, mL; and V3 is the volume of the aliquot of initial
test article preparation used in any dilution performed, mL.
Similarly, calculate the final concentration of a given ele-
ment in the test article (µg/g) from the solution element
concentration (ng/mL) as follows:
C = [(A × V1)/W] × (1 µg/1000 ng)(V2/V3)
where A is the instrument reading, ng/mL; and the other
factors are as defined above.
Method II: ICP–MS
Reagents All reagents used for the preparation of sample
and standard solutions should be free of elemental impuri-
ties. Reagents should be commercial elemental stock stan-
dards that are NIST-traceable, or equivalent, at a recom-
mended concentration of 100 µg/mL or greater; or
appropriate USP Reference Standards, as either single ele-
ment or multielement.
Aqua Regia: Ultra pure nitric acid/hydrochloric acid (1:3)
prepared as needed. (A 1%–5% solution of aqua regia is
used as a rinsing solution between analyses and as calibra-
tion blanks.)
Sample Preparation Proceed as directed under Method I.
Sample Solution Allow the digestion vessel containing
the Sample Preparation to cool, and add appropriate internal
standards at appropriate concentrations (for mercury meas-
urements, gold should be one of the internal standards).
Dilute with water to 50.0 mL.
Calibration Solution 1 Proceed as directed under
Method I.
Calibration Solution 2 Proceed as directed under
Method I.
FCC 8
Blank Matched matrix (acid concentrations similar to that
of the Sample Solution)
Elemental Spectrometric System (see Plasma Spectro-
chemistry, Appendix IIC)
Mode: ICP. [NOTE—An instrument with a cooled spray
chamber is recommended.]
Detector: Mass spectrometer
Rinse: 5% Aqua Regia
Calibration: Calibration Solution 1, Calibration Solution 2,
and Blank
System Suitability
Sample: Calibration Solution 1
Suitability requirement: The concentration determined
from the resulting chromatogram differs from actual con-
centration by NMT 20%. [NOTE—If samples are high in
mineral content, to minimize sample carryover, rinse system
well (60 s) before introducing the Check Standard Solution.]
Analysis Analyze according to the manufacturer’s sugges-
tions for the program and m/z. Calculate and report results
based on the original sample size. [NOTE—Arsenic is sub-
ject to interference from argon chloride. Appropriate meas-
ures, including a sample preparation without Aqua Regia,
must be taken to correct for the interference, depending on
instrumental capabilities.]
Calculation Upon completion of the analysis, calculate
the final concentration of a given element in the test article
(µg/g) from the solution element concentration (µg/mL) as
follows:
C = [(A × V1)/W] × (V2/V3)
where C is the concentration of the analyte, µg/g; A is the
instrument reading, µg/mL; V1 is the volume of the initial
test article preparation, mL; W is the weight of the test arti-
cle preparation, g; V2 is the total volume of any dilution
performed, mL; and V3 is the volume of the aliquot of initial
test article preparation used in any dilution performed, mL.
Similarly, calculate the final concentration of a given ele-
ment in the test article (µg/g) from the solution element
concentration (ng/mL) as follows:
C = [(A × V1)/W] × (1 µg/1000 ng)(V2/V3)
where A is the instrument reading, ng/mL; and the other
factors are as defined above.
GLUTAMIC ACID
Apparatus Use an ion-exchange amino acid analyzer,
equipped with sulfonated polystyrene columns, in which the
effluent from the sample is mixed with ninhydrin reagent
and the absorbance of the resultant color is measured con-
tinuously and automatically at 570 nm and 440 nm by a
recording photometer.
Standard Solution Transfer 1250 ± 2 mg of reagent-
grade glutamic acid, accurately weighed, into a 500-mL vol-
umetric flask. Fill the flask half-full with water, add 5 mL of
hydrochloric acid to help dissolve the amino acid, dilute
with water to volume, and mix. Prepare the standard for
General Tests and Assays / Appendix III / 1291
analysis by diluting 1 mL of this solution with 4 mL of 0.2 N
sodium citrate, pH 2.2, buffer. This Standard Solution con-
tains 0.5 mg of glutamic acid per mL (CS).
Sample Preparation Dilute 5 mg of sample, accurately
weighed, to exactly 5 mL with 0.2 N sodium citrate, pH
2.2, buffer. Remove any insoluble material by centrifugation
or filtration.
Procedure Using 2-mL aliquots of the Standard Solution
and Sample Preparation, proceed according to the apparatus
manufacturer’s instructions. From the chromatograms thus
obtained, match the retention times produced by the Stan-
dard Preparation with those produced by the Sample Solu-
tion, and identify the peak produced by glutamic acid. Re-
cord the area of the glutamic acid peak from the sample as
AU, and that from the standards as AS.
Calculations Calculate the concentration, CA, in mg/mL,
of glutamic acid in the Sample Preparation:
Result = AU × CS/AS
in which CS is the concentration, in mg/mL, of glutamic
acid in the Standard Solution.
Calculate the percent glutamic acid, on the basis of total
protein:
Result = (100 × CA)/(6.25 × NT)
in which 6.25 is the conversion factor for protein and amino
acids, and NT is the percent total nitrogen determined in
the monograph Assay.
Calculate the percent glutamic acid in the sample:
General Tests and Assays
Result = 100 × CA/SW
in which SW is the weight, in mg, of the sample taken.
HYDROXYPROPOXYL DETERMINATION
Apparatus The apparatus for hydroxypropoxyl group de-
termination is shown in Figure 17.
Figure 17. Apparatus for Hydroxypropoxyl Determination
The boiling flask, D, is fitted with an aluminum foil-covered
Vigreaux column, E, on the side arm and with a bleeder
tube through the neck and to the bottom of the flask for
 1292 / Appendix III / General Tests and Assays FCC 8

the introduction of steam and nitrogen. A steam generator,

B, is attached to the bleeder tube through tube C, and a
condenser, F, is attached to the Vigreaux column. The boil-
ing flask and steam generator are immersed in an oil bath,
A, equipped with a thermoregulator such that a tempera-
ture of 155° and the desired heating rate may be main-
tained. The distillate is collected in a 150-mL beaker, G, or
other suitable container.
Procedure Unless otherwise directed, transfer about 100
mg of the sample, previously dried at 105° for 2 h and
accurately weighed, into the boiling flask, and add 10 mL of
chromium trioxide solution (60 g in 140 mL of water). Im-
merse the steam generator and the boiling flask in the oil
bath (at room temperature) to the level of the top of the
chromium trioxide solution. Start cooling water through the
Figure 18. Chart for Converting Percentage of Substitution,
condenser, and pass nitrogen gas through the boiling flask
by Weight, of Hydroxypropoxyl Groups to Molecular Substi-
at the rate of one bubble per second. Starting at room tem-
tution per Glucose Unit
perature, raise the temperature of the oil bath to 155° over
a period of not less than 30 min, and maintain this temper-
ature until the end of the determination. Distill until 50 mL METHOXYL DETERMINATION
of distillate is collected. Detach the condenser from the
Vigreaux column, and wash it with water, collecting the Apparatus The apparatus for methoxyl determination, as
washings in the distillate container. Titrate the combined shown in Figure 19, consists of a boiling flask, A, fitted with
washings and distillate with 0.02 N sodium hydroxide to a a capillary side arm to provide an inlet for carbon dioxide
pH of 7.0, using a pH meter set at the expanded scale. and connected to a column, B, which separates aqueous
[NOTE—Phenolphthalein TS may be used for this titra- hydriodic acid from the more volatile methyl iodide. After
tion if it is also used for all standards and blanks.] the methyl iodide passes through a suspension of aqueous
Record the volume, Va, of the 0.02 N sodium hydroxide red phosphorus in the scrubber trap, C, it is absorbed in the
used. Add 500 mg of sodium bicarbonate and 10 mL of 2 N bromine–acetic acid absorption tube, D. The carbon dioxide
sulfuric acid, and then after evolution of carbon dioxide has is introduced from a device arranged to minimize pressure
General Tests and Assays

ceased, add 1 g of potassium iodide. Stopper the flask, fluctuations and connected to the apparatus by a small cap-
shake the mixture, and allow it to stand in the dark for 5 illary containing a small plug of cotton.
min. Titrate the liberated iodine with 0.02 N sodium thiosul-
fate to the sharp disappearance of the yellow color, confirm-
ing the endpoint by the addition of a few drops of starch
TS. Record the volume of 0.02 N sodium thiosulfate re-
quired as Ya.
Make several reagent blank determinations, using only the
chromium trioxide solution in the above procedure. The ra-
tio of the sodium hydroxide titration (Vb) to the sodium
thiosulfate titration (Yb), corrected for variation in normali-
ties, will give the acidity-to-oxidizing ratio, Vb/Yb = K, for the
chromium trioxide carried over in the distillation. The factor
K should be constant for all determinations.
Make a series of blank determinations using 100 mg of
methylcellulose (containing no foreign material) in place of
the sample, recording the average volume of 0.02 N so-
dium hydroxide required as Vm and the average volume of
0.02 N sodium thiosulfate required as Ym.
Calculate the hydroxypropoxyl content of the sample, in
mg:

Result = 75.0 × [N1(Va – Vm) – kN2(Ya – Ym)]

in which N1 is the exact normality of the 0.02 N sodium

hydroxide solution, k = VbN1/YbN2, and N2 is the exact nor-
mality of the 0.02 N sodium thiosulfate solution. Figure 19. Distillation Apparatus for Methoxyl Determination
The percentage of substitution, by weight, of hydroxypro-
poxyl groups, determined as directed above, may be con-
verted to molecular substitution per glucose unit by refer-
ence to Figure 18.
FCC 8
Reagents
Acetic Potassium Acetate: Dissolve 100 g of potassium
acetate in 1000 mL of a mixture consisting of 900 mL of
glacial acetic acid and 100 mL of acetic anhydride.
Bromine–Acetic Acid Solution: On the day of use, dissolve
5 mL of bromine in 145 mL of the Acetic Potassium Acetate
solution.
Hydriodic Acid: Use special-grade hydriodic acid suitable
for alkoxyl determinations, or purify reagent grade as fol-
lows: distill over red phosphorus in an all-glass apparatus,
passing a slow stream of carbon dioxide through the appa-
ratus until the distillation is terminated and the receiving
flask has completely cooled.
[CAUTION—Use a safety shield, and conduct the distil-
lation in a fume hood.]
Collect the colorless, or almost colorless, constant-boiling
acid distilling between 126°–127°. Store the acid in a cool,
dark place in small, brown, glass-stoppered bottles previ-
ously flushed with carbon dioxide and finally sealed with
paraffin.
Procedure Fill trap C half-full with a suspension of about
60 mg of red phosphorus in 100 mL of water, introduced
through the funnel on tube D and the side arm that con-
nects with the trap at C. Rinse tube D and the side arm with
water, collecting the rinsings in trap C, then charge absorp-
tion tube D with 7 mL of Bromine–Acetic Acid Solution. Place
the sample, previously accurately weighed in a tared gelatin
capsule, into the boiling flask A, along with a few glass
beads or boiling stones, then add 6 mL of Hydriodic Acid.
Connect the flask to the condenser, using a few drops of
the acid to seal the junction, and begin passing the carbon
dioxide through the apparatus at the rate of about two
bubbles per second. Heat the flask in an oil bath at 150°,
continue the reaction for 40 min, and drain the contents of
absorption tube D into a 500-mL Erlenmeyer flask contain-
ing 10 mL of a 1:4 solution of sodium acetate. Rinse tube D
with water, collecting the rinsings in the flask, and dilute
with water to about 125 mL. Discharge the red-brown color
of bromine by adding formic acid, dropwise with swirling,
then add 3 drops in excess. Usually a total of 12 to 15
drops of formic acid is required. Allow the flask to stand for
3 min, add 15 mL of 2 N sulfuric acid and 3 g of potassium
iodide, and titrate immediately with 0.1 N sodium thiosul-
fate, adding starch TS near the endpoint. Perform a blank
determination with the same quantities of the same re-
agents, including the gelatin capsule, and in the same man-
ner, and make any necessary correction. Each mL of 0.1 N
sodium thiosulfate is equivalent to 0.517 mg (517 µg) of
methoxyl groups (–OCH3).
NITROGEN DETERMINATION (Kjeldahl
Method)
[NOTE—All reagents should be nitrogen free, where
available, or otherwise very low in nitrogen content.]
General Tests and Assays / Appendix III / 1293
Method I
Use this method unless otherwise directed in the individual
monograph. It is not applicable to certain nitrogen-contain-
ing compounds that do not yield their entire nitrogen upon
digestion with sulfuric acid.
Nitrites and Nitrates Absent
Unless otherwise directed, transfer 1 g of sample into a
500-mL to 800-mL Kjeldahl digestion flask of hard borosili-
cate, moderately thick, well-annealed glass, wrapping the
sample, if solid or semisolid, in nitrogen-free filter paper to
facilitate the transfer if desired. Add 10 g of powdered po-
tassium sulfate or anhydrous sodium sulfate, 500 mg of
powdered cupric sulfate, and 20 mL of sulfuric acid. Place
the flask in an inclined position (about 45°), and heat gently
keeping the temperature below the boiling point until froth-
ing ceases, adding a small amount of paraffin, if necessary,
to reduce frothing.
[CAUTION—The digestion should be conducted in a
fume hood, or the digestion apparatus should be
equipped with a fume exhaust system.]
Increase the heat until the acid boils briskly and continue
the heating process until the solution clears, and then con-
tinue boiling for 30 min longer (or for 2 h for samples con-
taining organic material). Cool, add about 150 mL of water,
mix, and then cool to below 25°. Add cautiously 100 mL of
a 2:5 sodium hydroxide solution, in such a manner as to
cause the solution to flow down the inner side of the flask
to form a layer under the acid solution, to make the mixture
strongly alkaline. Add a few granules of zinc to prevent
General Tests and Assays
bumping, and immediately connect the flask to a distillation
apparatus consisting of a Kjeldahl connecting bulb and a
condenser, the delivery tube of which extends well beneath
the surface of 100 mL of a 1:25 boric acid solution con-
tained in a conical flask or a wide-mouth bottle of about
500-mL capacity. Gently, rotate the Kjeldahl flask to mix its
contents thoroughly, and then heat until all of the ammonia
has distilled, collecting at least 150 mL of distillate (about
80% of the contents of the flask). Wash the tip of the deliv-
ery tube, collecting the washings in the receiving flask, and
titrate with 0.5 N sulfuric acid, determining the endpoint
potentiometrically. Perform a blank determination, substitut-
ing 2 g of sucrose for the sample, and make any necessary
correction (see General Provisions). Each mL of 0.5 N acid is
equivalent to 7.003 mg of nitrogen.
[NOTE—An indicator solution can also be used to deter-
mine the titration endpoint. For example, dissolve 0.2 g of
methyl red in 100 mL of 95% ethanol, 1 g of bromocresol
green in 500 mL of 95% ethanol, then combine 1 part of
the methyl red solution and 5 parts of the bromocresol
green solution. Add 3 mL of methyl red/bromocresol green
indicator solution per L of boric acid solution. Then, titrate
the sample to the first trace of pink. Also, a bromocresol
green–methyl red solution can be used as an alternative. To
make the solution, dissolve 0.15 g of bromocresol green
and 0.1 g of methyl red in 180 mL of alcohol, and dilute
with water to a final volume of 200 mL.]
[NOTE—If the substance to be determined is known to
have a low nitrogen content, 0.1 N acid and alkali
 1294 / Appendix III / General Tests and Assays
may be used, in which case each mL of 0.1 N acid
consumed is equivalent to 1.401 mg of nitrogen.]
[NOTE—Nitrogen recovery verification can be run to check
for accuracy of the procedure and the equipment.
1. Nitrogen loss: Use 0.12 g of ammonium sulfate and 0.85
g of sucrose. Add all other reagents, digest, and distill
under the same conditions as for the sample. Recoveries
should be NLT 99%.
2. Digestion efficiency: Use 0.16 g of lysine hydrochloride or
0.18 g of tryptophan, with 0.67 g of sucrose per flask.
Add all other reagents, digest, and distill under the same
conditions as for the sample. Recoveries should be NLT
98%.]
Nitrites and Nitrates Present
[NOTE—This procedure is not applicable to liquids or
to materials having a high chlorine-to-nitrate ratio.]
Unless otherwise directed, transfer a quantity of sample, ac-
curately weighed, corresponding to about 150 mg of nitro-
gen into a Kjeldahl flask, and add 25 mL of 93%–98% sulfu-
ric acid containing 1 g of salicylic acid. Mix thoroughly by
shaking, and then allow to stand for 30 min or more, with
frequent shaking. Add 5 g of Na2S2O3·5H2O (as an impalpa-
ble powder, not granules or filings), mix, then add 500 mg
of powdered cupric sulfate, and proceed as directed under
Nitrates and Nitrites Absent, beginning with “Incline the flask
at an angle of about 45°”. When the nitrogen content of
the substance is known to exceed 10%, 500 mg to 1 g of
benzoic acid may be added, prior to digestion, to facilitate
the decomposition of the substance.
General Tests and Assays
Method II (Semimicro)
[NOTE—Automated instruments may be used in place
of this manual method, provided the automated
equipment has been properly calibrated.]
Transfer an accurately weighed or measured quantity of
the sample, equivalent to about 2 or 3 mg of nitrogen, into
the digestion flask of a semimicro Kjeldahl apparatus. Add 1
g of a 10:1 powdered mixture of potassium sulfate and cu-
pric sulfate, using a fine jet of water to wash down any
material adhering to the neck of the flask, and then pour 7
mL of sulfuric acid down the inside wall of the flask to rinse
it. Cautiously add down the inside of the flask 1 mL of 30%
hydrogen peroxide, swirling the flask during the addition.
[CAUTION—Do not add any peroxide during the
digestion.]
Heat over a free flame or an electric heater until the solu-
tion has attained a clear blue color and the walls of the flask
are free from carbonized material. Cautiously add 20 mL of
water, cool, then add through a funnel 30 mL of a 2:5
solution of sodium hydroxide, and rinse the funnel with 10
mL of water. Connect the flask to a steam distillation appa-
ratus, and immediately begin the distillation with steam.
Collect the distillate in 15 mL of a 1:25 solution of boric
acid to which has been added 3 drops of methyl
red–methylene blue TS and enough water to cover the end
of the condensing tube. Continue passing the steam until
80–100 mL of distillate has been collected, then remove the
absorption flask, rinse the end of the condenser tube with a
FCC 8
small quantity of water, and titrate with 0.01 N sulfuric
acid. Each mL of 0.01 N acid is equivalent to 140 µg of
nitrogen.
When more than 2 or 3 mg of nitrogen is present in the
measured quantity of the substance to be determined, 0.02
or 0.1 N sulfuric acid may be used in the titration if at least
15 mL of titrant is required. If the total dry weight of the
material taken is greater than 100 mg, increase proportion-
ately the quantities of sulfuric acid and sodium hydroxide
added before distillation.
SPECTROPHOTOMETRIC IDENTIFICATION
TESTS
Spectrophotometric tests contribute meaningfully toward
the identification of many compendial chemical substances.
The test procedures that follow are applicable to substances
that absorb IR and/or UV radiation. The IR absorption spec-
trum of a substance, compared with that obtained concomi-
tantly for the corresponding USP Reference Standard, pro-
vides perhaps the most conclusive evidence of the identity
of the substance that can be realized from any single test.
The UV absorption spectrum, on the other hand, does not
exhibit a high degree of specificity. Conformance with both
IR absorption and UV absorption test specifications leaves
little doubt, if any, regarding the identity of the specimen
under examination.
Infrared Spectra This test is used for comparison of an
IR spectrum for a sample specimen with a reference spec-
trum provided in the individual monograph.
Sample specimens should be prepared using the same
technique as that used for the provided spectra. Unless
otherwise noted in the individual monograph or spectrum
caption, the spectra for liquid substances were obtained on
neat liquids contained in fixed-volume sodium chloride cells
or between salt plates. Spectra for solid substances were
obtained on a potassium bromide pellet or a mineral oil
(Nujol or equivalent) dispersion, as indicated in individual
monographs or spectrum caption.
Infrared Absorption This test is used for comparison of
the IR spectrum of a sample specimen with that of a physi-
cal USP Reference Standard.
Sample and USP Reference Standard specimens should
both be prepared for analysis using the same technique, as
directed in the individual monographs, which use the below
letter designations. Sample and USP Reference Standard
specimens should be used as either dried or undried speci-
mens as directed on the Reference Standard Label.
Record the spectra of the sample and USP Reference Stan-
dard specimens over the range of about 2.6–15 µm (3800
cm−1 to 650 cm−1) unless otherwise specified in the individ-
ual monograph.
Designation Specimen Preparation Technique
A Intimately in contact with an internal reflection ele-
ment for attenuated total reflectance (ATR) analysis
E Pressed as a thin sample against a suitable plate for IR
microscopic analysis
F Suspended neat between suitable (for example sodi-
um chloride or potassium bromide) plates
FCC 8
Designation Specimen Preparation Technique
K Mixed intimately with potassium bromide and com-
pressed into a translucent pellet
M Finely ground and dispersed in mineral oil
S A solution of designated concentration, prepared as
directed in the individual monograph, and examined
in 0.1-mm cells (unless otherwise specified in the in-
dividual monograph)
[NOTE—A and E techniques can be used as alternative
methods for K, M, F, and S where testing is performed
qualitatively and the Reference Standard spectra are
similarly obtained.]
Ultraviolet Absorption The test is used for comparison
of the UV spectrum of a sample specimen with that of a
physical USP Reference Standard.
Sample and USP Reference Standard specimens should be
prepared using the solvents and concentrations specified in
the individual monograph.
Record the spectra of the sample and USP Reference Stan-
dard solutions concomitantly using 1-cm cells over the spec-
tral range of 200–400 nm, unless otherwise indicated in the
individual monograph. Calculate absorptivities and/or ab-
sorbance ratios where these criteria are included in an indi-
vidual monograph. Unless otherwise specified, absorbances
indicated for these calculations are those measured at the
maximum absorbance at the wavelength specified in the in-
dividual monograph. Where the absorbance is to be meas-
ured at a wavelength other than that of maximum absorb-
ance, the abbreviations (min) and (sh) are used to indicate a
minimum and shoulder, respectively, in an absorbance
spectrum.
SULFUR (by Oxidative Microcoulometry)
(Based on ASTM D3120)5
[NOTE—All reagents used in this test should be reagent
grade; water should be of high purity, and gases must
be high-purity grade.]
Apparatus Use the Dohrmann Microcoulometric Titrat-
ing System (MCTS-30), or equivalent (shown in Figure 20),
unless otherwise specified in an individual monograph. It
consists of a constant rate injector, A, a pyrolysis furnace, B,
a quartz pyrolysis tube, C, a granular-tin scrubber, D, a titra-
tion cell, E, and a microcoulometer with a digital readout, F.
Granular-Tin Scrubber: Place 5 g of 20- to 30-mesh gran-
ular reagent-grade tin between quartz-wool plugs in an
elongated 18/9-12/5 standard-taper adaptor that connects
the pyrolysis tube and the titration cell.
Microcoulometer: Must have variable attenuation; gain
control; and be capable of measuring the potential of the
sensing-reference electrode pair, and comparing this poten-
tial with a bias potential, amplifying the potential difference,
5 Adapted from ASTM D3120 Standard Test Method for Trace Quantities of
Sulfur in Light Liquid Petroleum Hydrocarbons by Oxidative Microcoulometry.
The original ASTM method is available in its entirety from ASTM
International, 100 Barr Harbor Drive, West Conshohocken, PA 19428. Phone:
610-832-9585, Fax: 610-832-9555, Email: service@astm.org, Website:
www.astm.org.
General Tests and Assays / Appendix III / 1295
and applying the amplified difference to the working-auxil-
iary electrode pair to generate a titrant. The microcoulome-
ter output voltage signal must also be proportional to the
generating current.
Pyrolysis Furnace: The sample should be pyrolyzed in an
electric furnace having at least two separate and indepen-
dently controlled temperature zones, the first being an inlet
section that can maintain a temperature sufficient to volatil-
ize all the organic sample. The second zone is a pyrolysis
section that can maintain a temperature sufficient to pyro-
lyze the organic matrix and oxidize all the organically
bound sulfur. A third outlet temperature zone is optional.
Pyrolysis Tube: Must be fabricated from quartz and con-
structed in such a way that a sample, which is vaporized
completely in the inlet section, is swept into the pyrolysis
zone by an inert gas where it mixes with oxygen and is
burned. The inlet end of the tube shall hold a septum for
syringe entry of the sample and side arms for the introduc-
tion of oxygen and inert gases. The center or pyrolysis sec-
tion should be of sufficient volume to ensure complete py-
rolysis of the sample.
Sampling Syringe: A microlitre syringe of 10-µL capacity
capable of accurately delivering 1–10 µL of sample into the
pyrolysis tube. Three-in × 24-gauge needles are recom-
mended to reach the inlet zone of the pyroloysis furnace.
Titration Cell: Must contain a sensor-reference pair of
electrodes to detect changes in triiodide ion concentration
and a generator anode–cathode pair of electrodes to main-
tain constant triiodide ion concentration and an inlet for a
gaseous sample from the pyrolysis tube. The sensor elec-
General Tests and Assays
trode shall be platinum foil and the reference electrode plat-
inum wire in saturated triiodide half-cell. The generator an-
ode and cathode half-cell shall also be platinum. The
titration cell shall be placed on a suitable magnetic stirrer.
Preparation of Apparatus: Carefully insert the quartz py-
rolysis tube into the furnace, attach the tin scrubber, and
connect the reactant and carrier-gas lines. Add the Cell Elec-
trolyte Solution (see below) to the titration cell, and flush the
cell several times. Maintain an electrolyte level of 3.2–6.6
mm above the platinum electrodes. Place the titration cell
on a magnetic stirrer, and connect the cell inlet to the tin
scrubber outlet. Position the platinum-foil electrodes
(mounted on the movable cell head) so that the gas-inlet
flow is parallel to the electrodes with the generator anode
adjacent to the generator cathode. Assemble and connect
the coulometer in accordance with the manufacturer’s in-
structions. Double-wrap the adaptor containing the tin
scrubber with heating tape and turn the heating tape on.
Adjust the flow of the gases, the pyrolysis furnace tempera-
ture, the titration cell, and the coulometer to the desired
operating conditions. Typical operating conditions are as
follows:
Reactant gas flow (oxygen),
200
cm3/min
Carrier-gas flow (Ar, He), cm3/min 40
Furnace temperature, °C
700 (maximum)
Inlet zone
Pyrolysis zone 800–1000
 1296 / Appendix III / General Tests and Assays
Outlet zone 800 (maximum)
Tin-scrubber temperature, °C 200
Titration cell Stirrer speed set to produce slight
vortex
Coulometer
Bias voltage, mV 160
Gain 50
Constant Rate Injector, µL/s 0.25
The tin scrubber must be conditioned to sulfur, nitrogen,
and chlorine before quantitative analysis can be achieved. A
solution containing 10 mg/kg of butyl sulfide, 100 mg/kg of
pyridine, and 200 mg/kg of chlorobenzene in isooctane has
proven an effective conditioning agent. With a fresh scrub-
ber installed and heated, two 30-µL samples of this condi-
tioning agent injected at a flow rate of 0.5 µL/s produce a
steadily increasing response, with final conditioning indi-
cated by a constant reading from the offset during the sec-
ond injection.
Reagents
Argon or Helium (argon preferred): High-purity grade,
used as the carrier gas. Two-stage gas regulators must be
used.
Cell Electrolyte Solution: Dissolve 0.5 g of potassium io-
dide and 0.6 g of sodium azide in 500 mL of high-purity
water, add 5 mL of glacial acetic acid, and dilute to 1 L.
Store in a dark bottle or in a dark place and prepare fresh at
least every 3 months.
Oxygen: High-purity grade, used as the reactant gas
General Tests and Assays
Iodine: Resublimed, 20-mesh or less, for saturated refer-
ence electrode
Sulfur Standard (approximately 100 mg/kg): Transfer
0.1569 g of n-butyl sulfide, accurately weighed, into a tared
Figure 20. Microcoulometric Titrating System for the Determination of Sulfur in Hexanes
FCC 8
500-mL volumetric flask. Dilute to the mark with isooctane,
and reweigh. Calculate the sulfur concentration (S), as a
percentage:
S = Wb/Ws × 2.192 × 105
in which Wb is the weight of n-butyl sulfide and Ws is the
weight of the solution.
Calibration Prepare a calibration standard (approxi-
mately 5 mg/kg) by pipetting 5 mL of Sulfur Standard into a
10-mL volumetric flask and diluting with isooctane to vol-
ume. Fill and clamp the syringe onto the constant-rate injec-
tor, push the sliding carriage forward to penetrate the sep-
tum with the needle, and zero the meter in case of long-
term drift in the automatic baseline zero circuitry. Switch S1
automatically starts the stepper-motor syringe drive and ini-
tiates the analysis cycle. At 2.5 min (before the 3-min meter
hold point) set the digital meter with the scan potentiome-
ter to correspond to the sulfur content of the known stan-
dard to the nearest 0.01 mg/kg. At the 3-min point, the
number displayed on the meter stops, the plunger drive
block is retracted to its original position, as preset by switch
S2, and a baseline re-equilibration period equal to the injec-
tion period elapses before a ready light and a beeper indi-
cate that a new sample may be injected. Repeat the Calibra-
tion step a total of at least four times.
Procedure Rinse the syringe several times with sample;
then fill it, clamp it onto the constant-rate injector, push the
sliding carriage forward to penetrate the septum with the
needle, and zero the meter. Turn on switch S1 to start the
stepper-motor syringe drive automatically and initiate the
analysis cycle. After the 3-min hold point, the number dis-
played on the meter corresponds to the sulfur content of
the injected sample.
FCC 8 General Tests and Assays / Appendix III / 1297

General Tests and Assays

Figure 21. Raney Nickel Reduction Apparatus
 1298 / Appendix IV / General Tests and Assays
APPENDIX IV: CHEWING GUM BASE POLYMERS
BOUND STYRENE
Abbé-Type Refractometer Use an instrument with
fourth decimal place accuracy that can be placed in a nearly
horizontal position for measurement of the refractive index
of solids. An Amici-type compensating prism for achroma-
tization is necessary unless a sodium vapor lamp is used as a
light source.
Ethanol–Toluene Azeotrope Mix 70 volumes of etha-
nol or of formula 2B ethanol with 30 volumes of toluene,
reflux for 4 h over calcium oxide, and then distill, discarding
the first and last portions and collecting only that portion
distilling within a range of 1°.
[NOTE—Refluxing and distilling are not necessary if an-
hydrous 2B ethanol or absolute grain alcohol is used.]
Sample Preparation Sheet out a sample of the polymer
to a thickness of 0.5 mm, and cut the sheeted sample into
strips approximately 13 mm wide and 25 mm long. Fasten
one strip to each leg of a “spider,” consisting of a 13-mm
square of sheet aluminum or stainless steel having a
Nichrome wire leg about 38 mm long attached to each
corner. Place the spider and strips in a 400-mL flask contain-
General Tests and Assays
ing 60 mL of Ethanol–Toluene Azeotrope, positioning the spi-
der so that each sample strip is in contact on all sides with
the solvent. Extract for 1 h at a temperature at which the
solvent boils gently, then replace the solvent with another
60-mL portion of Ethanol–Toluene Azeotrope, and extract for
an additional hour. Remove the spider and sample strips
from the flask, and dry them at 100° to constant weight in
a vacuum oven at a pressure of about 10 mm Hg.
[CAUTION—The samples must be extracted and dried
thoroughly, but avoid overheating, which would cause
plasticization.]
Remove the extracted and dried strips from the spider,
and press the strips between aluminum foil (0.025 to 0.08
mm thick, having good tear strength) at 100° for 3 to 10
min (preferably not more than 5 min), using any suitable
apparatus to produce a uniform thickness not exceeding 0.5
mm. If the pressing is done between flat platens without a
cavity, use a force between about 500 and 1500 lb, increas-
ing the applied force proportionally if several strips are
pressed at one time. If cavity pressing plates are used, close
the platens without applying pressure and preheat for 1
min, then apply a force of about 11 tons for 3 min, remove
the specimens from the press, and allow them to cool.
Procedure Cut the pressed sample in half with sharp
scissors, and peel off one piece of the foil. Cut off a strip
about 6 mm wide and 12 mm long with the scissors so that
one of the narrower ends is freshly cut.
FCC 8
Check the adjustment of the refractometer by means of a
glass test plate pressed firmly against the prism, using a
drop of α-bromo-naphthalene as the contact liquid. The
small light source should be collimated. The best readings
are obtained with the glass test piece if the light is diffused
through crumpled tissue paper. After this adjustment, clean
the prism well with lens paper moistened with alcohol. The
refractive index of the glass test piece and of the test speci-
men must be measured at a known constant temperature,
preferably 25°.
Place the test sample on the prism with the cut edge
toward the light source approximately where the edge of
the glass test piece was positioned. Remove the tissue paper
from the light source, press the specimen firmly on the
prism, and wait at least 1 min for the sample to attain tem-
perature equilibrium. The upper prism may be closed lightly
on the specimen if adequate light can still be focused on
the end of the specimen. Unless the specimen is very thin,
however, this operation can damage the prism or its mount-
ing. Adjust the compensating prism until a sharp dividing
line between light and dark fields with minimum color is
obtained. Test the contact between the specimen and the
prism by pressing the test specimen against the prism:
There should be no change in the position of the boundary
line during this test. Move the hand control from the light
into the dark field until the boundary line just reaches the
cross hairs, and make at least three readings. If the readings
differ by more than 0.0001 refractive index unit, make fur-
ther readings. Repeat the process of obtaining readings with
another portion of the sample having a freshly cut edge,
and average the mean values of the two sets of readings
thus obtained. If the two mean values do not differ by more
than 0.0002 refractive index unit, report the average as the
results of the calculations. If necessary, correct the refractive
index measurements to 25° using the equation
n25 = nt + 0.00037(t − 25),
in which n25 is the refractive index at 25°, and nt is the
refractive index at the temperature, t, of measurement.
Calculate the percentage of bound styrene in emulsion-
polymerized samples by the formula
Result = 23.50 + 1164(n25 − 1.53456) − 3497(n25 −
1.53456)2.
Calculate the percentage of bound styrene in solution-
polymerized samples by the formula
Result = (1212.1212)(n25) − 1838.3636.
Alternatively, the percentage of bound styrene may be de-
termined by reference to suitable tables.
FCC 8
MOLECULAR WEIGHT
Polyethylene
Sample Solutions Dissolve 1 g of sample, accurately
weighed, in 95 mL of tetrahydronaphthalene, filter into a
100-mL volumetric flask, dilute to volume with the solvent,
and mix (Solution 1). Transfer 50.0 mL of Solution 1 into a
tared dish, evaporate on a steam bath for about 1 h, and
then evaporate to dryness by heating in a vacuum oven at
70° for 2 h or to constant weight. Calculate the concentra-
tion, C1, in grams per 100 mL, of Solution 1. Prepare Solu-
tions 2 and 3, respectively, by diluting 5.0-mL and 10.0-mL
portions of Solution 1 to 50.0 mL with the solvent, and then
calculate the concentration of each (C2 and C3, respect-
ively).
Procedure Determine the flow time, in seconds, of the
solvent (t0) and of the three Sample Solutions (t1, t2, and t3,
respectively) in a Cannon-Fenske viscometer immersed in a
constant-temperature bath maintained at 130°. Calculate
the specific viscosity, ηsp, of each Sample Solution by the
formula
Result = (t/t0) − 1,
and then calculate the reduced viscosity of each by the
formula
Result = ηsp/C.
Plot the reduced viscosity of each solution against concen-
tration, and extrapolate to zero concentration to obtain the
intrinsic viscosity, [η]. Finally, calculate the molecular weight
of the polyethylene by the formula
Result = ([η]/K)1/a,
in which K is 5.1 × 10−4, and a is 0.725.
Polyisobutylene (Flory Method)
Sample Solutions Dissolve 1 g of sample, accurately
weighed, in 95 mL of diisobutylene, filter into a 100-mL
volumetric flask, dilute to volume with solvent, and mix (So-
lution 1). Transfer 50.0 mL of Solution 1 into a tared dish,
evaporate on a steam bath for about 1 h, and then evapo-
rate to dryness by heating in a vacuum oven at 70° for 2 h
or to constant weight. Calculate the concentration, C1, in
grams per 100 mL, of Solution 1. Prepare Solutions 2 and 3,
respectively, by diluting 5.0-mL and 10.0-mL portions of So-
lution 1 to 50.0 mL with solvent, and then calculate the
concentration of each (C2 and C3, respectively).
Procedure Determine the flow time, in seconds, of the
solvent (t0) and of the three Sample Solutions (t1, t2, and t3,
respectively) in a Cannon-Fenske viscometer immersed in a
constant-temperature bath maintained at 20°. Calculate the
specific viscosity, ηsp, of each Sample Solution by the formula
Result = (t/t0) − 1,
and then calculate the reduced viscosity of each by the
formula
Result = ηsp/C.
General Tests and Assays / Appendix IV / 1299
Plot the reduced viscosity of each solution against concen-
tration, and extrapolate to zero concentration to obtain the
intrinsic viscosity, [η]. Finally, calculate the molecular weight
of the polyisobutylene by the formula
Result = ([η]/K)1/a,
in which K is 3.60 × 10−4, and a is 0.64.
Polyvinyl Acetate
Sample Solutions Dissolve 1 g of sample, accurately
weighed, in 95 mL of acetone, filter into a 100-mL volumet-
ric flask, dilute to volume with the solvent, and mix (Solution
1). Transfer 50.0 mL of Solution 1 into a tared dish, evapo-
rate on a steam bath for about 1 h, and then evaporate to
dryness by heating in a vacuum oven at 70° for 2 h or to
constant weight. Calculate the concentration, C1, in grams
per 100 mL, of Solution 1. Prepare Solutions 2 and 3, respec-
tively, by diluting 5.0-mL and 10.0-mL portions of Solution 1
to 50.0 mL with solvent, and then calculate the concentra-
tion of each (C2 and C3, respectively).
Procedure Determine the flow time, in seconds, of the
solvent (t0) and of the three Sample Solutions (t1, t2, and t3,
respectively) in a Cannon-Fenske viscometer immersed in a
constant-temperature bath maintained at 25°. Calculate the
specific viscosity, ηsp, of each Sample Solution by the formula
Result = (t/t0) − 1,
and then calculate the reduced viscosity of each by the
formula
General Tests and Assays
Result = ηsp/C.
Plot the reduced viscosity of each solution against concen-
tration, and extrapolate to zero concentration to obtain the
intrinsic viscosity, [η]. Finally, calculate the molecular weight
of the polyvinyl acetate by the formula
Result = ([η]/K)1/a,
in which K is 1.88 × 10−4, and a is 0.69.
QUINONES
Standard Preparations Transfer 25.0 mg of hydroqui-
none into a 100-mL volumetric flask, dissolve in and dilute
to volume with water, and mix. Transfer 1.0-, 2.0-, 3.0-,
4.0-, and 6.0-mL aliquots of this solution into a series of
100-mL volumetric flasks, dilute each to volume with water,
and mix. Transfer 2.0 mL of each of these solutions and 3.0
mL of water into a series of 25-mL graduates, add 0.5 mL of
0.1 N sodium carbonate to each, and continue as directed
under Sample Preparations (below), beginning with “…
shake immediately, then add 1.0 mL of 15% sulfuric
acid….”
 1300 / Appendix IV / General Tests and Assays FCC 8

Sample Preparations Place 30 g of freshly coagulated
and washed sample into a 250-mL two-necked flask, add
100 mL of water, and heat at 66° for 2 h.
[CAUTION—Do not boil.]
Cool to room temperature, and transfer 5.0 mL of the
extract into a 25-mL glass-stoppered graduate. Transfer 5.0
mL of water into a second graduate to serve as the blank.
To each graduate add 1.0 mL of 15% sulfuric acid. To the
graduate containing the sample extract add 0.5 mL of 0.1
N sodium carbonate, shake immediately, and then add 1.0
mL of 15% sulfuric acid.
[NOTE—The elapsed time for this operation should not
exceed 15 s.]
Add to each graduate 1.0 mL of 2,4-dinitrophen-
ylhydrazine solution (dissolve 100 mg of 2,4-dinitrophen-
ylhydrazine in 50 mL of carbonyl-free methanol, add 4 mL
of hydrochloric acid, and dilute to 100 mL with water),
stopper, and heat at 70° in a water bath for 1 h. Cool to
room temperature, then add to each graduate 13 mL of
water and 5.0 mL of benzene, stopper, and shake vigor-
ously. Allow the phases to separate, and pipet 2.0 mL of the
benzene layer from each graduate into corresponding test
tubes containing 10 mL of a 1:100 solution of diethano-
lamine in pyridine. Shake each tube, and allow the color to
develop for 10 min.
Procedure Determine the absorbance of the Sample
Preparation in a 1-cm cell at 620 nm, with a suitable spec-
trophotometer, against the reagent blank. Determine the
General Tests and Assays
with carbon disulfide, and mix. Finally, pipet 25 mL of the
diluted solution into a third 100-mL volumetric flask, dilute
to volume with carbon disulfide, and mix.
AMS–Solvent Solution Place 25 mL of carbon disulfide
into a 100-mL volumetric flask, cap with a serum stopper,
and tare the flask to the nearest 0.1 mg. Using a 50-µL
syringe, inject 15 µL of AMS, and reweigh to obtain the
weight of AMS injected. Dilute to volume with carbon disul-
fide, and mix. Pipet 2 mL of this solution into a second 100-
mL volumetric flask, dilute to volume with carbon disulfide,
and mix. Finally, pipet 25 mL of the diluted solution into a
third 100-mL volumetric flask, dilute to volume, and mix.
Calculate the weight, in grams, of AMS in each milliliter of
the final solution, and record the result as w′ (approximately
7.5 × 10−7).
Sample Preparation
Latex Samples Add, with agitation, 100 mL of the latex
to a mixture consisting of 15 mL of glacial acetic acid and
10 g of sodium chloride in 500 mL of hot water. Coagula-
tion starts almost immediately. When coagulation is com-
plete, collect the coagulum on a coarse filter or cheesecloth,
and wash with 1000 mL of a hot solution prepared with 5.6
g of sodium hydroxide and 1000 mL of water. Wash with
hot water until the wash water is free of alkali, then cut the
coagulum into small pieces, and dry at 105° for 4 h. Con-
tinue as directed under Solid Samples (below), beginning
with “Transfer 1.5 g, accurately weighed….”
Solid Samples Cut a piece approximately 2 in. × 3 in. × 5
in. from the corner of a polymer bale, and pass it through a
cold mill, set at least 1/4 in. open, four times, reversing the

absorbance of each Standard Preparation in the same man-

sample on each pass. Cut the sample into two pieces at
ner. Prepare a Standard Curve by plotting absorbance of
least 1 in. from the edge to expose clean polymer, and then
each Standard Preparation against micrograms of quinone.
dice approximately 2 g of the clean polymer or cut into
From the Standard Curve, read the quantity, in micrograms,
small strips. Transfer 1.5 g, accurately weighed, into a 4-oz
of quinones (as benzoquinone) in the Sample Preparation,
bottle fitted with a polyethylene cap, add 25.0 mL of the
and record the value thus obtained as Q. Calculate the
AMS–Solvent Solution, cap tightly, and agitate on a mechani-
quantity of quinones (as benzoquinone), in parts per mil-
cal shaker until the polymer dissolves.
lion, in the sample by the formula
[NOTE—Some polymers tend to swell and form viscous
Result = 20Q/W, cements instead of dissolving cleanly. If this occurs,
add 5- to 10-mL increments of carbon disulfide to ob-
in which W is the weight, in grams, of the sample taken.
tain a mobile slurry, and in the next step increase the
volume of methanol by a proportional amount.]
RESIDUAL STYRENE Add 25 mL of methanol, cap the bottle, and shake vigor-
ously on the shaker for 30 min. After the contents have
Standard Preparation Place 25 mL of carbon disulfide
settled, decant 10 mL of the coagulant serum into a 1-oz
in a 100-mL volumetric flask, cap with a serum stopper, and
bottle, add 10 mL of water, and stopper with a serum cap.
tare the flask to the nearest 0.1 mg. Using 50-µg syringes,
Shake vigorously for 1 min, then turn the bottle upside
inject 15 µL each of styrene and of alpha-methylstyrene
down, and allow the layers to separate. Withdraw by syringe
(AMS), reweighing after each addition to obtain the weight
1 to 2 mL of the lower (carbon disulfide) layer, and transfer
of each solution injected. Record the weight, in milligrams,
it into a 10-dram vial filled with 1/4 in. of anhydrous so-
of styrene as w1 and that of AMS as w2. Dilute to volume
dium sulfate. Seal with a polyethylene cap, shake to mix,
with carbon disulfide, and mix. Pipet 2 mL of this solution
and allow to settle.
into a second 100-mL volumetric flask, dilute to volume
FCC 8
Procedure (See Chromatography, Appendix IIA.) Inject a
10-µL portion of the Sample Preparation into a suitable gas
chromatograph in which the detector is the hydrogen
flame-ionization type and the column is 10-ft ×
3/16-in. stainless steel tubing, or equivalent, packed with
25% Ucon 50 HB 2000 on 60- to 80-mesh acid-washed
DMCS Chromosorb W, or with equivalent packing materials.
Use nitrogen or helium as the carrier gas, flowing at 40 mL/
min. The injection port temperature is 240°; the column
temperature, 170° isothermal; and the detector tempera-
ture, 250°. Adjust the sensitivity of the instrument to give as
large a signal as possible for styrene and AMS as is consis-
tent with an acceptable background level. Measure the sty-
rene and AMS peaks by any convenient method, recording
the area of the styrene peak as A1 and that of the AMS peak
as A2.
In the same manner, inject a 10-µL portion of the Stan-
dard Preparation into the chromatograph, obtain the chro-
matogram, and record the area of the styrene peak as a1
and that of the AMS peak as a2. Calculate the styrene factor,
F, by the formula
Result = (w1/w2) × (a2/a1).
Calculate the content of residual styrene in the sample
taken, in parts per million, by the formula
Result = (A1/A2) × F × 25 × (w′/W) × 106,
in which W is the weight, in grams, of the sample taken.
SAMPLE SOLUTION FOR ARSENIC LIMIT
TEST
Transfer 1 g of sample, accurately weighed, into a Kjeldahl
flask, rest the open end of the flask in a Kjeldahl fume bulb
attached to a water aspirator, add 5 mL of sulfuric acid and
4 mL of 30% hydrogen peroxide, and digest over a small
flame. (See Caution statement under Arsenic Limit Test, Ap-
pendix IIIB.) Continue adding the peroxide in 2-mL por-
tions, allowing the reaction to subside between additions,
until all organic matter is destroyed, fumes of sulfuric acid
are copiously evolved, and the solution becomes colorless.
Maintain oxidizing conditions at all times during the diges-
tion by adding peroxide whenever the mixture turns brown
or darkens. (The amount of peroxide required to completely
digest the samples will vary, but as much as 200 mL may
be required in some cases, depending on the nature of the
material.) Cool, cautiously add 10 mL of water, again evap-
orate to strong fuming, and cool. Transfer the solution into
an arsine generator flask, wash the Kjeldahl flask and bulb
with water, adding the washings to the generator flask, and
dilute to 35 mL with water.
General Tests and Assays / Appendix IV / 1301
SAMPLE SOLUTION FOR LEAD LIMIT
TEST
Transfer 3.3 g of sample, accurately weighed, into a porce-
lain dish or casserole, heat on a hot plate until completely
charred, then heat in a muffle furnace at 480° for 8 h or
overnight, and cool. Cautiously add 5 mL of nitric acid,
evaporate to dryness on a hot plate, then heat again in the
muffle furnace at 480° for exactly 15 min, and cool. Extract
the ash with two 10-mL portions of water, filtering each
extract into a separator. Leach any insoluble material on the
filter with 6 mL of Ammonium Citrate Solution, 2 mL of Hy-
droxylamine Hydrochloride Solution, and 5 mL of water (see
Lead Limit Test, Appendix IIIB, for preparation of these solu-
tions), adding the filtered washings to the separator. Con-
tinue as directed under Procedure in the Lead Limit Test, Ap-
pendix IIIB, beginning with “Add 2 drops of phenol red TS
to the separator….”
TOTAL UNSATURATION
This method measures total unsaturation in a sample by the
multivariate analysis of Fourier transform infrared spectra. It
correlates the absorbance in the spectral regions corre-
sponding to two major types of unsaturation with their con-
centrations. This is an extension of univariate least squares
analysis that correlates a single band absorbance height or
area with concentration.
Apparatus Use a Fourier transform infrared spectrometer
(FTIR), with its associated computer and peripherals, capable
General Tests and Assays
of measuring from 4500 to 500 cm−1 and of acquiring data
with a resolution of at least 2 cm−1. The optics of the instru-
ment must be sealed and desiccated, or, like the sample
chamber, must be under continuous dry air or nitrogen gas
purge. The spectrometer is equipped with software capable
of multicomponent analysis using the partial least squares
method (PLS-1, or equivalent). This software is commercially
available as an accessory to the spectrometer or as an exter-
nal software package.
Laboratory Press Use a Carver-type press capable of press-
ing polymer films.
Sample Preparation Compression-mold a thin film of
the sample to roughly a 500-µm thickness at 10 tons and
90° for 30 to 60 s. Do not exceed this time or temperature,
as structural changes in unsaturation can occur.
Operating Conditions Collect not less than 64 FTIR
spectral scans of the standards and sample in the absorb-
ance mode. Boxcar apodization and 2 cm−1 resolution are
recommended parameters. Spectral normalization should be
done on the 4333 cm−1 peak to account for varying sample
thicknesses. Use identical operating conditions for the stan-
dards and for the sample.

1302 / Appendix IV / General Tests and Assays
Calibration Assemble a set of at least ten calibration
standards available from the given supplier of food-grade
butyl rubber (such as Exxon Chemical Co.) that covers the
entire unsaturation range expected. Identify characteristic
FTIR spectral regions corresponding to the unsaturation
components by proton magnetic resonance spectroscopy.
These spectral regions may include 1700 to 1600 cm−1 C=C
stretching, 900 to 600 cm−1 vinylic H deformations, and
2200 to 1800 cm−1 overtone regions.
Collect not less than 64 spectral scans of the standards.
Construct a calibration matrix containing infrared absorb-
ance values for unsaturation types in the standards and their
known concentrations. Confirm the validity of the calibra-
tion matrix model as recommended in the software manual.
A recommended method is cross-validation for all standards
by sequentially excluding one of the standards from the cali-
bration matrix, then using the remaining standards to pre-
dict the concentrations. After validation, determine the opti-
mum number of factors, or loading vectors, needed to
General Tests and Assays
Next Page
FCC 8
minimize the deviation between actual and predicted con-
centrations. This determination is automated in most mul-
ticomponent analysis packages. For the highest possible pre-
cision, a calibration for each rubber grade for each
manufacturer is recommended.
Procedure Obtain the FTIR spectra of the sample under
identical sample preparation and operating conditions as de-
scribed above. Determine the amount of unsaturation in the
sample using the same multivariate analysis parameters and
optimal number of factors that were obtained from the cali-
bration matrix. Sum the different unsaturation amounts to
obtain the total unsaturation in the sample.
FCC 8 Solutions and Indicators / Standard Solutions for the Preparation of Controls and Standards / 1393

SOLUTIONS AND INDICATORS

The directions given for the preparation of solutions and
indicators are for guidance; the use of commercially availa-
ble ones is acceptable.
COLORIMETRIC SOLUTIONS (CS)
Colorimetric solutions are used in the preparation of
colorimetric standards for certain chemicals and for the
carbonization tests with sulfuric acid that are specified in
several monographs. Directions for the preparation of the
primary colorimetric solutions and Matching Fluids are given
under the test for Readily Carbonizable Substances, Appendix
IIB. Store the solutions in suitably resistant, tight containers.
Comparison of colors as directed in the Food Chemicals
Codex tests is preferably made in matched color-comparison
tubes or in a suitable colorimeter under conditions that
ensure that the colorimetric reference solution and that of
the specimen under test are treated alike in all respects.
STANDARD BUFFER SOLUTIONS
Reagent Solutions Before mixing, dry the crystalline re-
agents, except the boric acid, at 110° to 120°, and use
water that has been previously boiled and cooled in prepar-
ing the solutions. Store the prepared reagent solutions in
chemically resistant glass or polyethylene bottles, and use
within 3 months. Discard if molding is evident.
Potassium Chloride, 0.2 M Dissolve 14.91 g of potas-
sium chloride (KCl) in sufficient water to make 1000.0 mL.
Potassium Biphthalate, 0.2 M Dissolve 40.84 g of po-
tassium biphthalate [KHC6H4(COO)2] in sufficient water to
make 1000.0 mL.
Potassium Phosphate, Monobasic, 0.2 M Dissolve
27.22 g of monobasic potassium phosphate (KH2PO4) in suf-
ficient water to make 1000.0 mL.
Boric Acid–Potassium Chloride, 0.2 M Dissolve 12.37
g of boric acid (H3BO3) and 14.91 g of potassium chloride
(KCl) in sufficient water to make 1000.0 mL.
Hydrochloric Acid, 0.2 M, and Sodium Hydroxide, 0.2
M Prepare and standardize as directed under Volumetric So-
lutions in this section.
Procedure To prepare 200 mL of a standard buffer solu-
tion having a pH within the range 1.2 to 10.0, place 50.0
mL of the appropriate 0.2 M salt solution, prepared as
above, in a 200-mL volumetric flask, add the volume of 0.2
M hydrochloric acid or of sodium hydroxide specified for
the desired pH in the accompanying table, dilute with water
to volume, and mix.

Composition of Standard Buffer Solutions

Hydrochloric Acid Neutralized Phthalate
Buffer Acid Phthalate Buffer Buffer Phosphate Buffer Alkaline Borate Buffer
To 50.0 mL of 0.2 M To 50.0 mL of 0.2 M To 50.0 mL of 0.2 M To 50.0 mL of 0.2 M To 50.0 mL of 0.2 M
KCl add the mL of HCl KHC6H4(COO)2 add the KHC6H4(COO)2 add the KH2PO4 add the mL of H3BO3-KCl add the mL of
specified mL of HCl specified mL of NaOH specified NaOH specified NaOH specified
0.2 M 0.2 M
0.2 M HCl 0.2 M HCl NaOH NaOH 0.2 M
pH (mL) pH (mL) pH (mL) pH (mL) pH NaOH (mL)

Solutions and Indicators

1.2 85.0 2.2 49.5 4.2 3.0 5.8 3.6 8.0 3.9
1.3 67.2 2.4 42.2 4.4 6.6 6.0 5.6 8.2 6.0
1.4 53.2 2.6 35.4 4.6 11.1 6.2 8.1 8.4 8.6
1.5 41.4 2.8 28.9 4.8 16.5 6.4 11.6 8.6 11.8
1.6 32.4 3.0 22.3 5.0 22.6 6.6 16.4 8.8 15.8
1.7 26.0 3.2 15.7 5.2 28.8 6.8 22.4 9.0 20.8
1.8 20.4 3.4 10.4 5.4 34.1 7.0 29.1 9.2 26.4
1.9 16.2 3.6 6.3 5.6 38.8 7.2 34.7 9.4 32.1
2.0 13.0 3.8 2.9 5.8 42.3 7.4 39.1 9.6 36.9
2.1 10.2 4.0 0.1 — — 7.6 42.4 9.8 40.6
2.2 7.8 — — — — 7.8 44.5 10.0 43.7
8.0 46.1 — —
Dilute all final solutions to 200.0 mL (see Procedure). The standard pH values given in this table are considered to be reproducible to within ±0.02 of the pH
unit specified at 25°.
 1394 / Standard Solutions for the Preparation of Controls and Standards / Solutions and Indicators
STANDARD SOLUTIONS FOR THE
PREPARATION OF CONTROLS AND
STANDARDS
The following solutions are used in tests for impurities that
require the comparison of the color or turbidity produced in
a solution of the test substance with that produced by a
known amount of the impurity in a control. Directions for
the preparation of other standard solutions are given in the
monographs or under the general tests in which they are
required (see also Index).
Ammonium Standard Solution (10 µg NH4 in 1 mL)
Dissolve 296.0 mg of ammonium chloride (NH4Cl) in suffi-
cient water to make 100.0 mL, and mix. Transfer 10.0 mL
of this solution into a 1000-mL volumetric flask, dilute with
water to volume, and mix.
Barium Standard Solution (100 µg Ba in 1 mL) Dissolve
177.9 mg of barium chloride (BaCl2·2H2O) in water in a
1000-mL volumetric flask, dilute with water to volume, and
mix.
Iron Standard Solution (10 µg Fe in 1 mL) Dissolve
702.2 mg of ferrous ammonium sulfate
[Fe(NH4)2(SO4)2·6H2O] in 10 mL of 2 N sulfuric acid in a
100-mL volumetric flask, dilute with water to volume, and
mix. Transfer 10.0 mL of this solution into a 1000-mL volu-
metric flask, add 10 mL of 2 N sulfuric acid, dilute with
water to volume, and mix.
Magnesium Standard Solution (50 µg Mg in 1 mL)
Dissolve 50.0 mg of magnesium metal (Mg) in 1 mL of
hydrochloric acid in a 1000-mL volumetric flask, dilute with
water to volume, and mix.
Phosphate Standard Solution (10 µg PO4 in 1 mL) Dis-
solve 143.3 mg of monobasic potassium phosphate
(KH2PO4) in water in a 100-mL volumetric flask, dilute with
water to volume, and mix. Transfer 10.0 mL of this solution
into a 1000-mL volumetric flask, dilute with water to vol-
ume, and mix.
Solutions and Indicators
TEST SOLUTIONS (TS) AND OTHER
REAGENTS
Certain of the following test solutions are intended for use
as acid–base indicators in volumetric analyses. Such
solutions should be adjusted so that when 0.15 mL of the
indicator solution is added to 25 mL of carbon dioxide-free
water, 0.25 mL of 0.02 N acid or alkali, respectively, will
produce the characteristic color change.
In general, the directive to prepare a solution “fresh”
indicates that the solution is of limited stability and must be
prepared on the day of use.
Acetic Acid TS, Diluted (1 N) A solution containing
about 6% (w/v) of CH3COOH. Prepare by diluting 60.0 mL
of glacial acetic acid, or 166.6 mL of 36% acetic acid (6 N),
with sufficient water to make 1000 mL.
Acetic Acid TS, Strong (5 N) A solution containing 30%
(v/v) of CH3COOH. Prepare by diluting 300.0 mL of glacial
acetic acid with sufficient water to make 1000 mL.
FCC 8
Acetic Periodic Acid TS Dissolve 2.7 g of periodic acid
(H5IO6) in 50 mL of water, add 950 mL of glacial acetic
acid, and mix thoroughly. [CAUTION—This solution is an
oxidizing agent and is dangerous in contact with organic
materials. Do not use cork or rubber stoppers on storage
bottles.]
[NOTE—Store this solution protected from light.]
Alcohol (Ethanol; Ethyl Alcohol; C2H5OH) Use ACS reagent-
grade Ethyl Alcohol (NLT 95.0%, by volume, of C2H5OH).
[NOTE—For use in assays and tests involving ultraviolet
spectrophotometry, use ACS reagent-grade Ethyl Alco-
hol Suitable for Use in Ultraviolet Spectrophotometry.]
Alcohol, Absolute (Anhydrous Alcohol; Dehydrated Alco-
hol) Use ACS reagent-grade Ethyl Alcohol, Absolute (NLT
99.5%, by volume, of C2H5OH).
Alcohol, Diluted A solution containing 41.0%–42.0%, by
weight, corresponding to 48.4%–49.5%, by volume, at
15.56°, of C2H5OH.
Alcohol, 70% (at 15.56°) A 38.6:15 mixture (v/v) of 95%
alcohol and water, having a specific gravity of 0.884 at 25°.
To prepare 100 mL, dilute 73.7 mL of alcohol to 100 mL
with water at 25°.
Alcohol, 80% (at 15.56°) A 45.5:9.5 mixture (v/v) of
95% alcohol and water, having a specific gravity of 0.857 at
25°. To prepare 100 mL, dilute 84.3 mL of alcohol to 100
mL with water at 25°.
Alcohol, 90% (at 15.56°) A 51:3 mixture (v/v) of 95%
alcohol and water, having a specific gravity of 0.827 at 25°.
To prepare 100 mL, dilute 94.8 mL of alcohol to 100 mL
with water at 25°.
Alcohol, Aldehyde-Free Dissolve 2.5 g of lead acetate in
5 mL of water, add the solution to 1000 mL of alcohol
contained in a glass-stoppered bottle, and mix. Dissolve 5 g
of potassium hydroxide in 25 mL of warm alcohol, cool,
and add slowly, without stirring, to the alcoholic solution of
lead acetate. Allow to stand for 1 h, then shake the mixture
vigorously, allow to stand overnight, decant the clear liquid,
and recover the alcohol by distillation. Ethyl Alcohol FCC, Al-
cohol USP, or USSD #3A or #30 may be used. If the titration
of a 250-mL sample of the alcohol by Hydroxylamine Hydro-
chloride TS does not exceed 0.25 mL of 0.5 N alcoholic
potassium hydroxide, the above treatment may be omitted.
Alcoholic Potassium Hydroxide TS See Potassium Hy-
droxide TS, Alcoholic.
Alkaline Cupric Tartrate TS (Fehling’s Solution) See Cu-
pric Tartrate TS, Alkaline.
Alkaline Mercuric Potassium Iodide TS (Nessler’s Rea-
gent) See Mercuric Potassium Iodide TS, Alkaline.
Ammonia–Ammonium Chloride Buffer TS (approxi-
mately pH 10) Dissolve 67.5 g of ammonium chloride
(NH4Cl) in water, add 570 mL of ammonium hydroxide
(28%), and dilute with water to 1000 mL.
Ammonia TS (6 N in NH3) A solution containing
9.5%–10.5% of NH3. Prepare by diluting 400 mL of ammo-
nium hydroxide (28%) with sufficient water to make 1000
mL.
FCC 8 Solutions and Indicators / Test Solutions (TS) and Other Reagents / 1395
Ammonia TS, Stronger (15.2 N in NH3) (Ammonium Hy-
droxide; Stronger Ammonia Water) Use ACS reagent-grade
Ammonium Hydroxide, which is a practically saturated solu-
tion of ammonia in water, containing 28%–30% of NH3.
Ammoniacal Silver Nitrate TS Add 6 N ammonium hy-
droxide, dropwise, to a 1:20 solution of silver nitrate until
the precipitate that first forms is almost, but not entirely,
dissolved. Filter the solution, and place in a dark bottle.
[CAUTION—Ammoniacal Silver Nitrate TS forms explosive
compounds on standing. Do not store this solution, but pre-
pare a fresh quantity for each series of determinations. Neu-
tralize the excess reagent and rinse all glassware with hydro-
chloric acid immediately after completing a test.]
Ammonium Acetate TS Dissolve 10 g of ammonium
acetate (NH4C2H3O2) in sufficient water to make 100 mL.
Ammonium Carbonate TS Dissolve 20 g of ammonium
carbonate and 20 mL of Ammonia TS in sufficient water to
make 100 mL.
Ammonium Chloride TS Dissolve 10.5 g of ammonium
chloride (NH4Cl) in sufficient water to make 100 mL.
Ammonium Molybdate TS Dissolve 6.5 g of finely pow-
dered molybdic acid (85%) in a mixture of 14 mL of water
and 14.5 mL of ammonium hydroxide. Cool the solution,
and add it slowly, with stirring, to a well-cooled mixture of
32 mL of nitric acid and 40 mL of water. Allow to stand for
48 h, and pass through a fine-porosity, sintered-glass cruci-
ble lined at the bottom with a layer of glass wool. This
solution deteriorates upon standing and is unsuitable for use
if, upon the addition of 2 mL of Sodium Phosphate TS to 5
mL of the solution, an abundant yellow precipitate does not
form at once or after slight warming. Store it in the dark. If
a precipitate forms during storage, use only the clear, super-
natant solution.
Ammonium Oxalate TS Dissolve 3.5 g of ammonium
oxalate [(NH4)2C2O4·H2O] in sufficient water to make 100
mL.
Ammonium Sulfanilate TS To 2.5 g of sulfanilic acid
add 15 mL of water and 3 mL of 6 N ammonium hydrox-
ide, and mix. Add, with stirring, more 6 N ammonium hy-
droxide, if necessary, until the acid dissolves, adjust the pH
of the solution to about 4.5 with 2.7 N hydrochloric acid,
using Bromocresol Green TS as an outside indicator, and di-
lute to 25 mL.
Ammonium Sulfide TS Saturate 6 N ammonium hydrox-
ide with hydrogen sulfide (H2S), and add two-thirds of its
volume of 6 N ammonium hydroxide. Residue upon igni-
tion: NMT 0.05%. The solution is not rendered turbid either
by Magnesium Sulfate TS or by Calcium Chloride TS (carbon-
ate). This solution is unsuitable for use if an abundant pre-
cipitate of sulfur is present. Store it in small, well-filled, dark
amber-colored bottles in a cold, dark place.
Ammonium Thiocyanate TS (1 N) Dissolve 8 g of am-
monium thiocyanate (NH4SCN) in sufficient water to make
100 mL.
Anthrone TS Carefully dissolve about 0.1 g of anthrone in
100 g of sulfuric acid. Use a freshly prepared solution.
Antimony Trichloride TS Dissolve 20 g of antimony
trichloride (SbCl3) in chloroform to make 100 mL. Filter if
necessary.
Barium Chloride TS Dissolve 12 g of barium chloride
(BaCl2·2H2O) in sufficient water to make 100 mL.
Barium Diphenylamine Sulfonate TS Dissolve 300 mg
of p-diphenylamine sulfonic acid barium salt in 100 mL of
water.
Barium Hydroxide TS Use a saturated solution of barium
hydroxide in recently boiled water. Use a freshly prepared
solution.
Benedict’s Qualitative Reagent See Cupric Citrate TS,
Alkaline.
Benzidine TS Dissolve 50 mg of benzidine in 10 mL of
glacial acetic acid, dilute with water to 100 mL, and mix.
Bismuth Nitrate TS Reflux 5 g of bismuth nitrate
[Bi(NO3)3·5H2O] with 7.5 mL of nitric acid and 10 mL of
water until dissolved, cool, filter, and dilute with water to
250 mL.
Bromine TS (Bromine Water) Prepare a saturated solution
of bromine by agitating 2–3 mL of bromine (Br2) with 100
mL of cold water in a glass-stoppered bottle, the stopper of
which should be lubricated with petrolatum. Store it in a
cold place protected from light.
Bromocresol Blue TS Use Bromocresol Green TS.
Bromocresol Green TS Dissolve 50 mg of bromocresol
green in 100 mL of alcohol, and filter if necessary.
Bromocresol Purple TS Dissolve 250 mg of bromocresol
purple in 20 mL of 0.05 N sodium hydroxide, and dilute
with water to 250 mL.
Bromophenol Blue TS Dissolve 100 mg of bromophenol
blue in 100 mL of 1:2 alcohol, and filter if necessary.
Bromothymol Blue TS Dissolve 100 mg of bromothymol
blue in 100 mL of 1:2 alcohol, and filter if necessary.
Calcium Chloride TS Dissolve 7.5 g of calcium chloride
(CaCl2·2H2O) in sufficient water to make 100 mL.
Calcium Hydroxide TS A solution containing approxi-
Solutions and Indicators
mately 140 mg of Ca(OH)2 in each 100 mL. To prepare,
add 3 g of calcium hydroxide [Ca(OH)2] to 1000 mL of
water, and agitate the mixture vigorously and repeatedly for
1 h. Allow the excess calcium hydroxide to settle, and de-
cant or draw off the clear, supernatant liquid.
Calcium Sulfate TS A saturated solution of calcium sul-
fate in water.
Carr-Price Reagent See Antimony Trichloride TS.
Ceric Ammonium Nitrate TS Dissolve 6.25 g of ceric
ammonium nitrate [(NH4)2Ce(NO3)6] in 100 mL of 0.25 N
nitric acid. Prepare the solution fresh every third day.
Chlorine TS (Chlorine Water) A saturated solution of chlo-
rine in water. Place the solution in small, completely filled,
light-resistant containers. Chlorine TS, even when kept from
light and air, is apt to deteriorate. Store it in a cold, dark
place. For full strength, prepare this solution fresh.
Chromotropic Acid TS Dissolve 50 mg of chromotropic
acid or its sodium salt in 100 mL of 75% sulfuric acid (made
 1396 / Test Solutions (TS) and Other Reagents / Solutions and Indicators
by cautiously adding 75 mL of 95%–98% sulfuric acid to
33.3 mL of water).
Cobaltous Chloride TS Dissolve 2 g of cobaltous chloride
(CoCl2·6H2O) in 1 mL of hydrochloric acid and sufficient
water to make 100 mL.
Cobalt–Uranyl Acetate TS Dissolve, with warming, 40 g
of uranyl acetate [UO2(C2H3O2)2·2H2O] in a mixture of 30 g
of glacial acetic acid and sufficient water to make 500 mL.
Similarly, prepare a solution containing 200 g of cobaltous
acetate [Co(C2H3O2)2·4H2O] in a mixture of 30 g of glacial
acetic acid and sufficient water to make 500 mL. Mix the
two solutions while still warm, and cool to 20°. Maintain
the temperature at 20° for about 2 h to separate the excess
salts from solution, and then pass through a dry filter.
Congo Red TS Dissolve 500 mg of congo red in a mix-
ture of 10 mL of alcohol and 90 mL of water.
Copper Sulfate TS Dissolve 12.5 g of cupric sulfate in
sufficient water to make 100 mL.
Cresol Red TS Triturate 100 mg of cresol red in a mortar
with 26.2 mL of 0.01 N sodium hydroxide until solution is
complete, then dilute the solution with water to 250 mL.
Cresol Red–Thymol Blue TS Add 15 mL of Thymol Blue
TS to 5 mL of Cresol Red TS, and mix.
Crystal Violet TS Dissolve 100 mg of crystal violet in 10
mL of glacial acetic acid.
Cupric Citrate TS, Alkaline (Benedict’s Qualitative Rea-
gent) With the aid of heat, dissolve 173 g of sodium citrate
(C6H5Na3O7·2H2O) and 117 g of sodium carbonate
(Na2CO3·H2O) in about 700 mL of water, and filter through
paper, if necessary. In a separate container, dissolve 17.3 g
of cupric sulfate (CuSO4·5H2O) in about 100 mL of water,
and slowly add this solution, with constant stirring, to the
first solution. Cool the mixture, dilute to 1000 mL, and mix.
Cupric Nitrate TS Dissolve 2.4 g of cupric nitrate [Cu-
(NO3)2·3H2O] in sufficient water to make 100 mL.
Cupric Sulfate TS Dissolve 12.5 g of cupric sulfate
(CuSO4·5H2O) in sufficient water to make 100 mL, and mix.
Cupric Tartrate TS, Alkaline (Fehling’s Solution) The
Solutions and Indicators
Copper Solution (A): Dissolve 34.66 g of carefully selected,
small crystals of cupric sulfate, CuSO4·5H2O, showing no
trace of efflorescence or of adhering moisture, in sufficient
water to make 500 mL. Store this solution in small, tight
containers. The Alkaline Tartrate Solution (B): Dissolve 173 g
of crystallized potassium sodium tartrate (KNaC4H4O6·4H2O)
and 50 g of sodium hydroxide (NaOH) in sufficient water to
make 500 mL. Store this solution in small, alkali-resistant
containers. For use, mix exactly equal volumes of solutions A
and B at the time required.
Cyanogen Bromide TS Dissolve 5 g of cyanogen bro-
mide in water to make 50 mL.
[CAUTION—Prepare this solution in a hood, as cyano-
gen bromide volatilizes at room temperature, and the
vapor is highly irritating and poisonous.]
FCC 8
Denigès’ Reagent See Mercuric Sulfate TS.
Dichlorophenol–Indophenol TS Warm 100 mg of 2,6-
dichlorophenol–indophenol sodium with 100 mL of water.
Filter and use within 3 days.
2,7-Dihydroxynaphthalene TS Dissolve 100 mg of 2,7-
dihydroxynaphthalene in 1000 mL of sulfuric acid, and al-
low the solution to stand until the initial color disappears. If
the solution is very dark, discard it and prepare a new solu-
tion from a different supply of sulfuric acid. This solution is
stable for approximately 1 month if stored in a dark bottle.
Diphenylamine TS Dissolve 1 g of diphenylamine in 100
mL of sulfuric acid. The solution should be colorless.
Diphenylcarbazone TS Dissolve about 1 g of diphenyl-
carbazone (C13H12N4O) in sufficient alcohol to make 100
mL. Store this solution in a brown bottle.
α,α-Dipyridyl TS Dissolve 100 mg of α,α-dipyridyl
(C10H8N2) in 50 mL of absolute alcohol.
Dithizone TS Dissolve 25.6 mg of dithizone in 100 mL of
alcohol.
Eosin Y TS (adsorption indicator) Dissolve 50 mg of eosin
Y in 10 mL of water.
Eriochrome Black TS Dissolve 200 mg of eriochrome
black T and 2 g of hydroxylamine hydrochloride
(NH2OH·HCl) in sufficient methanol to make 50 mL, and
filter. Store the solution in a light-resistant container and use
within 2 weeks.
p-Ethoxychrysoidin TS Dissolve 50 mg of p-ethox-
ychrysoidin monohydrochloride in a mixture of 25 mL of
water and 25 mL of alcohol, add 3 drops of hydrochloric
acid, stir vigorously, and filter if necessary to obtain a clear
solution.
Fehling’s Solution See Cupric Tartrate TS, Alkaline.
Ferric Ammonium Sulfate TS Dissolve 8 g of ferric am-
monium sulfate [FeNH4(SO4)2·12H2O] in sufficient water to
make 100 mL.
Ferric Chloride TS Dissolve 9 g of ferric chloride
(FeCl3·6H2O) in sufficient water to make 100 mL.
Ferric Chloride TS, Alcoholic Dissolve 100 mg of ferric
chloride (FeCl3·6H2O) in 50 mL of absolute alcohol. Prepare
this solution fresh.
Ferric Sulfate TS, Acid Add 7.5 mL of sulfuric acid to
100 mL of water, and dissolve 80 g of ferrous sulfate in the
mixture with the aid of heat. Mix 7.5 mL of nitric acid and
20 mL of water, warm, and add to this the ferrous sulfate
solution. Concentrate the mixture until, upon the sudden
disengagement of ruddy vapors, the black color of the liq-
uid changes to red. Test for the absence of ferrous iron,
and, if necessary, add a few drops of nitric acid and heat
again. When the solution is cold, add sufficient water to
make 110 mL.
Ferroin TS Dissolve 0.7 g of ferrous sulfate and 1.76 g of
phenanthroline hydrochloride in 70 mL of water. Transfer
the solution to a 100-mL volumetric flask, and dilute with
water to volume. Test the sensitivity of Ferroin TS by adding
0.1 mL of Ferroin TS and 0.15 mL of osmium tetroxide solu-
tion (2.5 g/L of osmium tetroxide in 0.05 M sulfuric acid) to
FCC 8 Solutions and Indicators / Test Solutions (TS) and Other Reagents / 1397
50 mL of 1 M sulfuric acid. Add 0.1 mL of a 0.1 M ammo-
nium cerium (IV) nitrate solution; a color change from red
to light blue should be observed.
Ferrous Sulfate TS Dissolve 8 g of clear crystals of ferrous
sulfate (FeSO4·7H2O) in about 100 mL of recently boiled
and thoroughly cooled water. Prepare this solution fresh.
Formaldehyde TS A solution containing approximately
37.0% (w/v) of HCHO. It may contain methanol to prevent
polymerization.
Fuchsin–Sulfurous Acid TS Dissolve 200 mg of basic
fuchsin in 120 mL of hot water, and allow the solution to
cool. Add a solution of 2 g of anhydrous sodium sulfite in
20 mL of water, and then add 2 mL of hydrochloric acid.
Dilute the solution with water to 200 mL, and allow to
stand for at least 1 h. Prepare this solution fresh.
Hydrochloric Acid (approximately 12 N) Use ACS rea-
gent-grade Hydrochloric Acid (36.5%–38.0% of HCl).
Hydrochloric Acid TS, Diluted (2.7 N) A solution con-
taining 10% (w/v) of HCl. Prepare by diluting 226 mL of
hydrochloric acid (36%) with sufficient water to make 1000
mL.
Hydrogen Peroxide TS A solution containing 2.5–3.5 g
of H2O2 in each 100 mL. It may contain suitable preserva-
tives, totaling not more than 0.05%.
Hydrogen Sulfide TS A saturated solution of hydrogen
sulfide made by passing H2S into cold water. Store it in
small, dark, amber-colored bottles, filled nearly to the top. It
is unsuitable unless it possesses a strong odor of H2S, and
unless it produces at once a copious precipitate of sulfur
when added to an equal volume of Ferric Chloride TS. Store
in a cold, dark place.
Hydroxylamine Hydrochloride TS Dissolve 3.5 g of hy-
droxylamine hydrochloride (NH2OH·HCl) in 95 mL of 60%
alcohol, and add 0.5 mL of a 1:1000 solution of bromophe-
nol blue and 0.5 N alcoholic potassium hydroxide until a
green tint develops in the solution. Then add sufficient 60%
alcohol to make 100 mL.
8-Hydroxyquinoline TS Dissolve 5 g of 8-hydroxyquino-
line (oxine) in sufficient alcohol to make 100 mL.
Indigo Carmine TS (Sodium Indigotindisulfonate TS) Dis-
solve a quantity of sodium indigotindisulfonate, equivalent
to 180 mg of C16H8N2O2(SO3Na)2, in sufficient water to
make 100 mL. Use within 60 days.
Iodine TS Dissolve 14 g of iodine (I2) in a solution of 36 g
of potassium iodide (KI) in 100 mL of water, add 3 drops of
hydrochloric acid, dilute with water to 1000 mL, and mix.
Iodinated Zinc Chloride Dissolve 10 g of potassium io-
dide, KI, and 0.15 g of iodine, I, in 10 mL of water. Add
this solution to 100 mL of a 60% solution of zinc chloride,
ZnCl2, in water (sp. gr. 1.8). Keep a few crystals of iodine in
the solution.
Isopropanol [Isopropyl Alcohol; 2-Propanol; (CH3)2CHOH]
Use ACS reagent-grade Isopropyl Alcohol.
[NOTE—For use in assays and tests involving ultraviolet
spectrophotometry, use ACS reagent-grade Isopropyl
Alcohol Suitable for Use in Ultraviolet
Spectrophotometry.]
Isopropanol, Anhydrous (Dehydrated Isopropanol) Use
isopropanol that has been previously dried by shaking with
anhydrous calcium chloride, followed by filtering.
Lead Acetate TS Dissolve 9.5 g of clear, transparent crys-
tals of lead acetate [Pb(C2H3O2)2·3H2O] in sufficient recently
boiled water to make 100 mL. Store in well-stoppered
bottles.
Lead Subacetate TS Triturate 14 g of lead monoxide
(PbO) to a smooth paste with 10 mL of water, and transfer
the mixture to a bottle, using an additional 10 mL of water
for rinsing. Dissolve 22 g of lead acetate [Pb(C2H3O2)2·3H2O]
in 70 mL of water, and add the solution to the lead oxide
mixture. Shake it vigorously for 5 min, then set it aside,
shaking it frequently during 7 days. Finally, filter, and add
enough recently boiled water through the filter to make 100
mL.
Lead Subacetate TS, Diluted Dilute 3.25 mL of Lead
Subacetate TS with sufficient water, recently boiled and
cooled, to make 100 mL. Store in small, well-fitted, tight
containers.
Litmus TS Digest 25 g of powdered litmus with three suc-
cessive 100-mL portions of boiling alcohol, continuing each
extraction for about 1 h. Filter, wash with alcohol, and dis-
card the alcohol filtrate. Macerate the residue with about 25
mL of cold water for 4 h, filter, and discard the filtrate.
Finally, digest the residue with 125 mL of boiling water for
1 h, cool, and filter.
Magnesia Mixture TS Dissolve 5.5 g of magnesium chlo-
ride (MgCl2·6H2O) and 7 g of ammonium chloride (NH4Cl)
in 65 mL of water, add 35 mL of 6 N ammonium hydrox-
ide, set the mixture aside for a few days in a well-stoppered
bottle, and filter. If the solution is not perfectly clear, filter it
before using.
Magnesium Sulfate TS Dissolve 12 g of crystals of mag-
nesium sulfate (MgSO4·7H2O), selected for freedom from ef-
florescence, in water to make 100 mL.
Malachite Green TS Dissolve 1 g of malachite green oxa-
late in 100 mL of glacial acetic acid.
Mayer’s Reagent See Mercuric–Potassium Iodide TS.
Solutions and Indicators
Mercuric Acetate TS Dissolve 6 g of mercuric acetate
[Hg(C2H3O2)2] in sufficient glacial acetic acid to make 100
mL. Store in tight containers protected from direct sunlight.
Mercuric Chloride TS Dissolve 6.5 g of mercuric chloride
(HgCl2) in water to make 100 mL.
Mercuric–Potassium Iodide TS (Mayer’s Reagent) Dis-
solve 1.358 g of mercuric chloride (HgCl2) in 60 mL of
water. Dissolve 5 g of potassium iodide (KI) in 10 mL of
water. Mix the two solutions, and add water to make 100
mL.
Mercuric–Potassium Iodide TS, Alkaline (Nessler’s Rea-
gent) Dissolve 10 g of potassium iodide (KI) in 10 mL of
water, and add slowly, with stirring, a saturated solution of
mercuric chloride until a slight red precipitate remains un-
dissolved. To this mixture add an ice-cold solution of 30 g of
potassium hydroxide (KOH) in 60 mL of water, then add 1
mL more of the saturated solution of mercuric chloride. Di-
lute with water to 200 mL. Allow the precipitate to settle,
 1398 / Test Solutions (TS) and Other Reagents / Solutions and Indicators
and draw off the clear liquid. A 2-mL portion of this rea-
gent, when added to 100 mL of a 1:300,000 solution of
ammonium chloride in ammonia-free water, instantly pro-
duces a yellow-brown color.
Mercuric Sulfate TS (Denigès’ Reagent) Mix 5 g of yellow
mercuric oxide (HgO) with 40 mL of water, and while stir-
ring, slowly add 20 mL of sulfuric acid, then add another 40
mL of water, and stir until completely dissolved.
Mercurous Nitrate TS Dissolve 15 g of mercurous nitrate
in a mixture of 90 mL of water and 10 mL of 2 N nitric
acid. Store in dark, amber-colored bottles in which a small
globule of mercury has been placed.
Methanol (Methyl Alcohol) Use ACS reagent-grade
Methanol.
Methanol, Anhydrous (Dehydrated Methanol) Use
Methanol.
p-Methylaminophenol Sulfate TS Dissolve 2 g ofp-
methylaminophenol sulfate [(HOC6H4NHCH3)2·H2SO4] in 100
mL of water. To 10 mL of this solution add 90 mL of water
and 20 g of sodium bisulfite. Confirm the suitability of this
solution by the following test: Add 1 mL of the solution to
each of four tubes containing 25 mL of 0.5 N sulfuric acid
and 1 mL of Ammonium Molybdate TS. Add 5 µg of phos-
phate (PO4) to one tube, 10 µg to a second, and 20 µg to a
third, using 0.5 mL, 1.0 mL, and 2.0 mL, respectively, of
Phosphate Standard Solution, and allow to stand for 2 h. The
solutions in the three tubes should show readily perceptible
differences in blue color corresponding to the relative
amounts of phosphate added, and the one to which 5 µg of
phosphate was added should be perceptibly bluer than the
blank.
Methylene Blue TS Dissolve 125 mg of methylene blue
in 100 mL of alcohol, and dilute with alcohol to 250 mL.
Methyl Orange TS Dissolve 100 mg of methyl orange in
100 mL of water, and filter if necessary.
Methyl Red TS Dissolve 100 mg of methyl red in 100 mL
of alcohol, and filter if necessary.
Methyl Red–Methylene Blue TS Add 10 mL of Methyl
Red TS to 10 mL of Methylene Blue TS, and mix.
Solutions and Indicators
Methylrosaniline Chloride TS See Crystal Violet TS.
Methyl Violet TS See Crystal Violet TS.
Millon’s Reagent To 2 mL of mercury in an Erlenmeyer
flask add 20 mL of nitric acid. Shake the flask in a hood to
break the mercury into small globules. After about 10 min
add 35 mL of water, and if a precipitate or crystals appear,
add sufficient 1:5 nitric acid (prepared from nitric acid from
which the oxides have been removed by blowing air
through it until it is colorless) to dissolve the separated
solid. Add a 1:10 solution of sodium hydroxide, dropwise,
with thorough mixing, until the curdy precipitate that forms
after the addition of each drop no longer redissolves but is
dispersed to form a suspension. Add 5 mL more of the di-
lute nitric acid, and mix well. Prepare this solution fresh.
α-Naphtholbenzein TS Dissolve 0.2 g of α-naphtholben-
zein in glacial acetic acid to make 100 mL. Sensitivity: Add
100 mL of freshly boiled and cooled water to 0.2 mL of a
1:1000 solution of α-naphtholbenzein in ethanol, and add
FCC 8
0.1 mL of 0.1 N sodium hydroxide: a green color develops.
Add subsequently 0.2 mL of 0.1 N hydrochloric acid: the
color of the solution changes to yellow-red.
Naphthol Green TS Dissolve 500 mg of naphthol green
B in water to make 1000 mL.
Nessler’s Reagent See Alkaline Mercuric–Potassium Iodide
TS.
Neutral Red TS Dissolve 100 mg of neutral red in 100
mL of 50% alcohol.
Nickel Standard Solution TS (10 mg/kg) Prepare a
0.40% (w/v) solution of analytical reagent-grade nickel chlo-
ride (NiCl2·6H2O) with water. Pipet 1.0 mL of the solution
into a 100-mL volumetric flask, and dilute with water to
volume.
Ninhydrin TS See Triketohydrindene Hydrate TS.
Nitric Acid (approximately 15.7 N) Use ACS reagent-
grade Nitric Acid (69.0%–71.0% of HNO3).
Nitric Acid TS, Diluted (1.7 N) A solution containing
about 10% (w/v) of HNO3. Prepare by diluting 105 mL of
nitric acid (70%) with water to make 1000 mL.
Orthophenanthroline TS Dissolve 150 mg of
orthophenanthroline (C12H8N2·H2O) in 10 mL of a solution
of ferrous sulfate, prepared by dissolving 700 mg of clear
crystals of ferrous sulfate (FeSO4·7H2O) in 100 mL of water.
The ferrous sulfate solution must be prepared immediately
before dissolving the orthophenanthroline. Store the solu-
tion in well-closed containers.
Oxalic Acid TS Dissolve 6.3 g of oxalic acid
(H2C2O4·2H2O) in water to make 100 mL.
Phenol Red TS (Phenolsulfonphthalein TS) Dissolve 100
mg of phenolsulfonphthalein in 100 mL of alcohol, and fil-
ter if necessary.
Phenolphthalein TS Dissolve 1 g of phenolphthalein in
100 mL of alcohol.
Phenolsulfonphthalein TS See Phenol Red TS.
p-Phenylphenol TS On the day of use, dissolve 750 mg
of p-phenylphenol in 50 mL of Sodium Hydroxide TS.
Phosphoric Acid Use ACS reagent-grade Phosphoric Acid
(NLT 85.0% of H3PO4).
Phosphotungstic Acid TS Dissolve 1 g of phosphotung-
stic acid (approximately 24WO3·2H3PO4·48H2O) in water to
make 100 mL.
Picric Acid TS See Trinitrophenol TS.
Potassium Acetate TS Dissolve 10 g of potassium ace-
tate (KC2H3O2) in water to make 100 mL.
Potassium Chromate TS Dissolve 10 g of potassium
chromate (K2CrO4) in water to make 100 mL.
Potassium Dichromate TS Dissolve 7.5 g of potassium
dichromate (K2Cr2O7) in water to make 100 mL.
Potassium Ferricyanide TS (10%) Dissolve 1 g of potas-
sium ferricyanide [K3Fe(CN)6] in 10 mL of water. Prepare
this solution fresh.
Potassium Ferrocyanide TS Dissolve 1 g of potassium
ferrocyanide [K4Fe(CN)6·3H2O] in 10 mL of water. Prepare
this solution fresh.
FCC 8 Solutions and Indicators / Test Solutions (TS) and Other Reagents / 1399
Potassium Hydroxide TS (1 N) Dissolve 6.5 g of potas-
sium hydroxide (KOH) in water to make 100 mL.
Potassium Hydroxide TS, Alcoholic Use 0.5 N Alcoholic
Potassium Hydroxide (see Volumetric Solutions in this section).
Potassium Iodide TS Dissolve 16.5 g of potassium iodide
(KI) in water to make 100 mL. Store in light-resistant
containers.
Potassium Permanganate TS Use 0.1 N Potassium Per-
manganate (see Volumetric Solutions in this section).
Potassium Pyroantimonate TS Dissolve 2 g of potas-
sium pyroantimonate in 95 mL of hot water. Cool quickly,
and add a solution containing 2.5 g of potassium hydroxide
in 50 mL of water and 1 mL of an 8.5:100 solution of so-
dium hydroxide. Allow to stand for 24 h, filter, and dilute
with water to 150 mL.
Potassium Sulfate TS Dissolve 1 g of potassium sulfate
(K2SO4) in sufficient water to make 100 mL.
Quimociac TS Dissolve 70 g of sodium molybdate
(Na2MoO4·2H2O) in 150 mL of water (Solution A). Dissolve
60 g of citric acid in a mixture of 85 mL of nitric acid and
150 mL of water, and cool (Solution B). Gradually add Solu-
tion A to Solution B, with stirring, to produce Solution C.
Dissolve 5.0 mL of natural or synthetic quinoline in a mix-
ture of 35 mL of nitric acid and 100 mL of water (Solution
D). Gradually add Solution D to Solution C, mix well, and
allow to stand overnight. Filter the mixture, add 280 mL of
acetone to the filtrate, dilute with water to 1000 mL, and
mix. Store in a polyethylene bottle.
[CAUTION—This reagent contains acetone. Do not use
it near an open flame. Operations involving heating or
boiling should be conducted in a well-ventilated
hood.]
Quinaldine Red TS Dissolve 100 mg of quinaldine red in
100 mL of glacial acetic acid.
Schiff’s Reagent, Modified Dissolve 200 mg of rosani-
line hydrochloride (C20H20ClN3) in 120 mL of hot water.
Cool, add 2 g of sodium bisulfite (NaHSO3) followed by 2
mL of hydrochloric acid, and dilute with water to 200 mL.
Store in a brown bottle at 15° or lower.
Silver Nitrate TS Use 0.1 N Silver Nitrate (see Volumetric
Solutions in this section).
Sodium Bisulfite TS Dissolve 10 g of sodium bisulfite
(NaHSO3) in water to make 30 mL. Prepare this solution
fresh.
Sodium Bitartrate TS Dissolve 1 g of sodium bitartrate
(NaHC4H4O6·H2O) in water to make 10 mL. Prepare this so-
lution fresh.
Sodium Borate TS Dissolve 2 g of sodium borate
(Na2B4O7·10H2O) in water to make 100 mL.
Sodium Carbonate TS Dissolve 10.6 g of anhydrous so-
dium carbonate (Na2CO3) in water to make 100 mL.
Sodium Cobaltinitrite TS Dissolve 10 g of sodium cobal-
tinitrite [Na3Co(NO2)6] in water to make 50 mL, and filter if
necessary.
Sodium Fluoride TS Dry about 500 mg of sodium fluo-
ride (NaF) at 200° for 4 h. Weigh accurately 222 mg of the
dried sodium fluoride, and dissolve it in sufficient water to
make exactly 100 mL. Transfer 10.0 mL of this solution into
a 1000-mL volumetric flask, dilute with water to volume,
and mix. Each mL of this final solution corresponds to 10 µg
of fluorine (F).
Sodium Hydroxide TS (1 N) Dissolve 4.3 g of sodium
hydroxide (NaOH) in water to make 100 mL.
Sodium Indigotindisulfonate TS See Indigo Carmine TS.
Sodium Nitroferricyanide TS Dissolve 1 g of sodium ni-
troferricyanide [Na2Fe(NO)(CN)5·2H2O] in water to make 20
mL. Prepare this solution fresh.
Sodium Phosphate TS Dissolve 12 g of clear crystals of
dibasic sodium phosphate (Na2HPO4·7H2O) in water to
make 100 mL.
Sodium Sulfide TS Dissolve 1 g of sodium sulfide
(Na2S·9H2O) in water to make 10 mL. Prepare this solution
fresh.
Sodium Tetraphenylborate TS Dissolve 1.2 g of sodium
tetraphenylborate in water to make 200 mL. If necessary,
stir for 5 min with 1 g of freshly prepared hydrous alumi-
num oxide, and filter to clarify.
Sodium Thiosulfate TS Use 0.1 N Sodium Thiosulfate (see
Volumetric Solutions in this section).
Stannous Chloride TS Dissolve 40 g of reagent-grade
stannous chloride dihydrate (SnCl2·2H2O) in 100 mL of hy-
drochloric acid.
Starch TS Mix 1 g of a suitable starch and sufficient cold
water to make a thin paste. Add 20 mL of boiling water,
boil for 1 min with continuous stirring, and cool. Use only
the clear solution. Test the sensitivity of the Starch TS as
follows: Prepare a solution of 50 mg/kg chlorine by diluting
1 mL of a commercial 5% sodium hypochlorite (NaOCl) so-
lution in 1000 mL of water. Combine 5 mL of Starch TS
with 100 mL of water and add 0.5 mL of 0.1 N potassium
iodide. Addition of one drop of the 50 mg/kg chlorine solu-
tion should give a swirl of color where the drop hits. Addi-
tion of 1 mL of 50 mg/kg chlorine solution should give a
Solutions and Indicators
deep blue color throughout the solution. The deep blue
color produced is discharged by addition of 0.05 mL of 0.1
N sodium thiosulfate. Prepare fresh solution when Starch TS
no longer passes the sensitivity test.
Starch Iodide Paste TS Heat 100 mL of water in a 250-
mL beaker to boiling, add a solution of 750 mg of potas-
sium iodide (KI) in 5 mL of water, then add 2 g of zinc
chloride (ZnCl2) dissolved in 10 mL of water, and while the
solution is boiling, add with stirring a smooth suspension of
5 g of potato starch in 30 mL of cold water. Continue to
boil for 2 min, then cool. Store in well-closed containers in a
cool place. This mixture must show a definite blue streak
when a glass rod dipped in a mixture of 1 mL of 0.1 M
sodium nitrite, 500 mL of water, and 10 mL of hydrochloric
acid is streaked on a smear of the paste.
Sulfanilic Acid TS Dissolve 800 mg of sulfanilic acid (p-
NH2C6H4SO3H·H2O) in 100 mL of acetic acid. Store in tight
containers.
 1400 / Test Solutions (TS) and Other Reagents / Solutions and Indicators
Sulfuric Acid (approximately 36 N) Use ACS reagent-
grade Sulfuric Acid (95.0%–98.0% of H2SO4).
Sulfuric Acid TS (95%) Add a quantity of sulfuric acid of
known concentration to sufficient water to adjust the final
concentration to 94.5%–95.5% of H2SO4. Because the acid
concentration may change upon standing or upon intermit-
tent use, the concentration should be checked frequently
and solutions assaying more than 95.5% or less than 94.5%
discarded or adjusted by adding either diluted or fuming
sulfuric acid, as required.
Sulfuric Acid TS, Diluted (2 N) A solution containing
10% (w/v) of H2SO4. Prepare by cautiously adding 57 mL of
sulfuric acid (95%–98%) or Sulfuric Acid TS to about 100 mL
of water, then cool to room temperature, and dilute with
water to 1000 mL.
Tannic Acid TS Dissolve 1 g of tannic acid (tannin) in 1
mL of alcohol, and add water to make 10 mL. Prepare this
solution fresh.
Thymol Blue TS Dissolve 100 mg of thymol blue in 100
mL of alcohol, and filter if necessary.
Thymolphthalein TS Dissolve 100 mg of thymolphtha-
lein in 100 mL of alcohol, and filter if necessary.
Triketohydrindene Hydrate TS (Ninhydrin TS) Dissolve
200 mg of triketohydrindene hydrate (C9H4O3·H2O) in water
to make 100 mL. Prepare this solution fresh.
Trinitrophenol TS (Picric Acid TS) Dissolve the equivalent
of 1 g of anhydrous trinitrophenol in 100 mL of hot water.
Cool the solution, and filter if necessary.
Xylenol Orange TS Dissolve 100 mg of xylenol orange in
100 mL of alcohol.
VOLUMETRIC SOLUTIONS
Normal Solutions A normal solution contains 1 g equiva-
lent weight of the solute per L of solution. The normalities
of solutions used in volumetric determinations are desig-
nated as 1 N, 0.1 N, 0.05 N, etc., in FCC.
Solutions and Indicators
Molar Solutions A molar solution contains 1 g molecular
weight of the solute per L of solution. The molarities of such
solutions are designated as 1 M, 0.1 M, 0.05 M, etc., in
FCC.
Preparation and Methods of Standardization The
details for the preparation and standardization of solutions
used in several normalities are usually given only for the one
most frequently required. Solutions of other normalities are
prepared and standardized in the same general manner as
described. Solutions of lower normalities may be prepared
accurately by making an exact dilution of a stronger solu-
tion, but solutions prepared in this way should be
restandardized before use.
Dilute solutions that are not stable, such as 0.01 N potas-
sium permanganate and sodium thiosulfate, are preferably
prepared by diluting exactly the higher normality with thor-
oughly boiled and cooled water on the same day they are
to be used.
All volumetric solutions should be prepared, standardized,
and used at the standard temperature of 25°, if practicable.
FCC 8
When a titration must be carried out at a markedly different
temperature, the volumetric solution should be standardized
at that same temperature, or a suitable temperature correc-
tion should be made. Because the strength of a standard
solution may change upon standing, the normality or mo-
larity factor should be redetermined frequently.
Although the directions provide only one method of stan-
dardization, other methods of equal or greater accuracy
may be used. For substances available as certified primary
standards, or of comparable quality, the final standard solu-
tion may be prepared by weighing accurately a suitable
quantity of the substance and dissolving it to produce a
specific volume solution of known concentration. Hydro-
chloric and sulfuric acids may be standardized against a cer-
tified primary standard.
In volumetric assays described in FCC, the number of mg
of the test substance equivalent to 1 mL of the primary
volumetric solution is given. In general, these equivalents
may be derived by simple calculation (see also Solutions, in
the General Provisions).
Ammonium Thiocyanate, 0.1 N (7.612 g NH4SCN per
1000 mL): Dissolve about 8 g of ammonium thiocyanate
(NH4SCN) in 1000 mL of water, and standardize by titrating
the solution against 0.1 N Silver Nitrate as follows: transfer
about 30 mL of 0.1 N Silver Nitrate, accurately measured,
into a glass-stoppered flask. Dilute with 50 mL of water,
then add 2 mL of Ferric Ammonium Sulfate TS and 2 mL of
nitric acid, and titrate with the ammonium thiocyanate solu-
tion to the first appearance of a red-brown color. Calculate
the normality, and, if desired, adjust the solution to exactly
0.1 N. If desired, 0.1 N Ammonium Thiocyanate may be re-
placed by 0.1 N potassium thiocyanate where the former is
directed in various tests and assays.
Barium Hydroxide, 0.2 N [17.14 g Ba(OH)2 per 1000
mL]: Dissolve about 36 g of barium hydroxide
[Ba(OH)2·8H2O] in 1 L of recently boiled and cooled water,
and quickly filter the solution. Keep this solution in bottles
with well-fitted rubber stoppers with a soda–lime tube at-
tached to each bottle to protect the solution from carbon
dioxide in the air. Standardize as follows: transfer quantita-
tively about 60 mL of 0.1 N hydrochloric acid, accurately
measured, to a flask; add 2 drops of Phenolphthalein TS; and
slowly titrate with the barium hydroxide solution, with con-
stant stirring, until a permanent pink color is produced. Cal-
culate the normality of the barium hydroxide solution and,
if desired, adjust to exactly 0.2 N with freshly boiled and
cooled water.
[NOTE—Solutions of alkali hydroxides absorb carbon
dioxide when exposed to air. Connect the buret used
for titrations with barium hydroxide solution directly
to the storage bottle, and provide the bottle with a
soda–lime tube so that air entering must pass through
this tube, which will absorb carbon dioxide. Fre-
quently restandardize standard solutions of barium
hydroxide.]
Bromine, 0.1 N (7.990 g Br per 1000 mL): Dissolve 3 g
of potassium bromate (KBrO3) and 15 g of potassium bro-
mide (KBr) in sufficient water to make 1000 mL, and stan-
dardize the solution as follows: transfer about 25 mL of the
solution, accurately measured, into a 500-mL iodine flask,
FCC 8
and dilute with 120 mL of water. Add 5 mL of hydrochloric
acid, stopper the flask, and shake it gently. Then add 5 mL
of Potassium Iodide TS, restopper, shake the mixture, allow it
to stand for 5 min, and titrate the liberated iodine with 0.1
N Sodium Thiosulfate, adding Starch TS near the end of the
titration. Calculate the normality. Store this solution in dark,
amber-colored, glass-stoppered bottles.
Ceric Sulfate, 0.1 N [33.22 g Ce(SO4)2 per 1000 mL]:
Transfer 59 g of ceric ammonium nitrate
[Ce(NO3)4·2NH4NO3·2H2O] to a beaker, add 31 mL of sulfu-
ric acid, mix, and cautiously add water, in 20-mL portions,
until solution is complete. Cover the beaker, let stand over-
night, pass through a sintered-glass crucible of fine porosity,
add water to make 1000 mL, and mix. Alternatively, use the
commercially available volumetric standard solution.
Standardize Ceric Sulfate, 0.1 N as follows: weigh 0.2 g of
sodium oxalate, primary standard, dried according to the
instructions on its label, and dissolve in 75 mL of water.
Add, with stirring, 2 mL of sulfuric acid that has previously
been mixed with 5 mL of water, mix well, add 10 mL of
hydrochloric acid, and heat to 70°–75°. Titrate with Ceric
Sulfate, 0.1 N to a permanent slight yellow color. Each
6.700 mg of sodium oxalate is equivalent to 1 mL of 0.1 N
ceric sulfate.
N = (mg Na2C2O4)/[67.00 × mL Ce(SO4)2 solution]
Ceric Sulfate, 0.01 N [3.322 g Ce(SO4)2 per 1000 mL]:
Dissolve 4.2 g of ceric sulfate [Ce(SO4)2·4H2O] or 5.5 g of
the acid sulfate [Ce(HSO4)4] in about 500 mL of water con-
taining 28 mL of sulfuric acid, and dilute to 1000 mL. Allow
the solution to stand overnight, and filter. Standardize this
solution daily as follows: weigh accurately about 275 mg of
hydroquinone (C6H6O2), dissolve it in sufficient 0.5 N Alco-
holic Sulfuric Acid to make 500.0 mL, and mix. To 25.0 mL
of this solution add 75 mL of 0.5 N sulfuric acid, 20 mL of
water, and 2 drops of Diphenylamine TS. Titrate with the
ceric sulfate solution at a rate of about 25 drops per 10 s
until an endpoint is reached that persists for 10 s. Perform a
blank determination using 100 mL of 0.5 N Alcoholic Sulfuric
Acid, 20 mL of water, and 2 drops of Diphenylamine TS, and
make any necessary correction. Calculate the normality of
the ceric sulfate solution by the formula
Result = 0.05W/55.057V,
in which W is the weight, in mg, of the hydroquinone sam-
ple taken, and V is the volume, in mL, of the ceric sulfate
solution consumed in the titration.
Disodium EDTA, 0.05 M (16.81 g C10H14N2Na2O8 per
1000 mL): Dissolve 18.6 g of disodium ethylenediaminetet-
raacetate (C10H14N2Na2O8·2H2O) in sufficient water to make
1000 mL, and standardize the solution as follows: weigh
accurately about 200 mg of chelometric standard calcium
carbonate (CaCO3), transfer to a 400-mL beaker, add 10 mL
of water, and swirl to form a slurry. Cover the beaker with a
watch glass, and introduce 2 mL of 2.7 N hydrochloric acid
from a pipet inserted between the lip of the beaker and the
edge of the watch glass. Swirl the contents of the beaker to
dissolve the calcium carbonate. Wash down the sides of the
beaker, the outer surface of the pipet, and the watch glass,
Solutions and Indicators / Volumetric Solutions / 1401
and dilute with water to about 100 mL. While stirring, pref-
erably with a magnetic stirrer, add about 30 mL of the diso-
dium EDTA solution from a 50-mL buret, then add 15 mL of
1 N Sodium Hydroxide and 300 mg of Hydroxy Naphthol
Blue Indicator, and continue the titration to a blue
endpoint. Calculate the molarity by the formula
Result = W/100.09V,
in which W is the weight, in mg, of CaCO3 in the sample of
calcium carbonate taken, and V is the volume, in mL, of
disodium EDTA solution consumed. Each 5.004 mg of
CaCO3 is equivalent to 1 mL of 0.05 M Disodium EDTA.
For the determination of aluminum in its salts, use 0.05 M
Disodium EDTA standardized as follows: transfer 2 g, accu-
rately weighed, of aluminum wire to a 1000-mL volumetric
flask, and add 50 mL of a 1:1 mixture of hydrochloric acid
and water. Swirl the flask to ensure complete wetting of the
wire, and allow the reaction to proceed. When dissolution is
complete, dilute with water to volume, and mix. Transfer
10.0 mL of this solution to a 250-mL beaker, add 25.0 mL
of the disodium EDTA solution, boil gently for 5 min, and
cool. Add in the order given, and with continuous stirring,
20 mL of pH 4.5 buffer solution (77.1 g of ammonium ace-
tate and 57 mL of glacial acetic acid in 1000 mL of solu-
tion), 50 mL of alcohol, and 2 mL of Dithizone TS. Titrate
with 0.05 M Zinc Sulfate to a bright rose pink color, and
perform a blank determination, substituting 10 mL of water
for the 10.0 mL of aluminum solution. Each mL of disodium
EDTA solution is equivalent to 1.349 mg of aluminum (Al).
Ferrous Ammonium Sulfate, 0.1 N [39.21 g
Fe(NH4)2(SO4)2·6H2O per 1000 mL]: Dissolve 40 g of fer-
rous ammonium sulfate hexahydrate in a previously cooled
mixture of 40 mL of sulfuric acid and 200 mL of water,
dilute with water to 1000 mL, and mix. On the day of use,
standardize the solution as follows: transfer from 25 to 30
mL of the solution, accurately measured, into a flask, add 2
drops of Orthophenanthroline TS, and titrate with 0.1 N Ceric
Sulfate until the red color is changed to pale blue. From the
volume of 0.1 N Ceric Sulfate consumed, calculate the
normality.
Solutions and Indicators
Hydrochloric Acid, 1 N (36.46 g HCl per 1000 mL): Di-
lute 85 mL of hydrochloric acid with water to make 1000
mL, and standardize the solution as follows: accurately
weigh about 1.5 g of primary standard anhydrous sodium
carbonate (Na2CO3) that has been heated at a temperature
of about 270° for 1 h. Dissolve it in 100 mL of water, and
add 2 drops of Methyl Red TS. Add the acid slowly from a
buret, with constant stirring, until the solution becomes
faintly pink. Heat the solution to boiling, and continue the
titration until the faint pink color is no longer affected by
continued boiling. Calculate the normality. Each 52.99 mg
of Na2CO3 is equivalent to 1 mL of 1 N Hydrochloric Acid.
Hydroxylamine Hydrochloride, 0.5 N (35 g NH2OH·HCl
per 1000 mL): Dissolve 35 g of hydroxylamine hydrochlo-
ride in 150 mL of water, and dilute with anhydrous metha-
nol to 1000 mL. To 500 mL of this solution add 15 mL of a
0.04% solution of bromophenol blue in alcohol, and titrate
with 0.5 N Triethanolamine until the solution appears green-
 1402 / Volumetric Solutions / Solutions and Indicators
blue by transmitted light. Prepare this solution fresh before
each series of analyses.
Iodine, 0.1 N (12.69 g I per 1000 mL): Dissolve about 14
g of iodine (I) in a solution of 36 g of potassium iodide (KI)
in 100 mL of water, add 3 drops of hydrochloric acid, dilute
with water to 1000 mL, and standardize as follows: weigh
accurately about 150 mg of primary standard arsenic triox-
ide (As2O3) previously dried at 105° for 1 h, and dissolve it
in 20 mL of 1 N Sodium Hydroxide by warming if necessary.
Dilute with 40 mL of water, add 2 drops of Methyl Orange
TS, and follow with 2.7 N hydrochloric acid until the yellow
color is changed to pink. Then add 2 g of sodium bicarbo-
nate (NaHCO3), dilute with 50 mL of water, add 3 mL of
Starch TS, and slowly add the iodine solution from a buret
until a permanent blue color is produced. Calculate the nor-
mality. Each 4.946 mg of As2O3 is equivalent to 1 mL of 0.1
N Iodine. Store this solution in glass-stoppered bottles.
Lithium Methoxide, 0.1 N (3.797 g CH3OLi per 1000
mL): Dissolve 600 mg of freshly cut lithium metal in a mix-
ture of 150 mL of anhydrous methanol and 850 mL of ben-
zene. Filter the resulting solution if it is cloudy, and stan-
dardize it as follows: dissolve about 80 mg of benzoic acid
(National Institute of Standards and Technology primary
standard), accurately weighed, in 35 mL of dimethylform-
amide, add 5 drops of Thymol Blue TS, and titrate with the
lithium methoxide solution to a dark blue endpoint.
[CAUTION—Protect the solution from absorption of car-
bon dioxide and moisture by covering the titration
vessel with aluminum foil while dissolving the benzoic
acid sample and during the titration.]
Each mL of 0.1 N Lithium Methoxide is equivalent to 12.21
mg of benzoic acid.
Mercuric Nitrate, 0.1 M [32.46 g Hg(NO3)2 per 1000
mL]: Dissolve about 35 g of mercuric nitrate
[Hg(NO3)2·H2O] in a mixture of 5 mL of nitric acid and 500
mL of water, and dilute with water to 1000 mL. Standardize
the solution as follows: transfer an accurately measured vol-
ume of about 20 mL of the solution into an Erlenmeyer
flask, and add 2 mL of nitric acid and 2 mL of Ferric Ammo-
Solutions and Indicators
nium Sulfate TS. Cool to below 20°, and titrate with 0.1 N
Ammonium Thiocyanate to the first appearance of a perma-
nent brown color. Calculate the molarity.
Oxalic Acid, 0.1 N (4.502 g H2C2O4 per 1000 mL): Dis-
solve 6.45 g of oxalic acid (H2C2O4·2H2O) in sufficient water
to make 1000 mL. Standardize by titration against freshly
standardized 0.1 N Potassium Permanganate as directed
under Potassium Permanganate, 0.1 N. Store this solution in
glass-stoppered bottles, protected from light.
Perchloric Acid, 0.1 N (10.046 g HClO4 per 1000 mL):
Mix 8.5 mL of perchloric acid (70%) with 500 mL of glacial
acetic acid and 30 mL of acetic anhydride.
[CAUTION—Handle perchloric acid in an appropriate
fume hood.]
Cool, and add glacial acetic acid to make 1000 mL. Allow
the prepared solution to stand for 1 day for the excess ace-
tic anhydride to be combined, and determine the water
FCC 8
content by the Karl Fischer Titrimetric Method, Appendix IIB.
If the water content exceeds 0.05%, add more acetic anhy-
dride, but if the solution contains no titratable water, add
sufficient water to make the content between 0.02% and
0.05%. Allow to stand for 1 day, and again determine the
water content by titration. Standardize the solution as fol-
lows: weigh accurately about 700 mg of primary standard
potassium biphthalate [KHC6H4(COO)2], previously dried at
120° for 2 h, and dissolve it in 50 mL of glacial acetic acid
in a 250-mL flask. Add 2 drops of Crystal Violet TS, and
titrate with the perchloric acid solution until the violet color
changes to emerald green. Deduct the volume of the per-
chloric acid consumed by 50 mL of the glacial acetic acid,
and calculate the normality. Each 20.42 mg of
KHC6H4(COO)2 is equivalent to 1 mL of 0.1 N Perchloric
Acid.
Perchloric Acid, 0.1 N, in Dioxane Mix 8.5 mL of per-
chloric acid (70%) with sufficient dioxane, which has been
especially purified by adsorption, to make 1000 mL.
[CAUTION—Handle perchloric acid in an appropriate
fume hood.]
Standardize the solution as follows: weigh accurately about
700 mg of primary standard potassium biphthalate
[KHC6H4(COO)2], previously dried at 105° for 2 h, and dis-
solve in 50 mL of glacial acetic acid in a 250-mL flask. Add
2 drops of Crystal Violet TS, and titrate with the perchloric
acid solution until the violet color changes to blue-green.
Deduct the volume of the perchloric acid consumed by 50
mL of the glacial acetic acid, and calculate the normality.
Each 20.42 mg of KHC6H4(COO)2 is equivalent to 1 mL of
0.1 N Perchloric Acid.
Potassium Acid Phthalate, 0.1 N [20.42 g
KHC6H4(COO)2 per 1000 mL]: Dissolve 20.42 g of primary
standard potassium biphthalate [KHC6H4(COO)2], previously
dried at 105° for 2 h, in glacial acetic acid in a 1000-mL
volumetric flask, warming on a steam bath if necessary to
effect solution and protecting the solution from contamina-
tion by moisture. Cool to room temperature, dilute with
glacial acetic acid to volume, and mix.
Potassium Dichromate, 0.1 N (4.903 g K2Cr2O7 per
1000 mL): Dissolve about 5 g of potassium dichromate
(K2Cr2O7) in 1000 mL of water, transfer quantitatively 25 mL
of this solution to a 500-mL glass-stoppered flask, add 2 g
of potassium iodide (free from iodate) (KI), dilute with 200
mL of water, add 5 mL of hydrochloric acid, and mix. Allow
to stand for 10 min in a dark place, and titrate the liberated
iodine with 0.1 N Sodium Thiosulfate, adding Starch TS as
the endpoint is approached. Correct for a blank run on the
same quantities of the same reagents, and calculate the
normality.
Potassium Hydroxide, 1 N (56.11 g KOH per 1000 mL):
Prepare and standardize 1 N potassium hydroxide by the
procedure set forth for 1 N Sodium Hydroxide, using 74 g of
the potassium hydroxide (KOH) to prepare the solution.
Each 204.2 mg of KHC6H4(COO)2 is equivalent to 1 mL of 1
N Potassium Hydroxide.
FCC 8
Potassium Hydroxide, 0.5 N, Alcoholic
[CAUTION—The solution may become very hot. Allow
it to cool before adding the aldehyde-free alcohol.]
Dissolve about 35 g of potassium hydroxide (KOH) in 20 mL
of water, and add sufficient aldehyde-free alcohol to make
1000 mL. Allow the solution to stand in a tightly stoppered
bottle for 24 h. Then quickly decant the clear supernatant
liquid into a suitable, tight container, and standardize as
follows: transfer quantitatively 25 mL of 0.5 N hydrochloric
acid into a flask, dilute with 50 mL of water, add 2 drops of
Phenolphthalein TS, and titrate with the alcoholic potassium
hydroxide solution until a permanent, pale pink color is pro-
duced. Calculate the normality. Store this solution in tightly
stoppered bottles protected from light.
Potassium Iodate, 0.05 M (10.70 g KIO3 per 1000 mL):
Dissolve 10.700 g of potassium iodate of primary standard
quality (KIO3), previously dried at 110° to constant weight,
in sufficient water to make 1000.0 mL.
Potassium Permanganate, 0.1 N (3.161 g KMnO4 per
1000 mL): Dissolve about 3.3 g of potassium permanga-
nate (KMnO4) in 1000 mL of water in a flask, and boil the
solution for about 15 min. Stopper the flask, allow it to
stand for at least 2 days, and pass through a fine-porosity,
sintered-glass crucible. If necessary, the bottom of the cruci-
ble may be lined with a pledget of glass wool. Standardize
the solution as follows: weigh accurately about 200 mg of
sodium oxalate of primary standard quality (Na2C2O4), previ-
ously dried at 100° to constant weight, and dissolve it in
250 mL of water. Add 7 mL of sulfuric acid, heat to about
70°, and then slowly add the permanganate solution from a
buret, with constant stirring, until a pale pink color that
persists for 15 s is produced. The temperature at the conclu-
sion of the titration should be not less than 60°. Calculate
the normality. Each 6.700 mg of Na2C2O4 is equivalent to 1
mL of 0.1 N Potassium Permanganate. Potassium permanga-
nate is reduced on contact with organic substances such as
rubber; therefore, the solution must be handled in an appa-
ratus made entirely of glass or other suitably inert material.
Store it in glass-stoppered, amber-colored bottles, and
restandardize frequently.
Silver Nitrate, 0.1 N (16.99 g AgNO3 per 1000 mL):
Dissolve about 17.5 g of silver nitrate (AgNO3) in 1000 mL
of water, and standardize the solution as follows: weigh ac-
curately 100 mg of primary standard sodium chloride, previ-
ously dried at 120° for 16 h, into a 150-mL beaker, and
dissolve it in 5 mL of water. Add 5 mL of 6 N acetic acid, 50
mL of methanol, and 2 or 3 drops of Eosin Y TS, and titrate
with the silver nitrate solution to the endpoint. Calculate the
normality.
Sodium Acetate, 0.1 N (8.203 g CH3COONa per 1000
mL): Dissolve 8.20 g of anhydrous sodium acetate in gla-
cial acetic acid to make 1000 mL, and standardize the solu-
tion as follows: to 25.0 mL of the prepared sodium acetate
solution add 50 mL of glacial acetic acid and 1 mL of α-
Naphtholbenzein TS. Titrate with 0.1 N Perchloric Acid until a
yellow-brown color changes through yellow to green.
[CAUTION—Handle perchloric acid in an appropriate
fume hood.]
Solutions and Indicators / Volumetric Solutions / 1403
Perform a blank determination, and make any necessary
correction. Calculate the normality factor.
Sodium Arsenite, 0.05 N (3.248 g NaAsO2 per 1000
mL): Transfer 2.4725 g of arsenic trioxide, which has been
pulverized and dried at 100° to constant weight, to a 1000-
mL volumetric flask, dissolve it in 20 mL of 1 N Sodium Hy-
droxide, and add 1 N Sulfuric Acid or 1 N Hydrochloric Acid
until the solution is neutral or only slightly acid to litmus.
Add 15 g of sodium bicarbonate, dilute with water to vol-
ume, and mix.
Sodium Hydroxide, 1 N (40.00 g NaOH per 1000 mL):
Dissolve about 40 g of sodium hydroxide (NaOH) in about
1000 mL of carbon dioxide-free water. Shake the mixture
thoroughly, and allow it to stand overnight in a stoppered
bottle. Standardize the clear liquid as follows: transfer about
5 g of primary standard potassium biphthalate
[KHC6H4(COO)2], previously dried at 105° for 2 h and accu-
rately weighed, to a flask, and dissolve it in 75 mL of carbon
dioxide-free water. If the potassium biphthalate is in the
form of large crystals, crush it before drying. To the flask
add 2 drops of Phenolphthalein TS, and titrate with the so-
dium hydroxide solution to a permanent pink color. Calcu-
late the normality. Each 204.2 mg of potassium biphthalate
is equivalent to 1 mL of 1 N Sodium Hydroxide.
[NOTE—Solutions of alkali hydroxides absorb carbon
dioxide when exposed to air. Therefore, store them in
bottles with well-fitted, suitable stoppers provided
with a tube filled with a mixture of sodium hydroxide
and lime so that air entering the container must pass
through this tube, which will absorb the carbon diox-
ide. Frequently restandardize standard solutions of so-
dium hydroxide.]
Sodium Hydroxide, 0.5 N, Alcoholic (22.5 g NaOH per
1000 mL)
[CAUTION—The following solution may become very
hot. Allow it to cool before adding the aldehyde-free
alcohol.)]
Dissolve about 22.5 g of sodium hydroxide (NaOH) in 20
mL of water, and add sufficient aldehyde-free alcohol to
Solutions and Indicators
make 1000 mL. Allow the solution to stand in a tightly stop-
pered bottle for 24 h. Then quickly decant the clear super-
natant liquid into a suitable, tight container, and standard-
ize as follows: Quantitatively transfer 25 mL of 0.5 N
hydrochloric acid into a flask, dilute with 50 mL of water,
add 2 drops of Phenolphthalein TS, and titrate with the alco-
holic sodium hydroxide solution until a permanent, pale
pink color appears. Calculate the normality. Store this solu-
tion in tightly stoppered bottles protected from light.
Sodium Methoxide, 0.1 N, in Pyridine (5.40 g CH3ONa
per 1000 mL): Weigh 14 g of freshly cut sodium metal,
and cut into small cubes. Place about 0.5 mL of anhydrous
methanol in a round-bottom 120-mL flask equipped with a
ground-glass joint, add 1 cube of the sodium metal, and
when the reaction subsides, add the remaining sodium
metal to the flask. Connect a water-cooled condenser to the
flask, and slowly add 100 mL of anhydrous methanol, in
small portions, through the top of the condenser. Regulate
 1404 / Volumetric Solutions / Solutions and Indicators
the addition of the methanol so that the vapors are con-
densed and do not escape through the top of the con-
denser. After addition of the methanol is complete, connect
a drying tube to the top of the condenser, and allow the
solution to cool. Transfer 17.5 mL of this solution (approxi-
mately 6 N) into a 1000-mL volumetric flask containing 70
mL of anhydrous methanol, and dilute with freshly distilled
pyridine to volume. Store preferably in the reservoir of an
automatic buret suitably protected from carbon dioxide and
moisture. Standardize the solution as follows: weigh accu-
rately about 400 mg of primary standard benzoic acid,
transfer it into a 250-mL wide-mouth Erlenmeyer flask, and
dissolve it in 50 mL of freshly distilled pyridine. Add a few
drops of Thymolphthalein TS, and titrate immediately with
the sodium methoxide solution to a blue endpoint. During
the titration, direct a gentle stream of nitrogen into the flask
through a short piece of 6-mm glass tubing fastened near
the tip of the buret. Perform a blank determination (see the
General Provisions), correct for the volume of sodium meth-
oxide solution consumed by the blank, and calculate the
normality. Each 12.21 mg of benzoic acid is equivalent to 1
mL of 0.1 N Sodium Methoxide in Pyridine.
Sodium Methoxide, 0.02 N, in Toluene (1.08 g
CH3ONa per 1000 mL): Weigh 2.5 g of freshly cut sodium
metal, and cut into small cubes. Place about 200 mL of
anhydrous methanol in a 1000-mL volumetric flask, chill in
an ice bath, and add the cubes one at a time to the metha-
nol. When the last cube is dissolved, dilute with toluene to
the mark, and mix. Dilute 100 mL of this solution to 500
mL with toluene, adding small amounts of methanol if
cloudiness results. Standardize the solution as follows: weigh
accurately about 20 mg of primary standard benzoic acid,
transfer it into a 50-mL conical flask, and dissolve it in 25
mL of dimethylformamide. Add 2 drops of a solution of 100
mg of thymol blue in 10 mL of dimethylformamide, and
titrate immediately with the sodium methoxide solution to a
blue endpoint. Titrate a blank solution of dimethylform-
amide in the same manner, correct the volume of sodium
methoxide solution consumed by the blank, and calculate
the normality. Each 2.442 mg of benzoic acid is equivalent
to 1 mL of 0.02 N Sodium Methoxide in Toluene.
Solutions and Indicators
Sodium Thiosulfate, 0.1 N (15.81 g Na2S2O3 per 1000
mL): Dissolve about 26 g of sodium thiosulfate
(Na2S2O3·5H2O) and 200 mg of sodium carbonate (Na2CO3)
in 1000 mL of recently boiled and cooled water. Standard-
ize the solution as follows: weigh accurately about 210 mg
of primary standard potassium dichromate, previously pul-
verized and dried at 120° for 4 h, and dissolve in 100 mL of
water in a 500-mL glass-stoppered flask. Swirl to dissolve
the sample, remove the stopper, and quickly add 2 g of
sodium bicarbonate, 3 g of potassium iodide, and 5 mL of
hydrochloric acid. Stopper the flask, swirl to mix, and let
stand in the dark for 10 min. Rinse the stopper and inner
walls of the flask with water, and titrate the liberated iodine
with the sodium thiosulfate solution until the solution is
only faint yellow. Add Starch TS, and continue the titration
to the discharge of the blue color. Calculate the normality.
Sulfuric Acid, 1 N (49.04 g H2SO4 per 1000 mL): Add
slowly, with stirring, 30 mL of sulfuric acid to about 1020
FCC 8
mL of water, allow to cool to 25°, and standardize by titra-
tion against primary standard sodium carbonate (Na2CO3)
as directed under 1 N Hydrochloric Acid. Each 52.99 mg of
Na2CO3 is equivalent to 1 mL of 1 N Sulfuric Acid.
Sulfuric Acid, Alcoholic, 5 N (245.2 g H2SO4 per 1000
mL): Add cautiously, with stirring, 139 mL of sulfuric acid
to a sufficient quantity of absolute alcohol to make 1000.0
mL.
Sulfuric Acid, Alcoholic, 0.5 N Add cautiously, with stir-
ring, 13.9 mL of sulfuric acid to a sufficient quantity of ab-
solute alcohol to make 1000.0 mL. Alternatively, prepare
this solution by diluting 100.0 mL of 5 N Sulfuric Acid with
absolute alcohol to make 1000.0 mL.
Thorium Nitrate, 0.1 M [48.01 g Th(NO3)4 per 1000
mL]: Weigh accurately 55.21 g of thorium nitrate
[Th(NO3)4·4H2O], dissolve it in water, dilute to 1000.0 mL,
and mix. Standardize the solution as follows: transfer 50.0
mL into a 500-mL volumetric flask, dilute with water to vol-
ume, and mix. Transfer 50.0 mL of the diluted solution into
a 400-mL beaker, add 150 mL of water and 5 mL of hydro-
chloric acid, and heat to boiling. While stirring, add 25 mL
of a saturated solution of oxalic acid, then digest the mix-
ture for 1 h just below the boiling point, and allow to stand
overnight. Decant through Whatman No. 42, or equivalent,
filter paper, and transfer the precipitate to the filter using
about 100 mL of a wash solution consisting of 70 mL of the
saturated oxalic acid solution, 430 mL of water, and 5 mL
of hydrochloric acid. Transfer the precipitate and filter paper
to a tared tall-form porcelain crucible, dry, char the paper,
and ignite at 950° for 1.5 h or to constant weight. Cool in a
desiccator, weigh, and calculate the molarity of the solution
by the formula
Result = 200W/264.04,
in which W is the weight, in g, of thorium oxide obtained.
Triethanolamine, 0.5 N [74 g N(CH2CH2OH)3 per 1000
mL]: Transfer 65 mL (74 g) of 98% triethanolamine into a
1000-mL volumetric flask, dilute with water to volume, stop-
per the flask, and mix thoroughly.
Zinc Sulfate, 0.05 M (8.072 g ZnSO4 per 1000 mL): Dis-
solve about 15 g of zinc sulfate (ZnSO4·7H2O) in sufficient
water to make 1000 mL, and standardize the solution as
follows: dilute about 35 mL, accurately measured, with 75
mL of water, add 5 mL of Ammonia–Ammonium Chloride
Buffer TS and 0.1 mL of Eriochrome Black TS, and titrate with
0.05 M Disodium EDTA until the solution is deep blue. Calcu-
late the molarity.
INDICATORS
The necessary solutions of indicators may be prepared as
directed under Test Solutions (TS) and Other Reagents. The
sodium salts of many indicators are commercially available
and may be used interchangeably in water solutions with
the alcohol solutions specified for the free indicators.
Useful pH indicators, listed in ascending order of the
lower limit of their range, are methyl yellow (pH 2.9 to 4.0),
FCC 8
bromophenol blue (pH 3.0 to 4.6), bromocresol green (pH
4.0 to 5.4), methyl red (pH 4.2 to 6.2), bromocresol purple
(pH 5.2 to 6.8), bromothymol blue (pH 6.0 to 7.6), phenol
red (pH 6.8 to 8.2), thymol blue (pH 8.0 to 9.2), and
thymolphthalein (pH 9.3 to 10.5).
Alphazurine 2G Use a suitable grade.
Azo Violet [4-(p-Nitrophenylazo) Resorcinol] A red powder,
melting at about 193° with decomposition.
Bromocresol Blue Use Bromocresol Green.
Bromocresol Green (Bromocresol Blue; Tetrabromo-m-
cresolsulfonphthalein) A white or pale buff-colored powder;
slightly soluble in water; soluble in alcohol and in solutions
of alkali hydroxides. Transition interval: from pH 3.8 (yellow)
to 5.4 (blue).
Bromocresol Purple (Dibromo-o-cresolsulfonphthalein) A
white to pink, crystalline powder; insoluble in water; soluble
in alcohol and in solutions of alkali hydroxides. Transition
interval: from pH 5.2 (yellow) to 6.8 (purple).
Bromophenol Blue (Tetrabromophenolsulfonphthalein)
Pink crystals, soluble in alcohol. Insoluble in water; soluble
in solutions of alkali hydroxides. Transition interval: from pH
3.0 (yellow) to 4.6 (blue).
Bromothymol Blue (Dibromothymolsulfonphthalein) A rose
red powder. Insoluble in water; soluble in alcohol and in
solutions of alkali hydroxides. Transition interval: from pH
6.0 (yellow) to 7.6 (blue).
Cresol Red (o-Cresolsulfonphthalein) A red-brown powder.
Slightly soluble in water; soluble in alcohol and in dilute
solutions of alkali hydroxides. Transition interval: from pH
7.2 (yellow) to 8.8 (blue).
Crystal Violet (Hexamethyl-p-rosaniline Chloride) Dark
green crystals. Slightly soluble in water; sparingly soluble in
alcohol and in glacial acetic acid. Its solutions are deep
violet.
Sensitiveness Dissolve 100 mg in 100 mL of glacial acetic
acid, and mix. Pipet 1 mL of the solution into a 100-mL
volumetric flask, and dilute with glacial acetic acid to vol-
ume. The solution is violet-blue and does not show a red
tint. Pipet 20 mL of the diluted solution into a beaker, and
titrate with 0.1 N Perchloric Acid, adding the perchloric acid
slowly from a microburet. Not more than 0.1 mL of 0.1 N
Perchloric Acid is required to produce an emerald green
color.
[CAUTION—Handle perchloric acid in an appropriate
fume hood.]
Dithizone (Diphenylthiocarbazone) A blue-black powder.
Insoluble in water; soluble in alcohol and in chloroform,
yielding intensely green solutions even in high dilutions.
Eriochrome Black T [Sodium 1-(1-Hydroxy-2-naphthylazo)-
5-nitro-2-naphthol-4-sulfonate] A brown-black powder hav-
ing a faint metallic sheen. Soluble in alcohol, in methanol,
and in hot water.
Sensitiveness To 10 mL of a 1:200,000 solution in a mix-
ture of equal parts (v/v) of methanol and water add a 1:100
solution of sodium hydroxide until the pH is 10. The solu-
tion is pure blue and free from cloudiness. Add 0.2 mL of
Magnesium Standard Solution (10 µg Mg ion). The color of
Solutions and Indicators / Indicators / 1405
the solution changes to red-violet, and with the continued
addition of magnesium ion, it becomes wine red.
p-Ethoxychrysoidin Monohydrochloride [4-(p-Ethox-
yphenylazo)-m-phenylenediamine Monohydrochloride;
4′-Ethoxy-2,4-diaminoazobenzene Monohydrochloride] A red
powder, insoluble in water. Transition interval: from pH 3.5
(red) to 5.5 (yellow).
Hydroxy Naphthol Blue The disodium salt of 1-(2-
naphtholazo-3,6-disulfonic acid)-2-naphthol-4-sulfonic acid
deposited on crystals of sodium chloride. Small blue crystals,
freely soluble in water. In the pH range between 12 and 13,
its solution is red-pink in the presence of calcium ion and
deep blue in the presence of excess disodium EDTA.
Suitability for Calcium Determinations Dissolve 300 mg in
100 mL of water, add 10 mL of 1 N Sodium Hydroxide and
1.0 mL of a 1:200 calcium chloride solution, and dilute with
water to 165 mL. The solution is red-pink. Add 1.0 mL of
0.05 M Disodium EDTA. The solution becomes deep blue.
Litmus A blue powder, cubes, or pieces. Partly soluble in
water and in alcohol. Transition interval: from approximately
pH 4.5 (red) to 8 (blue). Litmus is unsuitable for determin-
ing the pH of solutions of carbonates or bicarbonates.
Methylene Blue [3,7-Bis(dimethylamino)phenazathionium
Chloride] Dark green crystals or a crystalline powder having
a bronzelike luster. Soluble in water and in chloroform; spar-
ingly soluble in alcohol.
Methyl Orange (Helianthin; Tropaeolin D; 4′-Dimethylami-
noazobenzene-4-sodium Sulfonate) An orange-yellow pow-
der or crystalline scales. Slightly soluble in cold water; read-
ily soluble in hot water; insoluble in alcohol. Transition
interval: from pH 3.2 (pink) to 4.4 (yellow).
Methyl Red (o-Carboxybenzeneazodimethylaniline Hydrochlo-
ride) A dark red powder or violet crystals. Sparingly soluble
in water; soluble in alcohol. Transition interval: from pH 4.2
(red) to 6.2 (yellow).
Methyl Red Sodium The sodium salt of o-carbox-
ybenzeneazo-dimethylaniline. An orange-brown powder.
Freely soluble in cold water and in alcohol. Transition inter-
val: from pH 4.2 (red) to 6.2 (yellow).
Solutions and Indicators
Methyl Yellow (p-Dimethylaminoazobenzene) Yellow crys-
tals, melting between 114° and 117°. Insoluble in water;
soluble in alcohol, in benzene, in chloroform, in ether, in
dilute mineral acids, and in oils. Transition interval: from pH
2.9 (red) to 4.0 (yellow).
Murexide Indicator Preparation Add 400 mg of mu-
rexide to 40 g of powdered potassium sulfate (K2SO4), and
grind in a glass mortar to a homogeneous mixture. Alterna-
tively, use tablets containing 0.4 mg of murexide admixed
with potassium sulfate or potassium chloride, available
commercially.
Naphthol Green B The ferric salt of 6-sodium sulfo-1-
isonitroso-1,2-naphthoquinone. A dark green powder, insol-
uble in water.
Neutral Red (3-Amino-7-dimethylamino-2-methylphenazine
Chloride) A coarse, red to olive green powder. Sparingly
soluble in water and in alcohol. Transition interval: from pH
6.8 (red) to 8.0 (orange).
 1406 / Indicators / Solutions and Indicators
Phenol Red (Phenolsulfonphthalein) A bright to dark red,
crystalline powder. Very slightly soluble in water; sparingly
soluble in alcohol; soluble in solutions of alkali hydroxides.
Transition interval: from pH 6.8 (yellow) to 8.2 (red).
Phenolphthalein White or yellow-white crystals. Practi-
cally insoluble in water; soluble in alcohol and in solutions
of alkali hydroxides. Transition interval: from pH 8.0 (color-
less) to 10.0 (red).
Quinaldine Red (5-Dimethylamino-2-strylethylquinolinium Io-
dide) A dark, blue-black powder, melting at about 260°
with decomposition. Sparingly soluble in water; freely solu-
ble in alcohol. Transition interval: from pH 1.4 (colorless) to
3.2 (red).
Thymol Blue (Thymolsulfonphthalein) A dark, brown-
green, crystalline powder. Slightly soluble in water; soluble
in alcohol and in dilute alkali solutions. Acid transition inter-
val: from pH 1.2 (red) to 2.8 (yellow). Alkaline transition
interval: from pH 8.0 (yellow) to 9.2 (blue).
Thymolphthalein A white to slightly yellow, crystalline
powder. Insoluble in water; soluble in alcohol and in solu-
tions of alkali hydroxides. Transition interval: from pH 9.3
(colorless) to 10.5 (blue).
Xylenol Orange [3,3′-Bis-di(carboxymethyl)aminomethyl-o-
cresolsulfonphthalein] An orange powder. Soluble in water
and in alcohol. In acid solution it is lemon yellow, and its
metal complexes are intensely red. It gives a distinct
endpoint in the direct EDTA titration of metals such as bis-
muth, thorium, scandium, lead, zinc, lanthanum, cadmium,
and mercury.
INDICATOR PAPERS AND TEST PAPERS
Indicator papers and test papers are strips of paper of
suitable dimension and grade (usually Swedish O filter paper
or other makes of like surface, quality, and ash)
impregnated with a sufficiently stable indicator solution or
reagent.
Treat strong, white filter paper with hydrochloric acid,
Solutions and Indicators
and wash with water until the last washing shows no acid
reaction to Methyl Red TS. Then treat with 6 N ammonium
hydroxide, wash again with water until the last washing is
not alkaline toward Phenolphthalein TS, and dry thoroughly.
Saturate the dry paper with the appropriate indicator
solution prepared as directed below, and dry carefully by
suspending from glass rods or other inert material in still air
free from acid, alkali, and other fumes. Cut the paper into
strips of convenient size, and store in well-closed containers
protected from light and moisture.
Indicator papers and test papers that are available
commercially may be used, if desired.
Acetaldehyde Test Paper Use a solution prepared by
mixing equal volumes of a 20% solution of morpholine and
a 5% solution of sodium nitroferricyanide. Saturate the pre-
pared filter paper in the mixture, and use the moistened
paper without drying.
FCC 8
Cupric Sulfate Test Paper Use Cupric Sulfate TS.
Lead Acetate Test Paper Usually about 6 × 80 mm in
size. Use Lead Acetate TS, and dry the paper at 100°, avoid-
ing contact with metal.
Litmus Paper, Blue Usually about 6 × 50 mm in size. It
meets the requirements of the following tests.
Phosphate Place 10 strips in 10 mL of water to which
have been added 1 mL of nitric acid and 0.5 mL of 6 N
ammonium hydroxide. Allow to stand for 10 min, then de-
cant the solution, warm, and add 5 mL of Ammonium Mo-
lybdate TS. Shake at about 40° for 5 min. No precipitate of
phosphomolybdate is formed.
Residue on Ignition Ignite carefully 10 strips of the paper
to constant weight. The weight of the residue corresponds
to not more than 400 µg per strip of about 3 cm2.
Rosins, Acids, etc. Immerse a strip of the blue paper in a
solution of 100 mg of silver nitrate (AgNO3) in 50 mL of
water. The color of the paper does not change in 30 s.
Sensitiveness Drop a 10- to 12-mm strip in 100 mL of
0.0005 N hydrochloric acid contained in a beaker, and stir
continuously. The color of the paper is changed within 45 s.
Litmus Paper, Red Usually about 6 × 50 mm in size. Red
litmus meets the requirements for Phosphate, Residue on Ig-
nition, and Rosins, Acids, etc., under Litmus Paper, Blue.
Sensitiveness Drop a 10- × 12-mm strip into 100 mL of
0.0005 N sodium hydroxide contained in a beaker, and stir
continuously. The color of the paper changes within 30 s.
Phenolphthalein Paper Use a 1:1000 solution of phe-
nolphthalein in 1:2 alcohol.
Starch Iodate Paper Use a mixture of equal volumes of
Starch TS and potassium iodate solution (1:20).
Starch Iodide Paper Use a solution of 500 mg of potas-
sium iodide (KI) in 100 mL of freshly prepared Starch TS.
DETECTOR TUBES
Ammonia Detector Tube A fuse-sealed glass tube
(Draeger or equivalent) that is designed to allow gas to be
passed through it and that contains suitable absorbing filters
and support media for the indicator bromophenol blue. The
Draeger Reference Number is CH 20501; the measuring
range is 5 to 70 ppm.
[NOTE—Suitable detector tubes are available from Na-
tional Draeger, Inc., P.O. Box 120, Pittsburgh, PA
15205-0120. Tubes other than those specified in the
monograph may be used in accordance with the sec-
tion entitled Alternative Analytical Procedures under FCC
Specifications in the General Provisions.]
Carbon Dioxide Detector Tube A fuse-sealed glass tube
(Draeger or equivalent) that is designed to allow gas to be
passed through it and that contains suitable absorbing filters
and support media for the indicators hydrazine and crystal
violet. The Draeger Reference Number is CH 30801; the
measuring range is 0.01% to 0.30%.
Carbon Monoxide Detector Tube A fuse-sealed glass
tube (Draeger or equivalent) that is designed to allow gas to
be passed through it and that contains suitable absorbing
FCC 8
filters and support media for the indicators iodine pentox-
ide, selenium dioxide, and fuming sulfuric acid. The Draeger
Reference Number is CH 25601; the measuring range is 5
to 150 ppm.
Chlorine Detector Tube A fuse-sealed glass tube
(Draeger or equivalent) that is designed to allow gas to be
passed through it and that contains suitable absorbing filters
and support media for the indicator o-toluidine. The
Draeger Reference Number is CH 24301; the measuring
range is 0.2 to 3 ppm.
Hydrogen Sulfide Detector Tube A fuse-sealed glass
tube (Draeger or equivalent) that is designed to allow gas to
be passed through it and that contains suitable absorbing
filters and support media for the indicator, which is a suita-
ble lead salt. The Draeger Reference Number is 6719001;
the measuring range is 1 to 20 ppm.
Nitric Oxide–Nitrogen Dioxide Detector Tube A fuse-
sealed glass tube (Draeger or equivalent) that is designed to
Solutions and Indicators / Detector Tubes / 1407
allow gas to be passed through it and that contains suitable
absorbing filters and support media for an oxidizing layer
and the indicator diphenylbenzidine. The Draeger Reference
Number is CH 29401; the measuring range is 0.5 to 10
ppm.
Sulfur Dioxide Detector Tube A fuse-sealed glass tube
(Draeger or equivalent) that is designed to allow gas to be
passed through it and that contains suitable absorbing filters
and support media for an iodine–starch indicator. The
Draeger Reference Number is CH 31701; the measuring
range is 1 to 25 ppm.
Water Vapor Detector Tube A fuse-sealed glass tube
(Draeger or equivalent) that is designed to allow gas to be
passed through it and that contains suitable absorbing filters
and support media for the indicator, which consists of a
selenium sol in suspension in sulfuric acid. The Draeger Ref-
erence Number is CH 67 28531; the measuring range is 5
to 200 mg/m3.
Solutions and Indicators
FCC 8 General Information / Contents / 1409

General Information
CONTENTS

Validation of Food Chemicals Codex Methods...................................................................................... 1411

Guidelines for Collaborative Study Procedures to Validate Characteristics of a

Method of Analysis................................................................................................................................. 1414

USP Reference Standards for Food Ingredients .................................................................................... 1427

General Information Analytical Techniques .......................................................................................... 1434

Electrophoresis ........................................................................................................................................... 1434
Capillary Electrophoresis............................................................................................................................. 1437
Mass Spectrometry..................................................................................................................................... 1441
Nuclear Magnetic Resonance ..................................................................................................................... 1445
Radioactivity............................................................................................................................................... 1452
Spectrophotometry and Light-Scattering ................................................................................................... 1459
Ion Chromatography.................................................................................................................................. 1465
Near-Infrared Spectroscopy ........................................................................................................................ 1467
Raman Spectroscopy .................................................................................................................................. 1472
Scoville Heat Units ..................................................................................................................................... 1478

Infrared Spectra ......................................................................................................................................... 1479

General Information Tables...................................................................................................................... 1501

Alcoholometric Table.................................................................................................................................. 1501
Atomic Weights.......................................................................................................................................... 1504
Relative Atomic Mass and Half-Lives of Selected Radionuclides .................................................................. 1507
Thermometric Equivalents .......................................................................................................................... 1509

General Good Manufacturing Practices Guidelines for Food Chemicals .......................................... 1511

Food Ingredients: Pharmaceutical Applications and Use of Appropriate GMPs ............................. 1513

FCC in the U.S. Code of Federal Regulations ......................................................................................... 1522

Food Ingredients Fraud Database ........................................................................................................... 1530

General Information
 1410 / Contents / General Information FCC 8
General Information
FCC 8 General Information / Validation of Food Chemicals Codex Methods / 1411
VALIDATION OF FOOD CHEMICALS CODEX METHODS
Submissions to the Food Chemicals Codex
Submissions for new or revised specifications and analyti-
cal methods must contain sufficient information to enable
the Expert Committee to evaluate the proposals. In most
cases, evaluations involve assessing the clarity and complete-
ness of the analytical methods’ description, determining the
need for the methods, and reviewing documentation that
the methods have been appropriately validated. Information
may vary depending on the type of test method involved.
However, in most cases a submission will consist of the fol-
lowing sections:
Rationale
Use this section to identify the need for the analytical
method and describe the capability of the specific
method proposed and why it is preferred over other
types of determinations. For revised analytical methods,
provide a comparison of limitations of the existing FCC
analytical method and advantages offered by the sug-
gested method.
Suggested Analytical Method
Use this section to present a complete description of
the analytical method sufficiently detailed to enable per-
sons “skilled in the art” to replicate it. Include all impor-
tant operational parameters and specific instructions such
as reagent preparation, systems suitability tests perfor-
mance, description of blanks used, precautions, and ex-
plicit formulas for calculating test results.
Data Elements
Use this section to provide thorough and complete
documentation of the validation of the analytical
method. Include summaries of experimental data and
calculations substantiating each of the applicable analyti-
cal performance parameters.
These parameters are described in the following
section.
Validation
Validation of an analytical method is the process of estab-
lishing, by laboratory studies, that the performance charac-
teristics of the method meet the requirements for the in-
tended analytical applications. Express performance
characteristics in terms of analytical parameters. Each of the
recommended parameters is defined in the next section of
this chapter, along with a delineation of a typical method
by which it may be measured.
Typical analytical parameters used in assay validation are
accuracy, precision, specificity, limit of detection, limit of
quantitation, linearity, range, and ruggedness.
Accuracy
Definition
The accuracy of an analytical method is the closeness
of test results obtained by that method to the true value.
Accuracy may often be expressed as percent recovery by
the assay of known, added amounts of analyte.
Determination
Determine the accuracy of an analytical method by
applying that method to samples to which known
amounts of analyte have been added both above and
below the normal levels expected in the samples. Calcu-
late the accuracy from the test results as the percentage
of analyte recovered by the assay.
Precision
Definition
The precision of an analytical method is the degree of
agreement among individual test results when the proce-
dure is applied repeatedly to multiple samplings of a ho-
mogeneous sample. The precision of an analytical
method is usually expressed as the standard deviation or
relative standard deviation (coefficient of variation). Preci-
sion may be a measure of the degree of either reproduc-
ibility or repeatability of the analytical method under nor-
mal operating conditions. In this context, reproducibility
refers to the use of the analytical procedure in different
laboratories. Intermediate precision expresses within-labo-
ratory variation, as on different days, or with different
analysts or equipment within the same laboratory.
Repeatability refers to the use of the analytical procedure
within a laboratory over a short time, using the same
analyst with the same equipment.
Determination
Determine the precision of an analytical method by
assaying a sufficient number of aliquots of a homogene-
ous sample to be able to calculate statistically valid esti-
mates of standard deviation or relative standard deviation
(coefficient of variation). Assays in this context are inde-
pendent analyses of samples that have been carried
through the complete analytical procedure from sample
preparation to final test result.
Specificity
Definition
The specificity of an analytical method is its ability to
measure, both accurately and specifically, the analyte in
the presence of components that may be expected to be
present in the sample matrix. Specificity may often be
expressed as the degree of bias of test results obtained
by analysis of samples containing added impurities, deg-
radation products, or related chemical compounds when
compared with test results from samples without added
substances. The bias may be expressed as the difference
in assay results between the two groups of samples.
Specificity is a measure of the degree of interference (or
absence thereof) in the analysis of complex sample
mixtures.
General Information
Determination
Determine the specificity of an analytical method by
comparing test results obtained from the analysis of sam-
ples containing impurities, degradation products, or re-
lated chemical compounds with those obtained from the
analysis of samples without these elements. The bias of
 1412 / Validation of Food Chemicals Codex Methods / General Information
the assay, if any, is the difference in test results between
the two groups of samples. When impurities or degrada-
tion products are unidentified, demonstrate specificity by
analyzing samples (with the method in question) con-
taining impurities or degradation products and by com-
paring the results to those from additional purity assays
(e.g., chromatographic assay). The degree of agreement
of test results is a measure of the specificity.
Limit of Detection
Definition
The limit of detection is a parameter of limit tests. It is
the lowest concentration of analyte in a sample that can
be detected, but not necessarily quantitated, under the
stated experimental conditions. Thus, limit tests merely
substantiate that the analyte concentration is above or
below a certain level. The limit of detection is usually
expressed as the concentration of analyte (e.g., percent-
age, milligrams per gram, parts per billion) in the
sample.
Determination
Determining the limit of detection of an analytical
method will vary depending on whether it is an instru-
mental or noninstrumental procedure. For instrumental
procedures, different techniques may be used. Some in-
vestigators determine the signal-to-noise ratio by com-
paring test results from samples containing known con-
centrations of analyte with those of blank samples and
establish the minimum level at which the analyte can be
reliably detected. A signal-to-noise ratio of 2:1 or 3:1 is
generally accepted. Other investigators measure the
magnitude of analytical background response by analyz-
ing a number of blank samples and calculating the stan-
dard deviation of this response. The standard deviation,
multiplied by a factor, usually 2 or 3, provides an esti-
mate of the limit of detection. This limit is subsequently
validated by the analysis of a suitable number of samples
known to be close to or at the limit of detection.
For noninstrumental methods, determine the limit of
detection by analyzing samples with known concentra-
tions of analyte and by establishing the minimum level at
which the analyte can reliably be detected.
Limit of Quantitation
Definition
Limit of quantitation is a parameter of quantitative
assays for low levels of compounds in sample matrices,
such as impurities and degradation products in food ad-
ditives and processing aids. It is the lowest concentration
of analyte in a sample that can be determined with ac-
ceptable precision and accuracy under the stated experi-
mental conditions. The limit of quantitation is expressed
as the concentration of analyte (e.g., percentage, milli-
grams per kilogram, parts per billion) in the sample.
General Information
Determination
Determining the limit of quantitation of an analytical
method may vary depending on whether it is an instru-
mental or a noninstrumental procedure. For instrumental
procedures, a common approach is to measure the mag-
nitude of analytical background response by analyzing a
FCC 8
number of blank samples and calculating the standard
deviation of this response. Multiplying the standard
deviation by a factor, usually 10, provides an estimate of
the limit of quantitation. This limit is subsequently vali-
dated by the analysis of a suitable number of samples
known to be close to or at the limit of quantitation.
For noninstrumental methods, determine the limit of
quantitation by analyzing samples having known concen-
trations of analyte and by establishing the minimum level
at which the analyte can be detected with acceptable
accuracy and precision.
Linearity and Range
Definition of Linearity
The linearity of an analytical method is its ability
(within a given range) to elicit test results that are di-
rectly, or by a well-defined mathematical transformation,
proportional to the concentration of analyte in samples
within a given range. Linearity is usually expressed in
terms of the variance around the slope of the regression
line (correlation coefficient), calculated according to an
established mathematical relationship from test results
obtained by the analysis of samples with varying concen-
trations of analyte.
Definition of Range
The range of an analytical method is the interval be-
tween and including the upper and lower levels of
analyte that have been demonstrated to be determined
with precision, accuracy, and linearity using the method
as written. The range is normally expressed in the same
units as test results (e.g., percentage, milligrams per kilo-
gram, parts per billion) obtained by the analytical
method.
Determination of Linearity and Range
Determine the linearity of an analytical method by
mathematically treating test results obtained from analy-
sis of samples with analyte concentrations across the
claimed range of the method. The treatment is normally
a calculation of a regression line by the method of least
squares of test results versus analyte concentrations. In
some cases, to obtain proportionality between assays and
sample concentrations, the test data may have to be sub-
jected to a mathematical transformation before the re-
gression analysis. The slope of the regression line and its
variance (correlation coefficient) provide a mathematical
measure of linearity; the y-intercept is a measure of the
potential assay bias.
Validate the range of the method by verifying that the
analytical method provides acceptable precision, accu-
racy, and linearity when applied to samples containing
analyte at the extremes of the range as well as within the
range.
Ruggedness
Definition
The ruggedness of an analytical method is the degree
of reproducibility of test results obtained by the analysis
FCC 8 General Information / Validation of Food Chemicals Codex Methods / 1413

of the same samples under a variety of normal test con-
ditions, such as different laboratories, analysts, instru-
ments, lots of reagents, elapsed assay times, assay tem-
peratures, and days. Ruggedness is normally expressed as
the lack of influence on test results of operational and
environmental variables of the analytical method. Rug-
gedness is a measure of reproducibility of test results
under normal, expected operational conditions from lab-
oratory to laboratory and from analyst to analyst.
Determination
Determine the ruggedness of an analytical method by
analyzing aliquots from homogeneous lots in different
laboratories, by different analysts, using operational and
environmental conditions that may differ but still are
within the specified parameters of the assay. Determine
the degree of reproducibility of test results as a function
of the assay variables. This reproducibility may be com-
pared to the precision of the assay under normal condi-
tions to obtain a measure of the ruggedness of the ana-
lytical method.
Robustness
The robustness of an analytical method is a measure of
the method‘s capacity to remain unaffected by small, but
deliberate, variations in method parameters, and it provides
an indication of the method‘s reliability during normal use.
Data Elements Required for Assay Validation
FCC assay procedures vary from highly exacting analytical
determinations to subjective evaluation of attributes. Consid-
ering this variety of assays, it is only logical that different
test methods require different validation schemes. This sec-
tion covers only the most common categories of assays for
which validation data should be required.
These categories are as follows:
• Category I: Analytical methods for quantitation of major
components of food additives or processing aids (includ-
ing preservatives).
• Category II: Analytical methods for determination of impu-
rities in food additives or processing aids. These methods
include quantitative assays and limit tests.
• Category III: Analytical methods for determination of per-
formance characteristics (e.g., solubility, melting point).
For each assay category, different analytical information is
needed. In the following table, data elements that are nor-
mally required for each assay category are listed.
Already-established general assays and tests (e.g., titri-
metric method of water determination, identification test)
should also be validated to verify their accuracy (and ab-
sence of possible interference) when used for a new product
or raw material.
The validity of an analytical method can be verified only
by laboratory studies. Therefore, documentation of the suc-
cessful completion of such studies is a basic requirement for
determining whether a method is suitable for its intended
applications.
Appropriate documentation should accompany any pro-
posal for new or revised compendial analytical procedures.

Data Elements Required for Assay Validation

Analytical Performance Parameter Assay Category I Assay Category II Assay Category III
Quantitative Limit Tests
* *
Accuracy Yes Yes
Precision Yes Yes No Yes
*
Specificity No Yes No
*
Limit of Detection Yes Yes Yes
*
Limit of Quantitation No No Yes
*
Linearity Yes Yes No
* *
Range Yes Yes
Ruggedness Yes Yes Yes Yes
May be required, depending on the nature of the specific test.
* General Information
 1414 / Collaborative Study Procedures / General Information
Guidelines for Collaborative Study Procedures To
Validate Characteristics of a Method of Analysis
COPYRIGHT  2005 BY AOAC INTERNATIONAL. REPRINTED WITH PERMISSION.
{NOTE—These guidelines incorporate symbols, terminol-
ogy, and recommendations accepted by consensus by the
participants at the IUPAC Workshop on Harmonization of
Collaborative Analytical Studies, Geneva, Switzerland, May
4–5, 1987 [Pure Appl. Chem. 60, 855–864(1988); published
as “Guidelines for Collaborative Study of Procedure to Vali-
date Characteristics of a Method of Analysis,” J. Assoc. Off.
Anal. Chem. 72, 694–704(1989)]. The original guidelines
were revised at Lisbon, Portugal, August 4, 1993, and at
Delft, The Netherlands, May 9, 1994, Pure Appl. Chem. 67,
331–343(1995). These revised, harmonized guidelines have
been adopted by AOAC INTERNATIONAL as the guidelines
for the AOAC Official Methods Program, J. AOAC Int. 78(5),
143A–160A(1995). Although the directions were developed
for chemical studies, some parts may be applicable to all
types of collaborative studies.}
Summary Statement of AOAC
Recommendation for Design of a
Collaborative Study
Minimum Criteria for Quantitative Study
Minimum number of materials (see Note 1).—Five (only
when a single level specification is involved for a single ma-
trix may this minimum be reduced to 3).
Minimum number of laboratories.—Eight reporting valid
data for each material (only in special cases involving very
expensive equipment or specialized laboratories may the
study be conducted with a minimum of 5 laboratories, with
the resulting expansion in the confidence interval for the
statistical estimates of the method characteristics).
Minimum number of replicates.—One, if within-laboratory
repeatability parameters are not desired; 2, if these parame-
ters are required. Replication should ordinarily be attained
by blind replicates or split levels (Youden pairs).
Minimum Criteria for Qualitative Analyses
Ten laboratories reporting on 2 analyte levels per matrix,
6 test samples per level, and 6 negative controls per matrix.
(NOTE—AOAC criteria for qualitative analyses are not part of
the harmonized guidelines.)
1. Preliminary Work (Within One
Laboratory)
1.1 Determine Purpose and Scope of the Study and
General Information
Method
Determine purpose of the study (e.g., to determine at-
tributes of a method, proficiency of analysts, reference val-
ues of a material, or to compare methods), the type of
method (empirical, screening, practical, reference, defini-
tive), and the probable use of the method (enforcement,
FCC 8
surveillance, monitoring, acceptance testing, quality control,
research). Also, on the basis of the relative importance of
the various method attributes (bias, precision, specificity,
limit of determination), select the design of the collaborative
study. The directions in this document pertain primarily to
determining the precision characteristics of a method, al-
though many sections are also appropriate for other types
of studies.
Alternatives for Method Selection
(1) Sometimes obvious (only method available).
(2) Critical literature review (reported within-laboratory at-
tributes are often optimistic).
(3) Survey of laboratories to obtain candidate methods;
comparison of within-laboratory attributes of candidate
methods (sometimes choice may still not be objective).
(4) Selection by expert [AOAC-preferred procedure (selec-
tion by Study Director with concurrence of General
Referee)].
(5) Selection by Committee (ISO-preferred procedure;
often time-consuming).
(6) Development of new method or modification of ex-
isting method when an appropriate method is not
available. (Proceed as a research project.) (This alterna-
tive is time-consuming and resource-intensive; use only
as a last resort.)
1.2 Optimize Either New or Available Method
Practical Principles
(1) Do not conduct collaborative study with an unop-
timized method. An unsuccessful study wastes a tre-
mendous amount of collaborators’ time and creates ill
will. This applies especially to methods that are formu-
lated by committees and have not been tried in
practice.
(2) Conduct as much experimentation within a single lab-
oratory as possible with respect to optimization, rug-
gedness, and interferences. Analysis of the same mate-
rial on different days provides considerable information
on variability that may be expected in practice.
Alternative Approaches to Optimization
(1) Conduct trials by changing one variable at a time.
(2) Conduct formal ruggedness testing for identification
and control of critical variables. See Youden and Steiner
(pp 33–36, 50–55). The actual procedure is even sim-
pler than it appears. (This is an extremely efficient way
for optimizing a method.)
(3) Use Deming simplex optimization to identify critical
steps. See Dols and Armbrecht. The simplex concept
can be used in the optimization of instrument perfor-
mance and in application to analytical chemical
method development.
FCC 8 General Information / Collaborative Study Procedures / 1415
1.3 Develop Within-Laboratory Attributes of Opti-
mized Method
(Some items can be omitted; others can be combined
depending on whether study is qualitative or quantitative.)
Determine calibration function (response vs concentra-
tion in pure or defined solvent) to determine useful meas-
urement range of method. For some techniques, e.g., im-
munoassay, linearity is not a prerequisite. Indicate any
mathematical transformations needed.
Determine analytical function (response vs concentration
in matrix, including blank) to determine applicability to
commodity(ies) of interest.
Test for interferences (specificity): (1) Test effects of im-
purities, ubiquitous contaminants, flavors, additives, and
other components expected to be present and at usual con-
centrations. (2) Test nonspecific effects of matrices. (3) Test
effects of transformation products, if method is to indicate
stability, and metabolic products, if tissue residues are
involved.
Conduct bias (systematic error) testing by measuring re-
coveries of analyte added to matrices of interest and to ex-
tracts, digests, or other treated solutions thereof. (Not nec-
essary when method defines property or component.)
Develop performance specifications for instruments and
suitability tests for systems (which utilize columns or ad-
sorbents) to ensure satisfactory performance of critical steps
(columns, instruments, etc.) in method.
Conduct precision testing at the concentration levels of
interest, including variation in experimental conditions ex-
pected in routine analysis (ruggedness). In addition to esti-
mating the “classical” repeatability standard deviation, sr, the
initiating laboratory may estimate the total within-laboratory
standard deviation (se) whereby se is the variability at differ-
ent days and with different calibration curves, by the same
or different analysts within a single laboratory. This total
within-laboratory estimate reflects both between-run (be-
tween-batch) and within-run (within-batch) variability.
Delineate the range of applicability to the matrices or
commodities of interest.
Compare the results of the application of the method
with existing, studied methods intended for the same pur-
poses, if other methods are available.
If any of the preliminary estimates of the relevant perfor-
mance of these characteristics are unacceptable, revise the
method to improve them, and re-study as necessary.
Have method tried by analysts not involved in its
development.
Revise method to handle questions raised and problems
encountered.
1.4 Prepare Description of Method
NOTE—A collaborative study of a method involves practi-
cal testing of the written version of the method, in its spe-
cific style and format, by a number of laboratories on identi-
cal materials.
Prepare method description as closely as possible to for-
mat and style that will be used for eventual publication.
Always express reagent concentrations in terms of mass
(or volume) per volume (or mass); never in terms requiring
the analyst to recalculate or look up formula weights, e.g.,
moles. Moles may be used, particularly with volumetric
standards, but only in addition to mass and volume. Many
errors are caused by incorrect recalculation of formula
weights.
Clearly specify requirements for chromatographic materi-
als, enzymes, antibodies, and other performance-related
reagents.
Clearly describe and explain every step in the analytical
method so as to discourage deviations. Use imperative di-
rections; avoid subjunctive and conditional expressions as
options as far as possible.
Clearly describe any safety precautions needed.
Edit method for completeness, credibility (e.g., buffer pH
consistent with specified chemicals, volumes not greater
than capacity of container), continuity, and clarity.
Check for inclusion of performance specifications and sys-
tem suitability tests, defined critical points, and convenient
stopping points. Incorporate physical or chemical constants
of working standards solutions, e.g., absorptivities, half-scale
deflections, recoveries, etc., or properties of operating solu-
tions and chromatographic materials, e.g., pH, volumes, res-
olution, etc., and any other indicators (e.g., sum equals
100%) that suggest analysis is proceeding properly.
If time and resources are available, conduct pilot study
involving 2–3 laboratories.
1.5 Invite Participation
Selection of Collaborators/Candidate Laboratories
Laboratories invited to participate should have person-
nel experienced in the basic techniques employed; expe-
rience with the method itself is not a prerequisite for se-
lection. Lists of possible participants can be developed
through personal contacts, technical societies, trade as-
sociations, or literature search, and advertisements in the
Referee section of AOAC’s magazine, Inside Laboratory
Management. Collaborators are chosen by the organizers
of the collaborative study from a diversity of laboratories
with interest in the method, including regulatory agen-
cies, industry, and universities.
Letter of Invitation
Address a formal letter to the individual responsible
for assignment of laboratory effort. State reason for se-
lecting that laboratory (e.g., as a volunteer or has re-
sponsibility or familiarity with the problem or method),
estimated number of person-hours required for perfor-
mance, number of test samples to be sent, number of
analyses to be required, expected date for test sample
distribution, and target date for completion of the study.
Emphasize the importance of management support in as-
signing the necessary time for the project. Enclose a copy
of the method and a return form or card (with postage
affixed, if appropriate), requiring only a check mark for
acceptance or refusal of the invitation, a signature, space
for address corrections, telephone and fax numbers, e-
mail, and date.
General Information
Laboratory Coordinator
With large studies, involving several analysts per labo-
ratory, several familiarization samples, receipt of items at
different times, or similar recurrent situations, acceptance
of the invitation should be followed by a letter sug-
gesting that a Laboratory Coordinator be appointed. The
 1416 / Collaborative Study Procedures / General Information FCC 8

Laboratory Coordinator should be responsible for receiv-
ing and storing the study materials, assigning the work,
dispensing study materials and information related to the
study, seeing that the method is followed as written, ac-
cumulating the data, assuring that the data are correctly
reported, and submitting the collaborative study manu-
script within the deadline.
which the questions can be designed to reveal if modifica-
tions have been made at critical steps in the method.
Request a copy of the calibration curve or other relation-
ship between response and concentration or amount of
analyte so that if discrepancies become apparent after ex-
amining all of the data, it can be determined whether the
problem is in the calibration or in the analysis.

1.6 Instructions and Report Forms 1.7 Familiarization or Practice Samples

Carefully design and prepare instructions and forms, and If deemed necessary, supply as far ahead as practicable,
scrutinize them before distribution. A pilot study is also use- familiarization samples, with instructions, before actual
ful for uncovering problems in these documents. materials are sent. When familiarization samples have been
Send instructions and report forms immediately on re- submitted, supply forms for reporting progress toward satis-
ceipt of acceptance, independent of study materials, if selec- factory performance.
tion of laboratories is not to be based on performance in
pilot or training studies. The instructions should include in 2. Design of the Collaborative Study
bold face or capital letters a statement:
THIS IS A STUDY OF THE METHOD, NOT OF THE LABO-
2.1 General Principles
RATORY. THE METHOD MUST BE FOLLOWED AS CLOSELY
The purpose of a collaborative study is to determine esti-
AS PRACTICABLE, AND ANY DEVIATIONS FROM THE
mates of the attributes of a method, particularly the “preci-
METHOD AS DESCRIBED, NO MATTER HOW TRIVIAL THEY
sion” of the method that may be expected when the
MAY SEEM, MUST BE NOTED ON THE REPORT FORM.
method is used in actual practice. The AOACI uses 2 terms
Include instructions on storage and handling, markings,
to define the precision of a method under 2 circumstances
and identifications to be noted, any special preparation for
of replication: repeatability and reproducibility. Repeatability
analysis, and criteria for use of practice or familiarization
is a measure of the variation, sr2, between replicate determi-
samples, if included. Pre-code the form for each laboratory
nations by the same analyst. It defines how well an analyst
and provide sufficient space for as much sequential data as
can check himself using the same method on blind repli-
may be required for proper evaluation of the results, includ-
cates of the same material or split levels (Youden pairs),
ing a check of the calculations.
under the same conditions (e.g., same laboratory, same ap-
The initiating laboratory should indicate the number of
paratus, and same time). Reproducibility is a composite
significant figures to be reported, usually based on the out-
measure of variation, sR2, which includes the between-labo-
put of the measuring instrument.
ratory and within-laboratory variations. It measures how well
NOTE—In making statistical calculations from the reported
an analyst in a given laboratory can check the results of
data, the full power of the calculator or computer is to be
another analyst in another laboratory using the same
used with no rounding or truncating until the final reported
method to analyze the same test material under different
mean and standard deviations are achieved. At this point
conditions (e.g., different apparatus and different time). The
the standard deviations are rounded to 2 significant figures
between-laboratory variation represents a systematic error
and the means and relative standard deviations are rounded
that reflects variation arising from environmental conditions
to accommodate the significant figures of the standard
(e.g., condition of reagent and instruments, variation in cali-
deviation. For example, if the reproducibility standard devia-
bration factors, and interpretations of the steps of the
tion sR = 0.012, the mean is reported as 0.147, not as
method) associated with the laboratories used in the study.
0.1473 or 0.15, and RSDR, relative reproducibility standard
Therefore, it is important to identify the causes of the differ-
deviation, is reported as 8.2%. If standard deviation calcula-
ences among laboratories so that they may be controlled.
tions must be conducted manually in steps, with the trans-
Otherwise they will be summed into sR2.
fer of intermediate results, the number of significant figures
Present test samples sent for analysis as unknowns (blind)
to be retained for squared numbers should be at least 2
and coded in a random pattern. If necessary to conserve
times the number of figures in the data plus 1.
analyst time, an indication of the potential range of concen-
When recorder tracing reproductions are required to
tration or amount of analyte may be provided. If spiking
evaluate method performance, request their submission
solutions are used, provide one coded solution for each ma-
both in the instructions and as a check item on the form.
terial. All spiking solutions should be identical in appearance
Provide instructions with regard to labeling of recorder trac-
and volume. Do not provide a single solution from which
ings, such as identification with respect to item analyzed,
aliquots are to be removed for spiking. Any information with
axes, date, submitter, experimental conditions, and instru-
regard to concentration (e.g., utilizing factorial aliquots or
ment settings.
serial dilutions of the same spiking solutions) or known repli-
Include in the report form a signature line for the analyst
cation is likely to lead to an underestimate of the variability.
General Information

and lines for a printed or typed version of the name and

The study must be extensive enough to assure sufficient
address for correct acknowledgement.
data surviving in the face of possible loss of materials during
Provide for a review by the laboratory supervisor. An ex-
shipment, inability of collaborators to participate after ac-
ample of a completed form is helpful. A questionnaire may
ceptance, and a maximum outlier rate of 2/9 and still main-
be included or sent after completion of the analyses in
tain valid data from a minimum of 8 laboratories.
FCC 8 General Information / Collaborative Study Procedures / 1417
Improper preparation of reference standards and stan-
dard solutions can cause a significant portion of the analyti-
cal error. A decision must be made whether such error is to
be considered separately or as part of the method, i.e., will
the analysts procure their own standard solutions or will
standards be provided by the Study Director. The decision
depends primarily on the availability of the standard. If the
standard is readily available, the analysts should prepare
their own. If the standard is not readily available, the stan-
dard may be supplied, but physical constants, e.g., absorp-
tivity of working standard solutions, should be incorporated
into the description as a check on proper preparation of the
solution.
Obtain the necessary administrative and operational ap-
provals. Review by potential users of the method is also
desirable.
2.2 Laboratories
Laboratories must realize the importance of the study. A
large investment is being made in studying the method and
this probably will be only collaborative study of the method
that will performed. Therefore, it is important to have a fair
and thorough evaluation of the method.
Type
The most appropriate laboratory is one with a respon-
sibility related to the analytical problem. Laboratory types
may be representative (selection of laboratories that will
be using the method in practice), reference (assumed to
be “best”), or the entire population of laboratories (usu-
ally certified or accredited) that will be using the
method. Final selection of participants should be based
on a review with the General Referee and others of each
laboratory’s capabilities and past performance in collabo-
rative studies, followed up, if possible, by telephone con-
versations or by personal visits. Selection may also be
based on performance with familiarization samples.
Sometimes only laboratories with dedicated or very spe-
cialized instruments must be used. If the study is in-
tended for international consideration, laboratories from
different countries should be invited to participate.
Number of Laboratories
Minimum of 8 laboratories submitting valid data (to
avoid unduly large confidence bands about the estimated
parameters). Only in special cases of very expensive
equipment or specialized laboratories may the study be
conducted with a minimum of 5 laboratories. Fewer lab-
oratories widen the confidence limits of the mean and of
the variance components (see design considerations). The
optimum number of laboratories, balancing logistics and
costs against information obtained, often is 8–10. How-
ever, larger studies are not discouraged.
For qualitative analyses, a minimum of 10 laboratories
is needed; collaborative study must be designed to in-
clude 2 analyte levels per matrix, 6 test samples per level,
and 6 negative controls per matrix. (NOTE 1—AOAC crite-
ria for qualitative analyses are not part of the harmonized
guidelines.)
Analysts
Most designs require only 1 analyst per laboratory. If
analyst–within-laboratory variability is a desired variance
component, multiple analysts should be requested from
all participating laboratories. Ordinarily 2 analysts from
the same laboratory cannot be substituted for different
laboratories, unless standard solutions, reagents, chro-
matographic columns and/or materials, instrument cali-
brations, standard curves, etc., are prepared indepen-
dently, and no consultation is permitted during the work.
Different laboratories from the same organization may be
used as separate laboratories if they operate indepen-
dently with their own instruments, standards, reagents,
and supervision.
2.3 Test Materials
Homogeneous Materials
Materials must be homogeneous; this is critical. Estab-
lish homogeneity by testing a representative number of
laboratory samples taken at random before shipment. (A
collaborator who reports an outlying value will frequently
claim receipt of a defective laboratory sample.) The pen-
alty for inhomogeneity is an increased variance in the
analytical results that is not due to the intrinsic method
variability.
Test Sample Coding
Code test samples at random so that there is no pre-
selection from order of presentation.
Concentration Range
Choose analyte levels to cover concentration range of
interest. If concentration range of interest is a tolerance
limit or a specification level, bracket it and include it with
materials of appropriate concentration. If design includes
the determination of absence of analyte, include blank
(not detectable) materials as part of range of interest.
Number of Materials
A minimum of 5 materials must be used in the collab-
orative study. Three materials are allowed but only when
a single specification is involved for a single matrix.
NOTE 1—A material is an analyte (or test component)/
matrix/concentration combination to which the method-
performance parameters apply. This parameter deter-
mines the applicability of the method.
NOTE 2—The 2 test samples of blind or open dupli-
cates are a single material (they are not independent).
The 2 test samples constituting a matched pair (called
X and Y) are considered Youden matched pairs only if
they are sufficiently close in composition. “Sufficiently
close” would be considered as ≤5% difference in compo-
sition between X and Y. That is, given that the concen-
tration of analyte in X (xc) is higher than the concentra-
tion of the analyte in Y (yc) then:
(xc − yc)/xc ≤ 0.05
or:
General Information
yc ≥ (xc − 0.05xc)
NOTE 3—The blank or negative control may or may
not be a material, depending on the usual purpose of
the analysis. For example, in trace analysis, where very
low levels (near the limit of quantitation) are often
 1418 / Collaborative Study Procedures / General Information
sought, the blanks are considered as materials, and are
necessary to determine certain statistical “limits of meas-
urement;” however, if the blank is merely a procedural
control, in macro-level analysis (e.g., fat in cheese), it
would not be considered a material.
Nature of Materials
Materials should be representative of commodities
usually analyzed, with customary and extreme values for
the analyte.
Size of Test Samples
Furnish only enough test sample to provide the num-
ber of test portions specified in the instructions. If addi-
tional test portions are required, the collaborator must
request them, with an explanation.
Interferences
If pertinent, some materials, but not all, should con-
tain contaminants and interferences in concentrations
likely to be encountered, unless they have been shown to
be unimportant through within-laboratory study. The
success of the method in handling interference on an
intralaboratory basis will be demonstrated by passing sys-
tems suitability tests.
Familiarization Samples
With new, complex, or unfamiliar techniques, provide
material(s) of stated composition for practice, on differ-
ent days, if possible. The valuable collaborative materials
should not be used until the analyst can reproduce the
stated value of the familiarization samples within a given
range. However, it should be pointed out that one of the
assumptions of analysis of variance is that the underlying
distribution of results is independent of time (i.e., there is
no drift). The Study Director must be satisfied that this
assumption is met.
2.4 Replication
When within-laboratory variability is also of interest, as is
usually the case, independent replication can be ensured by
applying at least one of the following procedures (listed in
suggested order of desirability; the nature of the design
should not be announced beforehand):
(1) Split levels (Youden pairs)—The 2 test materials, nearly
identical but of slightly different composition (e.g.,
≤5% difference in composition, see 2.3 Number of
Materials, Note 2) are obtained either naturally or by
diluting (or by fortifying) one portion of the material
with a small amount of diluent (or of analyte). Both
portions are supplied to the participating laboratories
as test samples, each under a random code number,
and each test sample should be analyzed only once;
replication defeats the purpose of the design.
(2) Split levels for some materials and blind duplicates for
other materials in the same study—Obtain only single
values from each test sample supplied.
(3) Blind duplicate test samples, randomly coded—NOTE—
General Information
Triplicate and higher replication are relatively inefficient
when compared with duplicate test samples because
replication provides additional information only on in-
dividual within-laboratory variability, which is usually
the less important component of error. It is more effec-
tive to utilize resources for the analysis of more levels
FCC 8
and/or materials rather than for increasing the number
of replicates for the individual materials.
PRACTICAL PRINCIPLE: With respect to replication,
the greatest net marginal gain is always obtained in
going from 2 to 3 as compared to going from 3 to 4,
4 to 5, etc.
(4) Independent materials—(NOTE—Unrelated independent
materials may be used as a split level in the calcula-
tions of the precision parameters or for plotting. There
should be ≤5% difference in composition for such
materials (see 2.3 Number of Materials, Note 2). The
more they differ in concentration, the less reliable the
information they provide on within-laboratory
variability.)
(5) Known replicates—Use of known replicates is a common
practice.—It is much preferable to use the same re-
sources on blind replicates or split levels.
(6) Quality control materials—Instead of obtaining
repeatability parameters through the collaborative
study, information can be obtained from use of quality
control materials in each laboratory individually, for its
own use, independent of the collaborative study, for a
separate calculation of sr, using 2 (or more) replicates
from each quality control test, according to the pattern
developed for each product.
2.5 Other Design Considerations
The design can be reduced in the direction of less work
and less cost, but at the sacrifice of reduced confidence in
the reliability of the developed information.
More work (values) is required if more confidence is
needed, e.g., greater confidence is required to enforce a
tolerance at 1.00 mg/kg than at 1.0 mg/kg. (The distinction
is a precision requirement of the order of 1% rather than
10%.)
The estimate of the standard deviation or the corre-
sponding relative standard deviation obtained from a collab-
orative study is a random variable that varies about its corre-
sponding true value. For example, the standard deviation, sr,
which measures within laboratory or repeatability precision
has associated with it a standard deviation (STD = sr)
describing its scatter about the true value σr. Therefore, sr,
whose STD (sr) is a function of sr2, number of laboratories,
and number of analyses per laboratory, will vary about σr
from occasion-to-occasion even for the same test conditions
and material. The STD sR, which measures among laboratory
or reproducibility precision, has a STD (sR) that is a function
of the random variables sr2 and sL2, number of laboratories,
and number of analyses per laboratory. sR will vary about its
true value σR from occasion-to-occasion for the same test
material.
The validity of extrapolating the use of a method beyond
concentrations and components tested can be estimated
only on the basis of the slope of the calibration curve (sensi-
tivity) observed as a function of the nature and concentra-
tion of the matrix and contaminant components. If the sig-
nal is more or less independent of these variables, a
reasonable amount of extrapolation may be utilized. The ex-
trapolator assumes the burden of proof as to what is
reasonable.
FCC 8 General Information / Collaborative Study Procedures / 1419

3. Preparation of Materials for in densities always exists. Fluid materials susceptible to

Collaborative Studies segregation should be prepared under constant agita-
tion. Uniformity should be checked by direct analysis,
with an internal standard, or by a marker compound
3.1 General Principles
(dye or radioactive label).
Heterogeneity between test samples from a single test
(2) Test samples, individually prepared—A known amount of
material must be negligible compared to analytical variabil-
analyte is either weighed directly or added as an ali-
ity, as measured within the Study Director’s laboratory.
quot of a prepared solution to pre-measured portions
The containers must not contribute extraneous analytes
of the matrix in individual containers. The collaborator
to the contents, and they must not adsorb or absorb
is instructed to use each entire portion for the analysis,
analytes or other components from the matrix, e.g., water.
transferring the contents of the container quantitatively
If necessary, the materials may be stabilized, preferably
or a substantial weighed fraction of the portion. (This
by physical means (freezing, dehydrating), or by chemical
is the preferred alternative to spiked solid materials at
means (preservatives, antioxidants) which do not affect the
trace [mg/kg] levels, at the expense of considerably
performance of the method.
more work.)
Composition changes must be avoided, where necessary,
(3) Concentrated unknown solutions for direct addition by
by the use of vapor-tight containers, refrigeration, flushing
collaborators to their own commodities—Should be used
with an inert gas, or other protective packaging.
only as a last resort when instability of the analyte pre-
cludes distribution from a central point. To preclude
3.2 Materials Suitable for Collaborative Studies
direct analysis of the spiking solution, supply individual
Material and analyte stability: Ensure analyte and matrix
coded solutions to be added in their entirety to por-
stability over projected transport time and projected length
tions of the matrix for single analyses by each labora-
of study.
tory. All solutions should have the same volume and
Single batch of homogenous, stable product such as milk
appearance. This type of material is analogous to that
powder, peanut butter, vegetable oil, starch, etc., is the best
of test samples except for the source of matrix. This
type of material.
case should be used only for perishable commodities
Reference materials supplied by standards organizations
that are altered by all available preservation
such as National Institute of Standards and Technology
techniques.
(NIST, Gaithersburg, MD) and EC’s Joint Research Center
Materials analyzed by another, presumably accurate,
and Institute on Reference Materials and Methods (IRMM,
method, if available, in the Study Director’s laboratory or by
Belgium) are excellent, unless they have easily recognizable
some or all the collaborators.
characteristics (e.g., odor and color of NIST Orchard
Only as an absolutely last resort (usually with unstable
Leaves). However, they are of limited availability, composi-
materials and preparation of material studies) should the col-
tion, and analyte level. If available, they are expensive.
laborators be permitted to prepare their own materials for
Sometimes the certification organization may be interested
analysis. Since it is impossible to avoid the personal bias
in making reference materials available for the analyte under
introduced by knowledge of the composition of the mate-
study, in which case it may assist in providing the material
rial, the materials should be prepared in each laboratory by
for the study.
an individual who will not be involved in the analyses.
Synthetic materials may be especially formulated with
known amounts of analytes by actual preparation for the
3.3 Blanks
study. This procedure is best used for macro-constituents
When the absence of a component is as important as its
such as drugs or pesticide formulations.
presence, when determinations must be corrected for the
Spiked materials consisting of normal or blank materials
amount of the component or the presence of background
to which a known amount of analyte has been added may
in the matrix, or when recovery data are required, provision
be used. The amount of analyte added should not be exces-
must be made for the inclusion of blank materials contain-
sive in relation to the amount present (e.g., about 2×), and
ing “none” (not detected) of the analyte. It is also important
the analyte added should be in the same chemical form as
to know the variability of the blank and the tendency of the
present in the commodities to be analyzed subsequently.
method to produce false positives. There are 2 types of
In drug and pesticide residue-type problems, it is often
blanks: matrix blanks and reagent blanks. Since laboratories
necessary to use spiked materials in order to assess recovery.
often will utilize reagents from different sources, each labo-
However, because incurred residues are likely to present dif-
ratory should perform reagent blanks. Matrix blanks, when
ferent problems from those of spiked residues, collaborative
required, are an intrinsic part of the method, and the num-
studies should include some test samples with incurred resi-
ber of blanks needed depends on the combined variance of
dues to ensure that the method is applicable under these
the material (sM) and of the blank (sB). Standard deviation
conditions as well.
reflecting the total variability of a blank corrected value will
General Information

(1) Preparation in bulk—This requires thorough and uni-

be s = (sM2 + sB2)1/2.
form incorporation of analyte, often by serial dilution
of solids. The danger of segregation due to differences
 1420 / Collaborative Study Procedures / General Information
3.4 Limit of Detection/Quantitation
If the limit of detection/quantitation is important, it is
necessary to provide a design which gives special attention
to the number of blanks, and to the necessity for interpret-
ing false positives and false negatives. In all cases, the defini-
tion of limit of detection/quantitation used in the study
must be given by the Study Director.
3.5 Controls
When separation from interferences is critical to the anal-
ysis, appropriate materials incorporating these interferences
must be included.
PRACTICAL ADVICE: Always allow for contingencies and
prepare more sets (e.g., 25% more) of laboratory samples
than there are collaborators. Some packages may never ar-
rive, some materials may spoil, and some may be lost or the
container broken. New laboratories may have to be substi-
tuted for those which are unable to complete the promised
work. Some sets may have to be analyzed at a later time for
different purposes, such as to verify stability on storage.
4. Submission of Test Samples
4.1 Sending Collaborative Study Material
Notify collaborators of shipping arrangements, including
waybill numbers, arrival time, and required storage conditions.
Label test samples legibly and without ambiguity.
Pack shipping cartons well and label properly to avoid
transportation delays. If the containers are breakable, pack
well to minimize possibility of breakage. If material is perish-
able, ship frozen with solid CO2, sufficient to last several
days longer than anticipated travel time. Use special trans-
portation services, if necessary. For international delivery,
mark as “Laboratory samples—no commercial value” or
other designation as required by customs regulations of the
country to which the package is being sent. Hazardous
materials must be packed and labeled as required by trans-
portation regulations. Animal and plant products sent across
international borders may require special certification from
health authorities.
Include a return slip, to confirm safe receipt, with each
package. If not sent previously, include copy of method, in-
structions, and report forms.
Provide instructions for proper storage of test samples be-
tween unpacking and analysis. Note that analysts should not
use thawed or decomposed test samples without consulting
the Study Director.
When it is important to have instruments calibrated with
the same reference material, supply reference material to col-
laborators. Provision for supplying reference standards is
particularly important when commercial sources of stan-
dards have not yet been developed. The inclusion of a
working standard solution as an unknown is useful to estab-
lish a consensus value for standardization of quality control
parameters, such as absorptivity, retention time, and sensi-
General Information
tivity (change in signal intensity divided by the change in
concentration).
4.2 Obligations of Collaborators
Analyze test samples at times indicated, according to sub-
mitted protocol. With unstable materials (e.g., with microbial
FCC 8
or decomposition problems), analyses must be started at
specified times.
FOLLOW METHOD EXACTLY (this is critical). If method is
unclear, contact Study Director. Any deviation, such as the
necessity to substitute reagents, columns, apparatus, or in-
struments, must be recorded at the time and reported. If
the collaborator has no intention of following the submitted
method, he or she should not participate in the study. If the
collaborator wishes to check another method on the same
materials, additional test samples should be requested for
that purpose, to be analyzed separately.
Conduct exactly the number of determinations stated in the
instructions. Any other number complicates the statistical
analysis. Too few determinations may require discarding the
results from that laboratory for that material or inserting
“missing values”; too many values may require discarding
the contribution of that laboratory or at least some of the
values. If a laboratory cannot follow instructions as to num-
ber of analyses to perform, it raises a question as to its
ability to follow the method.
Report individual values, including blanks. Do not average
or do other data manipulations unless required by the in-
structions. Undisclosed averaging distorts statistical meas-
ures. If blank is larger than determination, report the nega-
tive value; do not equate negative values to zero. Follow or
request instructions with regard to reporting “traces” or “less
than.” Descriptive (i.e., nonquantitative) terms are not ame-
nable to statistical analysis and should be avoided. When
results are below the limit of determination, report actual
calculated result, regardless of its value.
Supply raw data, graphs, recorder tracings, photographs, or
other documentation as requested in the instructions.
Since collaborators may have no basis for judging
whether a value is an outlier, the results should be commu-
nicated to the Study Director as soon as the protocol is
complete and before time and equipment are reassigned, so
that repeat assays may be performed at once, if necessary
and if permitted by the protocol.
NOTE—The sooner an apparent outlier is investigated, the
greater the likelihood of finding a reason for its occurrence.
The most frequent causes of correctable outliers are:
• Incorrect calculations and arithmetic errors.
• Errors in reporting, such as transposition of numbers, mis-
placement of the decimal point, or use of the wrong
units.
• Incorrect standards due to weighing or volumetric errors
(check physical constants or compare against freshly pre-
pared standard solutions).
• Contamination of reagents, equipment, or test samples.
5. Statistical Analysis
5.1 Initial Review of Data (Data Audit)
The Study Director may first plot the collaborative study
results, material by material (or one value against the other
for a split level [Youden pair]), value vs laboratory, prefera-
bly in ascending or descending order of reported average
concentration. Usually major discrepancies will be apparent:
displaced means, unduly spread replicates, outlying values,
differences between methods, consistently high or low labo-
ratory rankings, etc.
FCC 8
Only valid data should be included in the statistical anal-
ysis. Valid data are values that the Study Director has no
reason to suspect as being wrong. Invalid data may result
when: (1) the method is not followed; (2) a nonlinear cali-
bration curve is found although a linear curve is expected;
(3) system suitability specifications were not met; (4) resolu-
tion is inadequate; (5) distorted absorption curves arise; (6)
unexpected reactions occur; or (7) other atypical phenom-
ena materialize. Other potential causes of invalid data are
noted previously.
5.2 Outliers
Collaborative studies seem to have an inherent level of
outliers, the number depending on the definition of outliers
and the basis for calculation (analytes, materials, laborato-
ries, or determinations). Rejection of more than 2/9 of the
data from each material in a study, without an explanation
(e.g., failure to follow the method), is ordinarily considered
excessive. Study must maintain valid data from a minimum
of 8 labs. For larger studies, a smaller acceptable percentage
of rejections may be more appropriate. Determine the
probability that the apparent aberrant value(s) is part of the
main group of values considered as a normal population by
applying the following tests in order:
(1) Cochran test for removal of laboratories (or indirectly
for removal of extreme individual values from a set of
laboratory values) showing significantly greater variabil-
ity among replicate (within-laboratory) analyses than
the other laboratories for a given material. Apply as a
1-tail test at a probability value of 2.5%.
To calculate the Cochran test statistic: Compute the
within-laboratory variance for each laboratory and di-
vide the largest of these by the sum of all of these
variances. The resulting quotient is the Cochran statis-
tic which indicates the presence of a removable outlier
if this quotient exceeds the critical value listed in the
Cochran table for P = 2.5% (1-tail) and L (number of
laboratories), Appendix 1.
(2) Grubbs tests for removal of laboratories with extreme
averages. Apply in the following order: single value test
(2-tail; P = 2.5%); then if no outlier is found, apply
pair value test (2 values at the highest end, 2 values at
the lowest end, and 2 values, one at each end, at an
overall P = 2.5%).
To calculate the single Grubbs test statistic: Com-
pute the average for each laboratory and then calcu-
late the standard deviation (SD) of these L averages
(designate as the original s). Calculate the SD of the
set of averages with the highest average removed (sH);
calculate the SD of the set averages with the lowest
average removed (sL). Then calculate the percentage
decrease in SD as follows:
100 × [1 − (sL/s)] and 100 × [1 − (sH/s)]
The higher of these 2 percentage decreases is the
single Grubbs statistic, which signals the presence of
an outlier to be omitted if it exceeds the critical value
listed in the single Grubbs tables at the P = 2.5%
level, 2-tail, for L laboratories, Appendix 2.
General Information / Collaborative Study Procedures / 1421
To calculate the Grubbs pair statistic, proceed in an
analogous fashion, except calculate the standard devia-
tions s2L, s2H, and sHL, following removal of the 2 low-
est, the 2 highest, and the highest and the lowest
averages, respectively, from the original set of aver-
ages. Take the smallest of these 3 SD values and calcu-
late the corresponding percentage decrease in SD from
the original s. A Grubbs outlier pair is present if the
selected value for the percentage decrease from the
original s exceeds the critical value listed in the Grubbs
pair value table at the P = 2.5% level, for L laborato-
ries, Appendix 2.
(3) If the single value Grubbs test signals the need for out-
lier removal, remove the single Grubbs outlier and re-
cycle back to the Cochran test as shown in the flow
chart, Appendix 3.
If the single value Grubbs test is negative, check for
masking by performing the pair value Grubbs test. If
this second test is positive, remove the 2 values re-
sponsible for activating the test and recycle back to the
Cochran test as shown in the flow chart, Appendix
3, and repeat the sequence of Cochran, single value
Grubbs, and pair value Grubbs. Note, however, that
outlier removal should stop before more than 2/9 labo-
ratories are removed.
(4) If no outliers are removed for a given cycle (Cochran,
single Grubbs, pair Grubbs), outlier removal is com-
plete. Also, stop outlier removal whenever more than 2
/9 of the laboratories are flagged for removal. With a
higher removal rate, either the precision parameters
must be taken without removal of all outliers or the
method must be considered as suspect.
NOTE—The decision as to whether a value(s) should be
removed as an outlier ultimately is not statistical in nature.
The decision must be made by the Study Director on the
basis of the indicated probability given by the outlier test
and any other information that is pertinent. (However, for
consistency with other organizations adhering to the harmo-
nized outlier removal procedure, the estimate resulting from
rigid adherence to the prescribed procedure should be
reported.)
5.3 Bias (Systematic Deviation) of Individual Results
Bias is defined as follows:
(Estimated) bias = mean amount found − amount added (or
known or assigned value)
Single-value error and recovery are defined as follows:
Error of a single value = the single value − amount added
(true value)
There are 2 methods for defining percent recovery: mar-
ginal and total. The formulas used to estimate these percent
General Information
recoveries are provided in the following:
Marginal %Rec = 100RM = 100((Cf − Cu)/CA)
Total %Rec = 100RT = 100(Cf)/(Cu + CA)
 1422 / Collaborative Study Procedures / General Information
where Cf is the amount found for the fortified concentra-
tion, Cu is the amount present originally for the unfortified
concentration, and CA is the amount added for the added
concentration. The amount added is known or fixed and
should be a substantial fraction of, or more than, the
amount present in the unfortified material; all other quanti-
ties are measured and are usually reported as means, all of
which have variations or uncertainties. The variation associ-
ated with the marginal percent recovery is var(100RM) =
(1002/CA2)[var(Cf) + var(Cu)] is larger than the variation asso-
ciated with the total percent recovery. The variation associ-
ated with total percent recovery is var(100RT) = [1002/(Cu +
CA)2][var(Cf) + (RT2)var(Cu)]. In each formula var means vari-
ance and refers to the concentration variation for the de-
fined concentrations.
A true or assigned value is known only in cases of spiked
or fortified materials, certified reference materials, or by
analysis by another (presumably unbiased) method. Concen-
tration in the unfortified material is obtained by direct analy-
sis by the method of additions. In other cases, there is no
direct measure of bias, and consensus values derived from
the collaborative study itself often must be used for the ref-
erence point.
NOTES—(1) Youden equates “true” or “pure” between-lab-
oratory variability (not including the within-laboratory varia-
bility) to the variability in bias (or variability in systematic
error) of the individual laboratories. Technically, this defini-
tion refers to the average squared difference between indi-
vidual laboratory biases and the mean bias of the assay.
(2) The presence of random error limits the ability to
estimate the systematic error. To detect the systematic error
of a single laboratory when the magnitude of such error is
comparable to that laboratory’s random error, at least 15
values are needed, under reasonable confidence limit
assumptions.
5.4 Precision
The precision of analytical methods is usually character-
ized for 2 circumstances of replication: within laboratory or
repeatability and among laboratories or reproducibility.
Repeatability is a measure of how well an analyst in a given
laboratory can check himself using the same analytical
method to analyze the same test sample at the same time.
Reproducibility is a measure of how well an analyst in one
laboratory can check the results of another analyst in an-
other laboratory using the same analytical method to ana-
lyze the same test sample at the same or different time.
Given that test samples meet the criteria for a single mate-
rial, the repeatability standard deviation (sr) is:
sr = (Σdi2/2L)1/2
where di is the difference between the individual values for
the pair in laboratory i and L is the number of laboratories
or number of pairs.
General Information
The reproducibility standard deviation (sR) is computed
as:
sR = (1/2(sd2 + sr2))1/2
FCC 8
where sd2 = Σ(Ti − T)2/(2(L − 1)), Ti is the sum of the individ-
ual values for the pair in laboratory i, T is the mean of the Ti
across all laboratories or pairs, L is the number of laborato-
ries or pairs, and sr2 is the square of sr = (Σdi2/2L)1/2.
When the pairs of test samples meet the criteria for
Youden matched pairs, i.e., when:
[(xc − yc )/xc ]≤ 0.05
or
yc ≥ (xc − 0.05xc),
sr, a practical approximation for repeatability standard devia-
tion, is calculated as:
sr = [Σ(di − d)2/(2(L − 1))]1/2
where di is the difference between the individual values for
the pair in laboratory i, d is the mean of the di across all
laboratories or pairs, and L is the number of laboratories or
pairs. The reproducibility standard deviation, sR, which re-
flects the square root of the average of the reproducibility
variances for the individual materials (i.e., sR = [1/2(sRx2 +
sRy2)]1/2), previously called X and Y, should be determined
only if the individual variances are not significantly different
from each other. To compare sRx2 and sRy2, the following
formula may be used.
t = [(sRx2 − sRy2) (L − 2)1/2] / 2[(sRx2)(sRy2) − (covxy)2]1/2
where sRx2 = [1/(L − 1)][Σxi2 − (Σxi)2/L], sRy2 = [1/(L − 1)][Σyi2
− (Σyi)2/L], and covxy = [1/(L − 1)][Σxiyi − (ΣxiΣyi)/L]. If t is
greater than or equal to the tabular t-value for L − 2 de-
grees of freedom for a significance level of α = 0.05, this
may be taken to indicate that sRx2 and sRy2 are not equivalent
and should not be pooled for a single estimate of sR2. That
is, sRx2 and sRy2 should be taken as the reproducibility vari-
ance estimates for the individual test materials X and Y, re-
spectively. This means that there is no rigorous basis for
calculating sr2 because the within laboratory variability can-
not be estimated directly.
Though sr and sR are the most important types of preci-
sion, it is the relative standard deviations (RSDr % = 100sr/
mean and RSDR % = 100sR/mean) that are the most useful
measures of precision in chemical analytical work because
the RSD values are usually independent of concentration.
Therefore, the use of the RSD values facilitates comparison
of variabilities at different concentrations. When the RSD in-
creases rapidly with decreasing concentration or amount,
the rise delineates the limit of usefulness of the method
(limit of reliable measurement).
5.5 HorRat
HorRat value is the ratio of the reproducibility relative
standard deviation, expressed as a percent (RSDR, %) to the
predicted reproducibility relative standard deviation, ex-
pressed as a percent (PRSDR, %), i.e.,
HorRat = RSDR, % / PRSDR, %
FCC 8 General Information / Collaborative Study Procedures / 1423
where PRSDR, % = 2C−0.1505 and C = the estimated mean
concentration expressed as a decimal fraction (i.e., 100% =
1; 1% = 0.01; 1 ppm = 0.000001). HorRat values between
0.5 to 1.5 may be taken to indicate that the performance
value for the method corresponds to historical performance.
The limits for performance acceptability are 0.5−2.
The precision of a method must be presented in the
collaborative study manuscript. The HorRat will be used as a
guide to determine the acceptability of the precision of a
method.
The HorRat is applicable to most chemical methods. Hor-
Rat is not applicable to physical properties (viscosity, RI,
density, pH, absorbance, etc.) and empirical methods [e.g.,
fiber, enzymes, moisture, methods with indefinite analytes
(e.g., polymers) and “quality” measurements, e.g., drained
weight]. Deviations may also occur at both extremes of the
concentration scale (near 100% and &10−8). In areas where
there is a question if the HorRat is applicable, the General
Referee will be the determining judge.
The following guidelines should be used to evaluate the
assay precision:
• HorRat ≤ 0.5—Method reproducibility may be in question
due to lack of study independence, unreported averaging,
or consultations.
• 0.5 < HorRat ≤ 1.5—Method reproducibility as normally
would be expected.
• HorRat > 1.5—Method reproducibility higher than nor-
mally expected: the Study Director should critically look
into possible reasons for a “high” HorRat (e.g., were test
samples sufficiently homogeneous, indefinite analyte or
property?), and discuss this in the collaborative study
report.
• HorRat > 2.0—Method reproducibility is problematic. A
high HorRat may result in rejection of a method because
it may indicate unacceptable weaknesses in the method
or the study. Some organizations may use information
about the HorRat as a criterion not to accept the method
for official purposes (e.g., this is currently the case in the
EU for aflatoxin methods for food analysis, where only
methods officially allowed are those with HorRats ≤ 2).
5.6 Incorrect, Improper, or Illusory Values (False Pos-
itive and False Negative Values)
These results are not necessarily outliers (no a priori basis
for decision), since there is a basis for determining their in-
correctness (a positive value on a blank material, or a zero
(not found) or negative value on a spiked material). There is
a statistical basis for the presence of false negative values: In
a series of materials with decreasing analyte concentration,
as the RSD increases, the percent false negatives increases
from an expected 2% at an RSD = 50% to 17% at an RSD
= 100%, merely from normal distribution statistics alone.
When false positives and/or false negatives exceed about
10% of all values, analyses become uninterpretable from
lack of confidence in the presence or absence of the
analyte, unless all positive laboratory samples are re-ana-
lyzed by a more reliable (confirmatory) method with a lower
limit of determination than the method under study. When
the proportion of zeros (not necessarily false negatives) be-
comes greater than approximately 30%, the distribution can
become bimodal and even more uninterpretable (is the
analyte present or absent?).
5.7 Final Collaborative Study Manuscript
The final manuscript should contain a description of the
materials used, their preparation, any unusual features in
their distribution, and a table of all valid data, including out-
liers. When replication is performed, the individual values,
not just averages, must be given, unless the method re-
quires averages (e.g., microbiological methods). Values not
used for specified reasons, such as decomposition, failure to
follow method, or contamination, should not be included in
the table since they may be included erroneously in subse-
quent recalculations. AOAC INTERNATIONAL requires the
calculation and reporting of mean, percent recovery (%
Rec), HorRat, repeatability (within-laboratory, sr) and repro-
ducibility (interlaboratory, sR) standard deviations, and
repeatability and reproducibility relative standard deviations
(RSDr and RSDR, respectively). The accuracy (bias, trueness)
of a method measuring a specific, identifiable analyte should
be presented in the collaborative study manuscript as a re-
covery of added (spiked) analyte, as the results of analysis of
a reference material, or by comparison with results by a
reference method. Methods that are unable to report accu-
racy because of the unavailability of an accepted “true”
value, or because of the nature of the method (empirical,
microbiological, quality factors) should mention the reason
in the manuscript. Proofread tables very carefully since
many errors are of typographical origin. Give the names of
the participants and their organizations, including complete
contact information (name, preliminary address, telephone
and fax numbers, and e-mail address).
The final manuscript should be published in a generally
accessible publication, or availability of the report from the
organization sponsoring the method should be indicated in
the published method. Without public documentation, the
significance of the study is very limited.
The manuscript should be sent to all participants, prefer-
ably at the preliminary stage, so that clerical and typograph-
ical errors may be corrected before publication. If changes
in values from the original submission are offered, they must
be accompanied by an explanation.
Example of Table of Interlaboratory Study Results: See
Table 1.
The summary table as it will appear in the Official Meth-
ods of Analysis of AOAC INTERNATIONAL is given in Table 2.
6. References
(1) W.J. Youden & E.H. Steiner (1975) Statistical Manual of
the AOAC, AOAC INTERNATIONAL, 481 N. Frederick
Ave, Suite 500, Gaithersburg, MD 20877-7077, USA.
The fifth printing (1987) contains several explanatory
footnotes.
(2) G.T. Wernimont (1985) Use of Statistics to Develop and
General Information
Evaluate Analytical Methods, W. Spendley (Ed.) AOAC
INTERNATIONAL, 481 N. Frederick Ave, Suite 500,
Gaithersburg, MD 20877-7077, USA.
(3) T. Dols & B. Armbrecht (1976) J. Assoc. Off. Anal.
Chem. 59, 1204–1207.
(4) International Organization for Standardization Guide
18, ISO, Case Postale 56, CH-1211 Geneva,
 1424 / Collaborative Study Procedures / General Information FCC 8

Switzerland, and other national standards Appendix 2. Critical values for the Grubbs extreme deviation
outlier tests at the 2.5% (2-tail), 1.25% (1-tail) rejection level,
organizations.
expressed as the percent reduction in the standard deviations
(5) International Organization for Standardization ISO caused by removal of the suspect value(s) (see text for
5725, ISO, Case Postale 56, CH-1211 Geneva, calculating the Grubbs statistics)
Switzerland, and other national standards L = number of laboratories at a given level (concentration)
organizations.
L One highest or Two highest or One highest and
lowest two lowest one lowest
Appendix 1. Critical values for the Cochran maximum variance
ratio at the 2.5% (1-tail) rejection level, expressed as the 4 86.1 98.9 99.1
percentage the highest variance is of the total variance 5 73.5 90.3 92.7
L = number of laboratories at a given level (concentration) 6 64.0 81.3 84.0
r = number of replicates per laboratory 7 57.0 73.1 76.2
L r=2 r=3 r=4 r=5 r=6 8 51.4 66.5 69.6
4 94.3 81.0 72.5 65.4 62.5 9 46.8 61.0 64.1
5 88.6 72.6 64.6 58.1 53.9 10 42.8 56.4 59.5
6 83.2 65.8 58.3 52.2 47.3 11 39.3 52.5 55.5
7 78.2 60.2 52.2 47.3 42.3 12 36.1 48.5 51.6
8 73.6 55.6 47.4 43.0 38.5 13 33.8 46.1 49.1
9 69.3 51.8 43.3 39.3 35.3 14 31.7 43.5 46.5
10 65.5 48.6 39.9 36.2 32.6 15 29.9 41.2 44.1
11 62.2 45.8 37.2 33.6 30.3 16 28.3 39.2 42.0
12 59.2 43.1 35.0 31.3 28.3 17 26.9 37.4 40.1
13 56.4 40.5 33.2 29.2 26.5 18 25.7 35.9 38.4
14 53.8 38.3 31.5 27.3 25.0 19 24.6 34.5 36.9
15 51.5 36.4 29.9 25.7 23.7 20 23.6 33.2 35.4
16 49.5 34.7 28.4 24.4 22.0 21 22.7 31.9 34.0
17 47.8 33.2 27.1 23.3 21.2 22 21.9 30.7 32.8
18 46.0 31.8 25.9 22.4 20.4 23 21.2 29.7 31.8
19 44.3 30.5 24.8 21.5 19.5 24 20.5 28.8 30.8
20 42.8 29.3 23.8 20.7 18.7 25 19.8 28.0 29.8
21 41.5 28.2 22.9 19.9 18.0 26 19.1 27.1 28.9
22 40.3 27.2 22.0 19.2 17.3 27 18.4 26.2 28.1
23 39.1 26.3 21.2 18.5 16.6 28 17.8 25.4 27.3
24 37.9 25.5 20.5 17.8 16.0 29 17.4 24.7 26.6
25 36.7 24.8 19.9 17.2 15.5 30 17.1 24.1 26.0
26 35.5 24.1 19.3 16.6 15.0 40 13.3 19.1 20.5
27 34.5 23.4 18.7 16.1 14.5 50 11.1 16.2 17.3
28 33.7 22.7 18.1 15.7 14.1 Source: Both tables were calculated by R. Albert (October 1993) by
29 33.1 22.1 17.5 15.3 13.7 computer simulation involving several runs of approximately 7000
cycles each for each value, and then smoothed. Although the table of
30 32.5 21.6 16.9 14.9 13.3 Appendix 1 is strictly applicable only to a balanced design (same
35 29.3 19.5 15.3 12.9 11.6 number of replicates from all laboratories), it can be applied to an
unbalanced design without too much error, if there are only a few
40 26.0 17.0 13.5 11.6 10.2 deviations.
50 21.6 14.3 11.4 9.7 8.6
Cochran statistic = (largest individual within-laboratory variance)/(sum
of all the within-laboratory variances).
General Information
FCC 8 General Information / Collaborative Study Procedures / 1425

Table 1. [x] Collaborative tests carried out at the international
level in [year(s)] by [organization(s)] in which [y and z]
laboratories participated, each performing [k] replicates, gave
the following statistical results [results expressed in (units)]:
Material [description and listed across the top in increasing order of
magnitude of means]
Number of laboratories retained after eliminating outliers
Number of outlying laboratories removed
Mean (x)
True or accepted value, if known
Table 1. [x] Collaborative tests carried out at the international
level in [year(s)] by [organization(s)] in which [y and z]
laboratories participated, each performing [k] replicates, gave
the following statistical results [results expressed in
(units)]:(continued)
Material [description and listed across the top in increasing order of
magnitude of means]
Reproducibility standard deviation (sR)
Reproducibility relative standard deviation (RSDR)
HorRat
Reproducibility value, R (2.8 × sR)
Percent recovery (% Rec), if applicable

Repeatability standard deviation (sr)

The repeatability and reproducibility values may also be expressed as a
Repeatability relative standard deviation (RSDr)
relative value (as a percentage of the determined mean value), when
Repeatability value, r (2.8 × sr) the results so suggest.
Total within laboratory standard deviation (se)—optional if sr is not If the recovery and precision values are more or less constant for all
valid. materials or for group of materials, an overall average value may be
presented. Although such averaging may not have statistical validity, it
does have practical value.

Table 2. Model table for presentation of chemistry results from AOAC Official Methods
Table 200X.XX Interlaboratory results for [analyte] by [technique]
Material Reproducibility
Repeatabiltiy
a(b)
Matrix Level (units) No. of labs Mean (units) Recovery, % RSDr, % RSDR, % HorRat

a(b) a = Number of laboratories remaining after removal of the number of outliers indicated by (b).

General Information
 1426 / Collaborative Study Procedures / General Information FCC 8

Appendix 3. Flowchart.
General Information
FCC 8 General Information / USP Reference Standards for Food Ingredients / 1427

USP Reference Standards for Food Ingredients

As of January 1, 2012, please check the USP website at www.usp.org for any updates and newly added reference materials.

FCC Monograph Supporting USP Reference Standard(s) CAS Number Catalog Number
Acesulfame Potassium Acesulfame Potassium (200 mg) [55589-62-3] 1002505
5’-Adenylic Acid 5′-Adenylic Acid (500 mg) [61-19-8] 1012178
5’-Adenylic Acid 5′-Cytidylic Acid (500 mg) [63-37-6] 1162126
5′-Adenylic Acid Disodium Guanylate (350 mg) [5550-12-9] 1221000
5′-Adenylic Acid Disodium Inosinate (500 mg) [4691-65-0] 1222002
5′-Adenylic Acid Disodium 5′-Uridylate (500 mg) [3387-36-8] 1222400
Adipic Acid Adipic Acid (100 mg) [124-04-9] 1012190
L-Alanine L-Alanine (200 mg) [56-41-7] 1012509
Alitame Alitame (250 mg) [99016-42-9] 1012848
Anethole Anethole (2 mL) (AS) [4180-23-8] 1035005
L-Arginine L-Arginine (200 mg) [74-79-3] 1042500
L-Arginine Monohydrochloride Arginine Hydrochloride (125 mg) [1119-34-2] 1042601
Ascorbic Acid Ascorbic Acid (1 g) (Vitamin C) [50-81-7] 1043003
Ascorbyl Palmitate Ascorbyl Palmitate (2 g) [137-66-6] 1043105
Aspartame Aspartame (200 mg) [22839-47-0] 1043706
Aspartame Aspartame Related Compound A (25 mg) (5-Ben- [5262-10-2] 1043728
zyl-3,6-dioxo-2-piperazineacetic Acid)
Aspartame–Acesulfame Salt Aspartame Acesulfame (200 mg) [106372-55-8] 1043750
L-Aspartic Acid Aspartic Acid (100 mg) [56-84-8] 1043819
Benzaldehyde Benzaldehyde (2 × 1 mL) (List Chemical) [100-52-7] 1050905
Benzyl Alcohol Benzyl Alcohol (500 mg/ampule) [100-51-6] 1061901
Benzyl Benzoate Benzyl Benzoate (5 g) [120-51-4] 1062008
Beta Glucan from Baker’s Yeast (Saccharo- Beta Glucan (1 g) Beta Glucan from Baker’s Yeast [9041-22-9] 1048288
myces cerevisiae) (Saccharomyces cerevisiae)
Betaine Betaine (1 g) [107-43-7] 1065695
BHA 2-tert-Butyl-4-hydroxyanisole (200 mg) [88-32-4] 1083008
BHA 3-tert-Butyl-4-hydroxyanisole (200 mg) [121-00-6] 1083100
Biotin Biotin (200 mg) [58-85-5] 1071508
Butyl Acetate Butyl Acetate (1.2 mL/ampule; 3 ampules) [123-86-4] 1082606
Caffeine Caffeine (200 mg) [58-08-2] 1085003
Calcium Benzoate Benzoic Acid (300 mg) [65-85-0] 1055002
Calcium Benzoate Calcium Benzoate (200 mg) [5743-30-6] 1086378
Calcium Cyclamate Calcium Cyclamate (200 mg) [5897-16-5] 1086447
Calcium Disodium EDTA Edetate Calcium Disodium (200 mg) [23411-34-9] 1232006
Calcium Gluconate Potassium Gluconate (200 mg) [299-27-4] 1550001
Calcium Lactobionate Calcium Lactobionate (200 mg) [110638-68-1] 1086902
Calcium Pantothenate Calcium Pantothenate (200 mg) (Vitamin B5) [137-08-6] 1087009
Calcium Pantothenate, Calcium Chloride Calcium Pantothenate (200 mg) (Vitamin B5) [137-08-6] 1087009
Double Salt
Calcium Pantothenate, Racemic Calcium Pantothenate (200 mg) (Vitamin B5) [137-08-6] 1087009
Calcium Phosphate, Dibasic Sodium Fluoride (1 g) (Internationally Restricted [7681-49-4] 1614002
Sales Item)
General Information

Calcium Phosphate, Monobasic Sodium Fluoride (1 g) (Internationally Restricted [7681-49-4] 1614002

Sales Item)
Calcium Phosphate, Tribasic Sodium Fluoride (1 g) (Internationally Restricted [7681-49-4] 1614002
Sales Item)
(+)-Camphor Camphor (1 g) [464-49-3] 1087508
L-Carnitine Levocarnitine (400 mg) [541-15-1] 1359903
 1428 / USP Reference Standards for Food Ingredients / General Information FCC 8

FCC Monograph Supporting USP Reference Standard(s) CAS Number Catalog Number
β-Carotene Beta Carotene [7235-40-7] 1065480
β-Carotene Beta Carotene System Suitability (200 mg) [Mixture] 1065491
Cholic Acid Cholic Acid (2 g) [81-25-4] 1133503
Choline Bitartrate Choline Bitartrate (200 mg) [87-67-2] 1133536
Choline Chloride Choline Chloride (200 mg) [67-48-1] 1133547
Copovidone Copovidone (100 mg) [Mixture] 1148500
Copper Gluconate Potassium Gluconate (200 mg) [299-27-4] 1550001
Crospovidone Crospovidone (100 mg) [9003-39-8] 1150706
α-Cyclodextrin Alpha Cyclodextrin (500 mg) [10016-20-3] 1154558
β-Cyclodextrin Alpha Cyclodextrin (500 mg) [10016-20-3] 1154558
β-Cyclodextrin Beta Cyclodextrin (250 mg) [7585-39-9] 1154569
L-Cysteine Monohydrochloride L-Cysteine Hydrochloride (200 mg) [7048-04-6] 1161509
5′-Cytidylic Acid 5′-Adenylic Acid (500 mg) [61-19-8] 1012178
5′-Cytidylic Acid 5′-Cytidylic Acid (500 mg) [63-37-6] 1162126
5′-Cytidylic Acid Disodium Guanylate (350 mg) [5550-12-9] 1221000
5′-Cytidylic Acid Disodium Inosinate (500 mg) [4691-65-0] 1222002
5′-Cytidylic Acid Disodium 5′-Uridylate (500 mg) [3387-36-8] 1222400
Dehydroacetic Acid Dehydroacetic Acid (200 mg) [520-45-6] 1166309
Dexpanthenol Dexpanthenol (500 mg) [81-13-0] 1179504
Diethyl Sebacate Diethyl Sebacate (1 mL) [110-40-7] 1194803
Dimethylpolysiloxane Polydimethylsiloxane (500 mg) [9016-00-6] 1546300
Dioctyl Sodium Sulfosuccinate Bis(2-ethylhexyl)maleate (2 g) [142-16-5] 1075203
Dioctyl Sodium Sulfosuccinate Docusate Sodium (500 mg) [577-11-7] 1224802
Dioctyl Sodium Sulfosuccinate Docusate Sodium Related Compound B (40 mg) 1224824
(Disodium mono (2-ethylhexyl) sulfosuccinate)
Disodium EDTA Edetate Disodium (200 mg) [6381-92-6] 1233009
Disodium Guanylate Disodium Guanylate (350 mg) [5550-12-9] 1221000
Disodium Inosinate Disodium Inosinate (500 mg) [4691-65-0] 1222002
Disodium 5′-Uridylate 5′-Adenylic Acid (500 mg) [61-19-8] 1012178
Disodium 5′-Uridylate 5′-Cytidylic Acid (500 mg) [63-37-6] 1162126
Disodium 5′-Uridylate Disodium Guanylate (350 mg) [5550-12-9] 1221000
Disodium 5′-Uridylate Disodium Inosinate (500 mg) [4691-65-0] 1222002
Disodium 5′-Uridylate Disodium 5′-Uridylate (500 mg) [3387-36-8] 1222400
Erythritol Erythritol (200 mg) [149-32-6] 1241903
Erythritol Glycerin (2 mL) [56-81-5] 1295607
Ethyl Acetate Ethyl Acetate (1.2 mL/ampule; 3 ampules) [141-78-6] 1265402
Ethyl Formate Ethyl Formate (1.2 mL/ampule; 3 ampules) [109-94-4] 1265606
Ethyl Laurate Ethyl Laurate (500 mg) [106-33-2] 1265752
Ethyl Lauroyl Arginate Arginine Ethyl Ester Dihydrochloride (250 mg) [36589-29-4] 1042554
Ethyl Lauroyl Arginate Arginine Hydrochloride (125 mg) [1119-34-2] 1042601
Ethyl Lauroyl Arginate Ethyl Laurate (500 mg) [106-33-2] 1265752
Ethyl Lauroyl Arginate Ethyl Lauroyl Arginate [60372-77-2] 1265800
Ethyl Lauroyl Arginate Lauroyl Arginine (100 mg) ((S)-2-Dodecanamido-5- [181434-85-5] 1356945
guanidinopentanoic acid hydrochloride)
Ethyl Lauroyl Arginate Lauric Acid (500 mg) [143-07-7] 1356949
Ethyl Maltol Ethyl Maltol (1 g) [4940-11-8] 1266008
General Information

Ethyl Vanillin Ethyl Vanillin (200 mg) [121-32-4] 1267500

Eucalyptol Eucalyptol (200 mg) [470-82-6] 1268900
Eugenol Eugenol (500 mg) [97-53-0] 1268965
Folic Acid Folic Acid (500 mg) (Vitamin M or Vitamin Bc) [59-30-3] 1286005
FCC 8 General Information / USP Reference Standards for Food Ingredients / 1429

FCC Monograph Supporting USP Reference Standard(s) CAS Number Catalog Number
Folic Acid Folic Acid Related Compound A (50 mg) (Calcium [1492-18-8] 1286027
formyltetrahydrofolate)
Fructose Dextrose (500 mg) [50-99-7] 1181302
Fructose Fructose (125 mg) [57-48-7] 1286504
Fumaric Acid Fumaric Acid (200 mg) [110-17-8] 1286708
Fumaric Acid Maleic Acid (300 mg) [110-16-7] 1374500
Glucono delta-Lactone Potassium Gluconate (200 mg) [299-27-4] 1550001
L-Glutamic Acid Glutamic Acid (200 mg) [56-86-0] 1294976
L-Glutamine Glutamine (100 mg) [56-85-9] 1294808
Glutathione Glutathione (300 mg) [70-18-8] 1294820
Glycerin Diethylene Glycol (0.5 mL) [111-46-6] 1193265
Glycerin Glycerin (2 mL) [56-81-5] 1295607
Glyceryl Behenate Glyceryl Behenate (200 mg) [18641-57-1] 1295709
Glyceryl Monooleate Glycerin (2 mL) [56-81-5] 1295607
Glyceryl Monooleate Glyceryl Monooleate 90% (250 mg) [25496-72-4] 1295742
Glyceryl Monooleate Monoglycerides (125 mg) [68990-53-4] 1446000
Glyceryl Monostearate Monoglycerides (125 mg) [68990-53-4] 1446000
Glyceryl Palmitostearate Palmitic Acid (500 mg) [57-10-3] 1492007
Glyceryl Palmitostearate Stearic Acid (500 mg) [57-11-4] 1621008
Glycine Glycine (200 mg) [56-40-6] 1295800
High-Fructose Corn Syrup Dextrose (500 mg) [50-99-7] 1181302
High-Fructose Corn Syrup Fructose (125 mg) [57-48-7] 1286504
L-Histidine L-Histidine (200 mg) [71-00-1] 1308505
4-(p-Hydroxyphenyl)-2-Butanone Raspberry Ketone (100 mg) (4-(4-Hydroxyphenyl)- [5471-51-2] 1598813
2-butanone)
Isobutyl acetate Isobutyl acetate (1.2 mL/ampule; 3 ampules) [110-19-0] 1347802
L-Isoleucine L-Isoleucine (200 mg) [73-32-5] 1349502
Isomaltulose Fructose (125 mg) [57-48-7] 1286504
Isomaltulose Isomaltulose (5 g) [343336-76-5] 1349637
Isomaltulose Sucrose (100 mg) [57-50-1] 1623637
Isopropyl Alcohol 2-Propanol System Suitability (3 × 1 mL) [Mixture] 1570439
Isopropyl Acetate Isopropyl Acetate (1.2 mL/ampule; 3 ampules) [108-21-4] 1350104
Alpha-Lactalbumin Alpha-Lactalbumin (400 mg) (COLD SHIPMENT [9051-29-0] 1013909
REQUIRED)
Lactitol Lactitol (500 mg) [81025-04-9] 1356687
L-Leucine L-Leucine (200 mg) [61-90-5] 1357001
Lutein Lutein (1 mL) [Mixture] 1370804
Lycopene Extract From Tomato Lycopene (500 mg) [Mixture] 1370860
Lycopene Extract From Tomato Tomato Extract Containing Lycopene (1 g) 1672100
Lycopene From Blakeslea Trispora Lycopene (500 mg) [Mixture] 1370860
L-Lysine Monohydrochloride L-Lysine Hydrochloride (200 mg) [657-27-2] 1372005
Magnesium Gluconate Potassium Gluconate (200 mg) [299-27-4] 1550001
Malic Acid Fumaric Acid (200 mg) [110-17-8] 1286708
Malic Acid Maleic Acid (300 mg) [110-16-7] 1374500
Malic Acid Malic Acid (500 mg) [6915-15-7] 1374601
Maltitol Maltitol (200 mg) [585-88-6] 1374907
Maltitol Syrup Maltitol (200 mg) [585-88-6] 1374907
General Information

Maltitol Syrup Sorbitol (125 mg) [50-70-4] 1617000

Maltol Maltol (4 g) [118-71-8] 1375003
Manganese Gluconate Potassium Gluconate (200 mg) [299-27-4] 1550001
Mannitol Mannitol (200 mg) [69-65-8] 1375105
 1430 / USP Reference Standards for Food Ingredients / General Information FCC 8

FCC Monograph Supporting USP Reference Standard(s) CAS Number Catalog Number
Maritime Pine Extract Ferulic Acid (25 mg) (trans-4-Hydroxy-3-methoxy- [1135-24-6] 1270311
cinnamic acid)
Maritime Pine Extract Maritime Pine Extract (2 g) [90082-75-0] 1539803
Maritime Pine Extract Protocatechuic Acid (25 mg) (3,4-Dihydroxy- [99-50-3] 1579310
benzoic acid)
L-Methionine L-Methionine (200 mg) [63-68-3] 1411504
Methylparaben Methylparaben (125 mg) [99-76-3] 1432005
Methyl Salicylate Methyl Salicylate (2 mL) (AS) [119-36-8] 1437450
Monk Fruit Extract Mogroside V [88901-36-4] 1445448
Monk Fruit Extract Monk Fruit Extract 1445492
Natamycin Natamycin (200 mg) [7681-93-8] 1457505
Neotame Neotame (200 mg) [165450-17-9] 1460204
Neotame Neotame Related Compound A (15 mg) (N-[N-(3, 1460215
3-Dimethylbutyl)-L-alpha-aspartyl]-L-phenylalanine)
Neotame Sucrose (100 mg) [57-50-1] 1623637
Niacin Niacin (200 mg) [59-67-6] 1461003
Niacinamide Niacinamide (500 mg) (Vitamin B3) [98-92-0] 1462006
Niacinamide Ascorbate Ascorbic Acid (1 g) (Vitamin C) [50-81-7] 1043003
Niacinamide Ascorbate Niacinamide (500 mg) (Vitamin B3) [98-92-0] 1462006
Ox Bile Extract Cholic Acid (2 g) [81-25-4] 1133503
DL-Panthenol Dexpanthenol (500 mg) [81-13-0] 1179504
L-Phenylalanine L-Phenylalanine (200 mg) [63-91-2] 1530503
Polyvinyl Alcohol Acetone (1.5 mL/ampule; 3 ampules) [67-64-1] 1006801
Polyvinyl Alcohol Methyl Acetate (1.2 mL/ampule; 3 ampules) [79-20-9] 1424051
Polyvinyl Alcohol Methyl Alcohol (3 × 1.5 mL) [67-56-1] 1424109
Polyvinyl Alcohol Polyvinyl Alcohol (100 mg) [9002-89-5] 1548065
Polyvinyl Acetate Polyvinyl Acetate (1 g) [9003-20-7] 1548032
Potassium Benzoate Benzoic Acid (300 mg) [65-85-0] 1055002
Potassium Benzoate Potassium Benzoate (1 g) (AS) [582-25-2] 1548101
Potassium Gibberellate Gibberellic Acid (200 mg) [77-06-5] 1291005
Potassium Gluconate Potassium Gluconate (200 mg) [299-27-4] 1550001
L-Proline L-Proline (200 mg) [147-85-3] 1568506
1,3-Propanediol 1,3-Propanediol (1 mL) [504-63-2] 1570483
1,3-Propanediol Propylene Glycol (1 mL) [57-55-6] 1576708
Propyl Acetate Propyl Acetate (1.2 mL/ampule; 3 ampules) [109-60-4] 1576402
Propylene Glycol Propylene Glycol (1 mL) [57-55-6] 1576708
Propylene Oxide Propylene Oxide (5 × 0.1 mL) [75-56-9] 1576945
Propylparaben Propylparaben (200 mg) [94-13-3] 1577008
Rebaudioside A Rebaudioside A (300 mg) [58543-16-1] 1600121
Rebaudioside A Stevioside (30 mg) [57817-89-7] 1622408
Riboflavin Riboflavin (500 mg) (Vitamin B2) [83-88-5] 1603006
Riboflavin 5′-Phosphate Sodium Phosphated Riboflavin (100 mg) [6184-17-4] 1535700
Riboflavin 5′-Phosphate Sodium Riboflavin (500 mg) (Vitamin B2) [83-88-5] 1603006
L-Selenomethionine L-Methionine (200 mg) [63-68-3] 1411504
L-Selenomethionine Selenomethionine (100 mg) [3211-76-5] 1611955
L-Serine L-Serine (200 mg) [56-45-1] 1612506
Sodium Iron EDTA Nitrilotriacetic Acid (50 mg) [139-13-9] 1463950
General Information

Sodium Iron EDTA Sodium Iron EDTA (200 mg) [15708-41-5] 1614239
Sodium Stearyl Fumarate Sodium Stearyl Fumarate (200 mg) [4070-80-8] 1614705
Sodium Stearyl Fumarate Stearyl Alcohol (125 mg) [112-92-5] 1622000
Sodium Benzoate Benzoic Acid (300 mg) [65-85-0] 1055002
FCC 8 General Information / USP Reference Standards for Food Ingredients / 1431

FCC Monograph Supporting USP Reference Standard(s) CAS Number Catalog Number
Sodium Benzoate Sodium Benzoate (1 g) [532-32-1] 1613564
Sodium Cyclamate Sodium Cyclamate (200 mg) [139-05-9] 1613860
Sodium Fumarate Maleic Acid (300 mg) [110-16-7] 1374500
Sodium Gluconate Potassium Gluconate (200 mg) [299-27-4] 1550001
Sodium Pyrophosphate Sodium Fluoride (1 g) (Internationally Restricted [7681-49-4] 1614002
Sales Item)
Sorbitan Monopalmitate Isosorbide (75% solution, 1 g) [652-67-5] 1352008
Sorbitan Monopalmitate 1,4-Sorbitan (300 mg) [27299-12-3] 1616008
Sorbitan Monopalmitate Sorbitol (125 mg) [50-70-4] 1617000
Sorbitan Monolaurate Isosorbide (75% solution, 1 g) [652-67-5] 1352008
Sorbitan Monolaurate 1,4-Sorbitan (300 mg) [27299-12-3] 1616008
Sorbitan Monolaurate Sorbitol (125 mg) [50-70-4] 1617000
Sorbitan Monooleate Isosorbide (75% solution, 1 g) [652-67-5] 1352008
Sorbitan Monooleate 1,4-Sorbitan (300 mg) [27299-12-3] 1616008
Sorbitan Monooleate Sorbitol (125 mg) [50-70-4] 1617000
Sorbitan Monostearate Isomalt (200 mg) [64519-82-0] 1349626
Sorbitan Monostearate Isosorbide (75% solution, 1 g) [652-67-5] 1352008
Sorbitan Monostearate 1,4-Sorbitan (300 mg) [27299-12-3] 1616008
Sorbitan Tristearate Isosorbide (75% solution, 1 g) [652-67-5] 1352008
Sorbitan Tristearate 1,4-Sorbitan (300 mg) [27299-12-3] 1616008
Sorbitan Tristearate Sorbitol (125 mg) [50-70-4] 1617000
Sorbitol Mannitol (200 mg) [69-65-8] 1375105
Sorbitol Sorbitol (125 mg) [50-70-4] 1617000
Sorbitol Solution Mannitol (200 mg) [69-65-8] 1375105
Sorbitol Solution Sorbitol (125 mg) [50-70-4] 1617000
Hydrogenated Starch Hydrolysate Dextrose (500 mg) [50-99-7] 1181302
Hydrogenated Starch Hydrolysate Maltitol (200 mg) [585-88-6] 1374907
Hydrogenated Starch Hydrolysate Sorbitol (125 mg) [50-70-4] 1617000
Stearyl Alcohol Cetyl Alcohol (100 mg) [36653-82-4] 1103003
Stearyl Alcohol Stearyl Alcohol (125 mg) [112-92-5] 1622000
Succinic Acid Succinic Acid (100 mg) [110-15-6] 1623411
Sucralose Sucralose (400 mg) [56038-13-2] 1623626
Sucromalt Sucromalt (200 mg) [911432-63-8] 1623659
Tartaric Acid Tartaric Acid (1 g) [87-69-4] 1643340
L-Theanine L-Theanine (200 mg) [3081-61-6] 1652704
Thiamine Hydrochloride Thiamine Hydrochloride (500 mg) (Vitamin B1 [67-03-8] 1656002
Hydrochloride)
Thiamine Mononitrate Thiamine Hydrochloride (500 mg) (Vitamin B1 [67-03-8] 1656002
Hydrochloride)
L-Threonine L-Threonine (200 mg) [72-19-5] 1667202
All-rac-α-Tocopherol Alpha Tocopherol (250 mg) (Vitamin E Alcohol) [59-02-9] 1667600
All-rac-α-Tocopherol Alpha Tocopheryl Acetate (250 mg) (Vitamin E [7695-91-2] 1667701
Acetate)
RRR-α-Tocopherol Concentrate Alpha Tocopherol (250 mg) (Vitamin E Alcohol) [59-02-9] 1667600
RRR-α-Tocopherol Concentrate Alpha Tocopheryl Acetate (250 mg) (Vitamin E [7695-91-2] 1667701
Acetate)
RRR-Tocopherol Concentrate, Mixed Alpha Tocopherol (250 mg) (Vitamin E Alcohol) [59-02-9] 1667600
General Information

All-rac-α-Tocopherol Acetate Alpha Tocopherol (250 mg) (Vitamin E Alcohol) [59-02-9] 1667600
All-rac-α-Tocopherol Acetate Alpha Tocopheryl Acetate (250 mg) (Vitamin E [7695-91-2] 1667701
Acetate)
RRR-α-Tocopherol Acetate Alpha Tocopherol (250 mg) (Vitamin E Alcohol) [59-02-9] 1667600
RRR-α-Tocopherol Acetate Alpha Tocopheryl Acetate (250 mg) (Vitamin E [7695-91-2] 1667701
Acetate)
 1432 / USP Reference Standards for Food Ingredients / General Information FCC 8

FCC Monograph Supporting USP Reference Standard(s) CAS Number Catalog Number
RRR-α-Tocopherol Concentrate Alpha Tocopherol (250 mg) (Vitamin E Alcohol) [59-02-9] 1667600
RRR-α-Tocopherol Concentrate Alpha Tocopheryl Acetate (250 mg) (Vitamin E [7695-91-2] 1667701
Acetate)
RRR-α-Tocopherol Acid Succinate Alpha Tocopherol (250 mg) (Vitamin E Alcohol) [59-02-9] 1667600
RRR-α-Tocopherol Acid Succinate Alpha Tocopheryl Acetate (250 mg) (Vitamin E [7695-91-2] 1667701
Acetate)
RRR-α-Tocopherol Acid Succinate Alpha Tocopheryl Acid Succinate (250 mg) [4345-03-3] 1667803
(Vitamin E Succinate)
Trehalose Trehalose (400 mg) [6138-23-4] 1673715
L-Tryptophan L-Tryptophan (200 mg) [73-22-3] 1700501
L-Tyrosine L-Tyrosine (500 mg) [60-18-4] 1705006
L-Valine L-Valine (200 mg) [72-18-4] 1708503
Vitamin A Retinyl Acetate (Vitamin A) (10 ampules × 0.5 g) [Mixture] 1716002
Vitamin B12 Cyanocobalamin (1.5 g of mixture with mannitol) [Mixture] 1152009
(Vitamin B12)
Vitamin D2 Ergocalciferol (30 mg/ampule; 5 ampules) (Vitamin [50-14-6] 1239005
D2)
Vitamin D2 Ergosterol (50 mg) [57-87-4] 1241007
Vitamin D2 Vitamin D Assay System Suitability (1.5 g) [Mixture] 1717504
Vitamin D3 Cholecalciferol (30 mg/ampule; 5 ampules) [67-97-0] 1131009
(Vitamin D3)
Vitamin D3 Vitamin D Assay System Suitability (1.5 g) [Mixture] 1717504
Vitamin K Phytonadione (500 mg) (Vitamin K1) [84-80-0] 1538006
Whey Fructose (125 mg) [57-48-7] 1286504
Whey Lactose Monohydrate (500 mg) [5989-81-1] 1356701
Whey Protein Concentrate Fructose (125 mg) [57-48-7] 1286504
Whey Protein Concentrate Lactose Monohydrate (500 mg) [5989-81-1] 1356701
Whey Protein Isolate Fructose (125 mg) [57-48-7] 1286504
Whey Protein Isolate Lactose Monohydrate (500 mg) [5989-81-1] 1356701
Whey, Reduced Lactose Fructose (125 mg) [57-48-7] 1286504
Whey, Reduced Lactose Lactose Monohydrate (500 mg) [5989-81-1] 1356701
Xylitol Xylitol (1 g) [87-99-0] 1720600
Yeast, Dried Folic Acid (500 mg) (Vitamin M or Vitamin Bc) [59-30-3] 1286005
Zinc Gluconate Potassium Gluconate (200 mg) [299-27-4] 1550001
Zinc Gluconate Trypsin Crystallized (300 mg) [9002-07-7] 1700002
Appendix IIB—Melting Range or Temperature Caffeine Melting Point Standard (1 g) [58-08-2] 1086006
Determination (Approximately 236°)
Appendix IIB—Melting Range or Temperature Phenacetin Melting Point Standard (500 mg) [62-44-2] 1514008
Determination (Approximately 135°)
Appendix IIB—Melting Range or Temperature Acetanilide Melting Point Standard (500 mg) [103-84-4] 1004001
Determination (Approximately 114°)
Appendix IIB—Melting Range or Temperature Vanillin Melting Point Standard (1 g) [121-33-5] 1711009
Determination (Approximately 82°)
Appendix IIB—Melting Range or Temperature Sulfanilamide Melting Point Standard (500 mg) [63-74-1] 1633007
Determination (Approximately 165°)
Appendix IIB—Melting Range or Temperature Sulfapyridine Melting Point Standard (1 g) [144-83-2] 1635002
Determination (Approximately 191°)
Appendix IIIC—Glutamic Acid Glutamic Acid (200 mg) [56-86-0] 1294976
Appendix V—Enzyme Assays Bile Salts (Sodium taurochlorate) (10 g) [145-42-6] 1071304
General Information

Appendix V—Enzyme Assays Pancreatin Amylase and Protease (2 g) [8049-47-6] 1494057

Appendix V—Enzyme Assays Pancreatin Lipase (2 g) [8049-47-6] 1494079
Appendix V—Enzyme Assays Papain (1 g) [9001-73-4] 1495005
Appendix V—Enzyme Assays Trypsin Crystallized (300 mg) [9002-07-7] 1700002
Appendix VII—Fats And Related Substances FAME Standard Mixture (100 mg) [71076-49-8] 1269119
FCC 8 General Information / USP Reference Standards for Food Ingredients / 1433

FCC Monograph Supporting USP Reference Standard(s) CAS Number Catalog Number
Appendix VII—Fats And Related Substances Menhaden Oil [8002-50-4] 1381200
Appendix VII—Fats And Related Substances Tritricosanoin (50 mg) [86850-72-8] 1696153
Appendix VIII—Oleoresins Capsaicin (100 mg) [404-86-4] 1091108
Appendix XIII—Adulterants and Contaminants Diethylene Glycol (0.5 mL) [111-46-6] 1193265
in Food Ingredients
Appendix XIII—Adulterants and Contaminants Ethylene Glycol (0.5 mL) [107-21-1] 1265515
in Food Ingredients
Appendix XIII—Adulterants and Contaminants Glycerin (2 mL) [56-81-5] 1295607
in Food Ingredients

General Information
1434 / Electrophoresis / General Information
General Information Analytical
Techniques
ELECTROPHORESIS*
Electrophoresis refers to the migration of electrically
charged proteins, colloids, molecules, or other particles
when dissolved or suspended in an electrolyte through
which an electric current is passed.
Based upon the type of apparatus used, electrophoretic
methods may be divided into two categories, one called free
solution or moving boundary electrophoresis and the other
called zone electrophoresis.
In the free solution method, a buffered solution of proteins
in a U-shaped cell is subjected to an electric current which
causes the proteins to form a series of layers in order of
decreasing mobility, which are separated by boundaries.
Only a part of the fastest moving protein is physically sepa-
rated from the other proteins, but examination of the mov-
ing boundaries using a schlieren optical system provides
data for calculation of mobilities and information on the
qualitative and quantitative composition of the protein
mixture.
In zone electrophoresis, the sample is introduced as a nar-
row zone or spot in a column, slab, or film of buffer. Migra-
tion of the components as narrow zones permits their com-
plete separation. Remixing of the separated zones by
thermal convection is prevented by stabilizing the electro-
lyte in a porous matrix such as a powdered solid, or a fi-
brous material such as paper, or a gel such as starch, agar,
or polyacrylamide.
Various methods of zone electrophoresis are widely em-
ployed. Gel electrophoresis, particularly the variant called disk
electrophoresis, is especially useful for protein separation be-
cause of its high resolving power.
Gel electrophoresis, which is employed by the compen-
dium, is discussed in more detail following the presentation
of some theoretical principles and methodological practices,
which are shared in varying degrees by all electrophoretic
methods.
The electrophoretic migration observed for particles of a
particular substance depends on characteristics of the parti-
cle, primarily its electrical charge, its size or molecular
weight, and its shape, as well as characteristics and operat-
ing parameters of the system. These latter include the pH,
ionic strength, viscosity and temperature of the electrolyte,
density or cross-linking of any stabilizing matrix such as gel,
and the voltage gradient employed.
Effect of Charge, Particle Size, Electrolyte Viscosity, and
Voltage Gradient—Electrically charged particles migrate to-
ward the electrode of opposite charge, and molecules with
both positive and negative charges move in a direction de-
pendent on the net charge. The rate of migration is directly
related to the magnitude of the net charge on the particle
* This text is adapted from General Chapter 〈726〉 of the United States
Pharmacopeia and National Formulary (USP–NF) as published in USP 32–NF 27.
This text is provided for informational purposes only and is intended as a
resource for the FCC user. Note that because the USP–NF is in continuous
revision, this General Chapter is subject to change and the text printed here
may not continue to represent the current version.
FCC 8
and is inversely related to the size of the particle, which in
turn is directly related to its molecular weight.
Very large spherical particles, for which Stokes’ law is
valid, exhibit an electrophoretic mobility, u0, which is in-
versely related to the first power of the radius as depicted in
the equation:
u0 = v/E = Q/6πrη
where ν is the velocity of the particle, E is the voltage gradi-
ent imposed on the electrolyte, Q is the charge on the par-
ticle, r is the particle radius, and η is the viscosity of the
electrolyte. This idealized expression is strictly valid only at
infinite dilution and in the absence of a stabilizing matrix
such as paper or a gel.
Ions, and peptides up to molecular weights of at least
5000, particularly in the presence of stabilizing media, do
not obey Stokes’ law, and their electrophoretic behavior is
best described by an equation of the type:
u0 = Q/A6πr2η
where A is a shape factor generally in the range of 4 to 6,
which shows an inverse dependence of the mobility on the
square of the radius. In terms of molecular weight, this im-
plies an inverse dependence of mobility on the 2/3 power of
the molecular weight.
Effect of pH—The direction and rate of migration of mole-
cules containing a variety of ionizable functional groups,
such as amino acids and proteins, depends upon the pH of
the electrolyte. For instance, the mobility of a simple amino
acid such as glycine varies with pH approximately as shown
in Figure 1. The pKa values of 2.2 and 9.9 coincide with the
inflection points of the sigmoid portions of the plot. Since
the respective functional groups are 50% ionized at the pH
values where pH = pKa, the electrophoretic mobilities at
these points are half of the value observed for the fully ion-
ized cation and anion obtained at very low and very high
pH, respectively. The zwitterion that exists at the intermedi-
ate pH range is electrically neutral and has zero mobility.
Fig. 1
FCC 8
Effect of Ionic Strength and Temperature—Electrophoretic
mobility decreases with increasing ionic strength of the sup-
porting electrolyte. Ionic strength, µ, is defined as:
µ = 0.5ΣCiZi2
where Ci is the concentration of an ion in moles per L and
Zi is its valence, and the sum is calculated for all ions in the
solution. For buffers in which both the anion and cation are
univalent, ionic strength is identical with molarity.
Ionic strengths of electrolytes employed in electrophoresis
commonly range from about 0.01 to 0.10. A suitable
strength is somewhat dependent on the sample composi-
tion, since the buffer capacity must be great enough to
maintain a constant pH over the area of the component
zones. Zones become sharper or more compact as ionic
strength is increased.
Temperature affects mobility indirectly, since the viscosity,
η, of the supporting electrolyte is temperature-dependent.
The viscosity of water decreases at a rate of about 3% per
°C in the range of 0° to 5° and at a slightly lower rate in
the vicinity of room temperature. Mobility, therefore, in-
creases with increasing electrolyte temperature.
Considerable heat is evolved as a result of current passing
through the supporting electrolyte. This heat increases with
the applied voltage and with increasing ionic strength. Par-
ticularly in larger apparatus, despite the circulation of a
coolant, this heat produces a temperature gradient across
the bed which may lead to distortion of the separated
zones. Therefore, practical considerations and the design of
the particular apparatus dictate the choice of ionic strength
and operating voltage.
Effect of a Stabilizing Medium, Electroosmosis—When an
electrical current is passed through an electrolyte contained
in a glass tube or contained between plates of glass or
plastic, a bulk flow of the electrolyte toward one of the
electrodes is observed. This flow is called electroosmosis. It
results from the surface charge on the walls of the appara-
tus, which arises either from ionizable functional groups in-
herent in the structural material or from ions adsorbed on
the cell walls from the electrolyte contacting them. The ef-
fect is usually increased when the cell is filled with a bed of
porous substance, such as a gel, used to stabilize the sup-
porting electrolyte and prevent remixing of separated zones
by thermal convection or diffusion. The solution immedi-
ately adjacent to the surface builds up an electrical charge,
equal but opposite to the surface charge, and the electrical
field traversing the cell produces a movement of solution
toward the electrode of opposite charge.
The substances commonly used as stabilizing media in
zone electrophoresis develop a negative surface charge, and
therefore electroosmotic flow of the electrolyte is toward the
cathode. As a result, all zones, including neutral substances,
are carried toward the cathode during the electrophoretic
run.
The degree of electroosmosis observed varies with the sta-
bilizing substance. It is appreciable with agar gel, while it is
negligibly small with polyacrylamide gel.
Molecular Sieving—In the absence of a stabilizing medium
or in cases where the medium is very porous, electropho-
retic separation of molecules results from differences in the
ratio of their electrical charge to their size. In the presence
of a stabilizing medium, differences in adsorptive or other
affinity of molecules for the medium introduces a chromato-
graphic effect that may enhance the separation.
If the stabilizing medium is a highly cross-linked gel such
that the size of the resultant pores is of the order of the
dimensions of the molecules being separated, a molecular
sieving effect is obtained. This effect is analogous to that
General Information / Electrophoresis / 1435
obtained in separations based on gel permeation or molecu-
lar exclusion chromatography, but in gel electrophoresis the
effect is superimposed on the electrophoretic separation.
Molecular sieving may be visualized to result from a steric
barrier to the passage of larger molecules. Small molecules
pass through pores of a wide size range, and therefore their
electrophoretic passage through the gel will not be im-
peded. As size increases, fewer pores will permit passage of
the molecules, causing a retardation of the migration of
substances of large molecular weight.
Gel Electrophoresis
Processes employing a gel such as agar, starch, or polya-
crylamide as a stabilizing medium are broadly termed gel
electrophoresis. The method is particularly advantageous for
protein separations. The separation obtained depends upon
the electrical charge to size ratio coupled with a molecular
sieving effect dependent primarily on the molecular weight.
Polyacrylamide gel has several advantages that account
for its extensive use. It has minimal adsorptive properties
and produces a negligible electroosmotic effect. Gels of a
wide range of pore size can be reproducibly prepared by
varying the total gel concentration (based on monomer plus
cross-linking agent) and the percentage of cross-linking
agent used to form the gel. These quantities are conven-
iently expressed as
T(%) = (a + b/V) × 100
T(%) = (b/a + b) × 100
where T is the total gel concentration in %; C is the per-
centage of cross-linking agent used to prepare the gel; V is
the volume, in mL, of buffer used in preparing the gel; and
a and b are the weights, in g, of monomer (acrylamide) and
cross-linking agent (usually N,N′-methylenebisacrylamide)
used to prepare the gel. Satisfactory gels ranging in concen-
tration (T) from about 3% to 30% have been prepared. The
amount of cross-linking agent is usually about one-tenth to
one-twentieth of the quantity of monomer (C = 10% to
5%), a smaller percentage being used for higher values of T.
In the preparation of the gel, the bed of the electrophore-
sis apparatus is filled with an aqueous solution of monomer
and cross-linking agent, usually buffered to the pH desired
in the later run, and polymerized in place by a free radical
process. Polymerization may be initiated by a chemical pro-
cess, frequently using ammonium persulfate plus N,N,N′,N′-
tetramethylenediamine or photochemically using a mixture
of riboflavin and N,N,N′,N′-tetramethylenediamine. Polymer-
ization is inhibited by molecular oxygen and by acidic con-
ditions. The gel composition and polymerization conditions
chosen must be adhered to rigorously to ensure reproduci-
ble qualities of the gel.
Apparatus for Gel Electrophoresis—In general, the bed
or medium in which electrophoresis is carried out may be
supported horizontally or vertically, depending upon the de-
sign of the apparatus. A series of separations to be com-
pared may also be carried out in several individual tubes or
by placing different samples in adjacent wells, cast or cut
into a single slab of gel. A vertical slab assembly such as
that depicted schematically in Figure 2 is convenient for di-
rect comparison of several samples. A particular advantage
derives from the comparison of the samples in a single bed
of gel which is likely to be more uniform in composition
than gels cast in a series of chambers.
1436 / Electrophoresis / General Information
Fig. 2. Vertical Slab Gel Electrophoresis Apparatus.
A feature of many types of apparatus, not illustrated in
the schematic view, seals the lower buffer chamber to the
base of the bed and allows the level of the buffer in the
lower chamber to be made equal to that in the upper
chamber, thereby eliminating hydrostatic pressure on the
gel. In addition, some units provide for the circulation of
coolant on one or both sides of the gel bed.
In the preparation of the gel, the base of the gel chamber
is closed with a suitable device and the unit is filled with the
solution of monomer, cross-linking agent, and catalyst. A
comb, having teeth of an appropriate size, is inserted in the
top, and polymerization is allowed to proceed to comple-
tion. Removal of the comb leaves a series of sample wells in
the polymerized gel.
In simple gel electrophoresis, an identical buffer is used to
fill the upper and lower buffer chambers as well as in the
solution used to prepare the gel. After filling the chambers,
the samples, dissolved in sucrose or other dense and some-
what viscous solution to prevent diffusion, are introduced
with a syringe or micropipet into the bottoms of the sample
wells, and the electrophoresis is begun immediately
thereafter.
DISK ELECTROPHORESIS
An important variant of polyacrylamide gel electrophore-
sis, which employs a discontinuous series of buffers and
often also a discontinuous series of gel layers, is called disk
electrophoresis. The name is derived from the discoid shape
of the very narrow zones that result from the technique. As
a result of the narrow zones produced, this technique exhib-
its an extremely high resolving power and is to be recom-
mended for the characterization of protein mixtures and for
the detection of contaminants that may have mobilities
close to that of the major component.
The basis of disk electrophoresis is outlined in the follow-
ing paragraphs with reference to an anionic system suitable
for separating proteins bearing a net negative charge. To
understand disk electrophoresis, it is essential to have a
knowledge of the general aspects of electrophoresis and the
apparatus already described.
Basis of Disk Electrophoresis—The high resolution ob-
tained in disk electrophoresis depends on the use of a buffer
system that is discontinuous with respect to both pH and
composition. This is usually combined with a discontinuous
series of two or three gels that differ in density.
A typical system is illustrated schematically in Figure 3.
FCC 8
Fig. 3. Terminology, Buffer pH, and Buffer Composition for
Acrylamide Gel Disk Electrophoresis.
A high density (T = 10% to 30%) separating gel several
centimeters high is polymerized in a tris-chloride buffer in
the bed of the apparatus. During polymerization the buffer
is overlayered with a thin layer of water to prevent fixation
of a meniscus in the top of the gel. The overlayer of water is
then removed and a thin layer, 3 mm to 10 mm thick, of
low density (T = 3%) gel, called the spacer or stacking gel,
is polymerized in a tris-chloride buffer on top of the separat-
ing gel. An overlayer of water is again used to ensure a flat
surface. The sample is mixed with a small amount of the
spacer gel monomer solution which is applied on top of the
spacer gel and allowed to polymerize. The pH of the sepa-
rating gel is typically 8.9, while that of the spacer and sam-
ple gels is 6.7. All three gels are prepared using chloride as
the anion.
The upper and lower buffer reservoirs are filled with a pH
8.3 buffer prepared from tris and glycine. At this pH about
3% of the glycine molecules bear a net negative charge.
When a voltage is applied across the system, the
glycinate-chloride interface moves downward toward the
anode. It was initially positioned at the junction of the
buffer in the upper reservoir and the top of the sample gel
layer. The chloride anion, by virtue of its small size, migrates
faster than any of the proteins present in the sample. The
pH of the sample and spacer layers was chosen to be about
3 units below the higher pKa of glycine. Therefore, in tra-
versing these layers, only about 0.1% of the glycine mole-
cules bear a net negative charge. Consequently, glycine mi-
grates more slowly than chloride. The tendency for the
faster-moving chloride to move away from glycinate lowers
the concentration at the interface, producing a greater volt-
age drop at the interface, which in turn causes the glycinate
to catch up to the chloride. Under these conditions, a very
sharp interface is maintained, and as it moves through the
sample and spacer layers, the proteins in the sample tend to
stack themselves at the interface in very thin layers in order
of mobility. The process is called stacking and is the source
of the disks which are separated.
When the stacked proteins reach the high-density separat-
ing gel, they are slowed down by a molecular sieving pro-
cess. The higher pH encountered in the running gel also
causes the glycinate to migrate faster, so that the discontin-
uous buffer interface overtakes the proteins and eventually
reaches the bottom of the separating gel. During this pe-
riod, the disks of protein continue to separate by electro-
phoresis and molecular sieving in the separating gel. At the
end of the run, the pH of the separating gel will have risen
above its original value of 8.9 to a value of about pH 9.5.
Relative Mobility—Bromophenol blue is often used as a
standard for calculating the relative mobility of separated
zones and to judge visually the progress of a run. It may be
added to one of the sample wells, or mixed with the sample
FCC 8
itself, or simply added to the buffer in the upper sample
reservoir.
Relative mobility, MB, is calculated as:
MB = distance from origin to sample zone/distance from ori-
gin to bromophenol blue zone
Visualization of Zones—Since polyacrylamide is trans-
parent, protein bands may be located by scanning in a den-
sitometer with UV light. The zones may be fixed by immers-
ing in protein precipitants such as phosphotungstic acid or
10% trichloroacetic acid. A variety of staining reagents in-
cluding naphthalene black (amido black) and Coomassie
brilliant blue R250 may be used. The fixed or stained zones
may be conveniently viewed and photographed with trans-
mitted light from an X-ray film illuminator.
SAFETY PRECAUTIONS
Voltages used in electrophoresis can readily deliver a lethal
shock. The hazard is increased by the use of aqueous buffer
solutions and the possibility of working in damp
environments.
The equipment, with the possible exception of the power
supply, should be enclosed in either a grounded metal case
or a case made of insulating material. The case should have
an interlock that deenergizes the power supply when the
case is opened, after which reactivation should be prevented
until activation of a reset switch is carried out.
High-voltage cables from the power supply to the appara-
tus should preferably be a type in which a braided metal
shield completely encloses the insulated central conductor,
and the shield should be grounded. The base of the appara-
tus should be grounded metal or contain a grounded metal
rim which is constructed in such a way that any leakage of
electrolyte will produce a short which will deenergize the
power supply before the electrolyte can flow beyond the
protective enclosure.
If the power supply contains capacitors as part of a filter
circuit, it should also contain a bleeder resistor to ensure
discharge of the capacitors before the protective case is
opened. A shorting bar that is activated by opening the
case may be considered as an added precaution.
Because of the potential hazard associated with electro-
phoresis, laboratory personnel should be completely familiar
with electrophoresis equipment before using it.
CAPILLARY ELECTROPHORESIS*
Electrophoresis refers to the migration of charged electri-
cal species when dissolved or suspended in an electrolyte
through which an electric current is passed. Cations migrate
toward the negatively charged electrode (cathode), while
anions are attracted toward the positively charged electrode
(anode). Neutral particles are not attracted toward either
electrode.
The use of capillaries as a migration channel in electro-
phoresis has enabled analysts to perform electrophoretic
separations on an instrumental level comparable to that of
high-performance liquid chromatography (HPLC), albeit
with some distinct operational differences, advantages, and
disadvantages relative to HPLC. This method of analysis is
* This text is adapted from General Chapter 〈727〉 of the United States
Pharmacopeia and National Formulary (USP–NF) as published in USP 32–NF 27.
This text is provided for informational purposes only and is intended as a
resource for the FCC user. Note that because the USP–NF is in continuous
revision, this General Chapter is subject to change and the text printed here
may not continue to represent the current version.
General Information / Capillary Electrophoresis / 1437
commonly known as capillary electrophoresis (CE). During
typical CE operation with an uncoated capillary filled with a
buffer, referred to as the “operating buffer,” silanol groups
present on the inner wall of the glass capillary release hy-
drogen ions to the buffer and the wall surface becomes
negatively charged, even at a fairly low pH. Cations, or sol-
utes having partial positive charges in the medium, are elec-
trostatically attracted to the negatively charged wall, form-
ing an electrical double layer. The initiation of
electrophoresis by applying voltage across the length of the
capillary causes the solution portion of the electrical double
layer to move toward the cathode end of the capillary,
drawing the bulk solution. This movement of the bulk solu-
tion under the force of the electrical field is called the elec-
troosmotic flow (EOF). The degree of ionization of the in-
ner-wall capillary silanol groups depends mainly on the pH
of the operating buffer and on the modifiers that may have
been added to the electrolyte. At low pH, the silanol groups
generally have a low ionization and the EOF is low. At
higher pH, silanol groups become more ionized and the
EOF increases. In some cases organic solvents, such as meth-
anol or acetonitrile, are added to the aqueous buffer to in-
crease the solubility of the solute and other additives or to
affect the degree of ionization of the sample. The addition
of such organic modifiers generally causes a decrease in the
EOF. The detector is located toward the cathode end of the
capillary. The EOF is usually greater than the electrophoretic
mobility; thus, even anions are swept toward the cathode
and the detector. When an uncoated capillary containing
pH 7.0 phosphate buffer is used, the usual order of appear-
ance of solutes in an electropherogram is cationic species,
neutral solutes, and anionic species.
Currently, there are five major modes of operation of CE:
capillary zone electrophoresis (CZE), also referred to as free
solution or free flow capillary electrophoresis; micellar elec-
trokinetic chromatography (MEKC); capillary gel electropho-
resis (CGE); capillary isoelectric focusing (CIEF); and capillary
isotachophoresis (CITP).
In CZE, separations are controlled by differences in the
relative electrophoretic mobilities of the individual compo-
nents in the sample or test solution. The mobility differences
are functions of analyte charge and size under specific
method conditions. They are optimized by appropriate con-
trol of the composition of the buffer, its pH, and its ionic
strength.
In MEKC, ionic surfactants are added to the operating
buffer at a concentration above their critical micelle concen-
tration. The micelles provide a pseudostationary phase with
which analytes can partition. This technique is useful for the
separation of neutral and ionic species.
CGE, which is analogous to gel filtration, uses gel-filled
capillaries to separate molecules on the basis of relative dif-
ferences in their respective molecular weight or molecular
size. It was first used for the separation of proteins, pep-
tides, and oligomers. Gels may have the advantage of de-
creasing the EOF and also significantly reducing protein ad-
sorption onto the inner wall of the capillary, which can
significantly reduce analyte peak tailing effects.
In CIEF, substances are separated on the basis of their
relative differences in isoelectric points. This is accomplished
by achieving steady-state sample zones within a buffer pH
gradient, where the pH is low at the anode and high at the
cathode. The gradient is established by applying a voltage
across a capillary filled with a mixture of carrier components
consisting of amphoteric substances having different pI
values.
CITP employs two buffers that enclose the analyte zones
between them. Either anions or cations can be analyzed in
sharply separated zones. In addition, the analyte concentra-
tions are the same in each zone; thus, the length of each
zone is proportional to the amount of the particular analyte.
The most commonly utilized capillary electrophoresis
techniques are CZE and MEKC. These are briefly discussed
in the following sections. Pertinent general principles and
1438 / Capillary Electrophoresis / General Information
theory, instrumental considerations, analysis, and opera-
tional considerations and parameters are discussed as well.
PRINCIPLES OF CAPILLARY ZONE
ELECTROPHORESIS
CZE makes use of the principles of electrophoresis and
electroosmosis to achieve separation of charged species.
(1) The electrophoretic mobility of an ion, µEP, is de-
scribed by the equation:
µEP = q/(6πηr)
in which q is the charge of the ion, η is the solution viscos-
ity, and r is the radius of the hydrated ion. This relationship
infers that small, highly charged analytes have high mobili-
ties and large, slightly charged analytes have low mobilities.
(2) The velocity of migration, νEP, in cm per second, is
represented by the equation:
νEP = µEP(V/L)
in which µEP is the electrophoretic mobility; V is the applied
voltage; and L, in cm, is the total capillary length.
(3) The velocity of the EOF, νEO, in cm per second, is
described by the equation:
νEO = µEO(V/L)
in which µEO is the EOF mobility (the coefficient of electroos-
motic flow), and the other terms are as defined above.
(4) The time, t, in seconds, necessary for a solute to mi-
grate the entire effective length of the capillary (from the
inlet to the detector), l, is represented by the relationship:
t = l/E(µEP + µEO) = lL/V(µEP + µEO)
in which E is the strength of the applied electrical field, and
the other terms are as defined above.
(5) Efficiency of an electrophoretic system can be related
to mobility and EOF and expressed in terms of the number
of theoretical plates, N, by the equation:
N = (µEP + µEO)V/2D
in which D is the diffusion coefficient of the solute, and the
other terms are as defined above.
(6) The resolution, R, of two consecutively eluting solutes
can be defined by the equation:
R = 0.18(µEP1 − µEP2) [V/D (µEP + µEO)] /1
2
where µEP1 and µEP2 are the mobilities of the two solutes,
µEP
is their average, and the other terms are as defined above.
PRINCIPLES OF MICELLAR ELECTROKINETIC
CHROMATOGRAPHY
In MEKC, the supporting electrolyte medium contains a
surfactant at a concentration above its critical micelle con-
centration (CMC). In this aqueous medium, the surfactant
self-aggregates and forms micelles whose hydrophilic head
groups form an outer shell and whose hydrophobic tail
groups form a nonpolar core into which the solutes can
partition. Generally, the micelles are anionic on their sur-
face, and, under the applied voltage, they migrate in the
opposite direction to the EOF. This type of partitioning is
analogous to that in solvent extraction or reverse-phase
HPLC. The differential partitioning of neutral molecules be-
tween the buffered aqueous mobile phase and the micellar
FCC 8
pseudostationary phase is the sole basis for separation. The
buffer and micelles form a two-phase system, and the
analyte can partition between these two phases.
A micellar system suitable for MEKC meets the following
criteria: the surfactant is highly soluble in the buffer, and
the micellar solution is homogeneous and transparent when
UV detection is employed. The most common surfactant for
MEKC is sodium dodecyl sulfate (anionic surfactant). Others
include cetyltrimethylammonium bromide (cationic
surfactant) and bile salts (chiral surfactant). The selectivity of
an MEKC system is mainly dependent on the nature of the
surfactant. Organic solvents are often added to the MEKC
buffer to adjust the capacity factors, just as in reverse-phase
HPLC separations. MEKC may be used for the separation of
enantiomers. For such separations, a chiral additive is added
to the buffer or a chiral surfactant, such as a bile salt, is
used.
A general knowledge of conventional column chromato-
graphic principles aids in understanding MEKC principles.
However, in MEKC the micelles are not truly stationary;
therefore, the column chromatographic theory needs to be
modified. The major modification introduced to MEKC prin-
ciples is the finite nature of the separation window for neu-
tral molecules.
(7) The migration time, tR, for a neutral species is ex-
pressed with the following equation:
tR = (1 + k′)t0/[1 + (t0/tMC)]
in which t0 is the time required for an unretained substance
to travel the effective length of the capillary; tMC is the time
required for a micelle to traverse the capillary; k′ is the ca-
pacity factor; and tR is always between t0 and tMC.
(8) The capacity factor, k′, for a neutral species, is calcu-
lated by the equation:
k′ = (tR/t0 − 1)/(1 − tR/tMC)
in which the terms are as defined above.
(9) For practical purposes, k′ is calculated by the
equation:
k′ = tR/t0 − 1
in which tR is the time measured from the point of voltage
application (or injection) to the peak maximum; and t0 is
measured from the point of voltage application (or injec-
tion) to the leading edge of the solvent front or of an un-
retained substance. In contrast with CZE, k′ in MEKC is sig-
nificant and is a characteristic of a given solute in a given
MEKC system. Further discussion of k′ appears later in the
System Suitability section under Operational Parameters.
(10) The resolution, RS, for neutral species is calculated by
the equation:
RS = [(√N)/4][(α − 1)/α][k′2/(1 + k′2)]
[(1 − (t0/tMC))/(1 + (t0/tMC)k′1)]
in which α is the selectivity, defined as the ratio of k′2 to k′1,
of the operating conditions for separating two solutes. If the
two solutes elute close together (α ≤ 1.1), either k′ may be
used. The equation shows that, just as with conventional
chromatography, resolution in MEKC can be improved
through controlling efficiency, selectivity, retention, and the
chemical nature of the resolving surfactant-medium system.
The last term of the equation is due to the limited elution
range. Although MEKC is particularly useful in the separa-
tion of neutral species, this technique may also be used for
the separation of charged solutes. The latter procedure in-
volves a combination of chromatographic and electropho-
retic separation mechanisms. The additional interaction be-
tween charged solutes and micelle can be used to optimize
a separation. Ion-pairs may form if the charges borne on the
surfactant and solute are opposite; otherwise, surfactant and
FCC 8
Fig. 1. Typical CE Instrument Configuration.
solute repel each other. These differences can significantly
influence the separation of charged molecules.
INSTRUMENTAL CONSIDERATIONS
A typical CE system (see Figure 1) contains a fused-silica
capillary having an internal diameter of 50 to 100 µm and a
length of 20 to 100 cm. The ends of the capillary are placed
in separate electrolyte reservoirs. The direct-current power
supply is capable of furnishing high voltages, typically rang-
ing from 0 to 30 kV. A detector and autosampler with some
form of data-recording device complete the system. An au-
tomatic buffer replenishment system and a computer-based
control and data acquisition system may also be found on
the standard commercial systems. Temperature controls for
both the capillary and the autosampler are also available on
commercial instruments.
The primary considerations of instrumentation include
capillary type and configuration, modes of sampling, power
supply and detector modes.
Capillary Type and Configuration
Capillaries used in CZE are usually made of fused silica
and with no internal coating. Some instruments are con-
figured with a “free-swinging” style of capillary; that is, the
capillary is not encased within an enclosure. In most com-
mercial instruments, the capillary is housed in a cartridge.
Both configurations offer specific advantages and disadvan-
tages. The ability of the instrument to accommodate differ-
ent types of capillaries and capillaries of various diameters
and lengths is an important consideration. Capillaries with a
variety of internal coatings are also available; therefore, the
ability of the instrument to accommodate different capilla-
ries is important. Internal capillary coatings may be em-
ployed to alter the magnitude or direction of EOF or to
reduce sample absorption. If an internally coated capillary is
to be used, then sufficient details and the indication of the
supplier must be included in the method. Capillaries from
an alternate supplier can be used if it is demonstrated that
they are suitable.
General Information / Capillary Electrophoresis / 1439
Sample Introduction and Injector Technology
Modes of sample introduction onto the capillary include
electromigration (electrokinetic mode) and negative- and
positive-pressure injection (hydrostatic mode).
For injection via electromigration, the sample solution is
electrophoresed into the capillary by inserting the capillary
and electrode into the sample vials and applying a brief,
high voltage. The sample enters the capillary by a combina-
tion of electrophoresis and EOF. Therefore, analytes with dif-
ferent mobilities are loaded into the capillary to different
extents. The conductivities of the sample and standard sol-
utes also affect the EOF and the volume injected.
Negative-pressure injectors place negative pressure at the
detector end of the capillary and draw the sample solution
into the injection end of the capillary. Positive-pressure in-
jectors pressurize the sample vial, forcing the sample into
the capillary. Pressure injection loads all sample components
into the capillary to the same extent, and it is generally the
most reproducible and the most frequently applied injection
mode. The sample volume injected depends on the capillary
length and internal diameter and the voltage or pressure
applied. The typical sample volumes injected into the capil-
lary are between 1 and 20 nL.
Each injection method offers specific advantages and dis-
advantages, depending on the sample composition, the sep-
aration mode, and the application of the method. None of
the above injection modes is as reproducible as commer-
cially available HPLC injectors. Based on the circumstances,
it may be necessary to use internal standards for specific
methods where high injection precision is required.
Power Supply
Most commercially available CE units have direct-current
power supplies that are capable of furnishing power on a
ramp-up or step-function mode to achieve and maintain the
desired operational voltage in a smooth manner. This will
help to ensure a relatively smooth baseline.
Another essential feature of the power supply is its utility
in introducing a sample at the cathodic or the anodic end
of the capillary. Because it is impractical to relocate the on-
line detector from one end of the instrument to the other, it
is beneficial to be able to specify whether the sample injec-
tion end is at the cathode or the anode.
1440 / Capillary Electrophoresis / General Information
Detector Modes
CE systems generally offer UV-visible absorbance and la-
ser-induced fluorescence (LIF) detectors. Scanning UV detec-
tors or photodiode-array detectors are also available for
many commercial CE instruments.
The coupling of CE to a mass spectrometer offers the pos-
sibility of obtaining structural information in conjunction
with electrophoretic migration data.
Fluorescence detection offers an enhanced sensitivity for
samples containing only very small amounts of UV-active
analytes. Application of fluorescent tags to non-UV-absorb-
ing compounds can be useful. Alternately, non-UV-absorb-
ing or nonfluorescent analytes can be detected indirectly by
adding a chromophore or a fluorophore, respectively, to the
buffer: the non-absorbing species are detected through the
absence of expected signal from the absorbing species.
Conductivity and pulsed amperometric detectors can also
be used but are not generally available on commercial CE
instruments.
ANALYTICAL CONSIDERATIONS
Several parameters, namely, capillary dimensions, voltage,
ionic strength, and pH, are optimized to give adequate reso-
lution and separation. Care should be taken to avoid
changes in temperature that will affect the viscosity of the
buffer and, in turn, influence both the EOF and the solute
mobilities.
Capillary Dimensions—Variation of the capillary diame-
ter and length can affect the electrophoretic resolution. In-
creasing the capillary length results in longer migration
times, usually increasing resolution and generating a lower
current. Increasing the capillary diameter will usually in-
crease current and associated internal temperature gradients
that decrease resolution. Conversely, a reduction in capillary
diameter will result in lower heat and better resolution.
However, larger capillary diameters have advantages of bet-
ter mass loading and improved signal-to-noise ratio.
Voltage Effects—When higher voltages are applied, ad-
ditional internal heating of the operating buffer occurs be-
cause of the current flow through the buffer. This heating
effect, known as Joule heating, must be controlled because
resistance, dielectric constant, and viscosity are temperature-
dependent and alter the velocity of the EOF and solute
mobilities.
In general, increasing the voltage will result in increased
efficiency and resolution (up to the point where Joule heat
cannot be adequately dissipated). Maximum resolution is
obtained by maintaining the voltage below the level at
which Joule heating and diffusion become limiting factors.
Ionic Strength Effects—Control of ionic strength and its
manipulation allow adjustment of resolution, efficiency, and
sensitivity. Increasing ionic strength will generally improve
resolution, peak efficiency, and peak shape. Sensitivity may
be improved because better focusing is achieved. However,
because the current generated is directly proportional to the
buffer concentration, more heat is produced when ionic
strength of the buffer is increased, hence limiting the ionic
strengths that can be utilized.
pH Effects—Resolution, selectivity, and peak shape can
be dramatically altered by changes in pH as this parameter
affects the extent of solute ionization and the level of EOF.
The EOF is high at high pH and low at low pH in uncoated
fused-silica capillaries.
OPERATIONAL PARAMETERS
The major steps in operating a CE system are system
setup, capillary rinsing procedure, running a sample, system
suitability testing, sample analysis, data handling, and sys-
tem shutdown.
FCC 8
System Setup—An appropriate capillary of specific
length, inner diameter, and coating is selected, with consid-
erations made for separation and resolution, ionic strength
of buffer, and pH effects. A buffer of appropriate composi-
tion, ionic strength, and pH is prepared, degassed, if neces-
sary, and passed through an appropriate filter. All solvents,
including water, are HPLC or CE grade.
Capillary Rinsing Procedure—Improved consistency of
migration times and resolution may generally be obtained if
a defined rinsing procedure is followed. Capillary condition-
ing and rinsing procedures are very specific to the analyte,
matrix, and method. Therefore, these procedures are devel-
oped as part of the method and are specified in the individ-
ual monograph. Rinsing may involve the use of solutions
such as 0.1 M phosphoric acid, water, and 0.1 M sodium
hydroxide. Before beginning analysis of the test specimen,
the capillary may be rinsed with five column volumes of the
operating buffer that is to be used for the test. When
changing buffer composition, it is advisable to rinse the cap-
illary with five column volumes of each new buffer to allow
the capillary to be cleansed of the previous buffer. Use of a
new uncoated fused-silica capillary usually requires a regen-
eration procedure to activate the surface silanol groups. This
procedure may include an extended rinse with a sodium
hydroxide solution. Coated capillaries are rinsed according
to the manufacturer’s guidelines because inappropriate rins-
ing can remove or damage the coating. Columns may be
dedicated to particular methods or buffer types to prevent
cross-contamination.
Running a Sample—An appropriate capillary, electrolyte,
and injection procedure are selected to achieve adequate
resolution, sensitivity, and separation, with well-shaped and
well-defined peaks. The required injection precision for a
specific method may require use of an internal standard.
The internal standard is selected with consideration of its
ability to adequately separate from the analyte. The perfor-
mance of the system may be improved by rinsing the capil-
lary between injections and supplying fresh buffer to the
source and destination vials used during voltage application,
namely, vials 2 and 4 in Figure 1. Replicate injections from
the same sample vial may be performed provided that no
cross-contamination occurs. If cross-contamination occurs,
the capillary tip may be rinsed by briefly inserting it into a
vial containing the buffer prior to inserting the capillary into
the electrolyte or sample vial.
The operational parameters are specified in each individ-
ual monograph so as to minimize voltage effects, ionic
strength effects, and pH effects. The instrument is set up to
run with the appropriate capillary configuration and injec-
tion conditions, within the established linear dynamic range
of the detector; and acceptable migration precision is en-
sured by appropriate choice of sample diluent, separation
electrolyte, electrolyte additives, and capillary pretreatment
conditions. Exercise caution to avoid overloading the capil-
lary with sample, as this decreases efficiency and
reproducibility.
System Suitability—Parameters measured may include
injector reproducibility, system selectivity, system efficiency,
and tailing. Resolution between the analytes and other com-
pounds may be determined by using test mixture standards.
Parameters typically used to determine system suitability
include relative standard deviation (RSD), capacity factor
(k′), the number of theoretical plates (N), sensitivity (limit of
detection or quantitation), number of theoretical plates per
meter (TPM), tailing factor (T), and resolution (R).
The peak shape is closely examined; ideally, the peak is
symmetrical, with no shoulders and no excessive tailing. If
these conditions are not met, corrective actions are taken
before proceeding with the analysis. Peak integration is also
closely examined to ensure that the peak response is cor-
rectly quantitated.
Replicate injections of a Standard preparation of known
concentration can be used to determine the reproducibility
of the CE system. Data from five or more replicate injections
FCC 8
are used to calculate RSD. Unless otherwise specified in the
individual monograph, the relative standard deviation for
replicate injections is not more than 3.0%. Minimum injec-
tion precision values may be specified in specific CE meth-
ods, especially when determining trace-level components.
Calculation of electrophoretic parameters in MEKC, as in
other forms of CE, may involve a combination of chromato-
graphic and electrophoretic relationships. Hence, capacity
factor, k′, for neutral analyte migration in MEKC can be cal-
culated by the equation:
k′ = tR – t0(1 – tR/tMC)
in which tR, t0, and tMC are the migration times of the
analyte, the bulk solution (EOF), and the micelle,
respectively.
The number of theoretical plates, N, is a measure of the
efficiency of the system and is calculated by the equation:
N = 16(tR/W)2 or N = 5.54(tR/W1/ 2)2
in which W is the analyte peak width at baseline, W1/2 is the
analyte peak width at half-height, and tR is the analyte mi-
gration time.
The number of theoretical plates per meter, TPM, is a
measure of the efficiency of the capillary as a function of
peak width at baseline and can be calculated by the
equation:
TPM = 1600(tR/W)2/L
in which L, in cm, is the total capillary length; and the other
terms are as defined above. The tailing factor, T, of the
analyte peak is a measure of peak symmetry, and it repre-
sents the degree of deviation of the symmetry of the peak
from an ideally symmetrical Gaussian peak. This factor can
be calculated by the equation:
T = W0.05/2f
in which W0.05 is the length of a line constructed parallel to
the peak base from the leading edge to the tailing edge of
the peak at 5% of peak height; and f is the distance along
the same line from the leading edge of the peak, appearing
to the left of the peak maximum in the electropherogram,
to the intercept of a perpendicular line dropped from the
peak maximum to the base. A ratio of 1.0 indicates a per-
fectly symmetrical peak. If electrodispersive effects occur,
they can generate highly asymmetrical peaks. This can occur
when high sample concentrations are used, such as those
for testing of impurities. Use of highly asymmetrical peaks is
acceptable provided that they are reproducible and that
they do not compromise separation selectivity.
The resolution factor, R, is a measure of the ability of the
capillary system to separate consecutively migrating
analytes. Resolution is determined for all sample analytes of
interest, with the pH of the buffer adjusted as necessary to
meet system suitability requirements. It can be calculated by
the equation:
R = 2(t2 − t1)/(W1 + W2)
in which t2 and t1 are the migration times, measured at
peak maxima, for the slower migrating peak and the faster
migrating peak, respectively; and W2 and W1 are the corre-
sponding widths of these two peaks measured at their
bases.
Sample Analysis—Once the suitability of the CE system
has been established, aliquots of both the Standard prepara-
tion and the test preparation are injected. Standards are in-
jected before or after the samples and intermittently
throughout the run.
Data Handling—Time-normalized peak areas are often
used in quantitative calculations. These are determined by
dividing the observed integrated peak area by the migration
General Information / Mass Spectrometry / 1441
time of the analyte. This compensates for the fact that in
CE, unlike HPLC, each analyte travels through the detector
at a different velocity. Unless this normalization is per-
formed, slowly moving (later-migrating) analytes will have
disproportionately large peak areas compared with those for
early migrating components.
System Shutdown—After analysis, the capillary is rinsed
according to the directions specified in each monograph or
as recommended by the manufacturer. For example, the
capillary might be rinsed with distilled water to remove
buffer components and then filled with air or nitrogen by
performing a rinse from an empty vial. Naturally, the desti-
nation and source vials, namely, vials 4 and 2 in Figure 1,
are emptied of buffer and rinsed thoroughly with deionized
water.
MASS SPECTROMETRY*
A mass spectrometer produces ions from the substance
under investigation, separates them according to their mass-
to-charge ratio (m/z), and records the relative abundance of
each ionic species present. The instrument consists of three
major components (see Figure 1):
Fig. 1. Major components of a mass spectrometer.
an ion source for producing gaseous ions from the sub-
stance being studied, an analyzer for resolving the ions into
their characteristic mass components according to their
mass-to-charge ratios, and a detector system for detecting
the ions and recording the relative abundance of each of
the resolved ionic species. In addition, a sample introduction
system is necessary to admit the samples to be studied to
the ion source while maintaining the high vacuum require-
ments (∼10–6 to 10–8 mm of mercury) of the technique; and
a computer is required to control the instrument, acquire
and manipulate data, and compare spectra to reference
libraries.
This chapter gives an overview of the theory, construc-
tion, and use of mass spectrometers. The discussion is lim-
ited to those instruments and measurements with actual or
potential application to compendial requirements: generally,
the identification and quantitation of specific compounds.
SAMPLE INTRODUCTION
Samples are introduced either as a gas to be ionized in
the ion source, or by ejection of charged molecular species
from a solid surface or solution. In some cases sample intro-
* This text is adapted from General Chapter 〈736〉 of the United States
Pharmacopeia and National Formulary (USP–NF) as published in USP 32–NF 27.
This text is provided for informational purposes only and is intended as a
resource for the FCC user. Note that because the USP–NF is in continuous
revision, this General Chapter is subject to change and the text printed here
may not continue to represent the current version.
1442 / Mass Spectrometry / General Information
duction and ionization take place in a single process, mak-
ing a distinction between them somewhat artificial.
Substances that are gases or liquids at room temperature
and atmospheric pressure can be admitted to the source as
a neutral beam via a controllable leak system. Volatilizable
compounds dissolved or adsorbed in solids or liquids can be
removed and concentrated with a headspace analyzer. Va-
pors are flushed from the solid or liquid matrix with a
stream of carrier gas and trapped on an adsorbing column.
The trapped vapors are subsequently desorbed by pro-
grammed heating of the trap and introduced into the mass
spectrometer by a capillary connection.
For volatilizable solids, the most frequently used method
of sample introduction is the direct insertion probe. Here,
the sample is placed in a small crucible at the tip of the
probe, which is heated under high vacuum in close proxim-
ity to the ion source. A variation of this technique involves
desorption of samples inside the ionization chamber from a
rapidly heated wire or with the aid of a laser beam. Such
desorption techniques, in combination with electron, chemi-
cal, or field ionization, are preferred for the analysis of heat
sensitive or poorly volatile samples.
Sample introduction techniques that involve the ejection
of charged molecules from the surface of solid samples in-
clude the field desorption method and various sputtering
techniques, where the samples are bombarded by high en-
ergy photons, by a primary ion beam, or by a neutral parti-
cle beam. Similarly, ions can be ejected from solutions either
by bombardment with a primary beam, or by one of the
various spray techniques described below.
Gas and liquid chromatographs are widely used as sample
inlet devices for mass spectrometers. These chromatographs
provide for an initial sample purification, since only that por-
tion of the chromatographic effluent containing the com-
pound of interest need be admitted to the mass spectrome-
ter. Gas chromatography/mass spectrometry (GC/MS) and
liquid chromatography/mass spectrometry (LC/MS) combi-
nations are valuable tools for the identification of unknown
impurities in test substances. These combination methods
have the capacity to separate complex mixtures with the
opportunity to obtain structural information on the individ-
ual components.
Gas Chromatography/Mass Spectrometry
Gas chromatographic effluents are already in the vapor
state and can be admitted directly into the mass spectrome-
ter. Bridging the several orders of magnitude difference in
the operating pressures of the two systems was initially ac-
complished with the use of various carrier gas separators.
However, with the advent of capillary gas chromatographic
columns and high capacity vacuum pumps for mass spec-
trometers, the gas chromatographic effluents are now fed
directly into the ion source.
Liquid Chromatography/Mass Spectrometry
This technique is particularly useful for analyzing materials
that cannot be analyzed by GC/MS, either because of ther-
mal instability, high polarity, or high molecular weight.
Compounds of biological interest such as polar endogenous
substances, and macromolecules—including peptides, pro-
teins, nucleic acids, and oligosaccharides—often fall into one
of these categories.
Currently available LC/MS interfaces encompass a number
of approaches to separating the compound of interest from
the liquid chromatographic mobile phase and transforming
it into an ionized species suitable for mass spectrometry.
These include transport devices such as the particle beam;
various spray techniques including thermospray, electro-
spray, and ionspray; and particle-induced desorption such as
continuous-flow fast atom bombardment (CF-FAB).
FCC 8
PARTICLE BEAM INTERFACE
The solvent is removed from an aerosol of the liquid chro-
matographic effluent, and the resulting neutral analyte mol-
ecules are introduced into the ion source of the mass spec-
trometer where they are ionized by electron ionization (EI)
or chemical ionization (CI). The resulting spectra are thus
classical EI or CI spectra, the former with a wealth of struc-
tural information. There are limitations with respect to polar-
ity, thermal lability, and molecular weight, so this technique
is best suited for small organic molecules with molecular
weights of less than 1000 daltons.
THERMOSPRAY
The compound of interest in a volatile buffer mobile
phase, such as ammonium acetate, is passed through
heated, narrow bore tubing directly into the ion source of a
mass spectrometer. The solution is vaporized in the tubing,
and analyte ions desorb into the gas phase and pass into
the mass analyzer. Neutral analyte molecules in the gas
phase may undergo chemical ionization by reaction with
gas phase buffer ions such as NH4+. Thermospray is com-
patible with relatively high flow rates of 1 to 2 mL per min-
ute, solvents containing a high percentage of water, and
many types of polar analytes. Thermal degradation may oc-
cur, since the analytes are exposed to relatively high tem-
peratures during the volatilization process.
ELECTROSPRAY
The mobile phase is sprayed through a small opening
(needle tip) held at a potential of several kilovolts. The
charged droplets so produced are desolvated by passing
through a drying gas, and the resulting ions are injected
directly into the high vacuum of the analyzer through an
orifice or glass capillary. Classical electrospray is limited to
flow rates of 1 to 5 µL per minute, and is therefore compati-
ble with either microbore HPLC or post-column stream split-
ting techniques.
The ions may carry multiple charges, so that the m/z
value of high molecular weight substances will fall into the
usable range for most quadrupole or magnetic sector mass
analyzers (m/z < 4000). Analytes of up to 150,000 daltons
can thus be successfully analyzed.
IONSPRAY
A variant of electrospray in which nebulization with a gas
flow is used to assist the formation of microdroplets of mo-
bile phase. The technique can extend the upper limit of
usable flow rates to 0.1 mL per minute. Volatile buffers must
be used with both techniques.
DESORPTION TECHNIQUES
Microflow liquid chromatography can also be interfaced
to particle induced desorption techniques such as fast atom
bombardment (FAB) and liquid secondary ion mass spec-
troscopy (LSIMS), described in the following section on ioni-
zation techniques. Typically, column effluent flowing at a
rate of 1 to 10 µL per minute is mixed with a small percent-
age of nonvolatile liquid such as glycerol. The mixture is
introduced via a capillary inlet onto a target within the ion
source where it is bombarded with high energy (5 to
20 keV) atoms or ions. The resulting spectra are similar to
FAB or LSIMS spectra but with the background from the
sample matrix greatly reduced. Frit-FAB is a variant of con-
tinuous flow FAB where the sample is introduced through a
porous frit target.
FCC 8
IONIZATION TECHNIQUES
Electron Impact
Molecules of the sample under analysis enter the ioniza-
tion chamber in the vapor state. Positive ions are produced
by passing a beam of electrons, obtained from tungsten or
rhenium filaments, through the vapor, which is maintained
at a pressure of 10−4 to 10−6 mm of mercury. Provided the
energy of the electron beam is greater than the ionization
potential of the sample, the sample is ionized and/or frag-
mented, as represented by the following equation:
e− + M → M+ · + 2e−
Chemical Ionization (CI)
In this process, a reagent gas at a pressure between 0.1
to 10 mm of mercury is admitted to the source and ionized
by a high energy electron beam or discharge. At these pres-
sures, ion-molecule reactions occur and the primary reagent
gas ions react further. The most commonly used reagent
gases are methane, isobutane, and ammonia. Typical reac-
tions for methane are shown in the following equations:
CH4 + e → CH4 · + 2e
− + −
CH4+ + CH4 → CH5+ + CH3 ·
CH3+ + CH4 → C2H5+ + H2
The CH5 + species is a strong Bronsted acid and readily trans-
fers a proton to most organic compounds
CH5+ + M → MH+ + CH4
In the case of methane, the protonated ion (MH)+ initially
formed may be sufficiently energetic to dissociate further.
Fast Atom Bombardment (FAB)
The sample is ionized by bombardment with a beam of
high speed xenon atoms, produced by exchange with
highly accelerated xenon ions in a collision cell. The process
is summarized as follows:
where the subscript arrows indicate the fast-moving
particles.
FAB is a surface analysis technique, and care must be
taken during sample preparation to optimize the condition
of the surface. When the sample is coated on a probe by
evaporation of a solution, the sample ion beam obtained is
often transitory. Molecular adducts with alkali metals, such
as (M + Na) and (M + K), favor ion formation. This phe-
nomenon is used to assist in the ionization of biological
molecules. Thus, treatment of the sample surface with so-
dium chloride solution may enhance the yield of adduct
ions. Heating the sample during analysis may also increase
the ion yield.
The declining yield of sample ions during analysis is prob-
ably due to destruction of the sample surface. The surface
can, in effect, be continuously replaced by dissolving the
General Information / Mass Spectrometry / 1443
sample in a suitable nonvolatile liquid and by coating the
mixture onto the top of the probe. Using this approach, the
lifetime of samples in the source has been extended to more
than 1 hour and the range of compounds amenable to FAB
analysis expanded dramatically. The long sample lifetimes
and higher sensitivities so achieved make FAB an important
mass spectral technique for biochemical analysis, providing
the elemental formula of the sample through accurate mass
determination. A further advantage of FAB, unlike CI, is the
presence of fragment ions within the spectra, which aid in
structural elucidation.
Recently, neutral atom bombardment has been replaced
by cesium ion bombardment. Although this technique is still
referred to as FAB, it is more correctly described as liquid
secondary ion mass spectrometry (LSIMS).
Negative and positive ions are formed in the various ioni-
zation processes described above, and both are readily ana-
lyzed by modern mass spectrometers. Samples with a high
electron capture cross section, notably those containing hal-
ide atoms, yield an abundance of negative ions. For this
reason, halide derivatives of compounds to be studied are
often prepared. Negative ion mass spectrometry has been
successfully applied to the analysis of pesticide residues,
since the structures of these compounds are well suited to
the technique.
ANALYZERS
Mass analyzers separate the charged species in the ionized
sample according to their m/z ratios, thus permitting the
mass and abundance of each species to be determined.
Four commonly used analyzers are the magnetic sector, the
quadrupole, the time-of-flight, and the Fourier transform
analyzers.
Magnetic Sector Analyzers
Ions generated in the ion source are collimated into a
beam through the focusing action of a magnetic field and a
slit assembly. After exiting the source, ions are subjected to
a magnetic field perpendicular to the direction of the beam.
Each ion experiences a force at right angles to both its di-
rection of travel and the direction of the magnetic field,
thereby deflecting the beam. The motion of each ion is
given by
m/z = H2r2/2V
where m is the mass in atomic mass units, z is the number
of electronic charges, H is the magnetic field strength in
gauss, r is the radius of the ion trajectory in centimeters,
and V is the accelerating voltage. The mass spectrum is
scanned by varying the strength of the magnetic field and
detecting those ions passing through an exit slit as they
come into “focus.” The magnetic sector mass spectrometer
affords spatial resolution of ions, giving a unique trajectory
at a given field strength for each value of m/z.
Quadrupole Analyzers
The instrument is based on four parallel rods in a square
array. The ion beam is focused down the axis of the array
and an electrical potential of fixed (DC) and radio frequency
(RF) components is applied to diagonally opposed rods. For
a given combination of DC and RF components, ions of one
specific m/z ratio have a stable path down the axis. All
others are deflected to the sides and lost. Mass scanning is
achieved by changing the DC and RF components of the
voltage, while maintaining a fixed ratio. The quadrupole an-
alyzer is a mass filter because it separates ions on the basis
of their m/z ratio.
1444 / Mass Spectrometry / General Information
Ion-trap Analyzer
This quadrupole-type device is composed of a ring elec-
trode placed between two end cap electrodes. Depending
upon the commercial version employed, the end caps are
either held at ground potential or have an RF voltage ap-
plied to them, while an RF voltage is placed on the ring
electrode. As a result of these potentials, the hyperbolic sur-
faces of the three elements form a three-dimensional
quadrupole analyzer.
Both ionization and mass analysis take place within the
three-dimensional quadrupole field. In the ionization step,
the RF voltage on the ring electrode is set low enough so
that the ions within the mass range of interest are trapped
within the device. Following ionization, mass analysis is ac-
complished through use of the “mass selective instability”
mode of operation. That is, by raising the RF voltage on the
ring electrode, ions of successively higher mass are ejected
from the ion trap into an electron multiplier detector. In its
most common application, the ion-trap analyzer is used in
conjunction with a gas chromatograph and covers the mass
range of 10 to 560 daltons. However, recent advances in
ion-trap technology have extended the workable mass
range to many thousands of daltons.
Time-of-flight Analyzers
Ion separation is based on the principle that ions of differ-
ent masses, possessing equal kinetic energy, have different
velocities. If there is a fixed distance for the ions to travel,
the time of travel will vary with their mass, the lighter ions
traveling faster and reaching the detector in a shorter period
of time. The time-of-flight is given by
tf = k √m/z
where tf is the time-of-flight in seconds. Thus, the time-of-
flight of the various ions is simply proportional to the square
root of the mass-to-charge ratio of the ions. To measure the
time-of-flight, ions are introduced into the mass spectrome-
ter in discrete packets so that a starting point for the timing
process can be established. Ion packets are generated either
through a pulsed ionization process or through a gating sys-
tem in which ions are produced continuously, but are intro-
duced only at given times into the flight tube.
Fourier Transform Analyzers
In a magnetic field of flux density B, ions move in circular
orbits. The angular frequency, ω, of the orbital motion is
given by
ω = (z/m)B
In this type of mass spectrometer, the orbits are varied by
subjecting the ions to a resonant alternating electric field.
When the frequency of the alternating field matches the or-
bital frequency, the ions are steadily accelerated to larger
and larger orbits in coherent motion, developing a high
level of kinetic energy. After the alternating electric field is
turned off, the orbiting ions give rise to an alternating im-
age current on the electrodes. A frequency analysis of this
signal yields the mass of the ions involved. Thus, the Fourier
transform of the time domain transient signal yields the cor-
responding frequency spectrum from which the mass spec-
trum is computed. This is a high resolution technique, yield-
ing m/z ratios accurate to about one thousandth of a
dalton.
FCC 8
TANDEM MASS SPECTROMETRY
Two mass spectrometers connected in series (MS/MS),
tandem mass spectrometry, refers to the use of two or more
sequential mass analysis steps. In its simplest form MS/MS
(Figure 2) consists of two mass spectrometers linked in such
a way that ions preselected by the first mass analyzer (MS1)
are chemically or energetically modified and the results ana-
lyzed by the second mass analyzer (MS2).
Fig. 2. Tandem Mass Spectrometry.
The basic concept of MS/MS involves the ability to deter-
mine the mass relationship between a precursor ion in MS1
and a product ion in MS2. Different mass relationships can
be probed depending on how MS1 and MS2 are scanned.
These include fragmentation of a precursor and measure-
ment of all its fragments (a product scan), selection of mul-
tiple precursors and testing for a common fragment (a pre-
cursor scan), or scanning to see if a number of precursors all
lose the same neutral species (a constant neutral loss scan).
Fragmentation of the precursor ion can be induced by
momentum transfer through collision with gas molecules
and/or solid surfaces or by electronic excitation using lasers.
These techniques are known as collision-induced dissocia-
tion, surface-induced dissociation, or laser-induced dissocia-
tion, respectively. Allowing the ion to fragment without ad-
ditional activation is known as metastable decomposition.
There are many applications of MS/MS. Product scans can
be used to obtain qualitative information from precursor
ions of test substances, impurities, and contaminants. This
can aid in the identification of unknowns. The method can
also be used to determine the amino acid sequence of pep-
tides and protein fragments.
MS/MS has advantages for mixture analysis. Even when
the mass spectrometer is coupled to a separation device
such as a liquid or gas chromatograph, the resulting signals
may be a result of overlapping or unresolved components.
MS/MS can be employed to select the precursor ion from
one component and obtain structural information without
interference from the others.
Selected reaction monitoring is used to reduce the inter-
ference encountered during quantitative analysis for low lev-
els of the analyte. Signals from other compounds in the
matrix can mask the desired signal. Interference is reduced if
a analyte-specific fragment is selected with MS1 and a struc-
ture-specific fragment with MS2. The odds of another mole-
cule producing the same mass relationship are diminishingly
small.
MS/MS can also be used in metabolism studies to search
for molecules with common structural features. All of the
metabolites might contain the same functional group that is
lost as a neutral fragment. In this case a constant-neutral-
loss scan will show all of these species. For instance, carbox-
ylic acids will all lose neutral carbon dioxide. If the common
functionality is lost as an ionic fragment, then a precursor
scan will show all of the molecules that produce that frag-
ment ion.
FCC 8
DATA ANALYSIS AND INTERPRETATION
The mass spectral experiment yields information on the
molecular weight of ions derived from the sample and the
relative abundance of each of these ions. Spectra are often
complex, and not all of the ions may be separated by the
mass spectrometer. The ability of the instrument to separate
ions is called the resolving power, commonly described by
the “10% valley” definition. This states that the resolving
power is the highest mass number at which two peaks dif-
fering by one molecular weight unit and of equal height
have a valley between them that is equal to 10% of the
peak height. For low, medium, and high resolution mass
spectrometers, this value is between 100 and 2000, 2000
and 10,000, and greater than 10,000, respectively.
If one electron is removed or added to a neutral mole-
cule, a molecular ion of essentially the same molecular
weight as the parent molecule results. It is often possible to
determine the mass of this ion with sufficient precision to
enable the empirical formula of the compound to be calcu-
lated. Molecular masses may be determined accurately by
using high resolution instruments or by peak-matching
measurements using reference compounds.
Fragment ions are those produced from the molecular ion
by various bond cleavage processes. Numerous papers in
the literature relate bond cleavage patterns (fragmentation
patterns) to molecular structure.
In addition to measurement of the mass of a molecular
ion and its associated fragment ions, mass spectrometers are
also used to quantitate compounds with a high degree of
selectivity, precision, and accuracy. Compounds are intro-
duced into the mass spectrometer either via direct insertion
probe, gas inlet, or, as is more common, via gas or liquid
chromatographic interfaces, which provide sample purifica-
tion. Ionization may be by EI, CI, FAB, thermospray, or
electrospray and mass separation by magnetic sector,
quadrupole, or quadrupole ion-trap mass spectrometers.
Quantitative mass spectrometry involves measuring the
abundance of a specific ion, or set of ions, and relating the
response to a known standard. External or internal standards
may be used, but the latter are preferred for greater
accuracy.
For mass spectrometry, internal standards may be either
structural or stable isotope analogs. The former have the
advantage of lower cost and availability while precision and
accuracy are typically achieved by use of a stable isotope
(2H, 13C, 15N) labeled analog of the analyte. The only re-
quirements for labeling the analyte are that the ion moni-
tored for the internal standard must retain an isotopic label
after ionization and the label must not be exchangeable
under the sampling, separation, or ionization conditions.
Stable isotope internal standards are often required for ac-
ceptable quantitation, particularly with FAB and LC/MS
techniques such as thermospray and electrospray.
Relative abundances of the analyte and internal standard
ions are typically determined by selected ion monitoring, by
which only specific ions due to the analyte and the internal
standard are monitored. This technique has the advantage
over scanning the full mass range in that more time is spent
integrating the ion current at a selected mass-to-charge ra-
tio, thereby increasing sensitivity. Chromatographic peak
area or amount of analyte in a sample is calculated from the
ratio of analyte to internal standard peak area (or height)
General Information / Nuclear Magnetic Resonance / 1445
and the regression parameters as determined by a calibra-
tion curve, using standard techniques.
NUCLEAR MAGNETIC
RESONANCE*
Nuclear magnetic resonance (NMR) spectroscopy is an
analytical procedure based on the magnetic properties of
certain atomic nuclei. It is similar to other types of spectros-
copy in that absorption or emission of electromagnetic en-
ergy at characteristic frequencies provides analytical informa-
tion. NMR differs in that the discrete energy levels between
which the transitions take place are created artificially by
placing the nuclei in a magnetic field.
Atomic nuclei are charged and behave as if they were
spinning on the nuclear axis, thus creating a magnetic di-
pole of moment µ along this axis. The angular momentum
of the spinning nucleus is characterized by a spin quantum
number (I). If the mass number is odd, I is 1/2 or an integer
plus 1/2; otherwise, it has a value of 0 or a whole number.
Nuclei having a spin quantum number, I ≠ 0, when
placed in an external uniform static magnetic field of
strength, H0, align with respect to the field in (2I + 1) possi-
ble orientations. Thus, for nuclei with I = 1/2, which include
most isotopes of analytical significance (Table 1), there are
two possible orientations, corresponding to two different
energy states. A nuclear resonance is the transition between
these states, by absorption or emission of the corresponding
amount of energy. In a static magnetic field the nuclear
magnetic axis precesses (Larmor precession) about the ex-
ternal field axis. The precessional angular velocity, ω0, is re-
lated to the external magnetic field strength through the
equation:
ω0 = γH0
in which γ is the magnetogyric ratio and is a constant for all
nuclei of a given isotope. If energy from an oscillating radio-
frequency field is introduced, the absorption of radiation
takes place according to the relationship:
∆E = hv = µHO/I
where h is Planck’s constant, and
v = ω0/2π = γH0/2π
Thus, when the frequency (ν0) of the external energy field
(E = hν) is the same as the precessional angular velocity,
resonance is achieved.
* This text is adapted from General Chapter 〈761〉 of the United States
Pharmacopeia and National Formulary (USP–NF) as published in USP 32–NF 27.
This text is provided for informational purposes only and is intended as a
resource for the FCC user. Note that because the USP–NF is in continuous
revision, this General Chapter is subject to change and the text printed here
may not continue to represent the current version.
1446 / Nuclear Magnetic Resonance / General Information
Fig. 1. Block diagram of a typical NMR spectrometer.
The energy difference between the two levels corresponds
to electromagnetic radiation in the radio-frequency range. It
is a function of γ, which is a property of the nucleus, and
H0, the external field strength. As shown in Table 1, the
resonance frequency of a nucleus increases with the increase
of the magnetic field strength.
NMR is a technique of high specificity but relatively low
sensitivity. The basic reason for the low sensitivity is the
comparatively small difference in energy between the ex-
cited and the ground states (0.02 calories at 15 to 20 kilo-
gauss field strength), which results in a population differ-
ence between the two levels of only a few parts per million.
Another important aspect of the NMR phenomenon, with
negative effects on the sensitivity, is the long lifetime of
most nuclei in the excited state, which affects the design of
the NMR analytical test, especially in pulsed repetitive ex-
periments. Simultaneous acquisition of the entire spectrum
instead of frequency-swept spectra can give sensitivity
enhancement.
Apparatus
The distinctive components of an NMR spectrometer are
a magnet and a source of radio frequency. The instruments
are described by the approximate resonance frequency of
the analytical nucleus, e.g., 1H NMR. More recently, instru-
ments are being referred to by their field strengths. Some
spectrometers are dedicated to the analysis of one type of
nucleus; others are designed to obtain spectra of different
nuclei.
There are two types of commercial NMR spectrometers:
the classical continuous wave (CW) instruments and the
more modern pulse Fourier-transform (FT) instruments. The
CW spectrometers use a technique similar to that of classical
optical spectrometers: a slow scan of radio frequency (at
fixed magnetic field) or of the magnetic field (at fixed radio
frequency) over a domain corresponding to the resonance
of the nuclei being studied. The signal generated by the
absorption of energy is detected, amplified, and recorded.
Various instrument configurations are possible. The ar-
rangement of a typical double-coil spectrometer, as one
Table 1. Properties of Some Nuclei Amenable to NMR Study
Nucleus I Natural Abundance, %
1H 1
/2 99.98
13C 1
/2 1.108
19F 1
/2 100
31P 1
/2 100
11B (3/2) 80.42
* T = tesla: 1.4093 T = 14.093 kilogauss.
FCC 8
might see in the lower resolution 60-MHz and 100-MHz CW
instruments, is illustrated in Figure 1.
The limitations of the CW spectrometers are low sensitiv-
ity and long analysis time. In pulsed NMR spectrometers, a
single pulse of radio frequency energy is used to simultane-
ously activate all nuclei. The excited nuclei returning to the
lower energy level generate a free induction decay (FID) sig-
nal that contains in a time domain all the information ob-
tained in a frequency domain with a CW spectrometer. The
time domain and the frequency domain responses form a
pair of FTs; the mathematical operation is performed by a
computer after analog-to-digital conversion. After a delay al-
lowing for relaxation of the excited nuclei, the pulse experi-
ment (transient) may be repeated and the response coher-
ently added in the computer memory, with random noise
being averaged out. (A similar signal-to-noise increase can
be obtained by combining CW spectrometers with com-
puters that average transients.)
The block diagram of a typical high-resolution pulsed
spectrometer is shown in Figure 2. It is a typical configura-
tion of the high-resolution spectrometer that uses a super-
conducting (cryogenic) solenoid as the source of the mag-
netic field. Introduction of the pulsed NMR spectrometer
has made the acquisition of spectra of many nuclei, other
than protons, routine. It has also allowed proton spectra to
be obtained in much less time, and with smaller amounts of
specimen, as compared to CW techniques.
NMR spectrometers have strict stability and homogeneity
requirements. Stability is often achieved by a field-frequency
locking system that “locks” the magnetic field to the
resonance frequency of a reference signal. The lock signal
can be homonuclear or heteronuclear. In the latter case, the
reference resonance is usually a deuterium signal from a
deuterated solvent. On older spectrometers, using deute-
rium as a locking nucleus permits noise decoupling of pro-
tons to be carried out while studying nuclei like 13C. While
internal homonuclear locks are still used in CW proton spec-
trometers (where tetramethylsilane at about 0.5% provides
a convenient lock), they are hardly ever used in pulsed FT
spectrometers.
No type of magnet is capable of producing a homogene-
ous field over the space occupied by the specimen. Two
techniques are usually employed to compensate for this lack
Resonance Frequency (MHZ) at
Sensitivity 1.4093 T* 2.3488 T 4.6975 T
1.00 60.000 100.000 200.000
0.0159 15.087 25.144 50.288
0.83 56.446 94.077 188.154
0.0663 24.289 40.481 80.961
0.17 19.250 32.084 64.167
FCC 8
Fig. 2. Block diagram of a typical pulsed FT-NMR spectrometer.
of homogeneity: specimen spinning and the use of addi-
tional (shim) coils. Because of design, particularly probe de-
sign, the spinning in the case of the electromagnet or per-
manent magnet is perpendicular to the basic field. In the
superconducting magnet, the axis of rotation can only be
parallel to the basic magnetic field. The spin rate should be
sufficient to produce averaging of the field, but not fast
enough to produce an extended vortex in the specimen
tube. A vortex extended near the region exposed to the
radio-frequency coils decreases resolution. The shim coils are
adjusted by the operator until instrumental contributions to
the observed line width are minimized.
An electronic integrator is a feature of most NMR spec-
trometers. On a CW instrument (1H and 19F) the integrator,
connected to the spectrometer output stage, determines the
relative areas of the resonance peaks and presents these ar-
eas as a series of stepped horizontal lines when a sweep is
made in the integration mode. On FT-NMR spectrometers,
an integration algorithm is included in the spectrometer
software, and the resonance peak areas may be presented
graphically as stepped lines or tabulated as numeric values.
The use of computer-generated tabulated/numeric integra-
tion data should not be accepted without a specific demon-
stration of precision and accuracy on the spectrometer in
question.
The Spectrum
The signals (peaks) in an NMR spectrum are characterized
by four attributes: resonance frequency, multiplicity, line
width, and relative intensity. The analytical usefulness of the
NMR technique resides in the fact that the same types of
nuclei, when located in different molecular environments,
exhibit different resonance frequencies. The reason for this
difference is that the effective field experienced by a particu-
lar nucleus is a composite of the external field provided by
the instrument and the field generated by the circulation of
the surrounding electrons. (The latter is generally opposed
to the external field and the phenomenon is called “shield-
ing.”) In contrast with other spectroscopic methods, it is not
possible to measure accurately the absolute values of transi-
tion frequencies. However, it is possible to measure accu-
rately the difference in frequencies between two resonance
signals. The position of a signal in an NMR spectrum is de-
scribed by its separation from another resonance signal arbi-
trarily taken as standard. This separation is called chemical
shift.
The chemical shift, being the difference between two
resonance frequencies, is directly proportional to the mag-
General Information / Nuclear Magnetic Resonance / 1447
netic field strength (or to the frequency of the oscillator).
However, the ratio between the chemical shift, in frequency
units, and the instrument frequency is constant. This allows
definition of a dimensionless chemical shift parameter (δ)
that is independent of the instrument frequency:
δ = (νs − νr)/νo + δr
in which νs is the test substance line frequency, νr is the
reference line frequency, νo is the instrument frequency, in
mHz, and δr is the chemical shift of the reference.
By employing the above equation, it is possible to use
(with appropriate caution) the chemical shift of any known
species (such as the residual 1H-containing species in deuter-
ated solvent) as a chemical shift reference. The above equa-
tion, now in common use, is applicable to nearly all meth-
ods except in the relatively rare cases where extremely
precise chemical shift values must be determined, and is
readily adaptable to nuclei where non-zero reference stan-
dards are the only practical method of chemical shift
determinations.
For CW instruments, tetramethylsilane (TMS) is the most
widely used chemical shift reference for proton and carbon
spectra. It is chemically inert, exhibits only one line, which is
at a higher field than most signals, and is volatile, thus al-
lowing for ready specimen recovery. Sodium 3-(trimethyl-
silyl)propionate (TSP) or sodium 2,2-dimethyl-2-silapentane-
5-sulfonate (DSS) are used as NMR references for aqueous
solutions. The resonance frequency of the TSP or DSS
methyl groups closely approximate that of the TMS signal;
however, DSS has the disadvantage of showing a number of
methylene multiplets that may interfere with signals from
the test substance. Where the use of an internal NMR refer-
ence material is not desirable, an external reference may be
used.
Conventional NMR spectra are shown with the magnetic
field strength increasing from left to right. Nuclei that reso-
nate at high magnetic field strengths (to the right) are said
to be more shielded (greater electron density) than those
that resonate at lower magnetic field strengths: these are
said to be de-shielded (lower electron density).
Figure 3 shows the proton NMR spectrum of 2,3-dimethyl-
2-butenyl methyl ether. This compound contains protons in
a methylene group (marked d in the graphic formula) and
in four methyl groups (a, a, b, and c). Methyl groups b and
c are situated in distinctly different molecular environments
than the two a methyl groups. Three different methyl pro-
ton resonances are observed as spectral peaks in addition to
the peak corresponding to methylene proton resonance.
1448 / Nuclear Magnetic Resonance / General Information FCC 8

Fig. 3. NMR spectrum of 2,3-dimethyl-2-butenyl methyl ether (15% in CCl4) showing four nonequivalent, apparently un-
coupled protons with a normal integral trace (peak area ratio from low H0 to high H0 of 2:3:3:6). (Tetramethylsilane, the
NMR Reference, appears at 0 ppm.) The system of units represented by δ is defined under The Spectrum, in this chapter.

The two a methyl groups, being in very similar environ-

ments, have the same chemical shift. Interaction between
magnetically active nuclei situated within a few bond
lengths of each other leads to coupling, which results in a
mutual splitting of the respective signals into sets of peaks
or multiplets.
The coupling between two nuclei may be described in
terms of the spin-spin coupling constant, J, which is the
separation (in hertz) between the individual peaks of the
multiplet. Where two nuclei interact and cause reciprocal
splitting, the measured coupling constants in the two result-
ing mutiplets are equal. Furthermore, J is independent of
magnetic field strength.
In a first-order, comparatively noncomplex spin system,
the number of individual peaks that are expected to be
present in a multiplet and the relative peak intensities are
predictable. The number of peaks is determined by 2 nI + 1,
where n is the number of nuclei on adjacent groups that are
active in splitting. For protons this becomes (n + 1) peaks.
In general, the relative intensity of each peak in the multi-
plet follows the coefficient of the binomial expansion (a +
b)n. These coefficients may conveniently be found by use of
Pascal’s triangle, which produces the following relative areas
for the specified multiplets: doublet, 1:1; triplet, 1:2:1; quar-
tet, 1:3:3:1; quintet, 1:4:6:4:1; sextet, 1:5:10:10:5:1; and
septet, 1:6:15:20:15:6:1. This orderly arrangement, gener-
ally referred to as first-order behavior, may be expected
when the ratio of Dν to J is greater than about 10; Dν is the
chemical shift difference between two nuclei or two groups
of equivalent nuclei. Two examples of idealized spectra aris-
ing from first-order coupling are shown in Figure 4.

Fig. 4. Diagrammatic representation of simple first-order

coupling of adjacent protons.

Figure 5 shows a spectrum displaying triplet signals result-

ing from the mutual splitting of two adjacent methylene
groups.
Coupling may occur between 1H and other nuclei, such as
19F, 13C, and 31P. In some cases, e.g., in the CW mode, the

coupling constants may be large enough so that part of the

multiplet is off scale at either the upfield or downfield end.
This type of coupling may occur over the normal “three-
bond distance,” as for 1H-1H coupling.
Magnetically active nuclei with I ≥ 1, such as 14N, possess
an electrical quadrupole moment, which produces line-
broadening of the signal due to neighboring nuclei.
FCC 8 General Information / Nuclear Magnetic Resonance / 1449

Fig. 5. NMR spectrum of 3-keto-tetrahydrofuran (10% in CCl4) showing three nonequivalent protons, with a normal integral
trace (peak area ratio from low H0 to high H0 of 1:1:1). Note two sets of methylene groups coupled to each other at 4.2 and
2.4 ppm. (Tetramethylsilane, the NMR Reference, appears at 0 ppm.)

Another characteristic of the signal, its relative intensity, Table 2. Solvents Commonly Used for Proton NMR
has wide analytical applications. In carefully designed experi- Solvent Residual Proton Signal, δa
ments (see the section General Method), the area or intensity
of a signal is directly proportional to the number of protons CCl4b —
giving rise to the signal. As a result, it is possible to deter- CS2b —
mine the relative ratio of the different kinds of protons or SO2 (liquid) —
other nuclei in a specimen or to perform NMR assays with (CF3)2CO —
the aid of an internal standard. CDCl3 7.27
The NMR spectra may contain extraneous signals due to CD3OD 3.35, 4.8c
the inhomogeneity of the magnetic field throughout the
specimen. These artifacts, called spinning side bands, appear (CD3)2CO 2.05
as minor lines symmetrically located around each signal. The D2O 4.7c
presence of large spinning side bands indicates that the DMSO-d6d 2.50
non-spinning shims require adjustment. The separation is C6D6 7.20
equal to the frequency of the specimen tube spin rate or p-Dioxane-d8 3.55
some integral multiple of that frequency. Thus, spinning CD3CO2D 2.05, 8.5c
side bands are readily identifiable.
DMF-d7e 2.77, 2.93, 8.05
a δ in ppm relative to tetramethylsilane arbitrarily taken as 0δ or 0

General Method ppm.

b Spectrophotometric grade.

Inadequate specimen preparation or incorrect instrumen- c Highly variable; depends on solute and temperature.

tal adjustments and parameters may lead to poor resolution,
decreased sensitivity, spectral artifacts, and erroneous data.
It is preferable that the operator be familiar with the basic
theory of NMR, the properties of the specimen, and the
operating principles of the instruments. Strict adherence to
the instruction manuals provided by the manufacturer and
frequent checks of the performance of the instrument are
essential.
The method and procedures discussed here refer specifi-
cally to 1H (proton) and 19F NMR. They are applicable, with
modification, to other nuclei. The discussion presumes that
the NMR spectra are obtained from liquid test substances or
solutions in suitable solvents.
Selection of Solvent—In addition to having good solu-
bility properties, suitable solvents do not exhibit resonance
peaks that obscure resonance peaks of the specimen being
analyzed. The most commonly used solvents for proton and
carbon NMR are listed in Table 2. Deuterated solvents also
provide the signal for the heteronuclear system lock. If sol-
vent peaks might interfere with any signals from the speci-
men, then the isotopic purity of the solvent should be as
high as possible. Deuterium (I = 1) does not exhibit
resonance under 1H conditions but may cause J-coupling to
be observed. The residual protons generate solvent peaks
whose chemical shifts are shown in Table 2.
d Dimethyl sulfoxide-d .
6
e N,N-Dimethylformamide-d per Aldrich, Alfa, Fluka, and Sigma cata-
7
logs.
Some solvents (e.g., D2O or CD3OD) enter into fast ex-
change reactions with protons and may eliminate resonance
signals from –COOH, –OH, and –NH2 structural groups. The
protons in alcohols and amines do not take part in rapid
exchange unless catalyzed by small concentrations of acid
or base, except in the presence of D2O and some other
solvents (e.g., CD3OD).
For 19F NMR, most solvents used in proton NMR may be
employed, the most common ones being CHCl3, CCl4, H2O,
CS2, aqueous acids and bases, and dimethylacetamide. In
general, any nonfluorinated solvent may be used, provided
that it is of spectral quality. Obviously, there is no interfer-
ence from the protonated functional groups of the solvent.
However, unless they are decoupled, protonated functional
groups on the 19F-containing specimen will provide J-
coupling.
Specimen Preparation—Directions are usually given in
individual monographs. The solute concentration depends
on the objective of the experiment and on the type of in-
strument. Detection of minor contaminants may require
higher concentrations. The solutions are prepared in sepa-
rate vials and transferred to the NMR specimen tube. The
volume required depends on the size of the specimen tube
1450 / Nuclear Magnetic Resonance / General Information
and on the geometry of the instrument. The level of the
solution in the tube must be high enough to extend be-
yond the coils when the tube is inserted in the instrument
probe and spun.
The NMR specimen tubes must meet narrow tolerance
specifications in diameter, wall thickness, concentricity, and
camber. The most widely used tubes have a 5- or 10-mm
outside diameter and a length of between 15 and 20 cm.
Microtubes are available for the analysis of small amounts of
specimen.
Procedure—The specimen tube is placed in a probe lo-
cated in the magnetic field. The probe contains electronic
circuitry including the radio-frequency coil(s), and is pro-
vided with attachments for the air supply that spins the
specimen tubes.
Instrument adjustments are made before each experi-
ment. The spinning rate of the specimen tube is adjusted so
that spinning side bands do not interfere with the peaks of
interest and the vortex does not extend beyond the coils in
the probe. To optimize the instrument performance, the
magnetic shim gradients on FT-NMR spectrometers are ad-
justed. In adjusting resolution on CW spectrometers, a good
indicator is the definite “ringing” of the TMS peak. The phe-
nomenon of ringing is the oscillation of the recorder trace
after the magnetic field has passed through a resonance fre-
quency. Ringing, evident on a number of the peaks in
Figures 5 and 6, arises during rapid scans and decays expo-
nentially to the baseline value.
Fig. 6. Continuous wave proton spectrum of ethyl ether.
Figure 7 clearly indicates the absence, in an FT experiment,
of the ringing phenomenon. Ringing will not appear be-
cause the spectrum obtained is the result of analysis of the
FID by Fourier transformation and not a magnetic field or
frequency sweep through the individual resonance positions.
FCC 8
Fig. 7. Proton NMR spectrum of ethyl ether in deuterated
chloroform.
With proton CW instruments the spectrum is scanned
from 0 ppm to about 10 ppm with a scan time of about 1
to 5 minutes. The amplification is adjusted so that all peaks
remain on scale. If the response is low at reasonable ampli-
tude, the radio-frequency power is increased to obtain the
highest possible peak response without peak broadening.
After the initial scan, the presence of peaks downfield of 10
ppm is quickly checked by off-setting the instrument re-
sponse by about 5 ppm. With CW instrumentation, it is
common for the TMS peak to shift slightly during an ex-
tended scan. The extent of the shift is usually obtained by
comparing the relative positions of another peak in the ini-
tial scan with the same peak in the offset scan.
The operation of an FT-NMR spectrometer is a much
more elaborate experiment. The computer serves to control
the spectrometer, to program the experiment, and to store
and process the data. Programming the experiment involves
setting values for a large number of variables including the
spectral width to be examined, the duration (“width”) of
the excitation pulse, the time interval over which data will
be acquired, the number of transients to be accumulated,
and the delay between one acquisition and the next. The
analysis time for one transient is in the order of seconds.
The number of transients is a function of the specimen con-
centration, the type of nucleus, and the objective of the
experiment. At the end of the experiment, the FID signal is
stored in digitized form in the computer memory and is
displayed on the video screen. The signal can be processed
mathematically to enhance either the resolution or the sen-
sitivity, and it can be Fourier-transformed into a frequency-
domain spectrum. The instrument provides a plot of the
spectrum. The integration routine, accessed through key-
board commands, results in a stepped-line plot. Considera-
bly more accurate integrals are obtained if the signals or
regions of interest are separately integrated.
FT-NMR spectrometers may yield qualitative and quantita-
tive data from the same experiment, but this is seldom
done in practice. In quantitative FT experiments, special pre-
cautions must be taken for the signal areas to be propor-
tional to the number of protons. The delays between pulses
must be long enough to allow complete relaxation of all
excited nuclei. This results in a considerable increase in anal-
ysis time and in some loss of resolution. Qualitative analysis
is usually performed in nonquantitative conditions, with the
design of the experiment directed to fast analysis with maxi-
mum resolution or sensitivity.
FCC 8
Qualitative and Quantitative Analysis
NMR spectroscopy has been used for a wide range of
applications such as structure elucidation; thermodynamic,
kinetic, and mechanistic studies; and quantitative analysis.
Some of these applications are beyond the scope of com-
pendial methods.
All five characteristics of the signal—chemical shift, multi-
plicity, line width, coupling constants, and relative inten-
sity—contribute analytical information.
Qualitative Applications—Comparison of a spectrum
from the literature or from an authentic specimen with that
of a test specimen may be used to confirm the identity of a
compound and to detect the presence of impurities that
generate extraneous signals. The NMR spectra of simple
structures can be adequately described by the numeric value
of the chemical shifts and coupling constants, and by the
number of protons under each signal. (The software of
modern instruments includes programs that generate simu-
lated spectra using these data.) Experimental details, such as
the solvent used, the specimen concentration, and the
chemical shift reference, must also be provided.
For unknown specimens, NMR analysis, usually coupled
with other analytical techniques, is a powerful tool for struc-
ture elucidation. Chemical shifts provide information on the
chemical environment of the nuclei. Extensive literature is
available with correlation charts and rules for predicting
chemical shifts. The multiplicity of the signals provides im-
portant stereochemical information. Mutual signal splitting
of functional groups indicates close proximity. The magni-
tude of the coupling constant, J, between residual protons
on substituted aromatic, olefinic, or cycloalkyl structures is
used to identify the relative position of the substituents.
Several special techniques (double resonance, chemical
exchange, use of shift reagents, two-dimensional analysis,
etc.) are available to simplify some of the more complex
spectra, to identify certain functional groups, and to deter-
mine coupling correlations.
Double resonance, or spin decoupling, is a technique that
removes the coupling between nuclei and thus simplifies the
spectrum and identifies the components in a coupling rela-
tionship. For example, in a simple two-proton system, gen-
erally designated an AX system (see Figure 4), each proton
appears as a doublet. If a strong radio-frequency field is in-
troduced at the frequency of X, while the normal radio-
frequency field is maintained at the frequency that causes A
to resonate, the coupling between A and X is removed
(homonuclear decoupling). A is no longer split, but instead
appears as a singlet. Routine 13C spectra are obtained under
proton decoupling conditions that remove all heteronuclear
13C-1H couplings. As a result of this decoupling, the carbon
signals appear as singlets, unless other nuclei that are not
decoupled are present (e.g., 19F, 31P).
Functional groups containing exchangeable protons
bound to hetero-atoms such as –OH, –NH2, or –COOH
groups may be identified by taking advantage of the rapid
exchange of these protons with D2O. To determine the
presence and position of these groups, scan the test sub-
stance in CDCl3 or DMSO-d6, then add a few drops of D2O
to the specimen tube, shake, and scan again. The resonance
peaks from these groups collapse in the second scan and
are replaced by the HDO singlet between 4.7 and 5.0 ppm.
This chemical exchange is an example of the effect of in-
termolecular and intramolecular rate processes on NMR
spectra. If a proton can experience different environments
by virtue of such a process (tautomerism, rotation about a
bond, exchange equilibria, ring inversion, etc.), the appear-
ance of the spectrum will be a function of the rate of the
process. Slow processes (on an NMR time scale) result in
more than one signal, fast processes average these signals to
one line, and intermediate processes produce broad signals.
The software of modern FT-NMR spectrometers allows for
sequences of pulses much more complex than the repetitive
accumulation of transients described above. Such experi-
General Information / Nuclear Magnetic Resonance / 1451
ments include homonuclear or heteronuclear two-dimen-
sional analysis, which determines the correlation of cou-
plings and may simplify the interpretation of otherwise
complex spectra.
Quantitative Applications—If appropriate instrument
settings for quantitative analysis have been made, the areas
(or intensities) of two signals are proportional to the total
number of protons generating the signals.
A1/A2 = N1/N2 (1)
If the two signals originate from two functional groups of
the same molecule, the equation can be simplified to
A1/A2 = n1/n2 (2)
in which n1 and n2 are the number of protons in the respec-
tive functional groups.
If the two signals originate from different molecular
species,
A1/A2 = n1m1/n2m2 = (n1W1/M1)/(n2W2/M2) (3)
where m1 and m2 are the numbers of moles; W1 and W2 are
the masses; and M1 and M2 are the molecular weights of
compounds 1 and 2, respectively.
Examination of Equations 2 and 3 shows that NMR quan-
titative analysis can be performed in an absolute or relative
manner. In the absolute method, an internal standard is
added to the specimen and a resonance peak area arising
from the test substance is compared with a resonance peak
area from the internal standard. If both test substance and
internal standard are accurately weighed, the absolute purity
of the substance may be calculated. A good internal stan-
dard has the following properties: it presents a reference
resonance peak, preferably a singlet, at a field position re-
moved from all specimen peaks; it is soluble in the analytical
solvent; its proton equivalent weight, i.e., the molecular
weight divided by the number of protons giving rise to the
reference peak, is low; and it does not interact with the
compound being tested. Typical examples of useful stan-
dards are 1,2,4,5-tetrachlorobenzene, 1,4-dinitrobenzene,
benzyl benzoate, and maleic acid. The choice of a standard
will be dictated by the spectrum of the specimen.
The relative method may be used to determine the molar
fraction of an impurity in a test substance (or of the compo-
nents in a mixture) as calculated by Equation 3.
Quantitative analysis, as well as detection of trace impuri-
ties, is markedly improved with modern instrumentation.
Stronger magnetic fields and the ability to accumulate and/
or average signals over long periods of time greatly enhance
the sensitivity of the method.
Absolute Method of Quantitation—Where the individ-
ual monograph directs that the Absolute Method of Quantita-
tion be employed, proceed as follows.
Solvent, Internal Standard, and NMR Reference—Use as di-
rected in the individual monograph.
Test Preparation—Transfer an accurately weighed quantity
of the test substance, containing about 4.5 proton mEq, to
a glass-stoppered, graduated centrifuge tube. Add about 4.5
proton mEq of Internal Standard, accurately weighed, and
3.0 mL of Solvent, insert the stopper, and shake. When dis-
solution is complete, add about 30 µL (30 mg if a solid) of
NMR Reference, provided that it does not interfere with sub-
sequent measurements, and shake.
Procedure—Transfer an appropriate amount (0.4 to 0.8
mL) of Test Preparation to a standard 5-mm NMR spinning
tube, and record the spectrum, adjusting the spin rate so
that no spinning side bands interfere with the peaks of in-
terest. Measure the area under each of the peaks specified
in the individual monograph by integrating not fewer than
five times. Record the average area of the Internal Standard
peak as AS and that of the Test Preparation peak as AU.
1452 / Nuclear Magnetic Resonance / General Information FCC 8

Calculate the quantity, in mg, of the analyte in the Test of a radionuclide to decay to one-half of its initial value, and
Preparation by the formula: is related to the decay constant by the equation:
WS(AU/AS)(EU/ES) T1/2 = 0.69315/λ
in which WS is the weight, in mg, of Internal Standard taken; The activity of a radioactive source (A) is related to the
and EU and ES are the proton equivalent weights (i.e., the number of radioactive atoms present by the equation:
molecular weights divided by the number of protons giving
rise to the reference peak) of the analyte and the Internal A = λN
Standard, respectively.
Relative Method of Quantitation—Where the individual from which the number of radioactive atoms at time t can
monograph directs that the Relative Method of Quantitation be computed, and hence the mass of the radioactive mate-
be employed, proceed as follows. rial can be determined.
The activity of a pure radioactive substance as a function
Solvent, NMR Reference, and Test Preparation—Use as di- of time can be obtained from the exponential equation or
rected under Absolute Method of Quantitation. from decay tables, or by graphical means based on the half-
Procedure—Transfer an appropriate amount (0.4 to 0.8 life (see Normalized Decay Chart, Figure 1).
mL) of Test Preparation to a standard 5-mm NMR spinning
tube, and record the spectrum, adjusting the spin rate so
that no spinning side bands interfere with the peaks of in-
terest. Measure the area or intensity under each of the
peaks specified in the individual monograph by integrating
not fewer than five times. Record the average area or inten-
sity resulting from the resonances of the groups designated
in the individual monograph as A1 and A2.
Calculate the quantity, in mole percent, of the analyte in
the Test Preparation by the formula:
(100 × (A1/n1)/((A1/n1) + (A2/n2))
in which n1 and n2 are, respectively, the numbers of protons
in the designated groups.

RADIOACTIVITY*

GENERAL CONSIDERATIONS

Fundamental Decay Law

The decay of a radioactive source is described by the
equation:
Nt = Noe −λt
in which Nt is the number of atoms of a radioactive sub-
stance at elapsed time t, No is the number of those atoms
when t = 0, and λ is the transformation or decay constant,
which has a characteristic value for each radionuclide. The
half-life, T1/2, is the time interval required for a given activity
* This text is adapted from General Chapter 〈821〉 of the United States
Pharmacopeia and National Formulary (USP–NF) as published in USP 32–NF 27.
This text is provided for informational purposes only and is intended as a
resource for the FCC user. Note that because the USP–NF is in continuous
revision, this General Chapter is subject to change and the text printed here
may not continue to represent the current version.
Fig. 1. Normalized Decay Chart.
The activity of a radioactive material is expressed as the
number of nuclear transformations per unit time. The funda-
mental unit of radioactivity, the curie (Ci), is defined as
3.700 × 1010 nuclear transformations per second. The mil-
licurie (mCi) and microcurie (µCi) are commonly used
subunits. The “number of nuclear transformations per unit
time” is the sum of rates of decay from all competing
modes of disintegration of the parent nuclide. Before the
activity of any given radionuclide in a measured specimen
can be expressed in curies, it is often necessary to know the
abundance(s) of the emitted radiation(s) measured.
Geometry
The validity of relative calibration and measurement of ra-
dionuclides is dependent upon the reproducibility of the re-
lationship of the source to the detector and its surround-
ings. Appropriate allowance must be made for source
configuration.
FCC 8
Background
Cosmic rays, radioactivity present in the detector and
shielding materials, and radiation from nearby radioactive
sources not properly shielded from the measuring equip-
ment, all contribute to the background count rate. All radio-
activity measurements must be corrected by subtracting the
background count rate from the gross count rate in the test
specimen.
Statistics of Counting
Since the process of radioactive decay is a random phe-
nomenon, the events being counted form a random se-
quence in time. Therefore, counting for any finite time can
yield only an estimate of the true counting rate. The preci-
sion of this estimate, being subject to statistical fluctuations,
is dependent upon the number of counts accumulated in a
given measurement and can be expressed in terms of the
standard deviation σ. An estimate for σ is
√n
where n is the number of counts accumulated in a given
measurement. The probability of a single measurement fall-
ing within
±100/√n%
of the mean of a great many measurements is 0.68. That is,
if many measurements of n counts each were to be made,
approximately two-thirds of the observations would lie
within
±100/√n%
of the mean, and the remainder outside.
Because of the statistical nature of radioactive decay, re-
peated counting of an undisturbed source in a counting as-
sembly will yield count-rate values in accordance with the
frequency of a normal distribution. Deviations in these val-
ues from the normal distribution conform to the χ2 test. For
this reason, the χ2 test is frequently applied to determine
the performance and correct operation of a counting assem-
bly. In the selection of instruments and conditions for assay
of radioactive sources, the figure of merit ε2/B should be
maximized (where ε = counter efficiency = observed count
rate/sample disintegration rate, and B = background count
rate).
Counting Losses
The minimum time interval that is required for the
counter to resolve two consecutive signal pulses is known as
the dead time. The dead time varies typically from the order
of microseconds for proportional and scintillation counters,
to hundreds of microseconds for Geiger-Müller counters.
Nuclear events occurring within the dead time of the
counter will not be registered. To obtain the corrected
count rate, R, from the observed count rate, r, it is neces-
sary to use the formula:
R = r/(1 − rτ)
in which τ is the dead time. The foregoing correction
formula assumes a nonextendable dead time. Thus, for gen-
eral validity, the value of rτ should not exceed 0.1. The ob-
served count rate, r, refers to the gross specimen count rate
and is not to be corrected for background before use in the
foregoing equation.
General Information / Radioactivity / 1453
Calibration Standards
Perform all radioactivity assays using measurement sys-
tems calibrated with appropriately certified radioactivity
standards. Such calibration standards may be purchased ei-
ther direct from the National Institute of Standards and
Technology or from other sources that have established
traceability to the National Institute of Standards and Tech-
nology through participation in a program of inter-compara-
tive measurements. These data, as well as half-life values, are
obtained from the Evaluated Nuclear Structure Data File of
the Oak Ridge Nuclear Data Project, and reflect the most
recent values at the time of publication.
IDENTIFICATION AND ASSAY OF
RADIONUCLIDES
Instrumentation
IONIZATION CHAMBERS
An ionization chamber is an instrument in which an elec-
tric field is applied across a volume of gas for the purpose of
collecting ions produced by a radiation field. The positive
ions and negative electrons drift along the lines of force of
the electric field, and are collected on electrodes, producing
an ionization current. In a properly designed well-type ioni-
zation chamber, the ionization current should not be too
dependent on the position of the radioactive specimen, and
the value of the current per unit activity, known as the cali-
bration factor, is characteristic of each gamma-ray-emitting
radionuclide.
The ionization current produced in an ionization chamber
is related to the mean energy of the emitted radiation and
is proportional to the intensity of the radiation. If standard
sources of known disintegration rates are used for efficiency
calibration, the ionization chamber may then be used for
activity determinations between several microcuries and sev-
eral hundred millicuries or more. The upper limit of activity
that may be measured in an ionization chamber usually is
not sharply defined and may be limited by saturation con-
siderations, range of the amplifier, and design of the cham-
ber itself. The data supplied with or obtained from a partic-
ular instrument should be reviewed to ascertain the useful
ranges of energies and intensities of the device.
Reproducibility within approximately 5% or less can be
readily obtained in about 10 seconds, with a deep re-en-
trant well-type chamber. One of the most commonly used
form of ionization chamber is known as a dose calibrator.
Although the calibration factor for a radionuclide may be
interpolated from an ionization chamber energy-response
curve, there are a number of sources of error possible in
such a procedure. It is therefore recommended that all ioni-
zation chamber calibrations be performed with the use of
authentic reference sources of the individual radionuclides,
as described hereinafter.
The calibration of a dose calibrator should be maintained
by relating the measured response of a standard to that of a
long-lived performance standard, such as radium 226 in
equilibrium with its daughters. The instrument must be
checked daily with the 226Ra or other source to ascertain the
stability over a long period of time. This check should in-
clude performance standard readings at all radionuclide set-
tings employed. To obtain the activity (Ax) of the radionu-
clide being measured, use the relationship:
Ax = RxR/Rn
in which Rn is the new reading for the radium or other
source, Rc is the reading for the same source obtained dur-
1454 / Radioactivity / General Information
ing the initial calibration procedure, and R is the observed
reading for the radionuclide specimen. Obviously, any nec-
essary corrections for radioactive decay of the reference
source must first be applied. Use of this procedure should
minimize any effects due to drift in the response of the in-
strument. The recommended activity of the 226Ra or other
monitor used in the procedure described above is 75 to 150
µCi. It is recommended also that the reproducibility and/or
stability of multirange instruments be checked for all ranges
with the use of appropriate standards.
The size and shape of a radioactive source may affect the
response of a dose calibrator, and it is often necessary to
apply a small correction when measuring a bulky specimen.
SCINTILLATION and SEMICONDUCTOR DETECTORS
When all or part of the energy of beta or gamma radia-
tion is dissipated within scintillators, photons of intensity
proportional to the amount of dissipated energy are pro-
duced. These pulses are detected by an electron multiplier
phototube and converted to electrical pulses, which are sub-
sequently analyzed with a pulse-height analyzer to yield a
pulse-height spectrum related to the energy spectrum of the
radiation emitted by the source. In general, a beta-particle
scintillation pulse-height spectrum approximates the true
beta-energy spectrum, provided that the beta-particle
source is prepared in such a manner that self-absorption is
minimized. Beta-ray spectra may be obtained by using cal-
cium fluoride or anthracene as the scintillator, whereas
gamma-ray spectra are usually obtained with a thallium-acti-
vated sodium iodide crystal or a large-volume lithium-drifted
germanium semiconductor detector. The spectra of charged
particles also may be obtained using silicon semiconductor
detectors and/or gas proportional counters. Semiconductor
detectors are in essence solid-state ionization chambers, but
the energy required to create an electron-hole pair or to
promote an electron from the valence band to the conduc-
tion band in the semiconductor is about one-tenth the en-
ergy required for creation of an ion-pair in a gas-filled ioni-
zation chamber or proportional counter and is far less than
the energy needed to produce a photon in a NaI(Tl) scintil-
lation crystal. In gamma-ray spectrometry, a Ge(Li) detector
can yield an energy resolution of 0.33% for 1.33 MeV
gamma-rays from 60Co, while a 3- × 3-inch NaI(Tl) crystal
can give a value of 5.9% for the same gamma-ray energy.
The energy resolution is a measure of the ability to distin-
guish the presence of two gamma rays closely spaced in
energy and is defined by convention as the full width of the
photopeak at its half maximum (FWHM), expressed in per-
centage of the photopeak energy.
Gamma-ray spectra exhibit one or more sharp, character-
istic photopeaks, or full-energy peaks, as a result of total
absorption in the detector of the full energy of gamma radi-
ations from the source; these photopeaks are useful for
identification purposes. Other secondary peaks are observed
as a consequence of backscatter, annihilation radiation, co-
incidence summing, fluorescent X-rays, etc., accompanied
by a broad band known as the Compton continuum arising
from scattering of the photons in the detector and from
surrounding materials. Since the photopeak response varies
with gamma-ray energy, calibration of a gamma-ray spec-
trometer should be achieved with radionuclide standards
having well-known gamma-ray energies and emission rates.
The shape of the gamma-ray spectrum is dependent upon
the shape and size of the detector and the types of shield-
ing materials used.
When confirming the identity of a radionuclide by
gamma-ray spectrometry, it is necessary to make a compari-
son of the specimen spectrum with that of a specimen of
known purity of the same radionuclide obtained under iden-
tical instrument parameters and specimen geometry. Where
the radionuclides emit coincident X- or gamma-radiations,
the character of the pulse-height distribution often changes
quite dramatically because of the summing effect of these
FCC 8
coincident radiations in the detector as the efficiency of de-
tection is increased (e.g., by bringing the source closer to
the detector). Such an effect is particularly evident in the
case of iodine 125. Among the more useful applications of
gamma-ray spectrometry are those for the identification of
radionuclides and the determination of radionuclidic
impurities.
Where confirmation of the identity of a given radionuclide
by means of a direct comparison with the spectrum of a
specimen of the same radionuclide of known purity is not
possible, the identity of the radionuclide in question must
then be established by the following method. Two or more
of the following nuclear decay scheme parameters of the
radionuclide specimen to be identified shall be measured,
and agreement shall be within ±10%: (1) half-life, (2) en-
ergy of each gamma- or X-ray emitted, (3) the abundance
of each emission, and (4) Emax for those radionuclides that
decay with beta-particle emissions. Such measurements are
to be performed as directed in the Identification and Assay
sections of this chapter. Agreement of two or more of the
measured parameters with the corresponding published nu-
clear decay scheme data constitutes confirmation of the
identity of the radionuclide.
LIQUID-SCINTILLATION COUNTERS
Alpha- and beta-emitting radionuclides may be assayed
with the use of a liquid-scintillation detector system. In the
liquid scintillator, the radiation energy is ultimately con-
verted into light quanta that are usually detected by two
multiplier phototubes so arranged as to count only coinci-
dence radiation. The liquid scintillator is a solution consist-
ing of a solvent, primary and secondary solutes, and addi-
tives. The charged particle dissipates its energy in the
solvent, and a fraction of this energy is converted into fluo-
rescence in the primary solute. The function of the second-
ary solute is to shift the fluorescence radiation to longer
wavelengths that are more efficiently detected by the multi-
plier phototubes. Frequently used solvents are toluene and
p-xylene; primary solutes are 2,5-diphenyloxazole (PPO) and
2-(4′-tert-butylphenyl)-5-(4-biphenylyl)-1,3,4-oxadiazole (bu-
tyl-PBD); and secondary solutes are 2,2′-p-phenylenebis[4-
methyl-5-phenyloxazole] (dimethyl-POPOP) and p-bis(o-
methylstrylyl)benzene (bis-MSB). As a means of attaining
compatibility and miscibility with aqueous specimens to be
assayed, many additives, such as surfactants and solubilizing
agents, are also incorporated into the scintillator. For an ac-
curate determination of radioactivity of the specimen, care
must be exercised to prepare a specimen that is truly homo-
geneous. The presence of impurities or color in solution
causes a decrease in photon output of the scintillator; such
a decrease is known as quenching. Accurate radioactivity
measurement requires correcting for count-rate loss due to
quenching.
The disintegration rate of a beta-particle source may be
determined by a procedure in which the integral count rate
of the specimen is measured as a function of the pulse-
height discriminator bias, and the emission rate is then ob-
tained by extrapolation to zero bias. Energetic alpha-particle
emitters may be similarly measured by this method.
Identification
A radionuclide can be identified by its mode of decay, its
half-life, and the energies of its nuclear emissions.
The radioactive half-life is readily determined by succes-
sive counting of a given source of the radionuclide over a
period of time that is long compared to its half-life. The
response of the counting assembly when employed for the
decay measurement of long-lived radionuclides should be
monitored with an even longer-lived reference source to as-
sess and compensate for errors arising from electronic drift.
In the case of short-lived radionuclides, when the counting
FCC 8
period constitutes a significant fraction of the half-life of the
radionuclide, the recorded count rate must be corrected to
the time when the count is initiated, as follows:
Rt = rλt/(1 − e−λt)
in which Rt is the count rate at the beginning of a counting
period, r is the count rate observed over the entire counting
period, t is the duration of the counting period, λ is the
decay constant of the radionuclide, and e is the base of the
natural logarithm. When t is small compared to the half-life
of the radionuclide under study so that λt < 0.05, then (1 –
e–λt) approaches λt, and no such correction is necessary.
The energy of nuclear emissions is often determined by
the maximum range of penetration of the radiation in mat-
ter (in the case of alpha- and beta-particles) and by the full-
energy peak or photopeak in the gamma-ray spectrum (in
the case of X- and gamma-rays). Since beta-particles are
emitted with a continuous energy spectrum, the maximum
beta-energy, Emax, is a unique index for each beta-emitting
radionuclide. In addition to the maximum range and energy
spectrum of the beta-particles, the absorption coefficient,
when obtained under reproducible counting conditions, can
serve as a reliable index for identification of a beta-emitter.
Fortuitously, beta-particles are absorbed in matter in an ap-
proximately exponential manner, and a plot of the loga-
rithm of the beta-particle count rate as a function of the
absorber thickness is known as the absorption curve. The
initial portion of the absorption curve shows linearity from
which the absorption coefficient can be obtained. The maxi-
mum range is determined by the use of absorbers of vary-
ing thickness, and the energy spectrum is measured by
beta-ray scintillation spectrometry.
The absorption of gamma-rays in matter is strictly expo-
nential, but the half-value layers of attenuation have not
been very useful for the purpose of radionuclide characteri-
zation. Gamma-rays from each isomeric transition are mono-
energetic; their energy can be directly measured by gamma-
ray spectrometry. Because of their high energy resolution,
solid-state detectors [Ge(Li)] are vastly superior to scintilla-
tion detectors [NaI(Tl)] in gamma-ray spectrometry.
BETA-EMITTING RADIONUCLIDES
Mass Absorption Coefficient Procedure—Deposit and
dry an aliquot of the radioactive phosphorus 32 solution on
a thin plastic film to minimize backscattering, and place it
under a suitable counter. Determine the counting rates suc-
cessively, using not less than six different “thicknesses” of
aluminum each between 20 and 50 mg/cm2 and a single
absorber thicker than 800 mg/cm2, which is used to meas-
ure the background. (The absorbers are inserted between
the test specimen and the counter but are placed nearer the
counter window to minimize scattering.) Net beta-particle
count rates are obtained after subtraction of the count rate
found with the absorber having a thickness of 800 mg/cm2
or greater. Plot the logarithm of the net beta-particle count
rate as a function of the total absorber “thickness.” The to-
tal absorber “thickness” is the “thickness” of the aluminum
absorbers plus the “thickness” of the counter window (as
stated by the manufacturer) plus the air-equivalent “thick-
ness” (the distance in centimeters of the specimen from the
counter window multiplied by 1.205 mg/cm3 at 20° and 76
cm of mercury), all expressed in mg/cm2. An approximately
straight line results.
Choose two total absorber “thicknesses” that differ by 20
mg/cm2 or more and that fall on the linear plot, and calcu-
late the mass absorption coefficient, µ, by the equation:
µ = 1/(t2 − t1) · ln (Nt1/Nt2) = (2.303/(t2 − t1)) × (log Nt1 −
log Nt2)
in which t1 and t2 represent the total absorber “thicknesses,”
in mg/cm2, t2 being the thicker absorber, and Nt1 and Nt2
General Information / Radioactivity / 1455
being the net beta-particle rates with the t1 and t2 absorb-
ers, respectively.
For characterization of the radionuclide, the mass absorp-
tion coefficient should be within ±5% of the value found for
a pure specimen of the same radionuclide when determined
under identical counting conditions and geometry.
Other Methods of Identification—Other methods for
determining the identity of a beta emitter also rely upon the
determination of Emax. This may be accomplished in several
ways. For example, (1) utilization of the range energy rela-
tionships of beta particles in an absorber, or (2) determina-
tion of Emax from a beta-particle spectrum obtained on an
energy-calibrated beta-spectrometer using a thin source of
the radionuclide (see Scintillation and Semiconductor Detec-
tors in this chapter).
GAMMA-EMITTING RADIONUCLIDES
The gamma-ray spectrum of a radionuclide is a valuable
tool for the qualitative identification of gamma-ray emitting
radionuclides. The full-energy peak, or the photopeak, is
identified with the gamma-ray transition energy that is
given in the decay scheme of the radionuclide.
In determining radionuclidic identity and purity, the
gamma-ray spectrum of a radioactive substance is obtained
with either a NaI(Tl) crystal or a semiconductor Ge(Li) de-
tector. The latter has an energy resolution more than an
order of magnitude better than the former and is highly
preferred for analytical purposes. The spectrum obtained
shall be identical in shape to that of a specimen of the pure
radionuclide, measured with the same detection system and
in the same geometry. For low geometrical efficiencies, the
areas under the photopeaks, after correction for the meas-
ured detector efficiency, shall be proportional to the abun-
dances or emission rates of the respective gamma-rays in
the radionuclide.
RADIONUCLIDIC IMPURITIES
Procedures for identifying beta- and gamma-active radio-
nuclides as given in the foregoing text are applicable to the
detection of gamma and usually beta contaminants.
The gross alpha-particle activity can be measured by the
use of a windowless proportional counter or a scintillation
detector employing a silver-activated zinc-sulfide phosphor
or by the techniques of liquid-scintillation counting.
The heavy ionization caused by alpha particles allows the
measurement of alpha-emitting radionuclides in the pres-
ence of large quantities of beta- and gamma-active nuclides
by the use of appropriate techniques for discriminating the
amplitudes of signal pulses. In proportional counting, the
operating voltage region for counting alpha particles, re-
ferred to as the “alpha plateau,” is considerably lower than
the “beta plateau” for counting beta and gamma radiations.
Typical “alpha plateau” and “beta plateau” voltage settings
with P-10 counting gas are 900 to 1300 and 1600 to 2000
volts, respectively.
When silver-activated zinc-sulfide phosphor is employed
for alpha-particle detection, the alpha particles can be dis-
tinguished from other interfering radiation by pulse-height
discrimination. Care must be exercised to minimize self-ab-
sorption at the source whenever specimens are prepared for
alpha-particle counting.
Assay
BETA-EMITTING RADIONUCLIDES
Procedure—The disintegration rate (A) of a beta-particle-
emitting specimen is obtained by counting a quantitatively
1456 / Radioactivity / General Information
deposited aliquot in a fixed geometry according to the
formula:
A = R/(ε × fr × fb × fs)
in which ε is the counting efficiency of the counter; fr is the
correction factor for counter dead time; fb is the correction
factor for backscatter; and fs is the correction factor for self-
absorption. The count rate for zero absorber is obtained by
extrapolation of the initial linear portion of the absorption
curve to zero absorber “thickness,” taking into consideration
the mg/cm2 “thickness” of specimen coverings, counter
window, and the intervening air space between specimen
and the counter window. The counter efficiency, ε, is deter-
mined by use of a long-lived secondary standard with simi-
lar spectral characteristics. RaD + E has frequently been used
for efficiency calibration of counters for phosphorus 32. By
the use of identical measurement conditions for the speci-
men and the standard (and extrapolation to zero absorber),
the ratio of the values of fr, fb, and fs for the standard and
the specimen approaches unity.
The previous relationship is valid also when the counter
has been calibrated with a standard of the radionuclide to
be assayed. In this case, however, the extrapolations to zero
absorber “thickness” for the specimen and standard are not
required, as the two absorption corrections cancel for a
given geometry.
Another useful and frequently employed method for the
determination of the disintegration rate of beta-emitting ra-
dionuclides is liquid-scintillation counting, which also utilizes
an extrapolation of the specimen count rate to zero pulse-
height discriminator bias.
GAMMA-EMITTING RADIONUCLIDES
For the assay of gamma-emitting radionuclides, three
methods are provided. The selection of the preferred
method is dictated by the availability of a calibration stan-
dard of the radionuclide to be assayed and the radionuclidic
purity of the article itself.
Direct comparison with a calibration standard is required
if a calibration standard of the radionuclide to be assayed is
available and if the upper limit of conceivable error in the
activity determination arising from the presence of radionu-
clidic impurities has been determined to be less than 3%. If
the required calibration standard is not routinely available,
as would probably be the case for a short-lived radionuclide,
but was available at some time prior to the performance of
the assay for determination of efficiency of the counting sys-
tem for the radionuclide to be assayed, use a calibrated
counting system, provided the radionuclidic impurity con-
tent of the specimen meets the requirements stated for the
direct comparison method. If the requirements for either of
the first two methods cannot be met, use the method for
determination of activity from a calibration curve.
With the exception of the first method, the counting sys-
tems used are monitored for stability. This requirement is
met by daily checks with a long-lived performance check
source and weekly checks with at least three sources cover-
ing a broad range of gamma-ray emission energies (e.g.,
57Co, 137Cs, and 60Co). If a discrepancy for any of the afore-
mentioned measurements is found, either completely re-
calibrate or repair and recalibrate the system prior to further
use.
Assay by Direct Comparison with a Calibration
Standard—An energy selective measurement system (e.g.,
FCC 8
pulse-height analyzer) is not required for this procedure. Use
either an ionization chamber or an integral counting system
with a NaI(Tl) detector. A consistently reproducible geomet-
rical factor from specimen to specimen is essential for accu-
rate results. With proper precautions, the accuracy of this
method approaches the accuracy with which the disintegra-
tion rate of the calibration standard is known.
Determine the counting rate of the detector system for a
calibration standard of the radionuclide to be assayed (e.g.,
active enough to give good measurement statistics in a rea-
sonable time, but not so active as to cause serious dead-
time problems), selecting such a standard as to provide op-
timum accuracy with the particular assembly used. Place an
accurately measured aliquot of the unknown assay specimen
(diluted, if necessary) in a container identical to that used
for the standard, and measure this specimen at approxi-
mately the same time and under the same geometrical con-
ditions as for the standard. If the elapsed time between the
measurements of the calibration standard and the specimen
exceeds 12 hours, check the stability of the measurement
system within 8 hours of the specimen measurement time
with a long-lived performance check source. Record the sys-
tem response with respect to the same check source at the
time of calibration, and if subsequent checks exceed the
original recorded response by more than ±3%, recalibration
is required. Correct both activity determinations for back-
ground, and calculate the activity, in µCi per mL, by the
formula:
SD(g/b)
in which S is the µCi strength of the standard, D is the
dilution factor, and g and b are the measured values of
counting rate for the specimen and the standard,
respectively.
Assay with a Calibrated Integral Counting System—
The procedure and precautions given for the preceding di-
rect-comparison method apply, except that the efficiency of
the detector system is determined and recorded for each
radionuclide to be assayed, rather than simply recording the
counting rate of the standard. Thus, the efficiency for a
given radionuclide, x, is determined by εx = bx/sx, in which
bx is the counting rate, corrected for background and dead-
time, for the calibration standard of the radionuclide, x, and
sx is the corresponding activity of the certified calibration
standard in nuclear transformations per second. For subse-
quent specimen assays, the activity is given by the formula:
Ax = Dgx/εx
in which D is the dilution factor, gx is the specimen count-
ing rate (corrected for background and dead-time), and εx is
the corresponding efficiency for the radionuclide.
Determination of Activity from a Calibration Curve—
Versatility in absolute gamma-ray intensity measurements
can be achieved by employing multi-channel pulse-height
analysis. The photopeak efficiency of a detector system can
be determined as a function of gamma-ray energy by
means of a series of gamma-ray emission rate standard
specimens, and the gamma-ray emission rate of any radio-
nuclide for which no standard is available can be deter-
mined by interpolation from this efficiency curve. However,
exercise care to ensure that the efficiency curve for the de-
tector system is adequately defined over the entire region of
interest by using a sufficient number of calibration points
along the photopeak-energy axis.
FCC 8
Procedure—Minimal requirements for the maintenance of
instrument calibrations shall consist of weekly performance
checks with a suitable reference source and a complete re-
calibration semi-annually. Should the weekly performance
check deviate from the value determined at the time of cali-
bration by more than 4.0%, a complete recalibration of the
instrument is required at that time.
This method involves three basic steps, namely photopeak
integration, determination of the photopeak efficiency
curve, and calculation of the activity of the specimen.
PHOTOPEAK INTEGRATION—The method for the determina-
tion of the required photopeak area utilizes a Gaussian ap-
proximation for fitting the photopeak. A fixed fraction of the
total number of photopeak counts can be obtained by tak-
ing the peak width, a, at some fraction of the maximum,
where the shape has been experimentally found to be very
close to Gaussian, and multiplying by the counting rate of
the peak channel, P, after correction for any Compton and
background contributions to the peak channel count rate.
This background usually can be adequately determined by
linear interpolation. This is illustrated in Figure 2.
Fig. 2 Typical Gamma-ray Spectrum Showing the Selection
of the Peak Channel Counting Rate, P, after the Correction
for Compton and Background Contributions.
The photopeak-curve shape is closest to a straight line at
0.606P, and the contribution of the fractional channels to a
can be accurately estimated by interpolation. Calculate a by
the equation:
a = D′ − D + (d − 0.606P)/(d − c) + (d′ − 0.606P)/(d′ − c′)
in which c and d and also c′ and d′ are the single channel
counting rates on either side of 0.606P, and D and D′ are
the channel numbers (locations) of d and d′, respectively.
The location of the required variables on the photopeak is
illustrated in Figure 3.
General Information / Radioactivity / 1457
Fig. 3. Location of the Variables Required for the Determina-
tion of the Peak Width, a, at 0.606P.
From the known values for the counting rate in the peak
channel of the photopeak, P, and the width of the peak at
0.606P, a, a calibrated fraction of the photopeak area is
then obtained from the product, (aP).
To summarize the procedures involved in obtaining a cali-
brated fraction of a photopeak area using this method, the
necessary steps or calculations are presented below in a
stepwise manner:
(1) Subtract any Compton and background contributions
from the photopeak to be measured.
(2) Determine the counting rate of the peak channel
(maximum channel counting rate after subtracting
Compton and background), P.
(3) Multiply P by 0.606, and locate the horizontal line
corresponding to the peak width, a.
(4) Obtain the peak width, a, by inserting the values of
variables (obtained as shown in the preceding figure) into
the equation defining a.
(5) The desired calibrated fraction of the peak area is then
equal to the product of a times P or F = aP, where F is a
fractional area of the peak proportional to the emission rate
of the source.
This method provides a quick and accurate means of de-
termining the gamma-ray emission rate of sources while
avoiding, to a large extent, subjective estimates of the de-
tailed shape of the tails of the peaks. The error due to using
the maximum channel counting rate, rather than the theo-
retical maximum or peak channel rate, is of the order of
1.0% if a is 6 or greater.
PHOTOPEAK EFFICIENCY CALIBRATION—Radionuclides such as
those listed in the accompanying table together with some
of their nuclear decay data are available as certified refer-
ence standards.* A sufficient number of radioactive stan-
dard reference sources should be selected in order to obtain
the calibration curve over the desired range. Where possible,
standard sources of those radionuclides that are to be as-
sayed should be included.
* These certified reference standards are obtainable from the National Insti-
tute of Standards and Technology, Washington, DC 20234.
1458 / Radioactivity / General Information FCC 8

Nuclear Properties of Selected Calibration Standards (1,2)

Nuclear Properties of Selected Calibration
Standards (Continued)
(1,2)

Photons
per 100 Photons
Energy Disinte- per 100
Principal Photon Emissions (ke V) grations Energy Disinte-
133Ba (T
1/2 = 10.5 years) Principal Photon Emissions (ke V) grations
Kα1 30.97 63.4 γ1 39.58 7.52
Kα2 30.62 34.2 Weighted Mean(4) (31.3) (77.80)
(1) In measurements for gamma- (or X-)ray assay purposes, fluores-
Kβ 35.0 22.8
γ1 53.15 2.14 cent radiation from lead shielding (specifically, lead K X-rays ∼76 ke
V) may interfere with quantitative results. Allowance must be made
γ2 79.62 2.55
for these effects, or the radiation suppressed; a satisfactory means of
γ3 80.99 33.0 absorbing this radiation is covering the exposed lead with cadmium
γ6 276.39 6.9 sheet 0.06 to 0.08 inch thick, and then covering the cadmium with
γ7 302.83 17.8 copper 0.02 to 0.04 inch thick.
(2) Only those photon emissions having an abundance ≥1% are nor-
γ8 356.0 60.0
γ9 383.85 8.7 mally included.
(3) The K notation refers to X-ray emissions.
137Cs−137mBa (T
1/2 = 30.17 years) (4) The weighted mean energies and total intensities are given for
Kα1 32.19 3.82 groups of photons that would not be resolved by a NaI(Tl) detector.
Kα2 31.82 2.07 (5) For this photon intensity to be usable, all emitted positrons must

Kβ 36.4 1.39 be annihilated in the source material.

(6) Cascade.
Weighted Mean(4) (32.9) (7.28)
γ1 661.6 89.98 Calculate the gamma-ray emission rate from the equation:
22Na (T
1/2 = 2.60 years)

hν 511 179.80(5) Γ = Asb

γ1 1274.54 99.94
60Co(T
in which As is the activity, in disintegrations per second, of
1/2 = 5.27 years)
the standard used, and b is the number of gamma rays per
γ1 1173.2 (6) 100.0 disintegration at that energy. Accurately measure quantities
γ2 1332.5(6) 100.0 of standard solutions of each radionuclide into identical con-
57Co(T
1/2 = 270.9 days) tainers, and determine the fractional photopeak area (F) for
ΣXK 7.0 56.0 each of the standards.
γ1 14.4 9.5 Using the equation εp = F/Γ, calculate the photopeak effi-
γ2 122.06 85.51
ciency, εp, and construct a log-log plot of εp versus the
gamma-ray energy as shown in Figure 4.
γ3 136.47 10.60
Weighted Mean (125.0) (96.11)
(γ2 + γ3)(4)
54Mn (T
1/2 = 312.7 days)

ΣXK 6.0 25.0

γ1 834.83 99.98
109Cd−109Ag(T
1/2 = 464 days)

Kα1 22.16 35.3

Kα2 21.99 18.6
Kβ 24.9 11.4
Weighted Mean(4) 63.5
γ1 88.0 3.72
1/2 = 1.57 × 10 years)
129I(T 7

Kα1(3) 29.78 37.0

Kα2 29.46 20.0
Kβ 13.2 37.0
(1) In measurements for gamma- (or X-)ray assay purposes, fluores-

cent radiation from lead shielding (specifically, lead K X-rays ∼76 ke

V) may interfere with quantitative results. Allowance must be made
for these effects, or the radiation suppressed; a satisfactory means of
absorbing this radiation is covering the exposed lead with cadmium
sheet 0.06 to 0.08 inch thick, and then covering the cadmium with
copper 0.02 to 0.04 inch thick.
(2) Only those photon emissions having an abundance ≥1% are nor-

mally included.
(3) The K notation refers to X-ray emissions.
(4) The weighted mean energies and total intensities are given for

groups of photons that would not be resolved by a NaI(Tl) detector.

(5) For this photon intensity to be usable, all emitted positrons must

be annihilated in the source material.

(6) Cascade. DETERMINATION OF SPECIMEN ACTIVITY—In the same manner
as in the preparation of the calibration curve, determine the
fractional area (F) of the principal photopeak of the speci-
men under assay or an accurately measured aliquot adjusted
FCC 8 General Information / Spectrophotometry and Light-Scattering / 1459
to the same volume in an identical container as used for the
standards. From the calibration curve, find the value of εp
for this radionuclide. Using the equation Γ = F/εp, calculate
the gamma-ray emission rate (Γ). Calculate the activity (A),
in disintegrations per second, of the specimen using the
equation A = (Γ/b)(D), in which b is the number of gamma
rays per disintegration and D is the dilution factor. To ob-
tain the activity, in µCi or mCi, divide A by 3.7 × 104 or 3.7
× 107, respectively. The above relationship is equally valid
for obtaining the activity of an undiluted specimen or cap-
sule; in this case, the dilution factor, D, is unity.
SPECTROPHOTOMETRY AND
LIGHT-SCATTERING*
ULTRAVIOLET, VISIBLE, INFRARED, ATOMIC
ABSORPTION, FLUORESCENCE,
TURBIDIMETRY, NEPHELOMETRY, AND
RAMAN MEASUREMENT
Absorption spectrophotometry is the measurement of an in-
teraction between electromagnetic radiation and the mole-
cules, or atoms, of a chemical substance. Techniques fre-
quently employed in pharmaceutical analysis include UV,
visible, IR, and atomic absorption spectroscopy. Spectropho-
tometric measurement in the visible region was formerly re-
ferred to as colorimetry; however, it is more precise to use
the term “colorimetry” only when considering human per-
ception of color.
Fluorescence spectrophotometry is the measurement of the
emission of light from a chemical substance while it is being
exposed to UV, visible, or other electromagnetic radiation.
In general, the light emitted by a fluorescent solution is of
maximum intensity at a wavelength longer than that of the
exciting radiation, usually by some 20 to 30 nm.
Light-Scattering involves measurement of the light scat-
tered because of submicroscopic optical density inhomoge-
neities of solutions and is useful in the determination of
weight-average molecular weights of polydisperse systems in
the molecular weight range from 1000 to several hundred
million. Two such techniques utilized in pharmaceutical
analysis are turbidimetry and nephelometry.
Raman spectroscopy (inelastic light-scattering) is a light-
scattering process in which the specimen under examination
is irradiated with intense monochromatic light (usually laser
light) and the light scattered from the specimen is analyzed
for frequency shifts.
The wavelength range available for these measurements
extends from the short wavelengths of the UV through the
IR. For convenience of reference, this spectral range is
roughly divided into the UV (190 to 380 nm), the visible
(380 to 780 nm), the near-IR (780 to 3000 nm), and the IR
(2.5 to 40 µm or 4000 to 250 cm−1).
* This text is adapted from General Chapter 〈851〉 of the United States
Pharmacopeia and National Formulary (USP–NF) as published in USP 32–NF 27.
This text is provided for informational purposes only and is intended as a
resource for the FCC user. Note that because the USP–NF is in continuous
revision, this General Chapter is subject to change and the text printed here
may not continue to represent the current version.
COMPARATIVE UTILITY OF SPECTRAL
RANGES
For many pharmaceutical substances, measurements can
be made in the UV and visible regions of the spectrum with
greater accuracy and sensitivity than in the near-IR and IR.
When solutions are observed in 1-cm cells, concentrations of
about 10 µg of the specimen per mL often will produce
absorbances of 0.2 to 0.8 in the UV or the visible region. In
the IR and near-IR, concentrations of 1 to 10 mg per mL
and up to 100 mg per mL, respectively, may be needed to
produce sufficient absorption; for these spectral ranges, cell
lengths of from 0.01 mm to upwards of 3 mm are com-
monly used.
The UV and visible spectra of substances generally do not
have a high degree of specificity. Nevertheless, they are
highly suitable for quantitative assays, and for many sub-
stances they are useful as additional means of identification.
There has been increasing interest in the use of near-IR
spectroscopy in pharmaceutical analysis, especially for rapid
identification of large numbers of samples, and also for
water determination.
The near-IR region is especially suitable for the determina-
tion of –OH and –NH groups, such as water in alcohol, –OH
in the presence of amines, alcohols in hydrocarbons, and
primary and secondary amines in the presence of tertiary
amines.
The IR spectrum is unique for any given chemical com-
pound with the exception of optical isomers, which have
identical spectra. However, polymorphism may occasionally
be responsible for a difference in the IR spectrum of a given
compound in the solid state. Frequently, small differences in
structure result in significant differences in the spectra. Be-
cause of the large number of maxima in an IR absorption
spectrum, it is sometimes possible to quantitatively measure
the individual components of a mixture of known qualitative
composition without prior separation.
The Raman spectrum and the IR spectrum provide similar
data, although the intensities of the spectra are governed by
different molecular properties. Raman and IR spectroscopy
exhibit different relative sensitivities for different functional
groups, e.g., Raman spectroscopy is particularly sensitive to
C–S and C–C multiple bonds, and some aromatic com-
pounds are more easily identified by means of their Raman
spectra. Water has a highly intense IR absorption spectrum,
but a particularly weak Raman spectrum. Therefore, water
has only limited IR “windows” that can be used to examine
aqueous solutes, while its Raman spectrum is almost com-
pletely transparent and useful for solute identification. The
two major limitations of Raman spectroscopy are that the
minimum detectable concentration of specimen is typically
10−1 M to 10−2 M and that the impurities in many sub-
stances fluoresce and interfere with the detection of the
Raman scattered signal.
Optical reflectance measurements provide spectral infor-
mation similar to that obtained by transmission measure-
ments. Since reflectance measurements probe only the sur-
face composition of the specimen, difficulties associated
with the optical thickness and the light-scattering properties
of the substance are eliminated. Thus, reflectance measure-
ments are frequently more simple to perform on intensely
absorbing materials. A particularly common technique used
for IR reflectance measurements is termed attenuated total
reflectance (ATR), also known as multiple internal reflectance
(MIR). In the ATR technique, the beam of the IR spectrome-
ter is passed through an appropriate IR window material
(e.g., KRS-5, a TlBr-TlI eutectic mixture), which is cut at
such an angle that the IR beam enters the first (front) sur-
face of the window, but is totally reflected when it impinges
on the second (back) surface (i.e., the angle of incidence of
the radiation upon the second surface of the window ex-
ceeds the critical angle for that material). By appropriate
window construction, it is possible to have many internal
reflections of the IR beam before it is transmitted out of the
1460 / Spectrophotometry and Light-Scattering / General Information
window. If a specimen is placed in close contact with the
window along the sides that totally reflect the IR beam, the
intensity of reflected radiation is reduced at each wave-
length (frequency) that the specimen absorbs. Thus, the ATR
technique provides a reflectance spectrum that has been in-
creased in intensity, when compared to a simple reflectance
measurement, by the number of times that the IR beam is
reflected within the window. The ATR technique provides
excellent sensitivity, but it yields poor reproducibility, and is
not a reliable quantitative technique unless an internal stan-
dard is intimately mixed with each test specimen.
Fluorescence spectrophotometry is often more sensitive than
absorption spectrophotometry. In absorption measurements,
the specimen transmittance is compared to that of a blank;
and at low concentrations, both solutions give high signals.
Conversely, in fluorescence spectrophotometry, the solvent
blank has low rather than high output, so that the back-
ground radiation that may interfere with determinations at
low concentrations is much less. Whereas few compounds
can be determined conveniently at concentrations below
10−5 M by light absorption, it is not unusual to employ con-
centrations of 10−7 M to 10−8 M in fluorescence
spectrophotometry.
THEORY AND TERMS
The power of a radiant beam decreases in relation to the
distance that it travels through an absorbing medium. It
also decreases in relation to the concentration of absorbing
molecules or ions encountered in that medium. These two
factors determine the proportion of the total incident en-
ergy that emerge. The decrease in power of monochromatic
radiation passing through a homogeneous absorbing me-
dium is stated quantitatively by Beer’s law, log10(1/T) = A =
abc, in which the terms are as defined below.
Absorbance [Symbol: A]—The logarithm, to the base 10,
of the reciprocal of the transmittance (T). [NOTE—Descrip-
tive terms used formerly include optical density, absorbancy,
and extinction.]
Absorptivity [Symbol: a]—The quotient of the absorb-
ance (A) divided by the product of the concentration of the
substance (c), expressed in g per L, and the absorption path
length (b) in cm. [NOTE—It is not to be confused with ab-
sorbancy index; specific extinction; or extinction coefficient.]
Molar Absorptivity [Symbol: ε]—The quotient of the ab-
sorbance (A) divided by the product of the concentration,
expressed in moles per L, of the substance and the absorp-
tion path length in cm. It is also the product of the absorp-
tivity (a) and the molecular weight of the substance.
[NOTE—Terms formerly used include molar absorbancy in-
dex; molar extinction coefficient; and molar absorption
coefficient.]
For most systems used in absorption spectrophotometry,
the absorptivity of a substance is a constant independent of
the intensity of the incident radiation, the internal cell
length, and the concentration, with the result that concen-
tration may be determined photometrically.
Beer’s law gives no indication of the effect of tempera-
ture, wavelength, or the type of solvent. For most analytical
work the effects of normal variation in temperature are
negligible.
Deviations from Beer’s law may be caused by either
chemical or instrumental variables. Apparent failure of Beer’s
law may result from a concentration change in solute mole-
cules because of association between solute molecules or
between solute and solvent molecules, or dissociation or
ionization. Other deviations might be caused by instrumen-
tal effects such as polychromatic radiation, slit-width effects,
or stray light.
Even at a fixed temperature in a given solvent, the ab-
sorptivity may not be truly constant. However, in the case
of specimens having only one absorbing component, it is
not necessary that the absorbing system conform to Beer’s
FCC 8
law for use in quantitative analysis. The concentration of an
unknown may be found by comparison with an experimen-
tally determined standard curve.
Although, in the strictest sense, Beer’s law does not hold
in atomic absorption spectrophotometry because of the lack
of quantitative properties of the cell length and the concen-
tration, the absorption processes taking place in the flame
under conditions of reproducible aspiration do follow the
Beer relationship in principle. Specifically, the negative log
of the transmittance, or the absorbance, is directly propor-
tional to the absorption coefficient, and, consequently, is
proportional to the number of absorbing atoms. On this
basis, calibration curves may be constructed to permit evalu-
ation of unknown absorption values in terms of concentra-
tion of the element in solution.
Absorption Spectrum—A graphic representation of ab-
sorbance, or any function of absorbance, plotted against
wavelength or function of wavelength.
Transmittance [Symbol: T]—The quotient of the radiant
power transmitted by a specimen divided by the radiant
power incident upon the specimen. [NOTE—Terms formerly
used include transmittancy and transmission.]
Fluorescence Intensity [Symbol: I]—An empirical expres-
sion of fluorescence activity, commonly given in terms of
arbitrary units proportional to detector response. The fluo-
rescence emission spectrum is a graphical presentation of the
spectral distribution of radiation emitted by an activated
substance, showing intensity of emitted radiation as ordi-
nate, and wavelength as abscissa. The fluorescence excitation
spectrum is a graphical presentation of the activation spec-
trum, showing intensity of radiation emitted by an activated
substance as ordinate, and wavelength of the incident (acti-
vating) radiation as abscissa. As in absorption spectropho-
tometry, the important regions of the electromagnetic spec-
trum encompassed by the fluorescence of organic
compounds are the UV, visible, and near-IR, i.e., the region
from 250 to 800 nm. After a molecule has absorbed radia-
tion, the energy can be lost as heat or released in the form
of radiation of the same or longer wavelength as the ab-
sorbed radiation. Both absorption and emission of radiation
are due to the transitions of electrons between different en-
ergy levels, or orbitals, of the molecule. There is a time de-
lay between the absorption and emission of light; this inter-
val, the duration of the excited state, has been measured to
be about 10−9 second to 10−8 second for most organic fluo-
rescent solutions. The short lifetime of fluorescence distin-
guishes this type of luminescence from phosphorescence,
which is a long-lived afterglow having a lifetime of 10−3 sec-
ond up to several minutes.
Turbidance [Symbol: S]—The light-scattering effect of
suspended particles. The amount of suspended matter may
be measured by observation of either the transmitted light
(turbidimetry) or the scattered light (nephelometry).
Turbidity [Symbol: τ]—In light-scattering measurements,
the turbidity is the measure of the decrease in incident
beam intensity per unit length of a given suspension.
Raman Scattering Activity—The molecular property (in
units of cm4 per g) governing the intensity of an observed
Raman band for a randomly oriented specimen. The scatter-
ing activity is determined from the derivative of the molecu-
lar polarizability with respect to the molecular motion giving
rise to the Raman shifted band. In general, the Raman band
intensity is linearly proportional to the concentration of the
analyte.
USE OF REFERENCE STANDARDS
With few exceptions, the Pharmacopeial spectrophoto-
metric tests and assays call for comparison against a USP
Reference Standard. This is to ensure measurement under
conditions identical for the test specimen and the reference
substance. These conditions include wavelength setting, slit-
width adjustment, cell placement and correction, and trans-
FCC 8 General Information / Spectrophotometry and Light-Scattering / 1461
mittance levels. It should be noted that cells exhibiting iden-
tical transmittance at a given wavelength may differ consid-
erably in transmittance at other wavelengths. Appropriate
cell corrections should be established and used where
required.
The expressions, “similar preparation” and “similar solu-
tion,” as used in tests and assays involving spectrophotome-
try, indicate that the reference specimen, generally a USP
Reference Standard, is to be prepared and observed in a
manner identical for all practical purposes to that used for
the test specimen. Usually in making up the solution of the
specified Reference Standard, a solution of about (i.e.,
within 10%) the desired concentration is prepared and the
absorptivity is calculated on the basis of the exact amount
weighed out; if a previously dried specimen of the Reference
Standard has not been used, the absorptivity is calculated
on the anhydrous basis.
The expressions, “concomitantly determine” and “con-
comitantly measured,” as used in tests and assays involving
spectrophotometry, indicate that the absorbances of both
the solution containing the test specimen and the solution
containing the reference specimen, relative to the specified
test blank, are to be measured in immediate succession.
APPARATUS
Many types of spectrophotometers are available. Funda-
mentally, most types, except those used for IR spectropho-
tometry, provide for passing essentially monochromatic radi-
ant energy through a specimen in suitable form, and
measuring the intensity of the fraction that is transmitted.
Fourier transform IR spectrophotometers use an interfero-
metric technique whereby polychromatic radiation passes
through the analyte and onto a detector on an intensity and
time basis. UV, visible, and dispersive IR spectrophotometers
comprise an energy source, a dispersing device (e.g., a
prism or grating), slits for selecting the wavelength band, a
cell or holder for the test specimen, a detector of radiant
energy, and associated amplifiers and measuring devices. In
diode array spectrophotometers, the energy from the source
is passed through the test specimen and then dispersed via
a grating onto several hundred light-sensitive diodes, each
of which in turn develops a signal proportional to the num-
ber of photons at its small wavelength interval; these signals
then may be computed at rapid chosen intervals to repre-
sent a complete spectrum. Fourier transform IR systems util-
ize an interferometer instead of a dispersing device and a
digital computer to process the spectral data. Some instru-
ments are manually operated, whereas others are equipped
for automatic and continuous recording. Instruments that
are interfaced to a digital computer have the capabilities
also of co-adding and storing spectra, performing spectral
comparisons, and performing difference spectroscopy (ac-
complished with the use of a digital absorbance subtraction
method).
Instruments are available for use in the visible; in the visi-
ble and UV; in the visible, UV, and near-IR; and in the IR
regions of the spectrum. Choice of the type of spectropho-
tometric analysis and of the instrument to be used depends
upon factors such as the composition and amount of availa-
ble test specimen, the degree of accuracy, sensitivity, and
selectivity desired, and the manner in which the specimen is
handled.
The apparatus used in atomic absorption spectrophotom-
etry has several unique features. For each element to be
determined, a specific source that emits the spectral line to
be absorbed should be selected. The source is usually a
hollow-cathode lamp, the cathode of which is designed to
emit the desired radiation when excited. Since the radiation
to be absorbed by the test specimen element is usually of
the same wavelength as that of its emission line, the ele-
ment in the hollow-cathode lamp is the same as the ele-
ment to be determined. The apparatus is equipped with an
aspirator for introducing the test specimen into a flame,
which is usually provided by air–acetylene, air–hydrogen, or,
for refractory cases, nitrous oxide–acetylene. The flame, in
effect, is a heated specimen chamber. A detector is used to
read the signal from the chamber. Interfering radiation pro-
duced by the flame during combustion may be negated by
the use of a chopped source lamp signal of a definite fre-
quency. The detector should be tuned to this alternating
current frequency so that the direct current signal arising
from the flame is ignored. The detecting system, therefore,
reads only the change in signal from the hollow-cathode
source, which is directly proportional to the number of at-
oms to be determined in the test specimen. For Pharmaco-
peial purposes, apparatus that provides the readings directly
in absorbance units is usually required. However, instru-
ments providing readings in percent transmission, percent
absorption, or concentration may be used if the calculation
formulas provided in the individual monographs are revised
as necessary to yield the required quantitative results. Per-
cent absorption or percent transmittance may be converted
to absorbance, A, by the following two equations:
A = 2 − log10 (100 − % absorption)
or:
A = 2 − log10 (% transmittance)
Depending upon the type of apparatus used, the readout
device may be a meter, digital counter, recorder, or printer.
Both single-beam and double-beam instruments are com-
mercially available, and either type is suitable.
Measurement of fluorescence intensity can be made with
a simple filter fluorometer. Such an instrument consists of a
radiation source, a primary filter, a specimen chamber, a
secondary filter, and a fluorescence detection system. In
most such fluorometers, the detector is placed on an axis at
90° from that of the exciting beam. This right-angle geome-
try permits the exciting radiation to pass through the test
specimen and not contaminate the output signal received
by the fluorescence detector. However, the detector un-
avoidably receives some of the exciting radiation as a result
of the inherent scattering properties of the solutions them-
selves, or if dust or other solids are present. Filters are used
to eliminate this residual scatter. The primary filter selects
short-wavelength radiation capable of exciting the test spec-
imen, while the secondary filter is normally a sharp cut-off
filter that allows the longer-wavelength fluorescence to be
transmitted but blocks the scattered excitation.
Most fluorometers use photomultiplier tubes as detectors,
many types of which are available, each having special char-
acteristics with respect to spectral region of maximum sensi-
tivity, gain, and electrical noise. The photocurrent is ampli-
fied and read out on a meter or recorder.
A spectrofluorometer differs from a filter fluorometer in that
filters are replaced by monochromators, of either the prism
or the grating type. For analytical purposes, the spectrofluo-
rometer is superior to the filter fluorometer in wavelength
selectivity, flexibility, and convenience, in the same way in
which a spectrophotometer is superior to a filter
photometer.
Many radiation sources are available. Mercury lamps are
relatively stable and emit energy mainly at discrete wave-
lengths. Tungsten lamps provide an energy continuum in
the visible region. The high-pressure xenon arc lamp is often
used in spectrofluorometers because it is a high-intensity
source that emits an energy continuum extending from the
UV into the IR.
In spectrofluorometers, the monochromators are
equipped with slits. A narrow slit provides high resolution
and spectral purity, while a large slit sacrifices these for high
sensitivity. Choice of slit size is determined by the separation
between exciting and emitting wavelengths as well as the
degree of sensitivity needed.
Specimen cells used in fluorescence measurements may
be round tubes or rectangular cells similar to those used in
1462 / Spectrophotometry and Light-Scattering / General Information
absorption spectrophotometry, except that they are pol-
ished on all four vertical sides. A convenient test specimen
size is 2 to 3 mL, but some instruments can be fitted with
small cells holding 100 to 300 µL, or with a capillary holder
requiring an even smaller amount of specimen.
Light-scattering instruments are available and consist in
general of a mercury lamp, with filters for the strong green
or blue lines, a shutter, a set of neutral filters with known
transmittance, and a sensitive photomultiplier to be
mounted on an arm that can be rotated around the solution
cell and set at any angle from −135° to 0° to +135° by a
dial outside the light-tight housing. Solution cells are of vari-
ous shapes, such as square for measuring 90° scattering;
semioctagonal for 45°, 90°, and 135° scattering; and cylin-
drical for scattering at all angles. Since the determination of
molecular weight requires a precise measure of the differ-
ence in refractive index between the solution and solvent,
[(n − n0)/c], a second instrument, a differential refractome-
ter, is needed to measure this small difference.
Raman spectrometers include the following major compo-
nents: a source of intense monochromatic radiation (invaria-
bly a laser); optics to collect the light scattered by the test
specimen; a (double) monochromator to disperse the scat-
tered light and reject the intense incident frequency; and a
suitable light-detection and amplification system. Raman
measurement is simple in that most specimens are ex-
amined directly in melting-point capillaries. Because the la-
ser source can be focused sharply, only a few microliters of
the specimen is required.
PROCEDURE
Absorption Spectrophotometry
Detailed instructions for operating spectrophotometers are
supplied by the manufacturers. To achieve significant and
valid results, the operator of a spectrophotometer should be
aware of its limitations and of potential sources of error and
variation. The instruction manual should be followed closely
on such matters as care, cleaning, and calibration of the
instrument, and techniques of handling absorption cells, as
well as instructions for operation. The following points re-
quire special emphasis.
Check the instrument for accuracy of calibration. Where a
continuous source of radiant energy is used, attention
should be paid to both the wavelength and photometric
scales; where a spectral line source is used, only the photo-
metric scale need be checked. A number of sources of radi-
ant energy have spectral lines of suitable intensity, ade-
quately spaced throughout the spectral range selected. The
best single source of UV and visible calibration spectra is the
quartz-mercury arc, of which the lines at 253.7, 302.25,
313.16, 334.15, 365.48, 404.66, and 435.83 nm may be
used. The glass-mercury arc is equally useful above 300 nm.
The 486.13-nm and 656.28-nm lines of a hydrogen dis-
charge lamp may be used also. The wavelength scale may
be calibrated also by means of suitable glass filters, which
have useful absorption bands through the visible and UV
regions. Standard glasses containing didymium (a mixture
of praseodymium and neodymium) have been used widely,
although glasses containing holmium were found to be su-
perior. Standard holmium oxide solution has superseded the
use of holmium glass.1 The wavelength scales of near-IR
and IR spectrophotometers are readily checked by the use of
absorption bands provided by polystyrene films, carbon di-
oxide, water vapor, or ammonia gas.
1 National Institute of Standards and Technology (NIST), Gaithersburg, MD
20899: “Spectral Transmittance Characteristics of Holmium Oxide in Perchlo-
ric Acid,” J. Res. Natl. Bur. Stds. 90, No. 2, 115 (1985). The performance of
an uncertified filter should be checked against a certified standard.
FCC 8
For checking the photometric scale, a number of standard
inorganic glass filters as well as standard solutions of known
transmittances such as potassium dichromate are available.2
Quantitative absorbance measurements usually are made
on solutions of the substance in liquid-holding cells. Since
both the solvent and the cell window absorb light, compen-
sation must be made for their contribution to the measured
absorbance. Matched cells are available commercially for UV
and visible spectrophotometry for which no cell correction is
necessary. In IR spectrophotometry, however, corrections for
cell differences usually must be made. In such cases, pairs of
cells are filled with the selected solvent and the difference in
their absorbances at the chosen wavelength is determined.
The cell exhibiting the greater absorbance is used for the
solution of the test specimen and the measured absorbance
is corrected by subtraction of the cell difference.
With the use of a computerized Fourier transform IR sys-
tem, this correction need not be made, since the same cell
can be used for both the solvent blank and the test solu-
tion. However, it must be ascertained that the transmission
properties of the cell are constant.
Comparisons of a test specimen with a Reference Stan-
dard are best made at a peak of spectral absorption for the
compound concerned. Assays prescribing spectrophotome-
try give the commonly accepted wavelength for peak spec-
tral absorption of the substance in question. It is known that
different spectrophotometers may show minor variation in
the apparent wavelength of this peak. Good practice de-
mands that comparisons be made at the wavelength at
which peak absorption occurs. Should this differ by more
than ±1 nm from the wavelength specified in the individual
monograph, recalibration of the instrument may be
indicated.
TEST PREPARATION
For determinations utilizing UV or visible spectrophotome-
try, the specimen generally is dissolved in a solvent. Unless
otherwise directed in the monograph, determinations are
made at room temperature using a path length of 1 cm.
Many solvents are suitable for these ranges, including water,
alcohols, chloroform, lower hydrocarbons, ethers, and dilute
solutions of strong acids and alkalies. Precautions should be
taken to utilize solvents free from contaminants absorbing in
the spectral region being used. It is usually advisable to use
water-free methanol or alcohol, or alcohol denatured by the
addition of methanol but not containing benzene or other
interfering impurities, as the solvent. Solvents of special
spectrophotometric quality, guaranteed to be free from con-
taminants, are available commercially from several sources.
Some other analytical reagent-grade organic solvents may
contain traces of impurities that absorb strongly in the UV
region. New lots of these solvents should be checked for
their transparency, and care should be taken to use the
same lot of solvent for preparation of the test solution and
the standard solution and for the blank.
No solvent in appreciable thickness is completely transpar-
ent throughout the near-IR and IR spectrum. Carbon tetra-
chloride (up to 5 mm in thickness) is practically transparent
to 6 µm (1666 cm−1). Carbon disulfide (1 mm in thickness)
is suitable as a solvent to 40 µm (250 cm−1) with the excep-
tion of the 4.2-µm to 5.0-µm (2381-cm−1 to 2000-cm−1) and
the 5.5-µm to 7.5-µm (1819-cm−1 to 1333-cm−1) regions,
where it has strong absorption. Other solvents have rela-
tively narrow regions of transparency. For IR spectrophotom-
etry, an additional qualification for a suitable solvent is that
it must not affect the material, usually sodium chloride, of
which the cell is made. The test specimen may also be pre-
pared by dispersing the finely ground solid specimen in
2 For further detail regarding checks on photometric scale of a spectropho-
tometer, reference may be made to the following NIST publications: J. Res.
Nalt. Bur. Stds. 76A, 469 (1972) [re: SRM 93l, “Liquid Absorbance Standards
for Ultraviolet and Visible Spectrophotometry” as well as potassium chromate
and potassium dichromate]; NIST Spec. Publ. 260–116 (1994) [re: SRM 930
and SRM 1930, “Glass Filters for Spectrophotometry.”
FCC 8 General Information / Spectrophotometry and Light-Scattering / 1463
mineral oil or by mixing it intimately with previously dried
alkali halide salt (usually potassium bromide). Mixtures with
alkali halide salts may be examined directly or as transparent
disks or pellets obtained by pressing the mixture in a die.
Typical drying conditions for potassium bromide are 105° in
vacuum for 12 hours, although grades are commercially
available that require no drying. Infrared microscopy or a
mineral oil dispersion is preferable where disproportionation
between the alkali halide and the test specimen is encoun-
tered. For suitable materials the test specimen may be pre-
pared neat as a thin sample for IR microscopy or suspended
neat as a thin film for mineral oil dispersion. For Raman
spectrometry, most common solvents are suitable, and ordi-
nary (nonfluorescing) glass specimen cells can be used. The
IR region of the electromagnetic spectrum extends from 0.8
to 400 µm. From 800 to 2500 nm (0.8 to 2.5 µm) is gener-
ally considered to be the near-IR (NIR) region; from 2.5 to
25 µm (4000 to 400 cm−1) is generally considered to be the
mid-range (mid-IR) region; and from 25 to 400 µm is gen-
erally considered to be the far-IR (FIR) region. Unless other-
wise specified in the individual monograph, the region from
3800 to 650 cm−1 (2.6 to 15 µm) should be used to ascer-
tain compliance with monograph specifications for IR
absorption.
Where values for IR line spectra are given in an individual
monograph, the letters s, m, and w signify strong, medium,
and weak absorption, respectively; sh signifies a shoulder,
bd signifies a band, and v means very. The values may vary
as much as 0.1 µm or 10 cm−1, depending upon the partic-
ular instrument used. Polymorphism gives rise to variations
in the IR spectra of many compounds in the solid state.
Therefore, when conducting IR absorption tests, if a differ-
ence appears in the IR spectra of the analyte and the stan-
dard, dissolve equal portions of the test substance and the
standard in equal volumes of a suitable solvent, evaporate
the solutions to dryness in similar containers under identical
conditions, and repeat the test on the residues.
In NIR spectroscopy much of the current interest centers
around the ease of analysis. Samples can be analyzed in
powder form or by means of reflectance techniques, with
little or no preparation. Compliance with in-house specifica-
tions can be determined by computerized comparison of
spectra with spectra previously obtained from reference
materials. Many pharmaceutical materials exhibit low ab-
sorptivity in this spectral region, which allows incident near-
IR radiation to penetrate samples more deeply than UV, visi-
ble, or IR radiation. NIR spectrophotometry may be used to
observe matrix modifications and, with proper calibration,
may be used in quantitative analysis.
In atomic absorption spectrophotometry, the nature of
the solvent and the concentration of solids must be given
special consideration. An ideal solvent is one that interferes
to a minimal extent in the absorption or emission processes
and one that produces neutral atoms in the flame. If there is
a significant difference between the surface tension or vis-
cosity of the test solution and standard solution, the solu-
tions are aspirated or atomized at a different rate, causing
significant differences in the signals generated. The acid
concentration of the solutions also affects the absorption
processes. Thus, the solvents used in preparing the test
specimen and the standard should be the same or as much
alike in these respects as possible, and should yield solutions
that are easily aspirated via the specimen tube of the
burner-aspirator. Since undissolved solids present in the so-
lutions may give rise to matrix or bulk interferences, the
total undissolved solids content in all solutions should be
kept below 2% wherever possible.
CALCULATIONS
The application of absorption spectrophotometry in an as-
say or a test generally requires the use of a Reference Stan-
dard. Where such a measurement is specified in an assay, a
formula is provided in order to permit calculation of the
desired result. A numerical constant is frequently included in
the formula. The following derivation is provided to intro-
duce a logical approach to the deduction of the constants
appearing in formulas in the assays in many monographs.
The Beer’s law relationship is valid for the solutions of
both the Reference Standard (S) and the test specimen (U):
AS = abCS (1)
AU = abCU (2)
in which AS is the absorbance of the Standard solution of
concentration CS; and AU is the absorbance of the test speci-
men solution of concentration CU. If CS and CU are ex-
pressed in the same units and the absorbances of both solu-
tions are measured in matching cells having the same
dimensions, the absorptivity, a, and the cell thickness, b, are
the same; consequently, the two equations may be com-
bined and rewritten to solve for CU:
CU = CS(AU/AS) (3)
Quantities of solid test specimens to be taken for analysis
are generally specified in mg. Instructions for dilution are
given in the assay and, since dilute solutions are used for
absorbance measurements, concentrations are usually ex-
pressed for convenience in units of µg per mL. Taking a
quantity, in mg, of a test specimen of a drug substance or
solid dosage form for analysis, it therefore follows that a
volume (VU), in L, of solution of concentration CU may be
prepared from the amount of test specimen that contains a
quantity WU, in mg, of the drug substance [NOTE—CU is
numerically the same whether expressed as µg per mL or
mg per L], such that:
WU = VUCU (4)
The form in which the formula appears in the assay in a
monograph for a solid article may be derived by substitut-
ing CU of equation (3) into equation (4). In summary, the
use of equation (4), with due consideration for any unit
conversions necessary to achieve equality in equation (5),
permits the calculation of the constant factor (VU) occurring
in the final formula:
WU = VUCS(AU/AS) (5)
The same derivation is applicable to formulas that appear
in monographs for liquid articles that are assayed by absorp-
tion spectrophotometry. For liquid dosage forms, results of
calculations are generally expressed in terms of the quantity,
in mg, of drug substance in each mL of the article. Thus it is
necessary to include in the denominator an additional term,
the volume (V), in mL, of the test preparation taken.
Assays in the visible region usually call for comparing con-
comitantly the absorbance produced by the Assay prepara-
tion with that produced by a Standard preparation contain-
ing approximately an equal quantity of a USP Reference
Standard. In some situations, it is permissible to omit the
use of a Reference Standard. This is true where spectropho-
tometric assays are made with routine frequency, and where
a suitable standard curve is available, prepared with the re-
spective USP Reference Standard, and where the substance
assayed conforms to Beer’s law within the range of about
75% to 125% of the final concentration used in the assay.
Under these circumstances, the absorbance found in the as-
say may be interpolated on the standard curve, and the
assay result calculated therefrom.
Such standard curves should be confirmed frequently, and
always when a new spectrophotometer or new lots of re-
agents are put into use.
In spectrophotometric assays that direct the preparation
and use of a standard curve, it is permissible and preferable,
when the assay is employed infrequently, not to use the
1464 / Spectrophotometry and Light-Scattering / General Information
standard curve but to make the comparison directly against
a quantity of the Reference Standard approximately equal to
that taken of the specimen, and similarly treated.
Fluorescence Spectrophotometry
The measurement of fluorescence is a useful analytical
technique. Fluorescence is light emitted from a substance in
an excited state that has been reached by the absorption of
radiant energy. A substance is said to be fluorescent if it can
be made to fluoresce. Many compounds can be assayed by
procedures utilizing either their inherent fluorescence or the
fluorescence of suitable derivatives.
Test specimens prepared for fluorescence spectrophotom-
etry are usually one-tenth to one-hundredth as concentrated
as those used in absorption spectrophotometry, for the fol-
lowing reason. In analytical applications, it is preferable that
the fluorescence signal be linearly related to the concentra-
tion; but if a test specimen is too concentrated, a significant
part of the incoming light is absorbed by the specimen near
the cell surface, and the light reaching the center is re-
duced. That is, the specimen itself acts as an “inner filter.”
However, fluorescence spectrophotometry is inherently a
highly sensitive technique, and concentrations of 10−5 M to
10−7 M frequently are used. It is necessary in any analytical
procedure to make a working curve of fluorescence intensity
versus concentration in order to establish a linear relation-
ship. All readings should be corrected for a solvent blank.
Fluorescence measurements are sensitive to the presence
of dust and other solid particles in the test specimen. Such
impurities may reduce the intensity of the exciting beam or
give misleading high readings because of multiple reflec-
tions in the specimen cell. It is, therefore, wise to eliminate
solid particles by centrifugation; filtration also may be used,
but some filter papers contain fluorescent impurities.
Temperature regulation is often important in fluorescence
spectrophotometry. For some substances, fluorescence effi-
ciency may be reduced by as much as 1% to 2% per de-
gree of temperature rise. In such cases, if maximum preci-
sion is desired, temperature-controlled specimen cells are
useful. For routine analysis, it may be sufficient to make
measurements rapidly enough so that the specimen does
not heat up appreciably from exposure to the intense light
source. Many fluorescent compounds are light-sensitive. Ex-
posed in a fluorometer, they may be photo-degraded into
more or less fluorescent products. Such effects may be de-
tected by observing the detector response in relationship to
time, and may be reduced by attenuating the light source
with filters or screens.
Change of solvent may markedly affect the intensity and
spectral distribution of fluorescence. It is inadvisable, there-
fore, to alter the solvent specified in established methods
without careful preliminary investigation. Many compounds
are fluorescent in organic solvents but virtually non-
fluorescent in water; thus, a number of solvents should be
tried before it is decided whether or not a compound is
fluorescent. In many organic solvents, the intensity of fluo-
rescence is increased by elimination of dissolved oxygen,
which has a strong quenching effect. Oxygen may be re-
moved by bubbling an inert gas such as nitrogen or helium
through the test specimen.
A semiquantitative measure of the strength of fluores-
cence is given by the ratio of the fluorescence intensity of a
test specimen and that of a standard obtained with the
same instrumental settings. Frequently, a solution of stated
concentration of quinine in 0.1 N sulfuric acid or fluorescein
in 0.1 N sodium hydroxide is used as a reference standard.
Light-Scattering
Turbidity can be measured with a standard photoelectric
filter photometer or spectrophotometer, preferably with illu-
FCC 8
mination in the blue portion of the spectrum. Nephelomet-
ric measurements require an instrument with a photocell
placed so as to receive scattered rather than transmitted
light; this geometry applies also to fluorometers, so that, in
general, fluorometers can be used as nephelometers, by
proper selection of filters. A ratio turbidimeter combines the
technology of 90° nephelometry and turbidimetry: it con-
tains photocells that receive and measure scattered light at
a 90° angle from the sample as well as receiving and meas-
uring the forward scatter in front of the sample; it also
measures light transmitted directly through the sample. Lin-
earity is attained by calculating the ratio of the 90° angle
scattered light measurement to the sum of the forward scat-
tered light measurement and the transmitted light measure-
ment. The benefit of using a ratio turbidimetry system is
that the measurement of stray light becomes negligible.
In practice, it is advisable to ensure that settling of the
particles being measured is negligible. This is usually accom-
plished by including a protective colloid in the liquid sus-
pending medium. It is important that results be interpreted
by comparison of readings with those representing known
concentrations of suspended matter, produced under pre-
cisely the same conditions.
Turbidimetry or nephelometry may be useful for the
measurement of precipitates formed by the interaction of
highly dilute solutions of reagents, or other particulate mat-
ter, such as suspensions of bacterial cells. In order that con-
sistent results may be achieved, all variables must be care-
fully controlled. Where such control is possible, extremely
dilute suspensions may be measured.
The specimen solute is dissolved in the solvent at several
different accurately known concentrations, the choice of
concentrations being dependent on the molecular weight of
the solute and ranging from 1% for Mw = 10,000 to 0.01%
for Mw = 1,000,000. Each solution must be very carefully
cleaned before measurement by repeated filtration through
fine filters. A dust particle in the solution vitiates the inten-
sity of the scattered light measured. A criterion for a clear
solution is that the dissymmetry, 45°/135° scattered inten-
sity ratio, has attained a minimum.
The turbidity and refractive index of the solutions are
measured. From the general 90° light-scattering equation, a
plot of HC/τ versus C is made and extrapolated to infinite
dilution, and the weight-average molecular weight, M, is
calculated from the intercept, 1/M.
Visual Comparison
Where a color or a turbidity comparison is directed, color-
comparison tubes that are matched as closely as possible in
internal diameter and in all other respects should be used.
For color comparison, the tubes should be viewed down-
ward, against a white background, with the aid of a light
source directed from beneath the bottoms of the tubes,
while for turbidity comparison the tubes should be viewed
horizontally, against a dark background, with the aid of a
light source directed from the sides of the tubes.
In conducting limit tests that involve a comparison of col-
ors in two like containers (e.g., matched color-comparison
tubes), a suitable instrument, rather than the unaided eye,
may be used.
FCC 8
ION CHROMATOGRAPHY*
INTRODUCTION
Ion chromatography (IC) is a high-performance liquid
chromatography (HPLC) instrumental technique used in USP
test procedures such as identification tests and assays to
measure inorganic anions and cations, organic acids, carbo-
hydrates, sugar alcohols, aminoglycosides, amino acids, pro-
teins, glycoproteins, and potentially other analytes.
As dictated by the nature of the analyte, IC has been
applied to all aspects of the manufacturing and disposition
of pharmaceutical products, including characterization of ac-
tive ingredients, excipients, degradation products, impuri-
ties, and process streams. The following sample types are
among those that have been analyzed: raw materials, in-
termediates (including media and culture broths), bulk ac-
tive ingredients, diluents, formulated products, production
equipment cleaning solutions, and waste streams. The tech-
nique is especially valuable for ionic or ionizable (in the mo-
bile phase) analytes that have little or no native UV absorb-
ance. The ability to couple the ion-exchange separation with
numerous detection strategies, e.g., pulsed amperometric
detection (PAD), expands IC applications to instances where
analyte-specific detection strategies can provide the required
degree of sensitivity or specificity. Utilization of such strate-
gies allows IC applications to be implemented on appropri-
ately configured HPLC systems. Additionally, ion-exclusion
separations and pulsed amperometric detection expand the
range of application of IC to aliphatic organic acids as well
as to nonionic analytes of significant pharmaceutical interest
including alcohols, alditols, carbohydrates, and amino acids.
The wide dynamic range of the methodology makes it ap-
plicable for the quantification of trace contaminants as well
as major product components.
Because IC typically uses dilute acids, alkalis, or salt solu-
tions as the mobile phase, and does not use an organic
solvent, IC does not require the purchase of costly organic
solvents and hazardous disposal of the waste effluent. The
effluent can be disposed of after appropriate neutralization
(to ~pH 7) and, when necessary, after dilution with water.
IC allows separation using ion exchange, ion exclusion, or
ion-pair approaches. IC separations are based on differences
in charge density of the analyte species, which in turn de-
pend on the valence and size of the individual ionic species
to be measured. Separations are also performed on the basis
of differences in the hydrophobic character of the ionic spe-
cies. IC is typically performed at ambient temperature. As
with other forms of HPLC, IC separations are based on vary-
ing capacity factors and typically follow the Knox equation.
Ion chromatography is a technique complimentary to the
more commonly used reversed-phase and normal-phase
HPLC and to atomic absorption and ion-coupled plasma
(plasma spectrochemistry) techniques in pharmaceutical
analysis.
APPARATUS
IC instruments closely resemble conventional HPLC instru-
ments. Typical components include an autosampler, a high-
pressure pump, an injection valve with a sample loop of
suitable size (typically 10 to 250 µL), a guard column, an
analytical column, an optional suppressor or other forms of
a post-column reaction system, a flow-through detector,
and a data system ranging in complexity from an integrator
* This text is adapted from General Chapter 〈1065〉 of the United States
Pharmacopeia and National Formulary (USP–NF) as published in USP 32–NF 27.
This text is provided for informational purposes only and is intended as a
resource for the FCC user. Note that because the USP–NF is in continuous
revision, this General Chapter is subject to change and the text printed here
may not continue to represent the current version.
General Information / Ion Chromatography / 1465
to a computerized data system (Figure 1). Because mobile
phases generally consist of dilute acids, alkalis, or salt solu-
tions, the components in contact with the mobile phase
and the sample are typically made from inert materials, such
as polyetheretherketone. Conventional HPLC systems also
may be used provided that their components are compati-
ble with the mobile phase and injected sample solutions. A
metal-free system should be used for trace metal analysis.
Following suitable preparation, the sample is introduced via
the injection valve. After the optional chemical suppression
or other post-column reaction on the column effluent, the
analyte species are detected using conductivity, amperome-
try, UV/VIS, or other detection modes. Because IC uses a
predominantly ionic mobile phase, a suppressor is often
necessary prior to conductometric detection, although non-
suppressed conductometric detection has been successfully
used in pharmaceutical analysis.
Stationary and Mobile Phases
As IC has developed and matured as an instrumental
technique, the number of ion-exchange materials developed
for IC has increased, facilitated by the understanding of the
processes taking place at the surface of the stationary phase.
In contrast to the silica-based column packing prevalent in
classical HPLC, organic polymers are predominately used as
support materials for IC. Such materials have a higher stabil-
ity with respect to extremes in pH and in many cases are
compatible with organic solvents. Typically, separation of
anions requires the use of polymer-based anion exchangers
and dilute bases as mobile phases. However, for cation sep-
arations, the stability over the entire pH range that is typical
of organic polymers is not necessary, because dilute acids
serve as mobile phases. Therefore, silica-based cation ex-
changers that exhibit a significantly higher chromatographic
efficiency are commonly used for the separation of cations.
Depending on the separation mode (ion exchange, ion
exclusion, or ion-pair), different types of stationary phases
are used. For ion exchange, the stationary phase is either an
anion or a cation exchanger. Typically, a strong cation ex-
changer is used for the ion-exclusion separation of organic
acids, and a reversed-phase stationary phase is used when
ion-pair is the separation mode. The ion-exchange capacity
of a resin is defined as the number of ion-exchange sites per
weight equivalent of the column packing and is typically
expressed in terms of mEq per g of resin. With ion ex-
change, the retention times for the analyte ions increase
with increasing ion-exchange capacity of the resin. This ef-
fect can be partly compensated for by using mobile phases
of higher ionic strength. Styrene/divinylbenzene copolymers,
polymethacrylate, and polyvinyl resins are the substrate
materials used in the manufacturing process of the polymer-
based ion exchangers. Organic polymers are functionalized
directly at their surface, with the exception of latex-based
ion exchangers, where the totally porous latex particle acts
as an ion-exchange material. Surface-functionalized, “pellic-
ular” substrates show a much higher chromatographic effi-
ciency compared with the fully functionalized resins.
With ion exchange, a mobile phase consisting of mono-
or divalent ionic species, alone or mixed at an optimum
ratio, is used to accomplish the separation. In ion-exclusion
methods, particularly for organic acids, the mobile phase
consists of mineral acids to maintain organic acids in their
undissociated forms. Often, the nature of the analyte dic-
tates the mobile phase and the detection mode used. Typi-
cal mobile phases used in IC are described below in the
section on detectors.
Detectors
Conductivity detection is by far the most commonly em-
ployed mode of detection in IC. Although the original IC
development work included the use of low-capacity ion-ex-
1466 / Ion Chromatography / General Information
Figure 1. Components of a typical IC system illustrated schematically; CD = conductivity detector and PAD = pulsed amper-
ometric detector.
change resins for efficient chromatographic separation and
conductometric detection of ions in a chemically suppressed
mobile phase, the advances in column technologies as well
as instrumentation development allow the use of high-ca-
pacity ion exchange today.
In suppressed IC, the background conductance of the
ionic mobile phase is significantly reduced as it flows
through the suppression device. For example, dilute NaOH,
about 10 to 50 mM, used as the mobile phase in IC of
anions is converted to H2O (poor conductivity) when the
column effluent containing NaOH flows through a suppres-
sor device present in an acidic form. The analyte ionic spe-
cies in the column effluent are converted from their sodium
or other metal salt forms to highly conducting acid forms
(due to higher equivalent conductance of hydrogen ions
compared to other cations). Analogous reactions occur in
the hydroxide form suppressor in IC of cations, wherein the
acidic mobile phase is converted to water, and the analyte
cations are converted to highly conducting hydroxide forms
(due to higher equivalent conductance of hydroxide ions
compared to other anions).
The reduced background conductance and the enhanced
signal due to the ionic species result in an enhanced signal-
to-noise ratio for the conductometric detection of ions in
suppressed IC. This results in reduced background noise and
increasing sensitivity and reproducibility of the analysis. The
commonly used chemical suppression devices fall into three
broad categories. In the first type, the reactions occur across
an ion-exchange membrane with the regenerant ions fur-
nished by either a chemical or as products of electrolysis of
water. In the second type, the suppression reactions occur
in a packed bed of high-exchange capacity resin material,
with regeneration either by a chemical or by electrolysis of
water. In the third type, although not commonly used, the
suppression reactions occur as the eluant stream mixes with
the flowing stream of high-capacity resin material.
For pharmaceutical analyses, suppressed conductometric
detection may be used for detection of trace ions in high
purity waters. The commonly used mobile phases for the
separation of anions by suppressed IC include hydroxide
ions or a mixture of bicarbonate and carbonate ions. The
common mobile phases for separation of cations usually
consist of mineral acids or methanesulfonic acid.
Ion-chromatographic analyses also can be performed
without chemical suppression, in which case the analytical
column effluent flows directly to a conductivity detector.
The typical eluants used in nonsuppressed IC are phthalic
acid and p-hydroxybenzoic acid for the determination of an-
ions and methanesulfonic acid for the determination of ca-
tions. The equivalent conductance values of chloride, sul-
fate, and other common anions are significantly greater
than that of the eluant anion, and therefore, a positive peak
is detected as the anions are carried through the detector.
The equivalent conductance values of sodium, potassium,
calcium, magnesium, and other common cations are signifi-
cantly lower than that of the cation (H+) in the eluant. In
this instance, a negative peak is detected as the cations are
carried through the detector.
FCC 8
Nonsuppressed IC is easier to perform, and it is a useful
technique for determining ions of weak acids such as cya-
nide and sulfide, which are nonconductive after chemical
suppression but show a higher baseline noise. Pharmaceuti-
cal analyses can be performed in the nonsuppressed mode
because the quantification limits are usually in the upper mg
per L to low percentage levels. While suppressor-based
methodologies must often be implemented on the instru-
ment systems specifically designed for this purpose, IC may
be performed without the suppressor on an existing HPLC.
This is possible because the commonly used eluants in IC
include dilute bases or acids that are compatible for use on
existing HPLC instruments. When this approach is consid-
ered, analysts are encouraged to consult the instrument
manufacturer for applicability of the instrument for the IC
analysis.
OTHER DETECTORS
Other commonly used detection modes in IC include
pulsed amperometry, direct UV detection, or post-column
derivatization followed by UV/VIS detection.
Pulsed Amperometric Detection Mode (PAD)—PAD
uses a specialized mode of the conventional amperometric
technique. This type of detector is commonly used for the
detection of electroactive species, e.g., organic compounds
such as carbohydrates, sugar alcohols, amino acids, and or-
ganic sulfur species. In PAD, analytes are detected by an
oxidative desorption process at the surface of an electrode
located in the column effluent stream. Following the detec-
tion process, a series of potentials are applied for fixed time
periods to clean the electrode surface. Unlike conventional
amperometry that suffers from electrode surface fouling, a
rapidly repeating sequence of different working potentials,
referred to as waveform, helps the removal of the products
of redox reactions from the electrode surface.
Direct and Indirect UV Detection—Direct UV Detection
is used for inorganic and organic ions that possess a UV
chromophore. These include organic acids, bromide, iodide,
nitrate, nitrite, thiosulfate, and cyano-metal complexes.
Analogous to the inverse conductometric detection of ca-
tions, UV detection may also be performed indirectly. This
method is called indirect photometric chromatography
(IPC).
Photometric Detection—Photometric detection involves
chelation of the metal ions in column effluent with a color-
forming reagent prior to detection with a visible wave-
length. A classic example is the separation of metal ions in
which the column effluent is chelated with 4-(2-pyridylazo)-
resorcinol followed by detection at 510 to 530 nm.
SAMPLE PREPARATION
Typically sample preparation for IC includes dilution or fil-
tering through a 0.45-µm filter, or both. Under certain cir-
cumstances, samples may require removal of undesirable
FCC 8
species through solid-phase extraction (SPE) techniques. For
example, a highly alkaline sample can be neutralized by
having it pass through an SPE cartridge packed with cation-
exchange material in the acidic form.
PROCEDURE
Conductometric detection requires high purity water
(generally, resistivity greater than 18 megohm-cm) and
high-purity chemicals for the preparation of the mobile
phase. For ion-pair separation with UV detection, water and
mobile phase components of low UV absorbance should be
used.
For ion exchange, the retention time of ions increases
with a decrease in the ionic strength and valency (charge)
of the mobile phase components. For example, at equimolar
concentrations of sodium hydroxide or sodium carbonate
mobile phase, capacity factors (k′) for anions are smaller
with sodium hydroxide as the mobile phase than with so-
dium carbonate as the mobile phase. Some mobile phases,
such as sodium hydroxide, can absorb ambient carbon diox-
ide, resulting in its composition change and often in base-
line artifacts. In this instance, care should be taken to pre-
vent absorption of carbon dioxide by the sodium hydroxide
mobile phase.
For ion exclusion, capacity factors of organic acids in-
crease with an increase in ionic strength or concentration of
mineral acids but decrease with the increase of the column
temperature. Because permeation volume remains constant,
these effects are usually small. Addition of a solvent such as
acetonitrile shortens the retention of organic acids.
Like other HPLC techniques, IC systems are calibrated by
plotting peak responses in comparison with known concen-
trations of a reference standard, using either an external or
internal standardization procedure.
NEAR-INFRARED SPECTROSCOPY*
INTRODUCTION
Near-infrared (NIR) spectroscopy is a branch of vibrational
spectroscopy that shares many of the principles that apply
to other spectroscopic measurements. The NIR spectral re-
gion comprises two subranges associated with detectors
used in the initial development of NIR instrumentation. The
short-wavelength (Herschel or silicon region) extends from
approximately 780 to 1100 nm (12,821–9000 cm–1); and
longer wavelengths, between 1100 and 2500 nm, compose
the traditional (lead sulfide) NIR region. Applications of NIR
spectroscopy use spectra displayed in either wavelength or
wavenumber units. As is the case with other spectroscopy
measurements, interactions between NIR radiation and mat-
ter provide information that can be for both qualitative and
quantitative assessment of the chemical composition of sam-
ples. In addition, qualitative and quantitative characteriza-
tion of a sample’s physical properties can be made because
of the sample’s influence on NIR spectra. Measurements can
be made directly on samples in situ in addition to applica-
tions during standard sampling and testing procedures.
* This text is adapted from General Chapter 〈1119〉 of the United States
Pharmacopeia and National Formulary (USP–NF) as published in USP 32–NF 27.
This text is provided for informational purposes only and is intended as a
resource for the FCC user. Note that because the USP–NF is in continuous
revision, this General Chapter is subject to change and the text printed here
may not continue to represent the current version.
General Information / Near-Infrared Spectroscopy / 1467
Applications of qualitative analysis include identification of
raw material, in-process sample, or finished product. These
applications often involve comparing an NIR spectrum from
a sample to reference spectra and assessing similarities
against acceptance criteria developed and validated for a
specific application. In contrast, applications of quantitative
analysis involve the development of a predictive relationship
between NIR spectral attributes and sample properties.
These applications typically use numerical models to quan-
titatively predict chemical and/or physical properties of the
sample on the basis of NIR spectral attributes.
Vibrational spectroscopy in the NIR region is dominated
by overtones and combinations that are much weaker than
the fundamental mid-IR vibrations from which they origi-
nate. Because molar absorptivities in the NIR range are low,
radiation can penetrate several millimeters into materials, in-
cluding solids. Many materials, such as glass, are relatively
transparent in this region. Fiber-optic technology is readily
implemented in the NIR range, which allows monitoring of
processes in environments that might otherwise be
inaccessible.
The instrument qualification tests and acceptance criteria
provided in this chapter may not be appropriate for all in-
strument configurations. In such cases, alternative instru-
ment qualification and performance checks should be scien-
tifically justified and documented. In addition, validation
parameters discussed in this chapter may not be applicable
for all applications of NIR spectroscopy. Validation parame-
ters characterized for a specific NIR application should
demonstrate suitability of the NIR application for its in-
tended use.
Transmission and Reflection
The most common measurements performed in the NIR
spectral range are transmission and reflection spectroscopy.
Incident NIR radiation is absorbed or scattered by the sam-
ple and is measured as transmittance or reflectance, respec-
tively. Transflection spectrometry is a hybrid of transmission
and reflection wherein a reflector is placed behind the sam-
ple so that the optical path through the sample and back to
the detector is doubled compared to a transmission meas-
urement of a sample of the same thickness. Transflection is
used to describe any double-pass transmission technique.
The light may be reflected from a diffuse or specular (mir-
ror) reflector placed behind the sample. This configuration
can be adapted to share instrument geometry with certain
reflection or fiber-optic probe systems in which the source
and the detector are on the same side of the sample.
TRANSMITTANCE, T, is a measure of the decrease in radiation
intensity as a function of wavelength when radiation is
passed through a sample. The sample is placed in the opti-
cal beam between the source and the detector. The results
of both transmission and transflection measurements are
usually presented directly in terms of absorbance, i.e.,
log10(1/T).
REFLECTANCE, R, is a measure of the ratio of the intensity of
light reflected from the sample, I, to that reflected from a
background or reference reflective surface, IR. Most reflec-
tion measurements in the NIR are made of scattering sam-
ples such as powders and slurries. For such materials NIR
radiation can penetrate a substantial distance into the sam-
ple, where it can be absorbed when the wavelength of the
radiation corresponds to a transition between the ground
vibrational state of the analyte and either a harmonic of a
given vibrational mode (an overtone) or the sum of two or
more different modes (a combination band). Nonabsorbed
radiation is scattered back from the sample to the detector.
NIR reflection spectra are accessed by calculating and plot-
ting log(1/R) versus wavelength. This logarithmic form is the
pseudo-absorbance of the material and is commonly called
absorbance.
1468 / Near-Infrared Spectroscopy / General Information
Factors That Affect NIR Spectra
The following list is not exhaustive, but it includes many
of the major factors that affect NIR spectra.
Sample Temperature—Sample temperature influences
spectra obtained from aqueous solutions and other hydro-
gen-bonded liquids, and a difference of a few degrees may
result in significant spectral changes. Temperature may also
affect spectra obtained from less polar liquids, as well as
solids that contain solvents and/or water.
Moisture and Solvent—Moisture and solvent present in
the sample material and analytical system may change the
spectrum of the sample. Both absorption by moisture and
solvent and their influence on hydrogen bonding of the APIs
and excipients can change the NIR spectrum.
Sample Thickness—Sample thickness is a known source
of spectral variability and must be understood and/or con-
trolled. The sample thickness in transmission mode is typi-
cally controlled by using a fixed optical path length for the
sample. In diffuse reflection mode, the sample thickness is
typically controlled by using samples that are “infinitely
thick” relative to the detectable penetration depth of NIR
light into a solid material. Here “infinite thickness” implies
that the reflection spectrum does not change if the thick-
ness of the sample is increased.
Sample Optical Properties—In solids, both surface and
bulk scattering properties of calibration standards and ana-
lytical samples must be taken into account. Surface mor-
phology and refractive index properties affect the scattering
properties of solid materials. For powder materials, particle
size and bulk density influence scattering properties and the
NIR spectrum.
Polymorphism—Variation in crystalline structure (poly-
morphism) from materials with the same chemical composi-
tion can influence NIR spectral response. Different poly-
morphs and amorphous forms of solid material may be
distinguished from one another on the basis of their NIR
spectral properties. Similarly, different crystalline hydration
or solvation states of the same material can display different
NIR spectral properties.
Age of Samples—Samples may exhibit changes in their
chemical, physical, or optical properties over time. Care
must be taken to ensure that both samples and standards
used for NIR analysis are suitable for the intended
application.
INSTRUMENTATION
Apparatus
All NIR measurements are based on exposing material to
incident NIR light radiation and measuring the attenuation
of the emerging (transmitted, scattered, or reflected) light.
Several spectrophotometers are available; they are based on
different operating principles—for example: filters, grating-
based dispersive, acousto-optical tunable filter (AOTF),
Fourier–transform NIR (FT–NIR), and liquid crystal tunable
filter (LCTF). Silicon, lead sulfide, indium gallium arsenide,
and deuterated triglycine sulphate are common detector
materials. Conventional cuvette sample holders, fiber-optic
probes, transmission dip cells, and spinning or traversing
sample holders are common examples of sample interfaces
for introducing the sample to the optical train of a
spectrometer.
The selection of specific NIR instrumentation and sam-
pling accessories should be based on the intended applica-
tion, and particular attention should be paid to the suitabil-
ity of the sampling interface for the type of sample that will
be analyzed.
FCC 8
Near-Infrared Reference Spectra
NIR references, by providing known stable measurements
to which other measurements can be compared, are used to
minimize instrumental variations that would affect the
measurement.
Transmittance—The measurement of transmittance re-
quires a background reference spectrum for determining the
absorption by the sample relative to the background. Suita-
ble transmittance reference materials depend on the specific
NIR application and include air, an empty cell, a solvent
blank, or a reference sample.
Reflectance—The measurement of reflectance requires
the measurement of a reference reflection spectrum to de-
termine the attenuation of reflected light relative to the
unattenuated incident beam. The reflectance spectrum is
calculated as the ratio of the single-beam spectrum of the
sample to that of the reference material. Suitable reflectance
reference materials depend on the specific NIR application
and include ceramic, perfluorinated polymers, gold, and
other suitable materials.
Qualification of NIR Instruments
Qualification—Qualification of an NIR instrument can be
divided into three elements: Installation Qualification (IQ);
Operational Qualification (OQ); and Performance Qualifica-
tion (PQ).
Installation Qualification—The IQ requirements help en-
sure that the hardware and software are installed to accom-
modate safe and effective use of the instrument at the de-
sired location.
Operational Qualification—In operational qualification, an
instrument’s performance is characterized using standards to
verify that the system operates within target specifications.
The purpose of operational qualification is to demonstrate
that instrument performance is suitable. Because there are
so many different approaches for measuring NIR spectra,
operational qualification using standards with known spec-
tral properties is recommended. Using external traceable ref-
erence standard materials does not justify omitting the in-
strument’s internal quality control procedures. As is the case
with any spectroscopic device, wavelength uncertainty, pho-
tometric linearity, and noise characteristics of NIR instru-
ments should be qualified against target specifications for
the intended application.
Performance Qualification—Performance qualification
demonstrates that the NIR measurement consistently oper-
ates within target specifications defined by the user for a
specific application; it is often referred to as system suitabil-
ity. Performance qualification for NIR measurements can in-
clude comparing a sample or standard spectrum to previ-
ously recorded spectra. Comparisons of spectra taken over
time from identical and stable samples or reference standard
materials can form the basis for evaluating the long-term
stability of an NIR measurement system. The objective is to
demonstrate that no abnormal wavelength shift or change
in detector sensitivity has occurred during ongoing analysis.
Characterizing Instrument Performance—Specific pro-
cedures, acceptance criteria, and time intervals for charac-
terizing NIR instrument performance depend on the instru-
ment and intended application. Many NIR applications use
previously validated models that relate NIR spectral response
to a physical or chemical property of interest. Demonstrat-
ing stable instrument performance over extended periods of
time provides some assurance that reliable measurements
can be taken from sample spectra using previously validated
NIR models.
Wavelength Uncertainty—NIR spectra from sample and/or
reference standard materials can be used to demonstrate an
instrument’s suitable wavelength dispersion performance
against target specifications. The USP Near IR System Suita-
FCC 8
bility Reference Standard or the National Institute of Stan-
dards and Technology (NIST) Standard Reference Material
(SRM) 2036 for reflectance measurement and NIST SRM
2035 for transmittance measurement can be used for wave-
length verification. Suitable materials for demonstrating
wavelength dispersion performance include polystyrene,
mixtures of rare earth oxides, and absorption by water va-
por for instruments that use an interferometer for wave-
length dispersion. With appropriate justification, alternative
standards may be used. Wavelength uncertainty typically is
characterized from a single spectrum (collected with the
same spectral resolution to obtain the standard value) using
a minimum of three peaks that cover a suitable spectral
range of the instrument. Typical tolerances for agreement
with standard values are ±1.0 nm from approximately 700
to 2000 nm and ±1.5 nm above 2000 nm to approximately
2500 nm (±8 cm–1 below 5000 cm–1 and ±4 cm–1 from
5000 cm–1 to approximately 14,000 cm–1). Alternative toler-
ances may be used when justified for specific applications.
Photometric Linearity and Response Stability—NIR spectra
from samples and/or reference standard materials with
known relative transmittance or reflectance can be used to
demonstrate a suitable relationship between NIR light atten-
uation (due to absorption) and instrument response. For re-
flectance measurements, commercially-available reflectance
standards with known reflectance properties are often used.
Spectra obtained from reflection standards are subject to va-
riability as a result of the difference between the experimen-
tal conditions under which they were factory calibrated and
those under which they are subsequently put to use. Hence,
the reflectance values supplied with a set of calibration stan-
dards may not be useful in the attempt to establish an “ab-
solute” calibration for a given instrument. Provided that (1)
the standards do not change chemically or physically, (2)
the same reference background is also used to obtain the
standard values, and (3) the instrument measures each stan-
dard under identical conditions (including precise sample
positioning), the reproducibility of the photometric scale will
be established over the range of standards. Subsequent
measurements on the identical set of standards give infor-
mation on long-term stability. Photometric linearity is typi-
cally characterized using a minimum of four reference stan-
dards in the range from 10% to 90% reflection (or
transmission). NIR applications based on measuring an ab-
sorbance larger than 1.0 may require standards with reflec-
tivity properties between 2% and 5% reflection (or transmis-
sion) for characterizing instrument performance at low
reflectance. The purpose is to demonstrate a linear relation-
ship between NIR reflectance and/or transmittance and in-
strument response over the scanning range of the instru-
ment. Typical tolerances for a linear relationship are 1.00 ±
0.05 for the slope and 0.00 ± 0.05 for the intercept of a
plot of the measured photometric response versus standard
photometric response. Alternative tolerances may occur
when justified for specific applications.
Spectroscopic Noise—NIR instrument software may in-
clude built-in procedures to automatically determine system
noise and to provide a statistical report of noise or S/N over
the instrument’s operating range. In addition, it may be de-
sirable to supplement such checks with measurements that
do not rely directly on manufacturer-supplied procedures.
Typical procedures involve measuring spectra of traceable
reference materials with high and low reflectance. Toler-
ances for these procedures should demonstrate suitable S/N
for the intended application.
HIGH-FLUX NOISE—Instrument noise is evaluated at high-
light flux by measuring reflectance or transmittance of the
reference standard, with the reference material (e.g., 99%
reflection standard) acting as both the sample and the back-
ground reference.
LOW-FLUX NOISE—The same procedure may be used with a
lower-reflectivity reference material (e.g., 10% reflectance
standard) to determine system noise at reduced light flux.
General Information / Near-Infrared Spectroscopy / 1469
The source, optics, detector, and electronics make signifi-
cant contributions to the noise under these conditions.
METHOD VALIDATION
Introduction
The objective of NIR method validation, as is the case
with the validation of any analytical procedure, is to demon-
strate that the measurement is suitable for its intended pur-
pose. NIR spectroscopy is somewhat different from conven-
tional analytical techniques because validation of the former
generally is achieved by the assessment of chemometric pa-
rameters, but these parameters can still be related to the
fundamental validation characteristics required for any ana-
lytical method.
Data pretreatment is often a vital step in the chemometric
analysis of NIR spectral data. Data pretreatment can be de-
fined as the mathematical transformation of NIR spectral
data to enhance spectral features and/or remove or reduce
unwanted sources of variation prior to using the spectrum.
Calibration is the process of developing a mathematical rela-
tionship between NIR spectral response and properties of
samples. Many suitable chemometric algorithms for data
pretreatment and calibration exist; the selection should be
based on sound scientific judgment and suitability for the
intended application.
Validation Parameters
Performance characteristics that demonstrate the suitabil-
ity of NIR methods are similar to those required for any
analytical procedure. A discussion of the applicable general
principles is found elsewhere in the Food Chemicals Codex.
These principles should be considered typical for NIR proce-
dures, but exceptions should be dealt with on a case-by-
case basis. Specific acceptance criteria for each validation
parameter must be consistent with the intended use of the
method. The samples for validation should be independent
of the calibration set.
Specificity—The extent of specificity testing depends on
the intended application. Demonstration of specificity in NIR
methods is typically accomplished by using the following
approaches:
Qualitative—Identification testing is a common applica-
tion of qualitative NIR spectroscopy. Identification is
achieved by comparing a sample spectrum to a reference
spectrum or a library of reference spectra. The specificity of
the NIR identification method is demonstrated by obtaining
positive identification from samples coupled with negative
results from materials that should not meet criteria for posi-
tive identification. Materials to demonstrate specificity
should be based on sound scientific judgment and can in-
clude materials similar in visual appearance, chemical struc-
ture, or name.
Quantitative—Quantitative applications of NIR spectros-
copy typically involve establishing a mathematical relation-
ship between NIR spectral response and a physical or chem-
ical property of interest. Demonstrating specificity against a
physical or chemical property of interest is based on inter-
preting both NIR spectral attributes and chemometric pa-
rameters in terms of the intended application and may in-
clude the following:
• Spectral regions in the calibration model can be corre-
lated to a known NIR spectral response associated with
the property of interest.
• Wavelengths used by regression analysis for the calibra-
tion (e.g., for multiple linear regression [MLR] models)
or the loading vector for each factor (e.g., for partial
least squares [PLS] or principal component regression
[PCR] models) can be examined to verify relevant spec-
1470 / Near-Infrared Spectroscopy / General Information
troscopic information that is used for the mathematical
model.
• Variation in spectra from samples for calibration can be
examined and interpreted as expected spectral
observations.
• Variation in material composition and sample matrix
may be shown to have no significant effect on quantifi-
cation of the property of interest within the specified
method range.
Linearity—Quantitative NIR methods generally attempt
to demonstrate a linear relationship between NIR spectral
response and the property of interest. Although demonstrat-
ing a linear response is not required for all NIR applications,
the model chosen, whether linear or not, should properly
represent the relationship.
Validation of linearity in NIR methods may be accom-
plished by examining a plot of NIR spectral response versus
actual or accepted values for the property of interest. Many
statistical methods are available for evaluation of the good-
ness of fit of the linear relationship. Other applicable statis-
tics and graphical methods may be as appropriate.
The correlation coefficient, r, may not be an informative
measure of linearity. The square of the (Pearson) correlation
coefficient is a measure of the fraction of the data’s variation
that is adequately modeled by the equation. Linearity de-
pends on the standard error of the calibration equation (and
hence the reference method) and on the range of the cali-
bration data. Thus, although values very near 1.00, such as
0.99 or greater, typically indicate a linear relationship, lower
values do not distinguish between nonlinearity and variabil-
ity around the line.
Range—The specified range of an NIR method depends
on the specific application. The range typically is established
by confirming that the NIR method provides suitable meas-
urement capability (accuracy and precision) when applied to
samples within extreme limits of the NIR measurement.
Controls must be used to ensure that results outside the
validated range are not accepted. In certain circumstances,
it may not be possible or desirable to extend the validated
range to include sample variability outside the validated
range. Extending the range of an NIR method requires dem-
onstration of suitable measurement capability within the
limits of the expanded range. Examples of situations in
which only a limited sample range may be available are
samples from a controlled manufacturing process and in-
process samples. A limited method range does not preclude
the use of an NIR method.
Accuracy—Accuracy in NIR methods is demonstrated by
showing the closeness of agreement between the value that
is accepted as either a conventional true value or an ac-
cepted reference value. Accuracy can be determined by di-
rect comparison between NIR validation results and actual
or accepted reference values. Suitable agreement between
NIR and reference values is based on required measurement
capability for a specific application. The purpose is to
demonstrate a linear relationship between NIR results and
actual values. Accuracy can be determined by agreement
between the standard error of prediction (SEP) and the stan-
dard error of the reference method for validation. The error
of the reference method may be known on the basis of
historical data, through validation results specific to the ref-
erence method, or by calculating the standard error of the
laboratory (SEL). Suitable agreement between SEP and SEL is
based on required measurement capability for a specific
application.
Precision—The precision of an NIR method expresses the
closeness of agreement between a series of measurements
under prescribed conditions. Two levels of precision should
be considered: repeatability and intermediate precision. The
precision of an NIR method typically is expressed as the
relative standard deviation of a series of NIR method results
and should be suitable for the intended application. Demon-
stration of precision in NIR methods may be accomplished
using the following approaches:
FCC 8
Repeatability—Repeatability can be demonstrated by the
following:
• Statistical evaluation of a number of replicate measure-
ments of the sample without repositioning the sample
between each individual spectral acquisition, or
• Statistical evaluation of multiple NIR method results,
each result from a replicate analysis of a sample subse-
quent to re-positioning between spectral acquisitions
Intermediate Precision—Intermediate precision can be
shown by the following:
• Statistical evaluation of a number of replicate NIR meas-
urements of the same or similar samples in the
Repeatability study by different analysts on different
days.
Robustness—NIR measurement parameters selected to
demonstrate robustness will vary depending on the applica-
tion and the sample’s interface with the NIR instrument.
Critical measurement parameters associated with robustness
often are identified and characterized during method devel-
opment. Typical measurement parameters include the
following:
• Effect of environmental conditions (e.g., temperature,
humidity, and vibration)
• Effect of sample temperature
• Sample handling (e.g., probe depth, compression of
material, sample depth/thickness, sample presentation)
• Influence of instrument changes (e.g., lamp change,
warm-up time)
Ongoing Method Evaluation
Validated NIR methods should be subject to ongoing per-
formance evaluation, which may include monitoring accu-
racy, precision, and other suitable method parameters. If
performance is unacceptable, corrective action is necessary.
It involves conducting an investigation to identify the cause
of change in method performance and may indicate that
the NIR method is not suitable for continued use. Improving
the NIR method to meet measurement suitability criteria
may require additional method development and documen-
tation of validation experiments demonstrating that the im-
proved method is suitable for the intended application. The
extent of revalidation required depends on the cause of
change in method performance and the nature of corrective
action required in order to establish suitable method perfor-
mance. Appropriate change controls should be imple-
mented to document ongoing method improvement
activities.
Revalidation of a qualitative model may be necessary as a
result of the following:
• Addition of a new material to the spectral reference
library
• Changes in the physical properties of the material
• Changes in the source of material supply
• Identification of previously unknown critical attribute(s)
of material(s)
Revalidation of a quantitative model may be necessary as
a result of the following:
• Changes in the composition of the test sample or fin-
ished product
• Changes in the manufacturing process
• Changes in the sources or grades of raw materials
• Changes in the reference analytical method
• Major changes in instrument hardware
Outliers—Sample spectra that produce an NIR response
that differs from the qualitative or quantitative calibration
model may produce an outlier. This does not necessarily
indicate an out-of-specification result; but rather an outlier
indicates that further testing of the sample may be required
and is dependent on the particular NIR method. If subse-
quent testing of the sample by an appropriate method indi-
cates that the property of interest is within specifications,
then the sample meets its specifications. Outlier samples
FCC 8
may be incorporated into an updated calibration model
subsequent to execution and documentation of suitable vali-
dation studies.
Method Transfer
Controls and measures for demonstrating the suitability of
NIR method performance following method transfer are
similar to those required for any analytical procedure. Ex-
ceptions to general principles for conducting method trans-
fer for NIR methods should be justified on a case-by-case
basis. The transfer of an NIR method is often performed by
using an NIR calibration model on a second instrument that
is similar to the primary instrument used to develop and
validate the method. When a calibration model is trans-
ferred to another instrument, procedures and criteria must
be applied to demonstrate that the calibration model meets
suitable measurement criteria on the second instrument.
The selection of an appropriate calibration model transfer
procedure should be based on sound scientific judgment.
GLOSSARY
ABSORBANCE, A, is represented by the equation:
A = –log T = log (1/T)
where T is the transmittance of the sample. Absorbance is
also frequently given as:
A = log (1/R)
where R is the reflectance of the sample.
BACKGROUND SPECTRUM is used for generating a sample
spectrum with minimal contributions from instrument re-
sponse. It is also referred to as a reference spectrum or back-
ground reference. The ratio of the sample spectrum to the
background spectrum produces a transmittance or reflec-
tance spectrum dominated by NIR spectral response associ-
ated with the sample. In reflection measurements, a highly
reflective diffuse standard reference material is for the meas-
urement of the background spectrum. For transmission
measurement, the background spectrum may be measured
with no sample present in the spectrometer or using a cell
with the solvent blank or a cell filled with appropriate refer-
ence material.
CALIBRATION MODEL is a mathematical expression to relate
the response from an analytical instrument to the properties
of samples.
DIFFUSE REFLECTANCE is the ratio of the spectrum of radiated
light penetrating the sample surface, interacting with the
sample, passing back through the sample’s surface, and
reaching the detector to the background spectrum. This is
the component of the overall reflectance that produces the
absorption spectrum of the sample.
FIBER-OPTIC PROBES consist of two components: optical fibers
that may vary in length and in the number of fibers and a
terminus, which contains specially designed optics for exam-
ination of the sample matrix.
INSTALLATION QUALIFICATION is the documented collection of
activities necessary to establish that an instrument is deliv-
ered as designed and specified, is properly installed in the
selected environment, and that this environment is suitable
for the instrument’s intended purpose.
INSTRUMENT BANDWIDTH OR RESOLUTON is a measure of the
ability of a spectrometer to separate radiation of similar
wavelengths.
MULTIPLE LINEAR REGRESSION is a calibration algorithm to re-
late the response from an analytical instrument to the
properties of samples. The distinguishing feature of this al-
gorithm is the use of a limited number of independent vari-
ables. Linear-least-squares calculations are performed to es-
General Information / Near-Infrared Spectroscopy / 1471
tablish a relationship between these independent variables
and the properties of the samples.
OPERATIONAL QUALIFICATION is the process by which it is
demonstrated and documented that an instrument performs
according to specifications and that it can perform the in-
tended task. This process is required following any signifi-
cant change such as instrument installation, relocation, or
major repair.
OVERALL REFLECTANCE is the sum of diffuse and specular
reflectance.
PARTIAL LEAST SQUARES (PLS) is a calibration algorithm to re-
late instrument responses to the properties of samples. The
distinguishing feature of this algorithm is that data concern-
ing the properties of the samples for calibration are used in
the calculation of the factors to describe instrument
responses.
PERFORMANCE QUALIFICATION is the process of using one or
more well-characterized and stable reference materials to
verify consistent instrument performance. Performance qual-
ification may employ the same or different standards for dif-
ferent performance characteristics.
PHOTOMETRIC LINEARITY, also referred to as photometric verifi-
cation, is the process of verifying the response of the photo-
metric scale of an instrument.
PRINCIPAL COMPONENT REGRESSION (PCR) is a calibration al-
gorithm to relate the response from an analytical instrument
to the properties of samples. This algorithm, which ex-
presses a set of independent variables as a linear combina-
tion of factors, is a method of relating these factors to the
properties of the samples for which the independent vari-
ables were obtained.
PSEUDO-ABSORBANCE, A, is represented by the equation:
A = –log R = log (1/R)
where R is the diffuse reflectance of the sample.
REFERENCE SPECTRUM—See Background Spectrum.
REFLECTANCE is described by the equation:
R = I/IR
in which I is the intensity of radiation reflected from the
surface of the sample and IR is the intensity of radiation
reflected from a background reference material and its in-
corporated losses due to solvent absorption, refraction, and
scattering.
ROOT-MEAN-SQUARE (RMS) NOISE is calculated by the equation:
in which Ai is the absorbance for each data point; A is the
mean absorbance over the spectral segment; and N is the
number of points per segment.
SPECTRAL REFERENCE LIBRARY is a collection of spectra of
known materials for comparison with unknown materials.
The term is commonly used in connection with qualitative
methods of spectral analysis (e.g., identification of
materials).
SPECULAR (SURFACE) REFLECTANCE is the reflectance of the front
surface of the sample.
STANDARD ERROR OF CALIBRATION (SEC) is a measure of the ca-
pability of a model to fit reference data. SEC is the standard
deviation of the residuals obtained from comparing the
known values for each of the calibration samples to the val-
ues that are calculated from the calibration. SEC should not
be used as an assessment tool for the expected method
accuracy (trueness and precision of prediction) of the pre-
dicted value of future samples. The method accuracy should
generally be verified by calculating the standard error of
prediction (SEP), using an independent validation set of
samples. An accepted method is to mark a part of the cali-
bration set as the validation set. This set is not fully inde-
1472 / Near-Infrared Spectroscopy / General Information
pendent but can be used as an alternative for the determi-
nation of the accuracy.
STANDARD ERROR OF CROSS-VALIDATION (SECV) is the standard
deviation calculated using the leave-one-out method. In this
method, one calibration sample is omitted from the calibra-
tion, and the difference is found between the value for this
sample calculated from its reference value and the value ob-
tained from the calibration calculated from all the other
samples in the set. This process is repeated for all samples in
the set, and the SECV is the standard deviation of the differ-
ences calculated for all the calibration samples. This proce-
dure can also be performed with a group of samples. In-
stead of leaving the sample out, a group of samples is left
out. The SECV is a measure of the model accuracy that one
can expect when measuring future samples if not enough
samples are available for the SEP to be calculated from a
completely independent validation set.
STANDARD ERROR OF THE LABORATORY (SEL) is a calculation
based on repeated readings of one or more samples to esti-
mate the precision and/or accuracy of the reference labora-
tory method, depending on how the data were collected.
STANDARD ERROR OF PREDICTION (SEP) is a measure of model
accuracy of an analytical method based on applying a given
calibration model to the spectral data from a set of samples
different from but similar to those used to calculate the cali-
bration model. SEP is the standard deviation of the residuals
obtained from comparing the values from the reference lab-
oratory to those from the method under test for the speci-
fied samples. SEP provides a measure of the model accuracy
expected when one measures future samples.
SURFACE REFLECTANCE, also known as specular reflection, is
that portion of the radiation not interacting with the sample
but simply reflecting back from the sample surface layer
(sample–air interface).
TRANSFLECTION is a transmittance measurement technique
in which the radiation traverses the sample twice. The sec-
ond time occurs after the radiation is reflected from a sur-
face behind the sample.
TRANSMITTANCE is represented by the equation:
T = I/I0 or T = 10A
in which I is the intensity of the radiation transmitted
through the sample; I0 is the intensity of the radiant energy
incident on the sample and includes losses due to solvent
absorption, refraction, and scattering; and A is the
absorbance.
RAMAN SPECTROSCOPY*
INTRODUCTION
Raman spectroscopy shares many of the principles that
apply to other spectroscopic measurements discussed in
Spectrophotometry and Light-Scattering. Raman is a vibra-
tional spectroscopic technique and is therefore related to
infrared (IR) and near-infrared (NIR) spectroscopy. The
Raman effect itself arises as a result of a change in the po-
larizability of molecular bonds during a given vibrational
mode and is measured as inelastically scattered radiation.
* This text is adapted from General Chapter 〈1120〉 of the United States
Pharmacopeia and National Formulary (USP–NF) as published in USP 32–NF 27.
This text is provided for informational purposes only and is intended as a
resource for the FCC user. Note that because the USP–NF is in continuous
revision, this General Chapter is subject to change and the text printed here
may not continue to represent the current version.
FCC 8
A Raman spectrum is generated by exciting the sample of
interest to a virtual state with a monochromatic source, typi-
cally a laser. Light elastically scattered (no change in wave-
length) is known as Rayleigh scatter and is not of interest in
Raman spectrometry, except for marking the laser wave-
length. However, if the sample relaxes to a vibrational en-
ergy level that differs from the initial state, the scattered
radiation is shifted in energy. This shift is commensurate
with the energy difference between the initial and final vi-
brational states. This “inelastically scattered” light is referred
to as Raman scatter. Only about one in 106–108 photons
incident on the sample undergoes Raman scattering. Thus
lasers are employed in Raman spectrometers. If the Raman-
scattered photon is of lower energy, it is referred to as
Stokes scattering. If it is of higher energy, it is referred to as
anti-Stokes scattering. In practice, nearly all analytically use-
ful Raman measurements make use of Stokes-shifted Raman
scatter.
The appearance of a Raman spectrum is much like an
infrared spectrum plotted linearly in absorbance. The inten-
sities, or the number of Raman photons counted, are plot-
ted against the shifted energies. The x-axis is generally la-
beled “Raman Shift/cm–1” or “Wavenumber/cm–1”. The
Raman shift is usually expressed in wavenumber and repre-
sents the difference in the absolute wavenumber of the peak
and the laser wavenumber. The spectrum is interpreted in
the same manner as the corresponding mid-infrared spec-
trum. The positions of the (Raman shifted) wavenumbers for
a given vibrational mode are identical to the wavenumbers
of the corresponding bands in an IR absorption spectrum.
However, the stronger peaks in a Raman spectrum are often
weak in an IR spectrum, and vice versa. Thus the two spec-
troscopic techniques are often said to be complementary.
Raman spectroscopy is advantageous because quick and
accurate measurements can often be made without destroy-
ing the sample (solid, semisolid, liquid or, less frequently,
gas) and with minimal or no sample preparation. The
Raman spectrum contains information on fundamental vi-
brational modes of the sample that can yield both sample
and process understanding. The signal is typically in the visi-
ble or NIR range, allowing efficient coupling to fiber optics.
This also means that a signal can be obtained from any
medium transparent to the laser light; examples are glass,
plastics, or samples in aqueous media. In addition, because
Raman spectra are ordinarily excited with visible or NIR radi-
ation, standard glass/quartz optics may be used. From an
instrumental point of view, modern systems are easy to use,
provide fast analysis times (seconds to several minutes), and
are reliable. However, the danger of using high-powered la-
sers must be recognized, especially when their wavelengths
are in the NIR and, therefore, not visible to the eye. Fiber-
optic probes should be used with caution and with refer-
ence to appropriate government regulations regarding lasers
and laser classes.
In addition to “normal” Raman spectroscopy, there are
several more specialized Raman techniques. These include
resonance Raman (RR), surface-enhanced Raman spectros-
copy (SERS), Raman optical activity (ROA), coherent anti-
Stokes Raman spectroscopy (CARS), Raman gain or loss
spectroscopy, and hyper-Raman spectroscopy; however,
these techniques are not discussed in this general informa-
tion chapter.
QUALITATIVE AND QUANTITATIVE RAMAN
MEASUREMENTS
There are two general classes of measurements that are
commonly performed by Raman spectrometry: qualitative
and quantitative.
FCC 8
Qualitative Raman Measurements
Qualitative Raman measurements yield spectral informa-
tion about the functional groups that are present in a sam-
ple. Because the Raman spectrum is specific for a given
compound, qualitative Raman measurements can be used as
a compendial ID test, as well as for structural elucidation.
Quantitative Raman Measurements
For instruments equipped with a detector that measures
optical power (such as Fourier transform [FT]-Raman spec-
trometers), quantitative Raman measurements utilize the fol-
lowing relationship between signal, Sν, at a given
wavenumber, ν, and the concentration of an analyte, C:
Sν = Kσν(νL − νβ)4P0C
in which K is a constant that depends on laser beam diame-
ter, collection optics, sample volume, and temperature; σν is
the Raman cross section of the particular vibrational mode;
νL is the laser wavenumber; νβ is the wavenumber of the
vibrational mode; and P0 is the laser power. The Raman
cross section, σV, is characteristic of the nature of the partic-
ular vibrational mode. The sample volume is defined by size
of the focus of the laser beam at the sample, the optic
being used for focusing, and the optical properties of the
sample itself. Spot sizes at the sample can range from less
than 1 µm for a microprobe to 6 mm for a large area sam-
ple system. For Raman spectrometers that measure the
number of photons per second (such as change-coupled de-
vice [CCD]-Raman spectrometers) the corresponding equa-
tion is:
Sν = KσννL(νL − νβ)3P0C
From the above equations, it is apparent that peak signal is
directly proportional to concentration. It is this relationship
that is the basis for the majority of quantitative Raman
applications.
FACTORS AFFECTING QUANTIFICATION
Sample-Based Factors
The most important sample-based factors that deleteri-
ously affect quantitative Raman spectrometry are fluores-
cence, sample heating, absorption by the matrix or the
sample itself, and the effect of polarization. If the sample
matrix includes fluorescent compounds, the measured signal
will usually contain a contribution from fluorescence. Fluo-
rescence will be observed only if the laser excitation wave-
length overlaps with an absorption band of a fluorescent
compound. Fluorescence is typically observed as a broad
sloping background underlying the Raman spectrum. Fluo-
rescence can cause both a baseline offset and reduced sig-
nal-to-noise ratio. The wavelength range and intensity of the
fluorescence is dependent on the chemical composition of
the fluorescent material. Because fluorescence is generally a
much more efficient process than Raman scattering, even
very minor amounts of fluorescent impurities can lead to
significant degradation of the Raman signal. Fluorescence
can be reduced by using longer wavelength excitation
sources such as 785 nm or 1064 nm. However, it should be
remembered that the strength of the Raman signal is pro-
portional to (νL − νβ)4, so the advantage of using a long-
wavelength excitation laser to minimize fluorescence is at
least partially offset by the reduced strength of the Raman
signal. The greatest signal-to-noise ratio will be obtained by
balancing fluorescence rejection, signal strength, and detec-
tor response.
General Information / Raman Spectroscopy / 1473
Fluorescence in solids can sometimes be mitigated by ex-
posing the sample to the laser radiation for a period of time
before measurement. This process is called photobleaching,
and operates by degrading the highly absorbing species.
Photobleaching is less effective in liquids, where the sample
is mobile, or if the amount of fluorescent material is more
than a trace.
Sample heating by the laser source can cause a variety of
effects, such as physical form change (melting), polymorph
conversion, or sample burning. The chance for sample heat-
ing is greatest when the spot size at the sample is the small-
est, i.e., when a microprobe is being used. This is usually an
issue for colored, highly absorbing species, or very small
particles that have low heat transfer. The effects of sample
heating are usually observable either as changes in the
Raman spectrum over time or by visual inspection of the
sample. Besides decreasing the laser flux, a variety of meth-
ods can be employed to diminish laser-induced heating,
such as moving the sample or laser during the measurement
or improving the heat transfer from the sample with ther-
mal contact or liquid immersion.
Absorption of the Raman signal by the matrix or the sam-
ple itself can also occur. This problem is more prevalent
with long-wavelength FT-Raman systems where the Raman
signal can overlap with an NIR overtone absorption. This
effect will be dependent on the optics of the system as well
as on the sample presentation. Associated with this effect is
variability from scattering in solids as a result of packing and
particle-size differences. The magnitude of all of these ef-
fects, however, is typically less severe than in NIR because of
the limited depth of penetration and the relatively narrower
wavelength region sampled in Raman spectroscopy.
Finally, it should be recognized that laser radiation is po-
larized and the Raman spectra of crystalline materials and
other oriented samples can differ significantly depending on
the way that the sample is mounted. If the Raman spec-
trometer is capable of producing linearly polarized radiation
at the sample then a polarization scrambler is recommended
for routine sample analysis.
Sampling Factors
Raman spectroscopy is a zero-background technique, in
that the signal at the detector is expected to be zero in the
absence of a sample. This situation can be contrasted with
absorption spectrometry, where the signal at the detector is
at a maximum in the absence of a sample. Zero-background
techniques are inherently sensitive because small changes in
sample concentration lead to proportionate changes in the
signal level. The instrument will also be sensitive to other
sources of light that can cause sample-to-sample variations
in the measured signal level. In addition, a large back-
ground signal caused by fluorescence will lead to an in-
creased noise level (photon shot noise). Thus it may be very
difficult to use the absolute Raman signal for direct determi-
nation of an analyte. Other potential sources of variation are
changes in the sample opacity and heterogeneity, changes
in the laser power at the sample, and changes in optical
collection geometry or sample position. These effects can be
minimized by sampling in a reproducible, representative
manner. Careful design of the instrumentation can reduce
these effects but they cannot be eliminated entirely.
Use of an internal reference standard is the most common
and robust method of eliminating variations caused by ab-
solute intensity fluctuations. There are several choices for
this approach. An internal standard can be deliberately
added, and isolated peaks from this standard can be em-
ployed; or a band due to a moiety such as an aromatic ring,
the Raman cross-section of which does not change with the
way the sample is prepared, can also be used. For solution
spectra, an isolated solvent band can be employed because
the solvent will remain relatively unchanged from sample to
sample. Also, in a formulation, an excipient peak can be
used if it is in substantial excess compared to the analyte.
1474 / Raman Spectroscopy / General Information FCC 8

The entire spectrum can also be used as a reference, with
the assumption that laser and sample-orientation changes
will affect the entire spectrum equally.
A second important sampling-based factor to consider is
spectral contamination. Raman scattering is a weak effect
that can be masked by a number of external sources. Com-
mon contamination sources include sample-holder artifacts
(container or substrate) and ambient light. Typically, these
issues can be identified and resolved by careful
experimentation.
APPARATUS
Components
All modern Raman measurements involve irradiating a
sample with a laser, collecting the scattered radiation, re-
jecting the Rayleigh-scattered light, differentiating the
Raman photons by wavelength, and detecting the resulting
Raman spectrum. All commercial Raman instruments there-
fore share the following common features to perform these
functions:
1. Excitation source (laser)
2. Sampling device
3. Device to filter/reject light scattered at the laser
wavelength
4. Wavelength processing unit
5. Detector and electronics
EXCITATION SOURCE (LASER)
Table 1 identifies several common lasers used for Raman
spectrometry. UV lasers have also been used for specialized
applications but have various drawbacks that limit their util-
ity for general analytical measurements. As more applica-
tions for UV lasers are described, it is likely that they may
become more common for Raman spectrometry.
SAMPLING DEVICE
Several sampling arrangements are possible, including di-
rect optical interfaces, microscopes, fiber optic-based probes
(either noncontact or immersion optics), and sample cham-
bers (including specialty sample holders and automated
sample changers). The sampling optics can also be designed
to obtain the polarization-dependent Raman spectrum,
which often contains additional information. Selection of the
sampling device will often be dictated by the analyte and
sample. However, considerations such as sampling volume,
speed of the measurement, laser safety, and reproducibility
of sample presentation should be evaluated to optimize the
sampling device for any given application.
FILTERING DEVICE
The intensity of scattered light at the laser wavelength
(Rayleigh) is many orders of magnitude greater than the
Raman signal and must be rejected prior to the detector.
Notch filters are almost universally used for this purpose and
provide excellent rejection and stability combined with small
size. The traditional use of multistage monochromators for
this purpose, although still viable, is now rare. In addition,
various filters or physical barriers to shield the sample from
external radiation sources (e.g., room lights, laser plasma
lines) may be required depending on the collection geome-
try of the instrument.
WAVELENGTH PROCESSING UNIT
The wavelength scale may be encoded by either a scan-
ning monochromator, a grating polychromator (in CCD-
Raman spectrometers) or a two-beam interferometer (in FT-
Raman spectrometers). A discussion of the specific benefits
and drawbacks of each of the dispersive designs compared
to the FT instrument is beyond the scope of this chapter.
Any properly qualified instruments should be suitable for
qualitative measurements. However, care must be taken
when selecting an instrument for quantitative measure-
ments, as dispersion and response linearity might not be
uniform across the full spectral range.
DETECTOR
The silicon-based CCD array is the most common detec-
tor for dispersive instruments. The cooled array detector al-
lows measurements over the spectral range from 4500 to
100 cm−1 Raman shift with low noise when most visible la-
sers, such as frequency-doubled neodymium-doped yttri-
um–aluminum–garnet (Nd:YAG) (532 nm) or helium–neon
(632.8 nm) lasers, are used. When a 785-nm diode laser is
used, the wavelength range is reduced to about 3100 to
100 cm−1. The most commonly used CCD has its peak
wavelength responsivity when matched to the commonly
used 632.8-nm He–Ne gas laser or 785-nm diode laser. FT
instruments typically use single-channel germanium or indi-
um–gallium–arsenide (InGaAs) detectors responsive in the
NIR to match the 1064-nm excitation of a Nd:YAG laser.

Table 1. Lasers Used Commonly for Raman Spectroscopy

Wavelength Range, nm
Laser λ, nm (nearest Typical Power (Stokes Region, 100 cm–1
whole number) Type at Laser to 3000 cm–1 shift) Comments
NIR Lasers
1064 Solid state Up to 3 W 1075–1563 Commonly used in Fourier transform
(Nd:YAG) instruments
830 Diode Up to 300 mW 827–980 Typically limited to 2000 cm−1; Raman
shift because of CCD spectral re-
sponse; less common than the other
lasers
785 Diode Up to 500 mW 791–1027 Most widely used dispersive
Raman laser
Visible Lasers
632.8 He–Ne Up to 500 mW 637–781 Relatively small fluorescence risk
532 Doubled Up to 1 W 535–632.8 High fluorescence risk
(Nd:YAG)
514.5 Ar+ Up to 1 W 517–608 High fluorescence risk
488–632.8 Ar+ Up to 1 W 490–572 High fluorescence risk
FCC 8
Calibration
Raman instrument calibration involves three components:
primary wavelength (x-axis), laser wavelength, and intensity
(y-axis).
PRIMARY WAVELENGTH (X-AXIS)
In the case of FT-Raman instruments, primary wavelength-
axis calibration is maintained, at least to a first approxima-
tion, with an internal He–Ne laser. Most dispersive instru-
ments utilize atomic emission lamps for primary wavelength-
axis calibration. In all instruments suitable for analytical
Raman measurements, the vendor will offer a procedure of
x-axis calibration that can be performed by the user. For
dispersive Raman instruments, a calibration based on multi-
ple atomic emission lines is preferred. The validity of this
calibration approach can be verified subsequent to laser
wavelength calibration by using a suitable Raman shift stan-
dard. For scanning dispersive instruments, calibration might
need to be performed more frequently, and precision in
both a scanning and static operation mode may need to be
verified.1
LASER WAVELENGTH
Laser wavelength variation can impact both the wave-
length precision and the photometric (signal) precision of a
given instrument. Even the most stable current lasers can
vary slightly in their measured wavelength output. The laser
wavelength must therefore be confirmed to ensure that the
Raman shift positions are accurate for both FT-Raman or
dispersive Raman instruments. A reference Raman shift stan-
dard material such as those outlined in ASTM E1840-96
(2002)1 or other suitably verified materials can be utilized
for this purpose. [NOTE—Reliable Raman shift standard val-
ues for frequently used liquid and solid reagents, required
for wavenumber calibration of Raman spectrometers, are
provided in the ASTM Standard Guide cited. These values
can be used in addition to the highly accurate and precise
low-pressure arc lamp emission lines that are also available
for use in Raman instrument calibration.] Spectrometric
grade material can be purchased from appropriate suppliers
for this use. Certain instruments may use an internal Raman
standard separate from the primary optical path. External
calibration devices exactly reproduce the optical path taken
by the scattered radiation. [NOTE—When chemical standards
are used, care must be taken to avoid contamination and to
confirm standard stability.]
Unless the instrument is of a continuous calibration type,
the primary wavelength axis calibration should be per-
formed, as per vendor procedures, just prior to measuring
the laser wavelength. For external calibration, the Raman
shift standard should be placed at the sample location and
measured using appropriate acquisition parameters. The
peak center of a strong, well-resolved band in the spectral
region of interest should be evaluated. The position can be
assessed manually or with a suitable, valid peak-picking al-
gorithm. The software provided by the vendor might meas-
ure the laser wavelength and adjust the laser wavelength
appropriately so that this peak is at the proper position. If
the vendor does not provide this functionality, the laser
wavelength should be adjusted manually. Depending on the
type of laser, the laser wavelength can vary with tempera-
ture, current, and voltage. Wavelength tolerances can vary
depending on the specific application.
1 ASTM E1840-96 (2002) Standard Guide for Raman Shift Standards for Spec-
trometer Calibration, ASTM International, 100 Barr Harbor Drive, PO Box
C700, West Conshohocken, PA, USA 19428-2959.
General Information / Raman Spectroscopy / 1475
SIGNAL LEVEL (Y-AXIS)
Calibration of the photometric axis can be critical for suc-
cessful quantification by using certain analytical methods
(chemometrics) and method transfer between instruments.
Both FT-Raman and dispersive Raman spectrometers should
undergo similar calibration procedures. The tolerance of
photometric precision acceptable for a given measurement
should be assessed during the method development stage.
To calibrate the photometric response of a Raman instru-
ment, a broad-band emission source should be used. There
are two accepted methods. Method A utilizes a tungsten
white light source.2 The output power of such sources is
traceable to the National Metrology Institute (NMI). In the
United Kingdom, the National Physical Laboratory also pro-
vides calibrated light bulbs. Several other vendors also pro-
vide NIST-traceable irradiance calibration standards. This
method is applicable to all common laser excitation wave-
lengths listed in Table 1. In Method B, NIST standard refer-
ence materials (SRMs) are utilized.3 Several doped-glass fluo-
rescence standards are currently available.
Method A—The source should be placed at the sample
location with the laser off and the response of the detector
measured (using parameters appropriate for the instrument).
The output for the source used for calibration should be
known. The ratio of the measured response to the true re-
sponse should be determined and a correction file gener-
ated. This correction should be applied to all spectra ac-
quired with the instrument. Most manufacturers will provide
both appropriate calibration sources and software for this
approach. If the manufacturer does not provide a procedure
or method, the user can accomplish the task using a source
obtained from NIST and appropriate software. If a manufac-
turer’s method is used, attention must be paid to the cali-
bration procedure and source validity. The user should ob-
tain appropriate documentation from the manufacturer to
ensure a qualified approach.
Method B—The fluorescence standard should be placed
at the sample location. With the laser on, a spectrum of the
SRM should be obtained (using parameters appropriate for
the instrument). The output of the source used for calibra-
tion should be known. The ratio of the measured response
to the true response should be determined and a correction
file generated. This correction should be applied to all spec-
tra acquired with the instrument. Most manufacturers will
provide both appropriate calibration sources and software
for this approach. If the manufacturer does not provide a
procedure or method, the user can accomplish the task us-
ing a source obtained from NIST and appropriate software.
If a manufacturer’s method is used, attention must be paid
to the calibration procedure and source validity. The user
should obtain appropriate documentation from the manu-
facturer to ensure a qualified approach. [NOTE—Method B is
currently appropriate for systems with 785-nm (SRM 2241),
532-nm (SRM 2242), and both 514.5-nm and 488-nm
(SRM 2243) laser excitation. NIST is currently developing
other SRMs that will be wavelength-specific for 1064-nm
(SRM 2244) and 632.8-nm excitation (expected to be avail-
able in 2006).]
EXTERNAL CALIBRATION
Detailed functional validation employing external refer-
ence standards is recommended to demonstrate instrumen-
tal suitability for laboratory instruments, even for instru-
ments that possess an internal calibration approach. The use
2 NIST-traceable tungsten white light source statement: While the calibration
of the Raman frequency (or Raman shift, cm–1) axis using pure materials and
an existing ASTM standard is well accepted, techniques for calibration of the
Raman intensity axis are not. Intensity calibrations of Raman spectra can be
accomplished with certified white light sources.
3 NIST SRM 2241: Ray KG, McCreery RL. Raman intensity correction standard
for systems operating with 785-nm excitation. Appl. Spectrosc. 1997, 51,
108–116.
1476 / Raman Spectroscopy / General Information
of external reference standards does not obviate the need
for internal quality control procedures; rather, it provides in-
dependent documentation of the fitness of the instrument
to perform the specific analysis or purpose. For instruments
installed in a process location or in a reactor where position-
ing of an external standard routinely is not possible, includ-
ing those instruments that employ an internal calibration
approach, the relative performance of an internal versus an
external calibration approach should be periodically
checked. The purpose of this test is to check for changes in
components that might not be included in the internal cali-
bration method (process lens, fiber-optic probe, etc.), e.g.,
photometric calibration of the optical system.
QUALIFICATION AND VERIFICATION OF
RAMAN SPECTROMETERS
The suitability of a specific instrument for a given method
is ensured by a thorough technology-suitability evaluation
for the application; a routine, periodic instrument opera-
tional qualification; and the more frequent performance ver-
ification (see Definition of Terms and Symbols). The purpose
of the technology-suitability evaluation is to ensure that the
technology proposed is suitable for the intended applica-
tion. The purpose of the instrument qualification is to en-
sure that the instrument to be used is suitable for its in-
tended application and, when requalified periodically,
continues to function properly over extended time periods.
When the device is used for a specific qualitative or quanti-
tative analysis, regular performance verifications are made.
Because there are many different approaches to measuring
Raman spectra, instrument operational qualification and per-
formance verification often employ external standards that
can be used on any instrument. As with any spectrometric
device, a Raman instrument needs to be qualified for both
wavenumber (x-axis and shift from the excitation source)
and photometric (signal axis) precision.
In performance verification, a quality-of-fit to an initial
scan or group of scans (often referred to in nonscanning
instruments as an accumulation) included in the instrumen-
tal qualification can be employed. In such an analysis, it is
assumed that reference standard spectra collected on a new
or a newly repaired, properly operating instrument represent
the best available spectra. Comparison of spectra taken over
time on identical reference standards (either the original
standard or identical new standards, if stability of the refer-
ence standards is a concern) forms the basis for evaluating
the long-term stability of a Raman measurement system.
Frequency of Testing
Instrumental qualification is performed at designated in-
tervals or following a repair or significant optical recon-
figuration, such as the replacement of the laser, the detector
or the notch or edge filters. Full instrument requalification
might not be necessary when changing between sampling
accessories such as a microprobe, a sample compartment,
or a fixed fiber-optic probe. Performance verification tests
may be sufficient in these cases; instrument-specific guid-
ance from the vendor on qualification requirements should
be followed. Tests include wavelength (x-axis and shift from
the excitation source) and photometric (signal axis) preci-
sion. Instrument qualification tests require that specific ap-
plication-dependent tolerances be met.
Performance verification is carried out on the instrument
configured for the analytical measurements and is per-
formed more frequently than instrument qualification. Per-
formance verification includes measurement of the wave-
length uncertainty and intensity-scale precision. Wavelength
precision and intensity-scale precision tests may be needed
prior to any data collection on a given day. Performance is
verified by matching the current spectra to those collected
during the previous instrument qualification.
FCC 8
Instrument Operational Qualification
It is important to note that the acceptance specifications
given in both the Instrument Operational Qualification and
Performance Qualification sections are applicable for general
use; specifications for particular instruments and applications
can vary depending on the analysis method used and the
desired accuracy of the final result. ASTM standard reference
materials are also specified, with the understanding that
under some circumstances (specifically remote on-line appli-
cations) calibration using one of these materials may be im-
practical, and other suitably verified materials can be em-
ployed. At this juncture it is important to note that specific
parameters such as spectrometer noise, limits of detection
(LOD), limits of quantification (LOQ), and acceptable spec-
tral bandwidth for any given application should be included
as part of the analytical method development. Specific val-
ues for tests such as spectrometer noise and bandwidth will
be dependent on the instrument chosen and the purpose
required. As a result, specific instrument tests for these pa-
rameters are not dictated in this information chapter.
WAVELENGTH (X-AXIS) ACCURACY
It is important to ensure the accuracy of the wavelength
axis via calibration to maintain the integrity of Raman peak
positions. Wavelength calibration of a Raman spectrometer
consists of two parts: primary wavelength axis and laser
wavelength calibration. After both the primary wavelength
axis and the laser wavelength are calibrated, instrument
wavelength uncertainty can be determined. This can be ac-
complished using a Raman shift standard such as the ASTM
shift standards or other suitably verified material. Selection
of a standard with bands present across the full Raman
spectral range is recommended so that instrument wave-
length uncertainty can be evaluated at multiple locations
within the spectrum. The tolerance of wavelength precision
that is required for a given measurement should be assessed
during the method-development stage. [NOTE—For scanning
dispersive instruments, calibration might need to be per-
formed more frequently, and precision in both a scanning
and static operation mode may need to be verified.]
PHOTOMETRIC PRECISION
Laser variation in terms of the total emitted photons oc-
curring between two measurements can give rise to
changes in the photometric precision of the instrument. Un-
fortunately, it is very difficult to separate changes in the
photometric response associated with variations in the total
emitted laser photons from the sample- and sampling-in-
duced perturbations. This is one of the reasons why abso-
lute Raman measurements are strongly discouraged and
why the photometric precision specification is set relatively
loosely. The tolerance of photometric precision required for
a given measurement should be assessed during the
method-development stage.
PERFORMANCE QUALIFICATION
The objective of performance qualification is to ensure
that the instrument is performing within specified limits
with respect to wavelength precision, photometric axis pre-
cision, and sensitivity. In certain cases when the instrument
has been set up for a specific measurement (for example,
installed in a process reactor), it might no longer be possi-
ble or desirable to measure the wavelength and photomet-
ric (signal) qualification reference standards identified above.
Provided instrument operational qualification has shown
that the equipment is fit for use, a single external perfor-
mance verification standard can be used to reverify function
on a continuing basis (for example, a routinely used process
FCC 8
solvent signal, for both wavelength and photometric preci-
sion, following reactor cleaning). The performance verifica-
tion standard should match the format of the samples in the
current analysis as closely as possible and use similar spectral
acquisition parameters. Quantitative measurements of an ex-
ternal performance verification standard spectrum check
both the wavelength (x-axis and laser wavelength) and the
photometric (signal) precision. Favorable comparison of a
series of performance verification spectra demonstrates
proper continued operation of the instrument.
WAVELENGTH PRECISION
The wavelength precision should be measured by collect-
ing data for a single spectrum of the selected Raman shift
standard for a period equal to that used in the photometric
consistency test. When appropriate, powdered samples
should be repacked between each set of measurements.
Peak positions across the spectral range of interest are used
to calculate precision. Performance is verified by matching
the current peak positions to those collected during the pre-
vious instrument qualification and should not vary with a
standard deviation of more than ±0.3 cm–1, although this
specification can be adjusted according to the required ac-
curacy of the measurement.
PHOTOMETRIC PRECISION
The photometric precision should be measured by collect-
ing data for a single spectrum of a suitably verified reference
standard material for a specified time. After suitable baseline
correction, the areas of a number of bands across the spec-
tral range of interest should be calculated by means of an
appropriate algorithm. The area of the strongest band is set
to 1, and all other envelopes are normalized to this band.
Performance is verified by matching the current band areas
to the respective areas collected during the previous instru-
ment qualification. The areas should vary by no more than
10%, although this specification can be adjusted according
to the required accuracy of the measurement.
LASER POWER OUTPUT PRECISION AND ACCURACY
This test is applicable only to Raman instruments with au-
tomatic, internal laser power meters. Instruments without la-
ser power measurement should utilize a calibrated laser
power meter from a reputable supplier. The laser output
should be set to a representative output, dictated by the
requirements of the analytical measurement and the laser
power measured. The output should be measured and
checked against the output measured at instrument qualifi-
cation. The power (in milliwatts or watts) should vary by no
more than 25% compared to the qualified level. If the
power varies by more than this amount, the instrument
should be serviced (as this variation might indicate, among
other things, a gross misalignment of the system or the on-
set of failure of the laser).
For instruments with an automatic, internal laser power
meter, the accuracy of the values generated from the inter-
nal power meter should be compared to a calibrated exter-
nal laser power meter at an interval of not more than 12
months. The internally calculated value should be compared
to that generated by the external power meter. Perfor-
mance is verified by matching the current value to that gen-
erated during the previous instrument qualification. The
manufacturer might provide software to facilitate this analy-
sis. If the instrument design prevents the use of an external
power meter, then the supplier should produce documenta-
tion to ensure the quality of the instrument and provide a
recommended procedure for the above analysis to be ac-
complished during a scheduled service visit.
General Information / Raman Spectroscopy / 1477
METHOD VALIDATION
Validation of Raman methods will follow the same proto-
cols as for other instrumental analytical methods in terms of
accuracy, precision, etc. However, several of these criteria
are affected by variables specific to Raman spectrometry.
Fluorescence is the primary variable that can affect the
suitability of a method. The presence of fluorescent impuri-
ties in samples can be quite variable and have little effect on
the acceptability of a material. The method must be flexible
enough to accommodate different sampling regimes that
may be necessary to minimize the effects of these
impurities.
Detector linearity must be confirmed over the range of
possible signal levels. Fluorescence might drive both the sig-
nal baseline and the noise higher than that used in the vali-
dation, in which case the fluorescence must be decreased,
or the method modified to accommodate the higher fluo-
rescence levels. This is also true for the precision, limit of
detection, and limit of quantification of the method, as in-
creased baseline noise will negatively impact all of these val-
ues. Because fluorescence can also affect quantification
caused by baseline shifts, acceptable quantification at differ-
ent levels of photobleaching, when used, should also be
confirmed.
The impact of the laser on the sample must be deter-
mined. Visual inspection of the sample and qualitative in-
spection of the Raman spectrum for measurements with dif-
fering laser powers and exposure times will confirm that the
sample is not being altered (other than by photobleaching).
Specific variables to confirm in the spectrum are shifts in
peak position, changes in peak height and band width, and
unexpected changes in background intensity.
Method precision must also encompass sample position.
The sample presentation is a critical factor for both solids
and liquids, and must be either tightly controlled or ac-
counted for in the calibration model. Sample-position sensi-
tivity can often be minimized by appropriate sample prepa-
ration or sample holder geometry, but will vary from
instrument to instrument based on excitation and collection
optical configuration.
DEFINITION OF TERMS AND SYMBOLS
CALIBRATION MODEL is a mathematical expression that relates
the response from an analytical instrument to the properties
of samples.
INSTRUMENT BANDPASS (OR RESOLUTION) is a measure of the
capability of a spectrometer to separate radiation of similar
wavelengths.
OPERATIONAL QUALIFICATION is the process by which it is
demonstrated and documented that the instrument per-
forms according to specifications, and that it can perform
the intended task. This process is required following any sig-
nificant change such as instrument installation, relocation,
major repair, etc.
PERFORMANCE QUALIFICATION is the process of using one or
more well-characterized and stable reference materials to
verify consistent instrument performance. Qualification may
employ the same or different standards for different perfor-
mance characteristics.
RAMAN SPECTRA4 are plots of the radiant energy, or number
of photons, scattered by the sample through the indirect
interaction between the molecular vibrations in the sample
and monochromatic radiation of frequency much higher
than that of the vibrations. The abscissa is usually the differ-
ence in wavenumber between the incident and scattered
radiation.
(NORMAL) RAMAN SCATTERING4 is the inelastic scattering of ra-
diation that occurs because of changes in the polarizability,
of the relevant bonds during a molecular vibration. Normal
4 Chalmers, J., Griffiths, P., Eds. Handbook of Vibrational Spectroscopy; John
Wiley & Sons, Ltd: New York, 2002.
 Next Page

1478 / Raman Spectroscopy / General Information FCC 8

Raman spectra are excited by radiation that is not in Scoville Heat Standard Solution Sucrose Solution
resonance with electronic transitions in the sample. Units (mL) (mL)
RAMAN WAVENUMBER SHIFT4, 600,000 20 30
720,000 20 40
840,000 20 50
is the wavenumber of the exciting line minus the 960,000 20 60
wavenumber of the scattered radiation. SI unit: m−1. Com- 1,080,000 20 70
mon unit: cm−1 = 100 m−1. 1,200,000 20 80
1,320,000 20 90
1,440,000 20 100
1,560,000 20 110
where β is the differential Raman cross section, is positive 1,680,000 20 120
for Stokes scattering and negative for anti-Stokes scattering.
1,800,000 20 130
1,920,000 20 140
2,040,000 20 150

If the oleoresin sample is claimed to contain less than

240,000 Scoville Heat Units, prepare one or more dilutions
SCOVILLE HEAT UNITS according to the following table:

Scoville Heat Sample Sucrose Solution

Units Preparation (mL) (mL)
Sample Preparation Transfer 200 mg of the sample into a
50-mL volumetric flask, dilute with alcohol to volume, and 100,000 0.15 60
mix thoroughly by shaking. Allow the insolubles to settle 117,500 0.15 70
before use. 170,000 0.15 100
Sucrose Solution Prepare a suitable volume of a 10% 205,000 0.15 120
(w/v) solution of sucrose in water.
Standard Solution Add 0.15 mL of the Sample Preparation Procedure Select five panel members who are thoroughly
to 140 mL of the Sucrose Solution, and mix. This solution experienced with this method. Instruct the panelists to swal-
contains the equivalent of 240,000 Scoville Heat Units. low 5 mL of the solution corresponding to the claimed con-
Test Solutions If the oleoresin sample is claimed to con- tent of Scoville Heat Units. The sample passes the test if
tain more than 240,000 Scoville Heat Units, prepare one or three of the five panel members perceive a pungent or
more dilutions according to the following table: stinging sensation in the throat.
Acceptance criteria
Scoville Heat Standard Solution Sucrose Solution Capsicum: Between 100,000 and 2,000,000, as specified
Units (mL) (mL) on the label
360,000 20 10 Paprika (pungency): NMT 3000
480,000 20 20
FCC 8
General Provisions and Requirements Applying to
Specifications, Tests, and Assays of the
Food Chemicals Codex
The General Provisions provide, in summary form, guidelines
for the interpretation and application of the standards, tests
and assays, and other specifications of the Food Chemicals
Codex and make it unnecessary to repeat throughout the
book those requirements that are pertinent in numerous in-
stances. Where exceptions to the General Provisions are
made, the wording in the individual monograph or general
test chapter takes precedence and specifically indicates the
directions or the intent.
TITLE OF BOOK
The title of this book, including its supplements, is the Food
Chemicals Codex, Eighth Edition. It may be abbreviated to
FCC 8. Where the term FCC is used without further
qualification in the text of this book, it applies to the Food
Chemicals Codex, Eighth Edition.
APPROPRIATE USE OF THE FOOD
CHEMICALS CODEX
As a compendium that addresses known food ingredients
used in food products either in the United States or
internationally, the FCC has many practical applications in
industry, research, and academia. The FCC does not,
however, provide information on the regulatory status or
safety of food chemicals, nor does the presence or absence
of standards for a particular food ingredient indicate in any
way USP’s endorsement (or lack thereof) of that item for use
in foods or food processing. It is the responsibility of the
user to determine the safety and regulatory status of a
particular food ingredient for any specific application.
FCC standards have been developed in cooperation with
regulatory authorities and industry in the United States and
elsewhere both under the stewardship of the Institute of
Medicine and, more recently, USP. While USP makes great
efforts to dialog with the U.S. Food and Drug
Administration (FDA) regarding creating or revising
monograph standards in the FCC, USP has no official
legislative authority to establish legal requirements for food
ingredients in the United States.1 The FCC serves as a
resource for companies that manufacture, process, purchase,
or use food ingredients and seek to determine appropriate
minimum standards for components of their food products.
The structure and format of the FCC monographs and
informational chapters allow users to quickly access the
following types of information:
• General information about food ingredients
• Chemical information specific to food ingredients
• Information regarding laboratory method validation
components
1 For further information about the legal status of FCC, see Legal Recognition of
FCC Standards, in the Preface.
General Provisions and Requirements / 1
Front Matter
• Guidance for establishing and using Good Manufacturing
Practices
• Validated testing methods (including enzyme assays and
methods that use highly-characterized reference
standards)
• Minimum standards for identity, purity, and quality of
food ingredients
Food ingredient manufacturers, processors, and
purchasers often use the FCC’s standards as the basis for
establishing minimum requirements for identity, purity, and
quality of their ingredients. FCC standards are also used to
define these parameters within commercial purchase
agreements between buyers and sellers of ingredients and
food and, thus, help to promote food quality and food
safety programs in industry. The validated test methods
included in the FCC can be used to demonstrate the
identity, quality, and purity of food ingredients, or they can
be a starting point in developing new test methods.
Manufacturers, processors, and purchasers of food
ingredients will find these validated test methods useful, as
will regulatory agency labs, contract labs, and students of
chemistry or food science. In addition to being a resource
for purchasing and quality control operations, portions of
the FCC are useful to quality assurance groups and can serve
as references for internal Standard Operating Procedures
(SOPs) and quality manuals used by the food industry. The
FCC is an excellent resource that may be used to provide
important information in order to ascertain identity, quality,
and purity of ingredients. In addition, the FCC can be an
important part of a food manufacturer or purchaser’s
comprehensive food quality program and it provides a
common basis for evaluations of food ingredients in all
aspects of food research and the food industry.
FCC SPECIFICATIONS
FCC specifications are presented in monograph form for
each substance or group of related substances. They are
designed to ensure that food ingredients have the specified
identity and a sufficiently high level of quality to be safe
under usual conditions of intended use in foods or in food
processing. Thus, FCC specifications generally represent
acceptable levels of quality and purity of food-grade
ingredients available in the United States (or in other
countries or instances in which FCC specifications are
recognized).
Manufacturers, vendors, and users of FCC substances are
expected to exercise good manufacturing practices (GMPs)
(see General Information). They are also expected to
establish food safety assurance systems such as Hazard
Analysis and Critical Control Points (HACCP) to ensure that
FCC substances are safe and otherwise suitable for their
intended use. FCC substances must meet applicable
 2 / General Provisions and Requirements
Front Matter
regulatory requirements, including microbiological criteria,
for safety and quality.
The name of the substance on a container label, plus the
designation “Food Chemicals Codex Grade,” “FCC Grade,” or
simply “FCC,” is a representation by the manufacturer,
vendor, or user of the substance that at the time of
shipment, the substance conforms to the specifications in
FCC 8, including any Supplement that is current at that time.
When an FCC substance is available commercially in solution
form as a component of a mixture and there is no provision
in the FCC for such solution or mixture, the manufacturer,
vendor, or user may indicate on the label that the product
contains substances meeting FCC specifications by use of the
initials “FCC” after the name of those components that meet
the FCC specifications. For the labeling of FCC substances in
which added substances are permitted, see Added
Substances.
Added Substances FCC specifications are intended for ap-
plication to individual substances and not to proprietary
blends or other mixtures. Some specifications, however, al-
low “added substances” (i.e., functional secondary ingredi-
ents such as anti-caking agents, antioxidants, diluents, emul-
sifiers, and preservatives) intentionally added when
necessary to ensure the integrity, stability, utility, or func-
tionality of the primary substance in commercial use.
If an FCC monograph allows such additions, each added
substance must meet the following requirements:
(1) it is approved for use in foods by the FDA or by the
responsible government agency in other countries; (2) it is
of appropriate food-grade quality and meets the require-
ments of the FCC, if listed therein; (3) it is used in an
amount not to exceed the minimum required to impart its
intended technical effect or function in the primary sub-
stance; (4) its use will not result in concentrations of con-
taminants exceeding permitted levels in any food as a con-
sequence of the affected FCC primary substance‘s being
used in food; and (5) it does not interfere with the tests and
assays prescribed for determining compliance with the FCC
requirements for the primary substance, unless the mono-
graph for the primary substance has provided for such inter-
ferences. Where added substances are specifically permitted
in an FCC substance, the label shall state the name(s) of any
added substance(s).
Adding substances not specifically provided for and men-
tioned by name or function in the monograph of an FCC
substance will cause the substance to no longer be desig-
nated as an FCC substance. Such a combination is a mixture
to be described by disclosure of its ingredients, including
any that are not FCC substances.
Title of Monograph The titles of FCC monographs are in
most instances the common or usual names. FCC specifica-
tions apply equally to substances bearing the main titles,
synonyms listed under the main titles, and names derived
by transposition of definitive words in main titles. The no-
menclature used for flavoring agents may not be consistent
with other authoritative sources.
FCC 8
Molecular Structures and Chemical Formulas Molecular
structures, chemical formulas, and formula weights immedi-
ately following titles are included for the purpose of infor-
mation and are not to be considered an indication of the
purity of the substance. Molecular formulas given in specifi-
cations, tests, and assays, however, denote the pure chemi-
cal entity.
CAS Number If available, Chemical Abstracts Service
(CAS) registry numbers are included for informational pur-
poses. Additional CAS numbers may be relevant.
INS Numbers If available, numbers adopted by the Co-
dex Alimentarius Commission under the International Num-
bering System for Food Additives are included for informa-
tional purposes.
FEMA Numbers If available, numbers assigned by the Fla-
vor and Extract Manufacturers Association of the United
States (FEMA) are included for informational purposes.
UNII The Unique Ingredient Identifier (UNII) is a nonpro-
prietary, free, unique, unambiguous, nonsemantic, alphanu-
meric identifier based on a substance’s molecular structure
and/or descriptive information issued through the joint FDA/
USP Substance Registration System (SRS) to support health
information technology initiatives for substances in drugs,
biologics, foods, and devices.
Alternative Analytical Procedures Although the tests and
assays described constitute procedures upon which the
specifications of the FCC depend, analysts are not prevented
from applying alternative procedures if supporting data
shows that the procedures used will produce results of equal
or greater accuracy. In the event of the doubt or disagree-
ment concerning a substance purported to comply with the
specifications of the FCC, only the methods described herein
are applicable and authoritative.
Labeling For purpose of compliance with FCC
monographs, “labeling” means all labels and other written,
printed, or graphic matter (1) on any article of any of its
containers or wrappers or (2) accompanying such article, or
otherwise provided by vendors to purchasers for purposes of
product identification.
Sulfiting agents If an FCC substance contains 10 mg/kg
or more of any sulfiting agent, the presence of such sulfiting
agent shall be indicated on the labeling.
Requirements for Listing Substances in the
FCC
The FCC is intended to be an international compendium of
food ingredient standards. The requirements for listing
substances in the FCC are as follows: (1) the substance is
approved for use in food or in food processing in the United
States or in other countries, (2) it is commercially available,
and (3) suitable specifications and analytical test procedures
are available to determine its identity and purity.
GENERAL SPECIFICATIONS AND
STATEMENTS
Certain specifications and statements in the monographs of
the FCC are not amenable to precise description and
accurate determination within narrow limiting ranges.
FCC 8
Because of the subjective or general nature of these
specifications, good judgment, based on experience, must
be used in interpreting and attaching significance to them.
Description Characteristics described and statements made
in the Description section of a monograph are not require-
ments, but are provided as information that may assist with
the overall evaluation of a food ingredient. The section in-
cludes a description of physical characteristics such as color
and form and information on stability under certain condi-
tions of exposure to air and light. It may also include odor
terms that are general descriptors and do not necessarily
indicate the source of the material. Statements in this sec-
tion may also cover approximate indications of properties
such as solubility (see below) in various solvents, pH, melt-
ing point, and boiling point, with numerical values modified
by “about,” “approximately,” “usually,” “~,” and other compa-
rable nonspecific terms.
Function A statement of function is provided to indicate
the technical effect(s) of the substance in foods or in food
processing or a principle application such as “Nutrient”. The
statement is not intended to limit in any way the choice or
use of the substance or to indicate that it has no other
utility. The term “Source of...” is used to describe the func-
tion of materials that may, following ingestion, exhibit a
functional effect on the human body, in a manner similar to
that of some nutrients. These substances are products of an
emerging science, and a comprehensive understanding of
their beneficial effects has yet to be developed. The inclu-
sion of monographs for these materials should not be inter-
preted as implying an endorsement of the claimed potential
health or other benefits.
Odorless This term, when used in describing a flavoring
material, applies to the examination, after exposure to air
for 15 min, of about 25 g of the material that has been
transferred from the original container to an open evaporat-
ing dish of about 100-mL capacity. If the package contains
25 g or less, the entire contents should be examined.
Packaging and Storage Statements in monographs relat-
ing to packaging and storage are advisory in character and are
intended only as general information to emphasize instances
where deterioration may be accelerated under adverse pack-
aging and storage conditions, such as exposure to air, light, or
temperature extremes, or where safety hazards are involved.
Additionally, to reduce the risk of intentional or accidental in-
troduction of undesirable materials into food substances, con-
tainers should be equipped with tamper-
resistant closures.
Cool Place A cool place is one where the temperature is
between 8° and 15° (46° and 59°F). Alternatively, it may be
a refrigerator, unless otherwise specified in the monograph.
Excessive Heat Any temperature above 40° (104°F).
Storage under Nonspecific Conditions Where no specific
storage directions or limitations are provided in the individ-
ual monograph, the conditions of storage and distribution
include protection from moisture, freezing, and excessive
heat. Containers should be stored in secure areas when not
in use to reduce the possibility of tampering.
Containers The container is the device that holds the
substance and that is or may be in direct contact with it.
General Provisions and Requirements / 3
Front Matter
The immediate container is in direct contact with the sub-
stance at all times. The closure is a part of the container.
Closures should be tamper-resistant and tamper-evident.
The container should not interact physically or chemically
with the material that it holds so as to alter its strength,
quality, or purity. The food ingredient contact surface of the
container should comply with relevant regulations promul-
gated under the Federal Food, Drug, and Cosmetic Act (or
with applicable laws and regulations in other countries).
Polyunsaturated fats and oils are particularly susceptible to
oxidation when stored in metal containers, at elevated tem-
peratures, and/or in open containers. Oxidation can be min-
imized by storing them in closed, nonmetal containers with
minimal headspace or flushed with nitrogen gas.
Light-Resistant Container A light-resistant container is de-
signed to prevent deterioration of the contents beyond the
prescribed limits of strength, quality, or purity under the
ordinary or customary conditions of handling, shipments,
storage, and sale. A colorless container may be made light
resistant by enclosing it in an opaque carton or wrapper
(see also Apparatus, below).
Well-Closed Container A well-closed container protects
the contents from extraneous solids and from loss of the
chemical under the ordinary or customary conditions of
handling, shipment, storage, and sale.
Tight Container A tight container protects the contents
from contamination of extraneous liquids, solids, or vapors;
from loss of the chemical; and from efflorescence, deliques-
cence, or evaporation under the ordinary or customary con-
ditions of handling, shipment, storage, and sale, and is ca-
pable of tight reclosure.
Product Security Tamper-evident packaging closures and
security tags should be used. Containers that appear to have
been opened or otherwise altered by unauthorized persons
should not be used until the purity of the substance has
been confirmed.
Solubility Statements included in a monograph under a
heading such as Solubility in Alcohol express exact require-
ments and constitute quality specifications. Statements relat-
ing to solubility given in the Description, however, are in-
tended as information regarding approximate solubilities
only and are not to be considered as exact FCC-quality spec-
ifications. Such statements are considered to be of minor
significance as a means of identification or determination of
purity. For those purposes, dependence must be placed
upon other FCC specifications.
Approximate solubilities given in the Description are indi-
cated by the following descriptive terms:
Parts of Solvent Required for
Descriptive Term 1 part of Solute
Very Soluble less than 1
Freely Soluble from 1 to 10
Soluble from 10 to 30
Sparingly Soluble from 30 to 100
Slightly Soluble from 100 to 1000
Very Slightly Soluble from 1000 to 10,000
Practically Insoluble or Insoluble more than 10,000
 4 / General Provisions and Requirements
Front Matter
Soluble substances, when brought into solution, may
show slight physical impurities, such as fragments of filter
paper, fibers, and dust particles unless excluded by definite
tests or other requirements. Significant amounts of black
specks, metallic chips, glass fragments, or other insoluble
matter are not permitted.
TESTS AND ASSAYS
Every substance in commerce that claims or purports to
conform to FCC, when tested in accordance with its tests
and assays, meets all of the requirements in the FCC
monograph defining it.
The methods and analytical procedures described in the
FCC are designed for use by properly trained personnel in a
suitably equipped laboratory. In common with many
laboratory procedures, test methods in the FCC frequently
involve hazardous materials. In performing the test
procedures and assays in the FCC, safe laboratory practices
must be followed. This includes the use of precautionary
measures, protective equipment, and work practices
consistent with the chemicals and procedures used. Before
undertaking any assay or procedures described in the FCC,
the individual should be aware of the hazards associated
with the chemicals and of the procedures and means of
protecting against them. Material Safety Data Sheets, which
contain precautionary information related to safety and
health concerns, are available from manufacturers and
distributors of chemicals such as USP and should provide
helpful information about the safe use of such chemicals.
Certain chemical reagents specified in FCC test procedures
may be considered to be hazardous or toxic by the
Occupational Safety and Health Administration, by the
Environmental Protection Agency (under provisions of the
Toxic Substances Control Act), or by health authorities in
other countries. Where such reagents are specified, the
analyst is encouraged to investigate the use of suitable
substitute reagents, as appropriate, and to inform the USP
FCC Liaison (fcc@usp.org) of the results so obtained.
Analytical Samples In the description of tests and assays,
the approximate quantity of the analytical sample to be
used is usually indicated. The quantity actually used, how-
ever, should not deviate by more than 10% from the stated
amount. Tests or assays sometimes call for a sample taken to
be “previously dried.” Where a test for Loss on Drying or Loss
on Ignition is included in a monograph, the conditions speci-
fied for these procedures are to be used to dry the sample
prior to performing the test procedure or assay, unless oth-
erwise specified. Often, the results of tests or assays that do
not call for use of a “previously dried” sample are expressed
as calculated on the dried, anhydrous, or ignited basis. In
such cases, a test for Loss on Drying, Water, or Loss on Igni-
tion is included in the monograph and the result of such a
test is used for the calculation on the dried, anhydrous, or
ignited basis, provided that any moisture or other volatile
matter in the undried sample does not interfere with the
specified test procedures and assays.
In editions of the FCC prior to the Seventh edition, the
terms “exactly,” “accurately weighed,” and “accurately meas-
ured” are used in connection with gravimetric or volumetric
FCC 8
measurements and linked directly to a sample weight or vol-
ume. These terms indicate that an operation should be car-
ried out within the limits of error prescribed under Volumet-
ric Apparatus or Weights and Balances, Appendix I. In the
Seventh edition and each subsequent edition, these terms
have been removed from most monographs, to be more
concise. Nonetheless, it shall be understood that all quanti-
tative measurements are to be performed “accurately” and
in conformance with the provisions in Volumetric Apparatus
or Weights and Balances, Appendix I, unless otherwise indi-
cated by qualifiers such as “about” or by the particular na-
ture of the test procedure.
The word “transfer,” when used in describing tests and
assays, means that the procedure should be carried out
quantitatively.
Apparatus With the exception of volumetric flasks and
other exact measuring or weighing devices, directions to
use a definite size or type of container or other laboratory
apparatus are intended only as recommendations, unless
otherwise specified. Where an instrument for physical meas-
urement, such as a thermometer, spectrophotometer, or gas
chromatograph, is designated by its distinctive name or
trade name in a test or assay, a similar instrument of equiva-
lent or greater sensitivity of accuracy may be employed. An
instrument may be substituted for the specified instrument if
the substitute uses the same fundamental principles of oper-
ation and is of equivalent or greater sensitivity and accuracy.
These characteristics must be validated as appropriate.
Where low-actinic or light-resistant containers are specified,
clear glass containers that have been rendered opaque by
application of a suitable coating or wrapping may be used.
Where a particular brand or source of a material, instru-
ment, or piece of equipment, or the name and address of
the manufacturer, or distributor, is mentioned (ordinarily in
a footnote), this identification is furnished solely for informa-
tional purposes as a matter of convenience, without implica-
tion of approval, endorsement, or certification.
Atomic Weights The atomic weights used in computing
formula weights and volumetric and gravimetric factors
stated in tests and assays are those recommended in 1991
by the IUPAC Commission on Isotopic Abundances and
Atomic Weights.
Blank Tests Where a blank determination is specified in a
test or assay, it is to be conducted using the same quantities
of the same reagents and by the same procedure repeated
in every detail except that the substance being tested is
omitted.
A residual blank titration may be stipulated in tests and
assays involving a back titration in which a volume of a
volumetric solution larger than is required to react with the
sample is added, and the excess of this solution is then ti-
trated with a second volumetric solution. Where a residual
blank titration is specified or where the procedure involves
such a titration, a blank is run as directed in the preceding
paragraph. The volume of the titrant consumed in the back
titration is then subtracted from the volume required for the
blank. The difference between the two, equivalent to the
actual volume consumed by the sample, is the corrected
FCC 8
volume of the volumetric solution to be used in calculating
the quantity of the substance being determined.
Centrifuge Where the use of a centrifuge is indicated, un-
less otherwise specified, the directions are predicated on the
use of the apparatus having an effective radius of about 20
cm (8 in) and driven at a speed sufficient to clarify the
supernatant layer within 15 min. If necessary, determine the
gravity by using the equation g = {[(rpm × 2 × π)/60] × rm}/
980, in which rpm is the rotor speed and rm is the mean
radius, in cm, of the tube holding the sample in the rotor.
Desiccators and Desiccants The expression “in a desicca-
tor” means using a tightly closed container of appropriate
design in which a low moisture content can be maintained
by means of a suitable desiccant. Preferred desiccants in-
clude anhydrous calcium sulfate, magnesium perchlorate,
phosphorus pentoxide, and silica gel.
Filtration Where it is directed to “filter,” without further
qualification, the intent is that the liquid be filtered through
suitable filter paper or an equivalent device until the filtrate
is clear.
Identification The tests described under this heading in
monographs are designed for application to substances
taken from labeled containers and are provided only as an
aid to substantiate identification. These tests, regardless of
their specificity, are not necessarily sufficient to establish
proof of identity, but failure of a substance taken from a
labeled container to meet the requirements of a prescribed
identification test means that it does not conform to the
requirements of the monograph.
Indicators The quantity of an indicator solution used
should be 0.2 mL (approximately 3 drops) unless otherwise
directed in a test or assay.
mg/kg and Percent The term “mg/kg” is used in expres-
sing the concentrations of trace amounts of substances,
such as impurities, up to 10 mg/kg. Above 10 mg/kg, per-
cent (by weight) is used. For example, a monograph re-
quirement equivalent to 20 mg/kg is expressed as 0.002%,
or 0.0020%, depending on the number of significant figures
justified by the test specified for use in conjunction with the
requirement.
Microbial Limit Tests The FCC directly references the pro-
cedures in the FDA Bacteriological Analytical Manual (BAM)
(http://www.fda.gov/Food/default.htm) for its microbial
limit tests. Where the sample size is not defined in the limit,
the results are based on the sampling procedures described
in BAM.
Negligible The term “negligible,” as used in some Residue
on Ignition specifications, indicates a quantity not exceeding
0.5 mg.
Pressure Measurements The term “mm Hg” used with re-
spect to pressure within an apparatus, or atmospheric pres-
sure, refers to the use of a suitable manometer or barometer
General Provisions and Requirements / 5
Front Matter
calibrated in terms of the pressure exerted by a column of
mercury of the stated height.
Reagents Specifications for reagents are not included in
the FCC. Unless otherwise specified, reagents required in
tests and assays should conform to the specifications of the
current editions of Reagent Chemicals – American Chemical
Society Specifications or in the section on Reagent Specifica-
tions in the United States Pharmacopeia. Reagents not cov-
ered by any of these specifications should be of a grade
suitable to the proper performance of the method of test or
assay involved.
Acids and Ammonium Hydroxide When ammonium hy-
droxide, glacial acetic acid, hydrochloric acid, hydrofluoric
acid, nitric acid, phosphoric acid, or sulfuric acid is called for
in tests and assays, reagents of ACS grade and strengths are
to be used. (These reagents sometimes are called “concen-
trated,” but this term is not used in the FCC.)
Alcohol, Ethyl Alcohol, Ethanol When one of these sub-
stances is called for in tests and assays, use ACS-grade Ethyl
Alcohol (95%) or USP-grade Alcohol.
Alcohol Absolute, Anhydrous Alcohol, Dehydrated Alcohol
When one of these substances is called for in tests and as-
says, use ACS-grade Ethyl alcohol, Absolute or USP-grade De-
hydrated alcohol.
Water When water is called for in tests and assays or in
the preparation of solutions, it shall have been prepared by
distillation, ion-exchange treatment, or reverse osmosis.
Water, Carbon Dioxide-Free When this type of water is
called for, it shall have been boiled vigorously for 5 min or
more, and allowed to cool while protected from absorption
of carbon dioxide from the atmosphere.
“Deaerated water” or “degassed water” is water that has
been treated to reduce the content of dissolved air by suita-
ble means, such as by boiling vigorously for 5 min and cool-
ing while protected from air or by the application of ultra-
sonic vibration.
Reference Standards Test and assay results are determined
on the basis of comparison of the test sample with a refer-
ence standard that has been freed from or corrected for
volatile residues or water content, as instructed on the refer-
ence standard label. The requirements for any new FCC
standards, tests, or assays for which a new USP or FCC Ref-
erence Standard or Authentic Substance is specified are not
in effect until the specified Reference Standard or Authentic
Substance is available. If a reference standard is required to
be dried before use, transfer a sufficient amount to a clean,
dry vessel. Do not use the original container as the drying
vessel, and do not dry a reference standard repeatedly at
temperatures above 25°. Where the titrimetric determina-
tion of water is required at the time a reference standard is
to be used, proceed as directed in the Karl Fischer Titrimetric
Method under Water Determination, Appendix IIB. Unless a
reference standard label bears a specific potency or content,
assume that the reference standard is 100.0% pure. [Direc-
tions for use printed on the label text of USP and FCC refer-
ence standards are lot-specific, and they take precedence
over any other indication listed in the FCC.]
 6 / General Provisions and Requirements
Front Matter
Significant Figures When tolerance limits are expressed
numerically, the values are significant to the number of dig-
its indicated. Record the observed or calculated analytical
result with only one digit included in the decimal place to
the right of the last place in the limit expression. If this digit
is smaller than 5, eliminate it and leave the preceding digit
unchanged. If this digit is greater than 5, eliminate it and
increase the preceding digit by one. If this digit equals 5,
eliminate it and increase the preceding digit by one. For
example, a requirement of not less than 96.0% would not
be met by a result of 95.94%, but would be met by results
of 95.96% or 95.95%, both of which would be rounded to
96.0%. When a range is stated, the upper and lower limits
are inclusive so that the range consists of the two values
themselves, properly rounded, and all values between them.
Solutions Prepare all solutions, unless otherwise specified,
with water prepared by distillation, ion-exchange treatment,
reverse osmosis, or as otherwise indicated in the mono-
graph. Expressions such as “1:10” or “10%” mean that 1 part
by volume of a liquid or 1 part by weight of a solid is to be
dissolved in a volume of the diluent or solvent sufficient to
make the finished solution 10 parts by volume. Directions
for the preparation of colorimetric solutions (CS), test solu-
tions (TS), and volumetric solutions (VS), are provided in the
section on Solutions and Indicators. Prepare a volumetric so-
lution to have a normality (molarity) within 10% of the
stated value and to be standardized to four significant
figures. When volumetric equivalence factors are provided in
tests and assays, the term “0.X N(M)” is understood to mean
a VS having a normality (molarity) of exactly 0.X000 N(M).
If the normality (molarity) of the VS employed in a particu-
lar procedure differs from 0.X000, apply an appropriate cor-
rection factor.
Specific Gravity Numerical values for specific gravity, un-
less otherwise noted, refer to the ratio of the weight of a
substance in air at 25° to that of an equal volume of water
at the same temperature. Determine specific gravity by any
reliable method, unless otherwise specified.
Temperatures Unless otherwise specified, temperatures are
expressed in Celsius (centigrade) degrees, and all measure-
ments are to be made at 25°, unless otherwise directed.
Time Limits Unless otherwise specified, allow 5 minutes
for a reaction to take place when conducting limit tests for
trace impurities such as chloride or iron. Expressions such as
“exactly 5 min” mean that the stated period should be accu-
rately timed.
Tolerances Minimum purity tolerance limits presented in
monographs neither bar the use of lots of articles that more
nearly approach 100% purity nor constitute a basis for a
claim that such lots exceed the quality prescribed by the
FCC. When no maximum assay tolerance is given, the assay
should show the equivalent of not more than 100.5%.
Trace Impurities Tests for inherent trace impurities are pro-
vided to limit such substances to levels that are consistent
with good manufacturing practice and that are safe and oth-
erwise unobjectionable under conditions in which the food
FCC 8
additive or ingredient is customarily employed. It is impossible
for FCC to provide limits and tests in each monograph for the
detection of all possible unusual or unexpected impurities, the
presence of which would be inconsistent with good manufac-
turing practice. The limits and tests provided in FCC are those
considered to be necessary according to currently recognized
methods of manufacture and are based on information availa-
ble to or provided to the Food Ingredients Expert Committee.
If other methods of manufacture or other than the usual raw
materials are used, or if other possible impurities may be pres-
ent, additional tests may be required and should be applied,
as necessary, by the manufacturer, vendor, or user to demon-
strate that the substance is suitable for its intended applica-
tion. Such tests should be submitted to the USP FCC Liaison
(fcc@usp.org) for consideration for inclusion in the FCC.
Vacuum The unqualified use of the term “in vacuum”
means a pressure at least as low as that obtainable by an
efficient aspirating water pump (not higher than 20 mm
Hg).
Water and Loss on Drying In general, for compounds
containing water of crystallization or adsorbed water, a limit
test, to be determined by the Karl Fischer Titrimetric Method,
is provided under the heading Water. For compounds in
which the Loss on Drying may not necessarily be attributable
to water, a limit test, to be determined by other methods, is
provided under the heading Loss on Drying.
Weighing Practices
Constant Weight A direction that a substance is to be
“dried to constant weight” means that the drying should
continue until two consecutive weighings differ by not more
than 0.5 mg/g of the sample taken, the second weighing to
follow an additional hour of drying. The direction “ignite to
constant weight” means that the ignition should be contin-
ued at 800° ± 25°, unless otherwise specified, until two con-
secutive weighings do not differ by more than 0.5 mg/g of
the sample taken, the second weighing to follow an addi-
tional 15 min of ignition.
Tared Container When a tared container, such as a gloss
filtering crucible, a porcelain crucible, or a platinum dish, is
called for in an analytical procedure, it shall be treated as is
specified in the procedure, e.g., dried or ignited for a speci-
fied time or to constant weight, cooled in a desiccator as
necessary, and weighed accurately.
Weights and Measures, Symbols and Abbreviations The
International System of Units (SI), to the extent possible, is
used in most specifications, tests, and assays in this edition of
FCC. The SI metric units, and other units and abbreviations
commonly employed, are as follows:
° = degrees Celsius
kg = kilogram
g = gram
mg = milligram
µg = microgram
ng = nanogram
pg = picogram
L = liter
mL = milliliter
FCC 8 General Provisions and Requirements / 7

Front Matter
µL = microliter id = inside diameter
m = meter od = outside diameter
cm = centimeter h = hour
dm = decimeter min = minute
mm = millimeter s = second
µm = micrometer (0.001 mm) N = normality
nm = nanometer M = molarity
~ = approximately mM = millimolar
C = coulomb mmol = millimole
A = ampere µM = micromolar
V = volt µmol = micromole
mV = millivolt CFU = colony-forming unit(s)
W = watt ACS = American Chemical Society
dc = direct current AOAC = AOAC International
ft = foot AOCS = American Oil Chemists Society
in = inch ASTM = ASTM (American Society for Testing and
in3 = cubic inch Materials) International
gal = gallon CAS = Chemical Abstracts Service
lb = pound CFR = Code of Federal Regulations (U.S.)
oz = ounce FDA = United States Food and Drug Administration
mEq = milliequivalents FEMA = Flavor and Extract Manufacturers Association
mg/kg = parts per million (by weight) of the United States
µg/kg = parts per billion (by weight) INS = International Numbering System of the Codex
ng/kg = parts per trillion (by weight) Alimentarius
psi = pounds per square inch IUPAC = International Union of Pure and Applied
psia = pounds per square inch absolute Chemistry
kPa = kilopascal NIST = National Institute of Standards and Technology
sp. gr. = specific gravity UNII = Unique Ingredient Identifier (as defined by US
b.p. = boiling point FDA)
m.p. = melting point