Analytical Chemistry 602037-Chapter 10 PDF

10/23/2019
TON DUC THANG UNIVERSITY

Faculty of Applied Sciences
Subject: ANALYTICAL CHEMISTRY

Code: 602037
Lecturer: PhD. HOANG THI THAI THANH
4/2/2019 602037 – Chapter 10: Chromatography 1
Chapter 10: Chromatography
1. Introduction to Chromatography
2. Classification
3. High performance liquid chromatography
4. Gas chromatography
5. Thin layer chromatography
6. Capillary electrophorosis
1
10/23/2019

• Chromatography is a technique in which the components of a

mixture are separated based on differences in the rates at which
they are carried through a fixed or stationary phase by a gaseous
or liquid mobile phase.
• Chromatography is a widely used method for the separation,

identification, and determination of the chemical components in
complex mixtures.

An eluent is a solvent used to

carry
the components of a mixture
through a stationary phase.
The mobile phase that exits the
column is termed the eluate.
The stationary phase in
chromatography is a phase that is
fixed
in place either in a column or on a
planar surface.
A chromatogram is a plot of some

function of solute concentration
versus elution time or elution
volume.
2
10/23/2019

2. Classification
• In column chromatography, the stationary phase is held in a

narrow tube, and the mobile phase is forced through the tube
under pressure or by gravity.
• In planar chromatography, the stationary phase is supported

on a flat plate or in the pores of a paper, and the mobile phase
moves through the stationary phase by capillary action or under
the influence of gravity.

2. Classification
3
10/23/2019

2. Classification
• The distribution constant for a solute in chromatography is

equal to the ratio of its molar concentration in the stationary phase
to its molar concentration in the mobile phase.
[A] in the stationary phase
[A] in the mobile phase

2. Classification
The dead time (void time), tM, is the time it takes for an unretained
species to pass through a chromatographic column.
Separations are based on the different times, tS, that components spend in
the stationary phase.
The retention time tR
4
10/23/2019

2. Classification
The average linear rate of solute migration:
The average linear velocity of the mobile phase molecules:
L is the length of the column packing
Migration Rates and Distribution Constants
The Retention Factor, k
Ideally, the retention factors

for analytes in a sample are
between 1 and 5.
5
10/23/2019
The selectivity factor, a, for solutes A and B is defined as the ratio of the
distribution constant of the more strongly retained solute (B) to the
distribution constant for the less strongly held solute (A).
Plate count or number of theoretical plates (N).
L: the length (usually in centimeters) of the

column packing
Plate height (H)
The efficiency of chromatographic columns

increases as the plate count N becomes greater
and as the plate height H becomes smaller.
6
10/23/2019

Gas Chromatography
7
10/23/2019

Gas Chromatography
Sample Injection System

For ordinary packed analytical columns, sample sizes range from a
few tenths of a microliter to 20 μL.
Capillary columns require samples that are smaller by a factor of
100 or more. For these columns, a sample splitter is often needed to
deliver a small known fraction (1:100 to 1:500) of the injected
sample, with the remainder going to waste.
Commercial gas chromatographs intended for use with capillary
columns incorporate such splitters, and they also allow for splitless
injection when packed columns are used.

Gas Chromatography
Column Configurations and Column Ovens

Length: 2 m to 60 m or more
Packed columns:
Capillary columns:
Temperature programming: increasing the column temperature
continuously or in steps during elution.
8
10/23/2019

Gas Chromatography
Chromatographic Detectors
9
10/23/2019

High-Performance Liquid Chromatography
• High-performance liquid chromatography, HPLC, is a type of

chromatography that combines a liquid mobile phase and a very
finely divided stationary phase. In order to obtain satisfactory flow
rates, the liquid must be pressurized to several hundred or more
pounds per square inch.
• The types of highperformance liquid chromatography are often

classified by the separation mechanism or by the type
of stationary phase. These include (1) partition, or liquid-liquid,
chromatography; (2) adsorption, or liquid-solid,
chromatography; (3) ion-exchange, or ion, chromatography; (4)
size-exclusion chromatography; (5) affinity chromatography;
and (6) chiral chromatography.

Applications of liquid
chromatography. Methods can be
chosen based on solubility and
molecular mass. In many cases, for
small molecules, reversed-phase
methods are appropriate. Techniques
toward the bottom of the diagram
are
best suited for high molecular mass
10
10/23/2019


• An isocratic elution in HPLC is one in which the solvent

composition remains constant.
• A gradient elution in HPLC is one in which the composition of

the solvent is changed continuously or in a series of steps.
• A guard column between the injector and the column removes
particulates and other solvent impurities.
11
10/23/2019


12
10/23/2019

• In normal-phase partition chromatography, the stationary

phase is polar and the mobile phase nonpolar.
• In normal-phase chromatography, the least polar analyte is eluted
first.
• In reversed-phase partition chromatography, the polarity of
these phases is reversed.
• In reversed-phase chromatography, the least polar analyte is eluted
last.
• Ion-pair chromatography is a subset of reversed-phase
chromatography in which easily ionizable species are separated on
reversed-phase columns.

• ChoiceofMobileandStationaryPhases
The order of polarities of common mobile phase
solvents are water > acetonitrile > methanol > ethanol >
tetrahydrofuran > propanol > cyclohexane > hexane.
13
10/23/2019


14
10/23/2019

High-Performance Liquid Chromatography: Ion
chromatography
• The conductivity detector is well suited for ion

chromatography.

High-Performance Liquid Chromatography: Ion
chromatography
What is exchanged?
Anion for anion and cation for cation
How are the ions exchanged?

Ions with higher affinity displace ions
of lower affinity to the stat. phase
Affinity increases as:

1. Charge increases
2. Size of (solvatized) ions decreases
3. Polarizability increases
30
15
10/23/2019

Stationary Phases in Ion-Exchange Chromatography
"Resins" or "Gels" that carry the ion-ex
changer surface.
Both are amorphous particles of organi
c material, but gels are softer.
Polystyrene resins:
made by co-polymerization of styrene
and vinyl-bearing molecules
Cellulose and dextran gels:

Dextran, cross-linked to glycerin, is cal
led (SephadexTM)
Polyacrylamide gel
31

Stationary Phases in Ion-Exchange Chromatography
• The benzene ring of the support can be modified to produce cation exchange
resin containing sulfonate group (SO3-) or an anion exchange resin containing
ammonium groups (NR3+). Ion exchangers are classified to strongly or weakly
acidic or basic. SO3- is strong because it remains ionized even in strong acidic
solutions. CO2- is weak because it becomes protonated at pH 4 and thus looses
its ion exchange capacity. Strongly basic quaternary ammonium salts remain
cationic at all pH values. Weakly basic tertiary anion exchangers are
deprotonated in moderately basic solutions and lose their ability to bind anions.
• The resin becomes more rigid and porous as cross linking increases. Lightly
cross linked resins permit rapid equilibration of solute between the inside and
outside of the particle, however they swell in water which decreases the density
of ion exchange sites and selectivity of the resin to different ions.
• Cellulose and dextran which are polymers of glucose possess larger pore size
and lower charge densities than polystyrene resins. They are well suited to
large molecules like proteins which may be irreversibly bound to resins because
they are highly charged.
32
16
10/23/2019
Examples of Ion-Exchange Resins
33

Size (or Molecular) Exclusion
Separation of mixtures according to

different molecule sizes of the
components: small molecules can
intrude into pores of the stationary phase
easily and are retained. But less easily
large molecules which are too big to
enter pores and are retained least.
Separation of biomolecules in aqueous

systems is called gel filtration
chromatography (GFC).
Separation of organic polymers in non-aqueous systems is called gel

permeation chromatography (GPC).
34
17
10/23/2019

Thin layer chromatography (TLC)
18
10/23/2019

Thin layer chromatography can be used to:

• Monitor the progress of a reaction
• Identify compounds present in a given substance
• Determine the purity of a substance

Stationery phase Description Application
Silica gel G Silica gel with average particle size Used in wide range
15µm containing ca 13% calcium pharmacopoeial test
sulfate binding agent
Silica gel G254 Silica gel G with fluorescence added Same application with Silica
gel G where visualization is
to be carried out under UV
light.
Cellulose Cellulose powder of less than 30µm Identification of

particle size. tetracyclines
19
10/23/2019

SOLVENT POLARITY INDEX
Heksana 0
Butanol 3.9
Chloroform 4.1
Methanol 5.1
Ethanol 5.1
Acetonitrile 5.8
Air 9.0

20
10/23/2019

Thin layer chromatography (TLC): DETECTION OF SPOT
1) Iodination-put the plate in which the spots face to the iodine

crystal and see what is the spot color changing
2) Ninhydrin:
-spesific identification of amino acid compounds.
- Ninhydrin solution will show a purple spot when it is
sprayed to the amino acid spot.
3) KMnO4
used to identify a reducing agent such as glucose, fructose,
vitamin C and others.
4) Alkaline tetrazolium blue
specificaly used for corticosteroid identification

Capillary electrophoresis
• Capillary electrophoresis is a separation technique

in which charged species are separated, based on
charge and size, by their different rates of migration
in an electric field. The capillary is made of
negatively charged fused silica inner wall which
forms an electrical double layer with cations in the
running buffer. These cations in the electrical
double layer move towards the negative cathode and
hence the overall direction of flow also known as
the electroosmotic movement is towards the cathode.
21
10/23/2019

Capillary electrophoresis
22

Analytical Chemistry 602037-Chapter 10 PDF

Încărcat de

Informații document

Titlu original

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

Analytical Chemistry 602037-Chapter 10 PDF

Încărcat de

Drepturi de autor:

Formate disponibile

10/23/2019

TON DUC THANG UNIVERSITY

Subject: ANALYTICAL CHEMISTRY

Lecturer: PhD. HOANG THI THAI THANH

4/2/2019 602037 – Chapter 10: Chromatography 1

Chapter 10: Chromatography

4/2/2019 602037 – Chapter 10: Chromatography 2

Chapter 10: Chromatography

• Chromatography is a technique in which the components of a

• Chromatography is a widely used method for the separation,

4/2/2019 602037 – Chapter 10: Chromatography 3

Chapter 10: Chromatography

An eluent is a solvent used to

A chromatogram is a plot of some

Chapter 10: Chromatography

• In column chromatography, the stationary phase is held in a

• In planar chromatography, the stationary phase is supported

4/2/2019 602037 – Chapter 10: Chromatography 5

Chapter 10: Chromatography

4/2/2019 602037 – Chapter 10: Chromatography 6

Chapter 10: Chromatography

• The distribution constant for a solute in chromatography is

[A] in the stationary phase

[A] in the mobile phase

4/2/2019 602037 – Chapter 10: Chromatography 7

Chapter 10: Chromatography

The retention time tR

4/2/2019 602037 – Chapter 10: Chromatography 8

Chapter 10: Chromatography

The average linear rate of solute migration:

The average linear velocity of the mobile phase molecules:

L is the length of the column packing

4/2/2019 602037 – Chapter 10: Chromatography 9

Chapter 10: Chromatography

Migration Rates and Distribution Constants

The Retention Factor, k

Ideally, the retention factors

4/2/2019 602037 – Chapter 10: Chromatography 10

Chapter 10: Chromatography

4/2/2019 602037 – Chapter 10: Chromatography 11

Chapter 10: Chromatography

Plate count or number of theoretical plates (N).

L: the length (usually in centimeters) of the

Plate height (H)

The efficiency of chromatographic columns

4/2/2019 602037 – Chapter 10: Chromatography 12

Chapter 10: Chromatography

4/2/2019 602037 – Chapter 10: Chromatography 13

Chapter 10: Chromatography

4/2/2019 602037 – Chapter 10: Chromatography 14

Chapter 10: Chromatography

Sample Injection System

4/2/2019 602037 – Chapter 10: Chromatography 15

Chapter 10: Chromatography

Column Configurations and Column Ovens

4/2/2019 602037 – Chapter 10: Chromatography 16

Chapter 10: Chromatography

4/2/2019 602037 – Chapter 10: Chromatography 17

Chapter 10: Chromatography

4/2/2019 602037 – Chapter 10: Chromatography 18

Chapter 10: Chromatography

• High-performance liquid chromatography, HPLC, is a type of

• The types of highperformance liquid chromatography are often