HPLC Theory

Practical High Performance
Liquid Chromatography
Course Number H5930A
Student Manual
Mass Spectrometry Data Systems
Capillary
Electrophoresis
s Gas Chromatography
Practical High Performance
H5930A
Student Manual
Manual Part Number H5930A-90000

Printed in January, 2001
Notice
The information contained in this document is subject to change without notice.
Agilent Technologies makes no warranty of any kind with regard to this material,
including but not limited to the implied warranties of merchantability and fitness
for a particular purpose.
Agilent Technologies shall not be liable for errors contained herein or for
incidental, or consequential damages in connection with the furnishing,
performance, or use of this material.
No part of this document may be photocopied or reproduced, or translated to
another program language without the prior written consent of Agilent
Technologies, Inc.
Agilent Technologies, Inc

11575 Great Oaks Way
Suite 100, MS 304B
Alpharetta, GA 30319
 2000 by Agilent Technologies, Inc.

All rights reserved
Printed in the United States of America
ii
Table Of Contents
INTRODUCTION TO HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ..............1

IN THIS SECTION YOU WILL LEARN: ............................................................................................2
HISTORICAL ASPECTS ...................................................................................................................3
SEPARATION PROCESS...................................................................................................................4
TYPICAL COLUMN PACKING SUPPORT ..........................................................................................5
TYPICAL COLUMN SUPPORTS – STYRENE DVB ............................................................................6
MODES OF LIQUID CHROMATOGRAPHY ........................................................................................7
DIFFERENCES IN GAS AND LIQUID CHROMATOGRAPHY ................................................................8
INSTRUMENTATION .......................................................................................................................9
CHROMATOGRAPHIC PARAMETERS .............................................................................................10
SEPARATION PARAMETERS .........................................................................................................11
REVIEW QUESTIONS ....................................................................................................................12
THE SEPARATION PROCESS..................................................................................................15
IN THIS SECTION YOU WILL LEARN: ..........................................................................................16
THE GOAL OF SEPARATION .........................................................................................................17
TOOLS FOR ACHIEVING A SEPARATION .......................................................................................18
RESOLUTION VALUES .................................................................................................................19
CALCULATE A RESOLUTION VALUE ............................................................................................20
FACTORS INFLUENCING RESOLUTION .........................................................................................21
RESOLUTION FACTORS ................................................................................................................22
SEPARATION SELECTIVITY ..........................................................................................................23
PARAMETERS WHICH WILL AFFECT SELECTIVITY ......................................................................24
COLUMN SELECTION ...................................................................................................................25
EFFECT OF TEMPERATURE ON SEPARATION ................................................................................26
CAPACITY FACTOR K’ .................................................................................................................27
CAPACITY FACTOR......................................................................................................................28
USEFUL SOLVENTS FOR REVERSED-PHASE CHROMATOGRAPHY .................................................29
SOLVENT STRENGTH: NORMAL PHASE ......................................................................................30
NORMAL PHASE: SOLVENT STRENGTH ......................................................................................31
EFFICIENCY .................................................................................................................................32
CALCULATING EFFICIENCY .........................................................................................................33
DISPERSION: EDDY DIFFUSION ..................................................................................................34
DISPERSION: LONGITUDINAL DIFFUSION ...................................................................................35
DISPERSION: MASS TRANSFER ...................................................................................................36
DISPERSION .................................................................................................................................37
TYPICAL FLOW RATES ................................................................................................................38
SMALL BORE AND MICROBORE COLUMNS ..................................................................................39
FAST OR HIGH SPEED COLUMNS .................................................................................................40
EXTRA-COLUMN BAND BROADENING ........................................................................................41
INCREASING RESOLUTION ...........................................................................................................42
PEAK SYMMETRY ........................................................................................................................43
WORKSHEETS ..............................................................................................................................44
MOBILE PHASE COMPOSITION – THE GENERAL ELUTION PROBLEM ...........................................46
MOBILE PHASE COMPOSITION – GRADIENT ELUTION .................................................................47
GRADIENT DEVELOPMENT ..........................................................................................................48
WORKSHEETS ..............................................................................................................................49
iii
PRACTICAL CONSIDERATIONS FOR GRADIENT ELUTION .............................................................52
PRACTICAL ASPECTS OF PERFORMING ANALYSES.....................................................53
IN THIS SECTION YOU WILL LEARN: ..........................................................................................54
MOBILE PHASE PREPARATION FILTRATION.................................................................................55
MOBILE PHASE DEGASSING ........................................................................................................56
VACUUM DEGASSING ..................................................................................................................57
SOLVENT MISCIBILITY ................................................................................................................58
MOBILE PHASE UV CUT-OFF .....................................................................................................59
COLUMN CARE ............................................................................................................................60
PRE-COLUMNS AND GUARD COLUMNS .......................................................................................61
SYRINGE WASH: HP 1090..........................................................................................................62
PRIMING ......................................................................................................................................63
COLUMN EQUILIBRATION ...........................................................................................................64
PREPARING SAMPLES: FILTERING ..............................................................................................65
PREPARING SAMPLES ..................................................................................................................66
WORKSHEET ...............................................................................................................................67
HPLC INSTRUMENTATION.....................................................................................................69
IN THIS SECTION, YOU WILL LEARN: .........................................................................................70
HPLC TUBING ............................................................................................................................71
FITTINGS .....................................................................................................................................72
FILTERS ............................................................................................................................... ........74
FUNCTIONS OF THE SDS..............................................................................................................75
MULTICHANNEL GRADIENT VALVE ............................................................................................76
DUAL PISTON PARALLEL PUMP...................................................................................................77
DUAL PISTON SERIES PUMP ........................................................................................................78
BALL VALVES .............................................................................................................................79
METERING PUMP SEALS AND PISTONS ........................................................................................80
DIAPHRAGM PUMP ......................................................................................................................81
SIEVES AND FILTERS ...................................................................................................................82
DAMPING UNIT ...........................................................................................................................83
1090 SDS....................................................................................................................................84
QUATERNARY PUMP ...................................................................................................................85
MANUAL INJECTION ....................................................................................................................86
AUTO-INJECTION SYSTEM ...........................................................................................................87
ROTOR SEALS .............................................................................................................................88
NECESSITY FOR MORE THAN ONE DETECTOR .............................................................................89
UV-VIS DETECTORS...................................................................................................................92
FLUORESCENCE DETECTION........................................................................................................98
REFRACTIVE INDEX DETECTION................................................................................................101
LIGHT SCATTERING DETECTION................................................................................................102
ELECTROCHEMICAL DETECTION ...............................................................................................103
CONDUCTIVITY DETECTION ......................................................................................................104
HPLC-MS.................................................................................................................................105
RADIOMETRIC DETECTORS .......................................................................................................107
WORKSHEET .............................................................................................................................108
HPLC TROUBLESHOOTING .................................................................................................111
IN THIS SECTION YOU WILL LEARN: ........................................................................................112
RECORD KEEPING .....................................................................................................................113
PROPER CARE OF THE HPLC.....................................................................................................114
PEAK RETENTION TIME AND PRECISION....................................................................................115
COMMON PUMP PROBLEMS .......................................................................................................116
PRESSURE PROBLEMS................................................................................................................117
BASELINE FLUCTUATIONS .........................................................................................................118
iv
NOISY BASELINE .......................................................................................................................119
MIXING PROBLEMS ...................................................................................................................120
MANUAL INJECTION VALVE ......................................................................................................121
AUTO-INJECTORS ......................................................................................................................122
GOOD COLUMN PRACTICES.......................................................................................................123
COLUMN FRIT REPLACEMENT ...................................................................................................124
COLUMN REGENERATION..........................................................................................................125
DETECTOR PERFORMANCE ........................................................................................................126
DETECTOR TIME CONSTANT .....................................................................................................127
DETECTOR HEAT EXCHANGERS ................................................................................................128
NOISY BASELINES .....................................................................................................................129
DRIFTING BASELINES ................................................................................................................130
GHOST PEAKS ...........................................................................................................................131
EXTRA-COLUMN DISPERSION ...................................................................................................132
PEAK SHAPE ..............................................................................................................................133
WORKSHEET .............................................................................................................................138
v
vi
Introduction to High Performance Liquid
Chromatography
Introduction to High Performance Liquid Chromatography
In This Section You Will Learn:
In This Section, You Will Learn
• The Historic Progression of Liquid Chromatography

• About the Separation Process
• Modes of HPLC
• The Basics of the HPLC Instrumentation
• About the Chromatogram
In this section you will learn how liquid chromatography has progressed
throughout this century. You will also learn the basic separation mechanism and
the main modes of high performance liquid chromatography. The layout of a
modern liquid chromatograph will be presented and the qualitative and
quantitative aspects of the chromatogram discussed.
2
Historical Aspects
Historical Aspects
Historical Aspects HC
3 CH
2
CH
3
CH
N N 3
• 1906 - Mikhail Semenovich Tswett Mg

(1872-1919)Calcium Carbonate,
Petroleum Ether N N
• 1940’s - Partition and Paper CH

3
Chromatography
• 1950’s - Gas, Thin-Layer, Gel Filtration
and Gradient Elution Chromatography 0
0
CH
3
•
0
1960’s - Introduction of Commercial 0 0
HPLC
CH CH CH
3 3 3
M.S. Tswett. Ber. Dtsch. Bot. Ges. 24: 384-393 (1906)
• Modern separation science began at the turn of the century with M.

Tswett’s separation of plant chlorophylls on a calcium carbonate
stationary phase with petroleum ether as the mobile phase. An apparatus
similar to the one above was used in the separation.
• In the 1940’s, Martin and Synge introduced the concept of partition
chromatography.
• During the 1950’s, gradient elution was introduced by Tiselius and the
theory of separation was described by Van Deemter.
• Finally, in the 1960’s, the first commercial liquid chromatographs were
introduced.
3
Separation Process
Separation Process
Separation Process
Stationary Phase
Mobile Phase
Chromatography is a separation process in which the components to be separated

are distributed between two phases, a stationary phase and a mobile phase.
Components of the sample mixture separate when they have differential migration
in the column. Differential migration depends on the equilibrium distribution of
the sample components between the stationary and mobile phase. Compounds
whose molecules are found to reside most of the time in the mobile phase will
elute first. Compounds whose molecules spend most of their time in the
stationary phase will move through the column more slowly and elute at later
retention times.
4
Typical Column Packing Support

OH
OH OH
O Si O OH
Si Si Si Si
O O O O
Silica Gel
Surface
Chemically Modified
Silica Gel
Pores CH
Si 3
Si
Si - O - Si - (CH2 )17 CH 3
CH
Si 3
Si
Si
Silica gel is commonly used as a stationary phase in adsorption chromatography

(normal-phase) and is the support for numerous chemically bonded stationary
phases. The surface of silica gel is covered with silanol groups which can interact
with molecules or serve as a reaction site for chemical bonding. Common bonded
phases include octadecysilyl (C-18), cyano, amino, C-8, C-4 or C-2.
5
Typical Column Supports – Styrene DVB
Typical Column Supports – Styrene DVB
Typical Column Supports - Styrene DVB
CH = CH 2 CH = CH 2 - CH 2- CH - CH2 - CH - CH2 - CH - CH2 -
CH = CH 2 - CH 2- CH - CH2 - CH - CH2 - CH - CH2 -

Styrene Divinyl Benzene
• Support for:
• Reversed and Normal Phases
• Ion Exchangers
• Size-Exclusion Chromatography
Cross-linked polystyrene is made from the copolymerization of styrene and

divinylbenzene. Polymer stationary phases such as styrene divinylbenzene are
stable in the pH range from 1-13 and can often withstand higher temperatures
than silica gel. They are most often used as supports for ion exchange columns or
size exclusion columns.
6
Modes of Liquid Chromatography
Types of Compounds Mode Stationary Phase Mobile Phase

Separated
Neutrals Reversed Phase C-18, C-8, C-4, C-2 Water/Organic

Weak Acids Modifiers
Weak Bases
Ionics, Bases, Acids Ion Pair C-18, C-8 Water/Organic
Ion Pair Reagent
Compounds insoluble Normal Phase Silica, Amino, Cyano Organics

in water, Organic Diol
isomers
Ionics Ion Exchange Anion or Cation Aqueous/Buffer
Inorganic Ions Exchange Resin Counter Ion
High MW Compounds Size Exclusion Polystyrene Silica Gel Filtration-

Polymers Aqueous
Gel Permeation-
Organic
The five major HPLC separation techniques are shown in the chart above. The
most widely used mode is reversed-phase. This technique has a wide application
range including neutrals, weak acids, weak bases, and ionics when used in
conjunction with an ion-pairing reagent. Normal- phase liquid chromatography
traditionally involved the use of bare silica or alumina columns and was known as
adsorption chromatography. Today bonded polar stationary phases are also
available. Ion-exchange is exclusively used for the separation of ions in solution.
Size exclusion separates molecules of high molecular weight based upon their
size.
7
Differences in Gas and Liquid Chromatography
LC
Only about 20% of known organic

compounds can be analyzed by GC.
GC
LC
GC
Polarity
0 2 4 6 8
10 10 10 10 10
Solute Molecular Weight
Gas Chromatography Liquid Chromatography
8
Instrumentation
Instrumentation
Instrumentation
Injector
Mixer
Pumps
Chromatogram
Column
Detector
Solvents
The components of a high performance liquid chromatograph include: solvent

reservoirs, a pumping system to provide accurate compositions, flows and the
pressure necessary to push the mobile phase through the tightly packed column, a
sample delivery mechanism which will not interrupt the flow of mobile phase, a
column where the separation takes place, and the detector to sense the presence of
individual sample components.
9
Chromatographic Parameters
t
R(B)
t R(A)
t = retention time
R
t 0= elution time of an unretained component
W = peak width at base

t
0
A B
Inject
Detector Response
W W
A B
Time
10
Sample components typically produce gaussian shaped peaks. Components

which are not retained by the stationary phase are said to elute at t0. Those
sample components that have some attraction for the stationary phase elute at later
retention times. Retention times provide the qualitative aspect of the
chromatogram. The chromatographic peak height or peak area may be related to
the quantity of analyte in the mixture when compared to standards of known
concentration.
10
Separation Parameters
• Column Stationary Phase Selection

• Column Length and Diameter
• Mobile Phase Composition
• Temperature
• Flow Rate
• Sample Size
11
The resolution of chromatographic peaks can be controlled by the selection of

proper separation parameters. Based upon the sample components’ molecular
structure, a column stationary phase is chosen. The length chosen may depend on
the difficulty of the separation. A mobile phase composition compatible with
both the samples and the stationary phase is selected and optimized to produce the
best separation possible. Mobile phase composition is the primary parameter used
to optimize an HPLC separation. Mobile phase selection may also depend upon
the detector parameters. The column temperature and flow rate are used as
secondary adjustments, fine tuning the chromatogram. The sample size is also an
important parameter as large injected masses or volumes can lead to loss of
resolution and degradation of peak shape.
11
Review Questions
Review Questions
Review Questions 1A
1. Name three differences in gas and liquid chromatography.
2. What is the most widely used mode of HPLC?
3. What mode may be used for separation of ions in solution?
12
12
Review Questions
Review Questions 1B
1. Name the parts of an HPLC instrument.
2. What is the symbol for an unretained component’s elution

time?
3. What is the difference between normal and reversed phase?
13
13
Review Questions
14
The Separation Process
• What factors influence the resolution between sample

components.
• How the capacity factor, selectivity and efficiency influence
resolution.
• How liquid chromatographic operating parameters affect each
resolution factor.
• When gradient elution can improve the separation.
In this section, the factors influencing the resolution of sample components will
be discussed. Included in this discussion are the capacity factor, selectivity and
efficiency. The relationship between the operating parameters such as mobile
phase composition, temperature and flow rate to resolution will also be discussed.
Finally, gradient elution will be explored.
16
The Goal of Separation
The Goal of Separation
The Goal of Separation: Resolution Between

Sample Components
t RA
A B
t RB
R - resolution
t - retention time of component B
t - retention time of component A
w - width at base of peak
w - width at half-height
0
t RB - t RA t RB - t RA
R=2 R=1.176
W A+ WB W 1/2A + W 1/2B
The most important goal of the chromatographer is to achieve adequate resolution

between all peaks in the chromatogram in a reasonable amount of time. A
quantitative measure of resolution between two adjacent chromatographic peaks
has been developed and appears above. The first equation describes the
resolution based upon the width at the base of each peak. The second equation
describes the resolution based upon the width at half-height.
17
Tools for Achieving a Separation
• Column Selection
– stationary phase, particle size, etc...
• Column Length
• Mobile phase composition
• Column Temperature
• Flow Rate
The experimental variables available to the liquid chromatographer to achieve

resolution between sample components include column selection, column length,
mobile phase composition, column temperature and flow rate. Column selection
includes choice of appropriate stationary phase, particle diameter, particle shape,
column diameter and column length. Mobile phase composition has the most
profound effect upon the spacing of chromatographic peaks and this is where
most development effort is usually focused. Column temperature and flow rate
are of secondary importance and are utilized to fine tune the separation.
18
Resolution Values
Resolution Values
Resolution Values
0.4
0.5
0.6 0.7
0.8 1.00 1.25
For equal peak areas, R of 1.5

gives baseline separation
The graphic above illustrates the resolution values for overlapping

chromatographic peaks. When two chromatographic peaks are baseline resolved,
the resolution value is 1.5. Quantification will not be precise when two adjacent
chromatographic peaks with resolution values of 1.25 or less are involved.
During method development, the analyst may wish to achieve a minimum
resolution value of 2 in order to insure robust method performance as the column
degrades or in case of minor alterations in experimental conditions.
19
Calculate a Resolution Value

t 1 t t 4
2
t
3
t 0
Width at
Ret Time half-height
t 1 0.913 .048
t .053
2 1.072
Calculate the resolution between the first two chromatographic

peaks.
Answer
20
Factors Influencing Resolution
x x k’
R = 1/4 N -1
1 + k’
Efficiency Selectivity Capacity
N: Total number of theoretical plates available; column efficiency.

k: Capacity factor, the peak retention function.
a: the relative separation of the peaks; the selectivity function.
The degree of resolution between two chromatographic peaks is dependent upon

three factors. The first term, efficiency can be varied with flow rate and column
length. This term reflects how much dispersion takes place within a
chromatographic peak. The second term, selectivity, illustrates how well the
chromatographic system chosen can distinguish between sample components.
Selectivity is dependent upon stationary phase selection, mobile phase selection
and column temperature, among others. The final term is related to the capacity
factor and is primarily influenced by mobile phase composition. The discussion
will elaborate on each of these factors.
21
Resolution Factors
Resolution Factors
Resolution Factors
Capacity Selectivity
Efficiency
Capacity, selectivity and efficiency are illustrated graphically in the

chromatogram presented above.
22
Separation Selectivity
x x k’
R = 1/4 N -1
1 + k’
To change selectivity:
Change Mobile Phase Composition.
Change to Different Mobile Phase.
Change Mobile Phase pH.
k’ B
Change Column Temperature.
= Use Special Chemical Effects.
k’ A
Change Stationary Phase.
A B A B
= 1.04 = 1.22
1 2 1 2
The separation selectivity is quantitatively given by which is simply a measure of

the spacing between the apex of two chromatographic peaks. A selectivity value
of 1 indicates that no separation took place and the k’ values are identical.
Increasing selectivity is a very useful way to increase the resolution, as it does not
necessarily involve a concomitant increase in analysis time. Increasing the
selectivity, however, can be more difficult as it usually involves a change in the
actual mobile phases used, column selected or the addition of modifiers to the
mobile phase.
23
Parameters Which Will Affect Selectivity
Change mobile phase

Stronger Weaker
composition Solvents Solvents
IPA/Water ACN/Water MeOH/Water
DECREASING INCREASING
Change stationary phase

Not
Easily Easily
Overloaded Overloaded C-18
C-2
Low Organic High Organic

Stationary Phase Stationary Phase
10
Notice that changing the actual mobile phase constituents can be a powerful way
to change the chromatographic selectivity. Notice that different mobile phase
compositions will actually cause different spacing between the apex of the
chromatographic peaks. Probably, the most significant changes in selectivity can
be realized with a change in stationary phase. The example given illustrates
possible selectivity differences between a C-8 column and a C-18 column.
Although both columns are essentially a straight chain hydrocarbon bonded to the
surface of the silica gel, one will usually find greater selectivity with the greater
carbon content.
24
Column Selection
Column Selection
Column Selection
Types of Mode Stationary Mobile Phase
Compounds Phase
Separated
Neutrals Reversed-Phase C-18, C-8, C-4, Water/Organic Phase
Weak Acids C-2
Weak Bases
Ionics, Bases, Ion-Pair C-18, C-8 Water/Organic Ion-Pair
Acids Reagent
Compounds Normal -Phase Silica, Amino, Organics

Insoluble in Cyano Diol
Water, Organic
Isomers
Ionics Ion Exchange Anion or Cation Aqueous/Buffer
Inorganic Ions Exchange Counter Ion
Resin
High MW Size Exclusion Silica Gel Filtration-Aqueous

Compounds Styrene- Gel Permeation-Organic
Polymers Divinylbenzene
11
The most significant way to change the selectivity is to change the stationary
phase. Listed above are the five most frequently used modes of liquid
chromatography. The most widely used mode is reversed-phase. An off-shoot of
reversed-phase liquid chromatography is ion-pair chromatography for the
separation of strong acids and bases. Normal-phase liquid chromatography
traditionally involved the use of silica columns, but now popular bonded phases
such as cyano, amino, and diol are also available. Ion exchange chromatography
is utilized solely for the separation of ions. Size exclusion separates compounds
based upon their size such as polymers and biomolecules. Selectivity may be
improved by simply changing the type of column used, but staying within the
same mode of liquid chromatography. An example would be using a cyano
column in reversed-phase as opposed to a C-18.
25
Effect of Temperature on Separation
40º C
AB
65º C
B
A
0 5 10
Time in
Minutes
12
Column selectivity may also be altered with a change in column temperature

although this experimental variable does not produce the dynamic changes
associated with mobile or stationary phase changes. Raising the column oven
temperature will increase the efficiency and decrease the retention time of solutes.
Occasionally, as in the example presented, peak elution order may change as the
result of a change in temperature. In addition to separation improvements, the use
of a column oven will produce better retention time precision.
26
Capacity Factor k’
x x k’
R = 1/4 N -1
1 + k’
Capacity factor is characteristic of a specific compound

at a given mobile phase composition, temperature, and
column type.
Capacity factor is equal to the number of moles in the
stationary phase divided by the number of moles n the
mobile phase.
tR2
tR1
tO k’ = tR - tO
tO
Inject
13
The capacity factor is related to the ratio of the total number of moles of a given
component in the stationary phase versus those in the mobile phase for any given
equilibration. A higher k’ value indicates that the sample is highly retained and
has spent a significant amount of time interacting with the stationary phase. The
capacity factor is characteristic of a specific compound at a given mobile phase
composition, temperature, and column type. One may use the capacity factor
instead of retention time to identify components qualitatively. The value is
independent of flow rate making day-to-day fluctuations less troublesome.
27
Capacity Factor
Capacity Factor
Capacity Factor
Weaker Solvent Composition

mAU
60% Acetonitrile
40% Water
The single most important way to change the
capacity of a chromatographic peak is to change
2 4 6 8 10 the mobile phase composition.
Increasing the strength of the mobile phase
Time (min.)
decreases the capacity factor of the eluents.
For reversed phase, an increase of 10%
Stronger Solvent Composition organic decreased k’ for each
chromatographic peak by a factor of 2 or 3.
80% Acetonitrile
mAU
20% Water
2 4 6 8 10
Time (min.)
14
The capacity factor is usually changed by modifying the mobile phase

composition. The example provided is that of a reversed-phase separation on a C-
18 column. In reversed-phase, a weak mobile phase will be more polar than a
strong mobile phase. A weaker mobile phase composition is produced by
increasing the amount of water in the mobile phase, thus increasing the retention
of components. Conversely, an increase of 10% organic decreases the k’ for each
component by a factor of 2 or 3.
28
Useful Solvents for Reversed-Phase Chromatography
Useful Solvents for Reversed-Phase

Chromatography
Useful Solvents for Reversed-Phase

Chromatography
• Water
• Methanol Elution
• Acetonitrile Strength
• Isopropanol
• Dioxane
• Tetrahydrofuran
15
Reversed-phase mobile phases generally consist of mixtures of water or aqueous

buffer with various water -miscible organic solvents. The stronger the organic
mixed with water, the faster sample components will elute from the column. One
hundred percent organic will flush a reversed-phase column.
29
Solvent Strength: Normal Phase
Solvent Strength: Normal Phase
Solvent Strength - Normal Phase
Solvent Strength ∈º
Silica Alumina
Fluoroalkanes -0.2 -0.25
n-Pentane 0.0 0.0

1-Chlorobutane 0.20 0.26
Xylene 0.24 0.26

Toluene 0.28 0.29
Benzene 0.20 0.32
Chloroform 0.26 0.40

Methylene Chloride 0.32 0.42
Tetrahydrofuran 0.44 0.57

Acetonitrile 0.50 0.65
Methanol 0.7 0.95
16
The elutropic series for normal-phase liquid chromatography is provided above.

Solvents with larger solvent strength values will cause sample components to
elute more quickly from the column. In the adsorption chromatography model,
strong mobile phases are strongly adsorbed to the stationary phase. Sample
molecules will have little ability to knock these mobile phase molecules from the
substrate and therefore sample molecules elute quickly. When a mobile phase
molecule is weak enough to be displaced from the stationary phase, sample
molecules are retained and a separation occurs.
30
Normal Phase: Solvent Strength
Normal Phase: Solvent Strength
Normal Phase - Solvent Strength
HC CH
3 3
X+T+B X = Xylene
CH 3 T
T = Toluene X
B
B = Benzene
∈°= 0.42
Methylene ∈°= 0.00
Mobile Phase
Chloride n-Pentane
17
To separate xylene, toluene, and benzene, a mobile phase should be chosen that is
less strongly adsorbed to the stationary phase. Pentane with a relative elution
strength of 0.0 is weak enough to be displaced by these sample components.
Methylene chloride with an elution strength of 0.42 is more strongly retained than
any of the sample components so no separation occurs.
31
Efficiency
Efficiency
Efficiency
x k’
R = 1/4 N x -1
1 + k’
Low
Response
Detector
Efficiency
Inject
High
Efficiency
Time
18
Another one of the factors that influence resolution is the column efficiency.
Column efficiency is expressed as N, or plate number. In an ideal
chromatographic system, a chromatographic peak would appear as a vertical line
in the chromatogram. In reality, dispersion occurs causing the peak to take on a
guassion shape. The better the column efficiency (less dispersion) the easier it
will be to achieve resolution between chromatographic peaks.
32
Calculating Efficiency
t
R
W
1/2
Inject
2
tR tR 2
= 2 ∏ hptr
2
N = 16 = 5.54
WB W 1/2 A
HETP = L
B
Time
N
N: Efficiency
HETP: Height Equivalent to a Theoretical Plate
L: Column Length
hp: Peak Height
A: Peak Area
19
To calculate the column efficiency use one of the equations presented here. Make
certain the chromatographic peak chosen has a k’ value greater than 2. A typical
plate number for a new 4.6 X 100 mm column with 5 um particles is 8 or 9000
plates. The number of theoretical plates is proportional to the column length. The
HETP, or height equivalent to a theoretical plate, is also a measure of the column
efficiency, which describes the efficiency of a given column for unit length of
column.
33
Dispersion: Eddy Diffusion
Dispersion: Eddy Diffusion
Dispersion - Eddy Diffusion
Pack Columns
Carefully
Initial
Use Narrow Mesh Band
Range Width
H A = Eddy Diffusion - The

E Multi-Path Effect
T
P Final
A Band
Width
Linear Velocity
20
Dispersion of a chromatographic peak occurs as a result of differing migration

rates through the column. Differing migration rates are a result of physical
processes, such as eddy diffusion. The A-term results from inhomogeneity of
flow path velocities around stationary phase particles. The A-term can be
considered independent of linear velocity. To diminish dispersion resulting from
this term, columns should be carefully packed using a narrow mesh range.
Smaller particles will also decrease this effect, as well as smaller column
diameters.
34
Dispersion: Longitudinal Diffusion
Dispersion: Longitudinal Diffusion
Dispersion - Longitudinal Diffusion
Small Effect in LC
Significant at Low Flow Rates
H
E
T B
P
Linear
Velocity
21
7KH% WHUPRUORQJLWXGLQDOGLIIXVLRQWHUPGHILQHVWKHHIIHFWRIUDQGRP
molecular motion on dispersion. Although not as serious a consideration in LC,
this term becomes more significant at lower linear velocities.
35
Dispersion: Mass Transfer
Dispersion: Mass Transfer
Dispersion - Mass Transfer
C = Mass Transfer Between Phases

Reduce Effect with Low Flow Rate
Reduce Effect with Small Particles
H
E
T
P Mobile
.u Phase
C
Stagnant
Linear Mobile
Velocity Stationary Phase
Phase
22
Dispersion due to mass transfer has both a component relating to the sample
molecules interaction with the stationary phase as well as a component relating to
the sample molecules interaction within the mobile phase. The stationary phase
interaction requires a finite rate of equilibration between the sample molecule and
the stationary phase. The mobile phase interaction relates to the diffusion of
analytes. The structure of the stationary phase is porous. The mobile phase
within these pores is on the whole stagnant. Once a sample molecule finds itself
with a stagnant pore, the only way for it to rejoin the other sample molecules in
the mobile phase is for the molecule to diffuse out of the pore. This term is
adversely affected at higher flow rates.
36
Dispersion
Dispersion
Dispersion
The Van Deemter
Equation
A = Eddy Diffusion (Multi-Path Effect)

B = Random Molecular Diffusion
C = Mass Transfer Between Phases
B + C .u
HETP
B + u
u h=A
C. u
Linear Velocity
23
The total effect of the three terms, eddy diffusion, longitudinal diffusion, and
mass transfer is additive. The graphic illustrates that there is an optimal linear
velocity for each chromatographic column indicated by the dip in the graph.
Typically, one operates the column at a linear velocity just above the dip in the
curve.
37
Typical Flow Rates
Typical Flow Rates
Typical Flow Rates
mm i.d.(5um particles) mL/min
4.6 1-2
3.0 0.4-0.8
2.1 0.2-0.4
1.0 0.05-0.09
2
i.d.( µ bore)
flow rate
flow rate = i.d. (analytical) (analytical)
24
From the previous discussion, it is apparent that each chromatographic column

will have an optimal operating flow rate. Typical flow rates presented on the
basis of column diameter are shown above. Flow rate as an experimental
variable produces only small changes in resolution and is used for fine tuning the
chromatogram.
38
Small Bore and Microbore Columns

dp = 10 µm Conventional
4.6 1.00
mm mL/min
dp = 5 µm
4.6
mm
0 2 4 6 8 10
Microbore
2.1 0.01
mm dp = 5 µm mL/min
or
1.0
mm 100 mm
200 mm
0 2 4 6 8 10
Advantages Disadvantages
• Decreased solvent consumption. • Instrumentation must have very low
• Good for trace analysis if sample extra-column volume.
amount is limited. • Frits must be changed more
• Easy flow rate conversion to change frequently.
method from analytical column to
mircrobore column. 2
i.d. (bore) flow rate
flow rate( bore) = (analytical)
i.d.(analytical)
25
A conventional HPLC column is 4.6 mm i.d. with 5 um particles. Small bore (2.1
mm i.d.) or microbore columns (1.0 mm i.d. or <) may be utilized with the
following advantages: 1) decreased solvent consumption; and, 2) decreased peak
dispersion resulting in better peak signal to noise ratio. A 4.6 mm i.d. column
method may be transcribed to smaller internal diameter column using the flow
rate conversion provided. Some disadvantages include the need for low dead
volume instrumentation and that fact that the column inlet frits clog more easily.
39
Fast or High Speed Columns

4.6 dp= 10 µm
mm Conventional
1.00 mL/min
4.6 dp= 5 µm
mm
0 2 4 6 8 10
4.6 dp= 3 µm
mm High Speed
60 mm 5.00 mL/min
100 mm
200 mm
Advantages 0 0.5 1.0
• Very short analysis time.

• Easy flow rate conversion to change
analytical method to fast column
method.
flow rate = particle diameter (analytical) flow rate (analytical)
(fast)
particle diameter (fast)
Disadvantages
• Instrumentation must have very low
extra-column volume.
• Time constant and cell volume of
detector must be low; signal acquisition
and integration must be fast.
26
High speed columns have typically a 4.6 mm internal diameter, but utilize 3 um
SDUWLFOHV$ PSDUWLFOHFROXPQPDLQWDLQVKLJKHIILFLHQFLHVHYHQDWKLJKIORZ
rates. As a result, analyses may be run at flow rates from 3-5 mL/min shortening
the analysis time. These columns are typically 60mm in length due to the
increased back pressure. Instrumentation with low dead-volume characteristics is
required because of reduced dispersion.
40
Extra-Column Band Broadening
• Sample Volume
• Volume of Connective Tubing
• Detector Volume
• Detector Electronic Time Constant
27
Dispersion that occurs outside of the column is termed extra column band
broadening. In a well designed HPLC, this dispersion is negligible as compared
to internal band broadening. Chromatographers must endeavor not to add extra
lengths of tubing or wide diameter tubing to the liquid chromatograph between
the injector and detector. One must also be careful to match HPLC fittings and
set appropriate time constants on detectors.
41
Increasing Resolution
Increase k’
Increase Selectivity
Increase Efficiency
28
In summary, the three factors which influence resolution between

chromatographic peaks are capacity, efficiency, and selectivity. The most
effective way to increase the capacity factor is to change the mobile phase
composition. Selectivity can be increased through mobile phase composition or
component changes, column stationary phase changes or temperature adjustment.
Column efficiency can be increased by lengthening the column, using the
appropriate flow rate, and increasing the column temperature.
42
Peak Symmetry
Peak Symmetry
Peak Symmetry
S = B/A
A B
10% of peak height
Excellent Acceptable Unacceptable Awful

S = 1.0 - 1.05 S = 1.2 S=2 S=4
29
Ideally, chromatographic peaks should be guassion in shape. However, most of

the time they have a tail. The calculation above is used to mathematically
describe peak symmetry. Values greater than 3 are not acceptable.
43
Worksheets
Worksheets
Worksheet
The analysis was performed on a 100 x

4.6 mm i.d., 10 um, C-8 column.
The flow rate was 2 mL/min with
70/30 IPA/water.
List ways to improve the separation.
30
44
Worksheets
Worksheet
1. What happens to resolution when k’ is increased?
2. What happens to resolution when the column efficiency is

increased?
3. What parameters affect column efficiency?
4. What parameters affect k’?
31
45
Mobile Phase Composition – The General Elution Problem
Mobile Phase Composition – The General Elution

Problem
Mobile Phase Composition - The General Elution

Problem
Isocratic elution of an homologous series
(wide range of k’ values)
Poor resolution of early eluting peaks.

Increase in peak width and decrease
in peak height for later eluting peaks.
Long analysis times.
32
When a single mobile phase composition is used for an analysis, analytes may
elute over a wide range of k’ values. Early eluting peaks may not be completely
separated and late eluting peaks are broad, therefore losing peak detectability due
to their decreased peak height. Long analysis times are also characteristic of such
separations.
46
Mobile Phase Composition – Gradient Elution
Mobile Phase Composition – Gradient Elution
Mobile Phase Composition - Gradient Elution
• "Ideal" chromatogram of an
homologous series.
• Optimum overall resolution.
• Equal band widths for all peaks.
• Shorter analysis times.
Other Uses:
• To quickly check unknown
samples.
• To clean the column.
33
Gradient elution is the solution to the general elution problem. When using a
gradient elution program, the initial solvent strength is selected to give an elution
strength capable of eluting the early chromatographic peaks with adequate
resolution. The elution strength is increased in a predetermined way in order to
bring other peaks off the column with optimum overall resolution, increased peak
height, and shortened analysis times.
47
Gradient Development
10% 100%
Organic Organic
Consider:
• Choice of Organic Solvent
• Initial Composition of Mobile Phase
• Ramp Rate
• Gradient shape
• Flow Rate
• Column Re-equilibration
• No Peaks k’ < 2
• No Peaks k’ > 10
34
A scouting run can be the initial step during gradient development. A linear
gradient is run from 5-10% organic to 100% organic over a set time period, then
the composition is held for some additional time to insure all sample components
have eluted. The results are examined to determine the proper initial gradient
composition and gradient profile.
48
Worksheets
Worksheets
Worksheet - How can this gradient be

improved?
10% Linear Gradient 100%

Organic Organic
1. What is the problem with this gradient?
2. How would you improve it?
35
49
Worksheets

improved?

Organic Organic
1. What is wrong with this chromatogram?
2. How can it be improved?
36
50
Worksheets

improved?

Organic Organic
1. What is wrong with this chromatogram?
2. How can the chromatogram be Improved?
37
51
Practical Considerations for Gradient Elution
• Solvents must be pure or ghost

Ghost Peaks
peaks will occur.
• Make certain the buffer is soluble at
final gradient mobile phase
composition.
• Allow time for column
reconditioning between runs.
• Different LC models will have
0% MeOH 100%
different delay volumes.
38
Solvents utilized in gradient elution must be pure. Water quality is of particular

importance. Impurities are retained on the column while the composition of the
mobile phase is weak. As the elution strength is increased, the impurities appear
as peaks in the chromatogram. To avoid precipitation in the instrument, test that
the buffer is soluble in the final mobile phase composition. Finally, to increase
retention time precision, make certain that adequate re-equilibration time is
allowed between each chromatographic run.
52
Practical Aspects of Performing Analyses
• Mobile phase preparation
• Column care
• Sample preparation
54
Mobile Phase Preparation Filtration
• Always use HPLC grade solvent

Vacuum
• Change HPLC grade water daily.
– Prevents particulate matter from
damaging the instrument or
column head.
• Use at least 0.5 mm filters.
– Organics - PTFE
– Water - Nylon
– Inorganic Membrane Filter
Filtration
Apparatus
All solvents used on the HPLC should be at HPLC grade. These solvents are pre-
filtered and purified to have minimal absorbance in the UV. After dissolving
buffers and additives, the mobile phase should be filtered with at least a 0.5 um
filter to remove particulate matter, which may damage the instrument or column.
The apparatus shown is one possible device for filtration. A vacuum is applied to
pull solvent through the filter housed inside the screw cap. The reservoir is
plastic coated to avoid implosion. Handle the filters with tweezers and make
certain the filtration apparatus is clean at all times. Nylon is a good filter for
aqueous mobile phases, while PTFE is an excellent filter for most organic
solvents. Inorganic membranes are resistant to a wide range of HPLC solvents.
55
Mobile Phase Degassing
• Purpose
– Removes dissolved oxygen and
nitrogen from the mobile phase
• Methods
– He sparging
– On-line vacuum degassing
– Refluxing
– Vacuum filtration
– Ultrasonication
Degassing the mobile phase is an important step before beginning an HPLC

analysis because water and lower molecular weight alcohols dissolve relatively
large amounts of air. Degassing removes these dissolved gasses from the mobile
phase. The dissolved gasses can result in bubble formation in the pumps or
detector. Dissolved oxygen can quench fluorescence detection. Helium can be
used to sparge mobile phases because it has a low solubility and thus can " knock
out" other dissolved gasses. Boiling premixed solvents is not recommended
because the more volatile component is lost more rapidly changing the
composition.
56
Vacuum Degassing
Vacuum Degassing
Vacuum Degassing
The best way to remove dissolved gasses from the mobile phase is vacuum
degassing. The mobile phase is pulled through gas permeable tubing coiled
within a vacuum chamber on the way to the pump. Besides adequately removing
dissolved gasses, the other advantages include: real-time degassing and less
expense as helium is not required.
57
Solvent Miscibility
Solvent Miscibility
Solvent Miscibility
Name
Acetic Acid
Acetone
Acetonitrile
Benzene Immiscible
Butyl Alcohol
Carbon Tetrachloride
Miscible
Chloroform
Cyclohexane
Cyclopentane
Dichloroethane
2-Propanol is an
Dichloromethane
Dimethylformamide
Dimethyl Sulfoxide
excellent intermediate
Dioxan
Ethylacetate
Ethyl Alcohol
solvent
Di-Ethylether
Heptane
Hexane
Methyl Alcohol
Methylethyl Ketone
I-Octane
Pentane
I-Propyl Alcohol
Di-Propylether
Tetrachloroethane
Tetrahydrofuran
Toluene
Trichloroethane
W ater
Xylene
leth l
e
C ropy oho
ne
an
er
ol
l
ro e
ho
clo ane
He ne r
eto id
CH etha
Be nitrile
te
Eth ho
Ca lcoh
e
hlo tan
et
lc
ate eth
c lo rm
Ac Ac
He yleth
ME Alco
tyl e
C h on T
Eth ceta
Di- yl A
A c ne
Di- Alco
Cy hex
B u en
Cy rofo
Dic pen
DM 2Cl 2
loro
Pe ne
I-P ne
Tri ne
Me ne
A
Cl
c
eto
n
SO
ne
nz
rop
eti
pta
yla
r
rb
xa
c ta
nta
lue
l
xa
K
F
lo
thy
ch
yl
2
P
le
F
Ac
2H
DM
Dio
Eth
I-O
To
TH
Xy
W
Not all common HPLC solvents are miscible. If immiscible solvents are mixed
several problems may result such as an unstable baseline, fluctuating pressures
and high pressure. If you are uncertain about the last solvent system used in your
HPLC, flush the flow path with isopropanol. This solvent is miscible with most
common HPLC solvents. To move from a normal-phase separation to a reversed-
phase separation, remove the normal phase column, add a capillary tube in its
place and flush the liquid chromatograph with isopropanol. After, you may
proceed with the reversed-phase analysis.
58
Mobile Phase UV Cut-Off
Mobile Phase UV Cut-Off
Mobile Phase UV-Cutoff
Solvent UV Cutoff (nm)
Acetonitrile 190
Water 190 UV cutoff is the
Cyclohexane 195 wavelength at which
Hexane 200 absorbance equals 1 AU.
Methanol 210
Ethanol 210
Diethyl Ether 220
Dichloromethane 220
Chloroform 240
Carbon Tetrachloride 265
Tetrahydrofuran 280 (210)
Toluene 280
Listed above are the UV-cutoffs for common HPLC solvents. When utilizing a
UV detector, care should be taken not to use the solvent below or near its UV-
cutoff or an unacceptable noise level will result limiting your detectability. For
instance, if you were monitoring a compound at 220 nm, you would select
acetonitrile over methanol because acetonitrile’s UV-cutoff is lower than
methanol resulting in better detection performance. Other factors must be
addressed for different detectors. For instance, when using a mass spectrometer
with a particle beam interface as the detector, one must consider the molecular
weight of the mobile phase and additives.
59
Column Care
Column Care
Column Care
á Filter all Solvents and Samples á Store Column in Appropriate

Solvent
á Use a Guard Column á Do Pay Attention to the Safe
Operating pH Range of the
Column
á Flush to Remove Buffers at End of á Do Not Pressure or Solvent
Use Shock the Column
á Cap When Not in Use á Do Not Operate Silica or Bonded

Phases for Extended Periods at
High Temperatures
By following the manufacturers recommendations and applying the suggestions

above, you may extend the lifetime of your analytical HPLC columns. To prevent
clogging of the column inlet frit and damage to the column bed, Filter all solvents
and samples. Guard columns, positioned between the injector and analytical
column will extend the life of your analytical column by trapping particulate
matter and strongly retained sample components. Make certain that your column
is flushed and free of buffers and damaging additives before storage. Caps the
ends of the column firmly to prevent the column from drying out. The normal
operating range of silica based bonded phase columns is from pH 2 to 8. Silica is
soluble in the ppm range at pH 7.5 and above. Silica columns will degrade more
quickly when operated at elevated temperatures.
60
Pre-Columns and Guard Columns
Pre-Columns and Guard Columns
Pre-columns and Guard Columns
Mobile phase Guard column

from
pump
Pre-column
Injector
Analytical
column
Pre-column acts on mobile phase.
Alternative: Polymer analytical columns
Guard column acts on sample.
To detector
Pre-columns are positioned prior to the injector and serve to condition the mobile
phase. The lifetime of a silica column may be extended because the pre-column
will saturate the mobile phase with dissolved silica before the mobile phase ever
reaches the analytical column. Extreme pH’s, high ionic strength, or high mobile
phase polarity all contribute to dissolution of silica. Guard columns are placed
between the injector and the analytical column. Guard columns should be the
same stationary phase and internal diameter as the analytical column, but they are
very short. These columns protect the analytical column from impurities and
particulates. Many are sold as cartridges to facilitate frequent replacement.
61
Syringe Wash: HP 1090
Syringe Wash: HP 1090
Syringe Wash 1090
You should perform a syringe wash:

• Daily
• After changing mobile phase
composition
• When you experience
reproducibility problems
• When air has been found in the
solvent delivery system
Normal Mode Wash Mode
10
For best reproducibility of peak area and height, an HP 1090 requires a syringe
wash on at least a daily basis. Syringe washes should be performed any time the
mobile phase composition changes, when you perform any maintenance on the
auto-injector area, or as part of your start up procedure each day. The syringe
wash function simply removes air bubbles from the syringe. The wash function
has nothing to do with sample contamination. The sample loop is continuously
flushed during normal operation. Syringe washes are not required on the HP 1050
or HP 1100 series HPLC’s.
62
Priming
Priming
Priming
Purge Valve
Priming the HPLC Pump
Flow
to waste
Removes air bubbles from the solvent

composition.
Allows easy solvent system change.
11
When a liquid chromatograph has been idle, there is always the possibility that air
has managed to permeate the flow path. Priming the liquid chromatograph
involves pumping each channel at 100% composition and high flow rate until
steady pressure and flow is obtained. The mobile phase will forcibly expel any
trapped air. Priming on a daily basis, when the mobile phase is changed, or when
maintenance work is required will lead to more reproducible peak areas and
retention times. With the HP 1090, the capillary to the column should be
disconnected and the end placed into a beaker before priming. For the HP 1050
or 1100, the purge valve may be opened and the flow channeled to waste.
63
Column Equilibration
• Assures reproducible results

• 5-10 column volumes for equilibration of Reversed-phase columns
12
Before you begin an analysis, the column must be equilibrated with the mobile
phase. Reversed-phase columns using a simple mobile phase without buffers and
modifiers require only 5-10 column volumes for equilibration. Some applications
may take much longer. A column, which has not been equilibrated properly, will
exhibit irreproducible retention times. Other symptoms of an unequilibrated
column are unstable pressure and a drifting baseline.
64
Preparing Samples: Filtering
Preparing Samples: Filtering
Preparing Samples - Filtering
• Nylon - hydrophilic nature works with aqueous and solvent

based samples, autoclavable to 121ºC, pH range 3-12, no
concentrated acids.
• PTFE- a hydrophobic membrane which is highly resistant to
solvents, acids, and alkalies. This filter is generally used for
non-aqueous samples. pH range 1-14.
• Cellulose Acetate- good filter for aqueous biological samples
with very low protein retention. pH range 4-8.
• PVDF- highly resistant to most solvents, exhibits low protein
binding. pH 2-12.
• Ultrafilter Membranes- molecular weight cut-off filters for
biological samples.
• Nitrocellulose- exhibits high protein retention.
• Solid Phase Extraction.
13
Samples should be filtered prior to injection. Sample particulates will cause

blockages in the capillary tubing, particularly at the point of injection, and in the
column inlet frit. Many HPLC suppliers sell a variety of filter products, which are
application and mobile phase dependent. The list above can provide you with a
starting point. Do not forget solid phase extraction, which can be useful for
removal of strongly retained sample components that may damage the analytical
column. Solid phase extraction may also be utilized for isolation and
concentration of a particular set of sample components.
65
Preparing Samples
Preparing Samples
Preparing Samples
• Dissolve the sample in the mobile phase or in a solvent weaker than the
mobile phase.
• The sample volume should be kept as small as possible.
Sample in Mobile Phase Sample in Stronger Solvent
14
Ideally, the sample should be dissolved in the mobile phase or in a solvent weaker
than the mobile phase for best chromatographic results. If the sample is dissolved
in a stronger solvent than the mobile phase, and large injection volumes are used,
chromatographic peaks will become broad and begin to have a doublet
appearance. Sample volumes should be kept as small as possible in order to avoid
loss of resolution due to volume overloading. The injection volume limitations
are related to the column internal diameter. For instance, a 2.1mm i.d. column
VKRXOGKDYHLQMHFWLRQYROXPHV ORUOHVV
66
Worksheet
Worksheet
Worksheet
1. You are running a routine analysis when you notice a periodic perturbation
in the baseline. The pressure reading is fluctuating up and down. What is
the problem? How would you correct it?
2. You decide to run a reversed-phase analysis on an instrument in your lab.

The previous operator does not indicate the solvents last used in the
instrument. You place water in channel A and turn on the pump. The
pressure increases at rapid rate and becomes variable. You cannot get a
stable baseline. Suggest a possible reason for this dilemma.
15
67
Worksheet
68
HPLC Instrumentation
In This Section, You Will Learn:
In This Section, You Will Learn:
In This Section, You Will Learn About the

Following HPLC Components:
Injector
Mixer
• Tubing and fittings
Pumps
• Solvent Delivery Systems
Chromatogram • Injection Systems
• Detectors
Column
Detector
Solvents
In this section, you will learn about the components of a high performance liquid
chromatograph including fittings and tubing, solvent delivery systems, injectors,
and detectors.
70
HPLC Tubing
HPLC Tubing
HPLC Tubing
• Stainless Steel
– Commonly 1/16" OD with various internal diameters.
• Teflon
– Good for pressures up to 1000 psi. Commonly used from the
solvent reservoirs to the pump.
• PEEK
– Can be used to replace stainless steel tubing when a metal-free
environment is desired. 1000-8000 psi
• Tefzel
– Pressures up to 3500 psi for metal-free analysis.
HPLC tubing is most commonly stainless steel or plastic. Most stainless steel
tubing is 1/16 inch o.d. with varying internal diameters. HP uses stainless steel
tubing with an internal diameter down to 0.12 mm. Stainless steel handles the
high pressures of HPLC well and is more robust. Teflon tubing is often used for
the connections from the mobile phase reservoir to the pump. The internal
diameter will be sufficiently large to deliver solvent to the pump without drawing
air. PEEK (polyetheretherketone) may be used in place of stainless steel when the
analyst desires to limit sample contact with metal ions. Tefzel tubing can also be
used when the analyst wants to limit sample exposure to stainless steel.
71
Fittings
Fittings
Fittings
Swagelok Waters
0.090 in. 0.130 in.
Rheodyne
Parker
0.170 in.
0.090 in.
Valco Uptight
0.090 in.
0.080 in.
Troubleshooting LC Fittings, Part II. J. W. Dolan and P. Upchurch.

LC/GC Magazine 6:788 (1988)
There are many different fittings for stainless steel tubing which are not
necessarily interchangeable. The distance from the swaged ferrule to the end of
the tubing varies from manufacturer to manufacturer. When incompatible fittings
are mixed, undesirable peak dispersion may result. HP HPLC instrumentation
utilizes swagelock stainless steel fittings. For a leak free connection, tighten the
fitting with your fingers, then using the wrench, turn the fitting one quarter turn.
Over tightening the fitting may cause damage.
72
Fittings
Fittings
Other HPLC Fittings
Finger Tight Fittings Zero Dead-Volume Union

• Universal, the fitting does not attach • Connect two pieces of tubing.
permanently to the tubing. • without any dead volume between
• Convenient, no wrenches. the tubing.
• Not usually interchangeable from
one manufacturer to another.
Finger tight fittings have become very popular. They are nearly universal because
the ferrule is not swaged permanently to the tubing. It also very convenient not to
have to get out the wrenches every time you have to change a column. Unions are
used to connect two pieces of tubing together. A zero dead-volume union will
connect two pieces of tubing together without adding any additional dead volume.
Most manufacturers of zero dead-volume unions do not butt the tubing from one
capillary directly up against the other capillary. Instead, a thin web is used
between the two pieces of tubing.
73
Filters
Filters
Filters
Guard
column
Injector
Precolumn Analytical
Filter Column
Solvent Inlet Filter Precolumn Filter

• Stainless Steel with 10 micron • Used between the injector and
porosity. guard column.
• Removes particles from solvent. • 2 to 0.5 micron.
• Removes particulates from sample
and injector wear.
• Must be well designed to prevent
dispersion.
Particulates in the mobile phase may damage the pumping system. Commonly, a
10 micron solvent inlet frit is placed in the mobile phase reservoir to trap
particulates. The solvent inlet filter should be replaced or cleaned on a periodic
basis or the required flow through the filter may not be possible resulting in
mobile phase composition changes and air in the pump. A precolumn filter may
be placed between the injector and the analytical column to catch particles in the
sample and particles from injection valve wear. The filter usually consists of a
0.5 to 2 micron frit held in a cartridge. The frit can be easily replaced when the
system pressure rises.
74
Functions of the SDS
Functions of the SDS
Functions of the Solvent Delivery System
The solvent delivery system has three basic functions:
1. Provide accurate and constant flow.

2. Provide accurate mobile phase compositions.
3. Provide the force necessary to push the mobile phase through
the tightly packed column.
A solvent delivery system must provide accurate and reproducible flow and
composition. It must also provide the force necessary to push the mobile phase
through the tightly packed column. The next slides will illustrate some of the
ways to accomplish this task. In addition, the solvent delivery system should not
produce pressure pulsations. The addition of a damping unit is usually customary.
75
Multichannel Gradient Valve
Multichannel Gradient Valve
Multi-channel Gradient Valve
• Determines mobile phase composition.

• Largest solvent plug fills first.
• HP 1090 PV5, HP 1050 quaternary pump, and the HP 1100 quaternary
pump.
The purpose of a multi-channel gradient valve is to produce the solvent

composition. This valve provides a fixed volume packet to a pump. For example,
in the case of the HP 1090 PV5 system, the flow is split into 89ul packets.
Therefore, if the desired composition were to be 80% A and 20% B, the valve
would remain open on the A channel for 71.2 µl then close and deliver 17.8 µl of
B.
76
Dual Piston Parallel Pump
P
Check
Valves
Rotary
Switching
Valve
Pumphead
Piston
A B
Single Combined
Piston Delivery
Delivery
B
P
A
Piston ’A’ Advancing

Piston B Retracting
A dual piston parallel pump is designed to deliver a continuous flow of mobile

phase to the column by operating 180 degrees out of phase. The destructive
interference of alternating pump pulses dampness the total pulsation. While one
piston of the pump is delivering solvent to the column, the other retracts to refill
the solvent chamber. An example of a dual piston pump is the HP 1090 metering
pump.
77
Dual Piston Series Pump
Dual Piston Series Pump
Dual Piston Pump in Series
• First piston chamber is twice the

size of the second.
• Provides constant flow and pressure
necessary to get through column.
10
An alternative method for delivery of a pulse free mobile phase is the dual piston
series pump. The first solvent chamber is twice the volume of the second solvent
chamber. While the second piston delivers mobile phase to the column, the first
piston chamber refills. When the second piston chamber is empty, the first piston
moves forward and not only refills the second chamber, but also continues to
deliver mobile phase to the column.
78
Ball Valves
Ball Valves
Ball Valves for Reciprocating Piston Pumps
Gold Seal
Sapphire
Insert
Ruby Ball
Spring
Insert
11
The purpose of a ballvalve or check valve is to provide unidirectional flow. The

elements of a ballvalve include a sapphire seat, a ruby ball and a spring for
tension. When a piston is drawing solvent from the mobile phase reservoir, the
ruby ball on the inlet side is pulled upward allowing mobile phase to fill the
solvent chamber. The ruby ball on the outlet side of the chamber is pulled down
against the sapphire seat so that solvent which has already been displaced to the
column will not be pulled back into chamber. When the piston is on the forward
stroke, the mobile phase will push the outlet ruby ball away from the sapphire seat
The force of the mobile phase will push the ruby ball on the inlet side into the
sapphire seat preventing flow to the reservoir.
79
Metering Pump Seals and Pistons
1 1 = Piston
2 = Support Rings
2
3 = Seal Keepers
3
4 4 = Seals
5
5 = Wear Retainers
12
The pistons found in these pumps are typically made from man-made sapphire.
They should be examined on a periodic basis for scratches. Piston pumps also
contain piston seals which should be replaced on a periodic basis to maintain
retention time and peak area reproducibility. Some styles of piston pumps also
contain wear retainers so that seal wear material will be trapped and not damage
other parts of the instrument.
80
Diaphragm Pump
Diaphragm Pump
Diaphragm Pump
+
Pressure
Override
Oil Valve
Check
Valve
Piston
Inlet Ball
Valve
Stainless Steel Outlet ball
Diaphragm Valve
13
The reciprocating piston/diaphragm pump has the advantage of removing the

piston and piston seal from the harmful mobile phase and placing them in a
lubricating environment. One side of a gold coated stainless steel diaphragm
contains oil while the other side comes in contact with the mobile phase. When a
reciprocating piston places pressure on the diaphragm from the oil side, the
diaphragm bulges downward forcing solvent from under the diaphragm out onto
the column. As the reciprocating piston retracts, the diaphragm bulges upward
allowing solvent to fill the space underneath the diaphragm. The HP 1090
contains such a pump to provide the high pressures necessary to force the mobile
phase through the tightly packed column. It operates at 10Hz.
81
Sieves and Filters
Sieves and Filters
Sieves and Filters
HP 1090
Sieves and Filters are used to protect

other parts of the LC from pump seal material.
Filter
)
OR
Z
Z
OR
)
Sieve
Ball-Valve Housings
HP 1050
14
Most solvent delivery systems contain an in-line solvent filter or frit before the
injector. The purpose of this filter or sieve is to collect pump seal particles of the
pump seals as they break off so that they don’t damage the injection valve or in
the case of the HP 1090 the ball-valves. These in-line filters must be replaced on
a regular basis.
82
Damping Unit
Damping Unit
Damping Unit
Damping
Pump
Unit Ripple
2%
P /P
Pressure
• Filled with compressible liquid separated from mobile phase by a membrane.

• Pressure ripples from high pressure pump are reduced to < 2% of original
value.
15
The purpose of the damping unit is to reduce pressure pulsations caused by the
action of the pumps to a minimum. A damping unit consists of a diaphragm
separating the mobile phase from a compressible liquid. In the HP 1090, the
diaphragm is placed after the diaphragm pump. In the dual piston series pump,
the diaphragm is located between the first and second piston chambers.
83
1090 SDS
1090 SDS
Solvent Delivery System - 1090
High-Pressure
Diaphragm Pump
Low Pressure
Compliance • Metering pump for each solvent
reservoir - for composition and
flow.
Switching
• Mixing in the Low Pressure
Valve
Damping Unit Compliance.
• Diaphragm pump for high pressure
• Damping unit.
Dual-syringe metering pump
16
The 1090 can be used to illustrate all of the components of a solvent delivery
system working together. Each mobile phase reservoir has its own parallel piston
pump (metering pump) for metering mobile phase flow and composition. These
pumps are operating at low pressures. Mixing occurs in the low pressure
compliance. After mixing, the mobile phase flows through a sieve then an inlet
ball valve into the diaphragm or booster pump. Here the pressure necessary to
force the mobile phase through the column is supplied. On the outlet side of this
pump, there is another ball-valve and then a damping unit to reduce pressure
pulsations.
84
Quaternary Pump
Quaternary Pump
Solvent Delivery System

Quatenary Pumping System
• Multichannel gradient valve for

composition.
• Dual piston series pump for flow
and pressure.
• Damping unit.
17
A quaternary solvent delivery system consists of a multi-channel gradient valve

for mobile phase composition control. The mobile phase then flows through an
active inlet check-valve and then into the first chamber of the dual piston series
pump. A ball-valve is placed at the outlet of the first chamber to provide
unidirectional flow. The mobile phase then flows through a damping unit and
into the second chamber of the dual piston series pump. A frit is located at the
outlet of the dual piston’s second chamber. This pump provides the high pressure
and the proper flow rate.
85
Manual Injection
Manual Injection
Manual Injection
Load
Inject
Sample
Syringe
To
Waste
To
Column To
Column
Sample
From Loop
Pump (Fixed Volume)
18
Manual injection valves are typically six-port valves with a sample loop across
one pair of the ports. The loop is filled with sample while the mobile phase
bypasses this part of the valve. The valve is then switched to the inject position
and the contents of the sample loop are carried onto the column. To prevent
concentration gradients, five times the sample volume is typically injected.
Internal sample loops are found on injection valves with injection volumes less
than 5 ml. Flushing a manual valve is extremely important to prevent blockages
and sample carry-over. Do not use GC syringes in an LC. This practice will lead
to a scratched rotor seal.
86
Auto-injection System
Auto-injection System
Auto-Injection Systems
Injecting a Sample
Pre-Run Prepare to Inject
Load Sample Inject & Run
19
Auto-injectors can be operated by compressed air or electronically actuated. They

can also be fixed or variable volume. For illustrative purposes, the HP 1050 auto-
injector is shown. Prior to injection, the mobile phase flow is through the valve,
metering device, sample loop, needle, needle seat, needle seat capillary, back to
the valve and onto the column. For an injection, the valve switches so that the
mobile phase flow will bypass the auto-injector. The needle arm rises and a vial
is placed under the needle. The needle is forced down into the vial and the
metering device is pulled back to draw sample into the needle and the loop. After
the appropriate volume is drawn, the needle rises and the vial is returned. The
needle moves into the seat and the valve is again switched to allow flow through
the auto-injector delivering sample to the column.
87
Rotor Seals
Rotor Seals
Rotor Seals
Align notch with

pin
20
A rotor seal is located inside injector valves. The seal is a disk with grooves to
direct the mobile phase flow path. These seals need to be replaced on a periodic
basis or irreproducible injection volumes will result due to cross-port leaks.
88
Necessity for More than One Detector
Necessity for More than One Detector -

Sensitivity
e
e
en
en
ne
yr
th
yle
p
ne
an
d)
en
er
or
re
-c
yr
i)p
py
flu
23
p
ne
gh
e)
k)
a)
ne
(1
o(
o(
o(
ne
se
o(
no
le
nz
nz
nz
nz
re
ry
ry
de
Ch
Be
Py
Be
Pe
Be
Be
In
UV-signal
WL 248/411
WL 241/394
WL 270/388
WL 247/504
WL 302/420
Fluorescence
PAH’s extracted from soil;

Sup.LC-PAH 150x4.6mm;
Solv.: H2O/CH3OH= 10:90
21
There are a number of useful detectors for HPLC. While the most popular
detectors are UV-VIS absorbance based, other detectors are needed. UV-VIS
detectors are generally less sensitive than fluorescence or electrochemical based
detectors.
89
Necessity for More than One Detector -

Selectivity
Flecainide in
Serum
UV signal
FL signal
Therapeutic concentration: 1.8mg/l, 20ul injected

UV and fluorescence signal
22
From time to time, it may be necessary to identify a trace component in a complex

matrix. An instrument such as a fluorescence, electrochemical, or mass selective
detector may be required to effectively quantitate the sample component when it
cannot be separated from other components chromatographically. The selective
detector can be programmed for a specific property of a compound or compound
class.
90
Necissity for More than One Detector
Necissity for More than One Detector
Necessity for More than One

Detector - Qualitative Information
Qualitative Information
Chlortoluron ?
Atrazine ?
Take peak spectrum (UV) Take peak spectrum (MS)
200
58
215
44
172
68
132 138 158

96 104
60 80 100 120 140 160 180 200 220
Wavelength (nm)
Mass/Charge
23
Often, a detector is needed which will help identify unknown compounds. For
gas chromatographic analysis, the mass spectrometer is such a detector.
Qualitative analysis is not yet that routine in HPLC. The diode array, mass
spectrometer and infrared detectors, however, are becoming increasingly useful.
91
UV-VIS Detectors
UV-VIS Detectors
Beer’s Law
Absorbance Io
log _____ = A = abc
Detectors I
Fixed
Wavelength
Flow I o = Incident Radiation Intensity
Cell
Light Slits
Source I = Transmitted Radiation Intensity
I
A = Absorbance
Lenses a = molar absorptivity

Io
Detector b = path length
Elements
c = solute concentration
24
UV-Vis detectors are the most commonly used detectors in HPLC. Solutes which
absorb UV or visible radiation (typically 190 - 600 nm) can be detected. The
degree of absorption is a function of the molar absorptivity of the sample
molecule, the path length of the detector flow cell and the solute concentration.
The solute concentration is directly proportional to the absorbance allowing
quantification. UV-Vis detectors can routinely achieve detection of only a few
nanograms. They have a large linear dynamic range and are very robust.
92
UV-VIS Detectors
UV-VIS Detectors
Chromophores
Chromophore Structure max(nm)
Amine -NH2 195

Ethylene -C=C- 190
Ketone C=O 195
Ester -COOR 205
Aldehyde -CHO 210
Carboxyl -COOH 200-210
Nitro -NO2 310
Phenyl - 202,255
Naphthyl - 220,275
25
When utilizing a UV-Vis spectrophotometer, it is often advantageous to work at

the absorbance maximum of a component or compound class being analyzed. It
is more important, however to work at the wavelength which will provide you
with the best possible signal to noise ratio. Make certain that background
interferences, such as absorbance of mobile phase component, do not degrade
your signal to noise. While many compounds absorb at or near 254 nm, it is
advantageous to have a variable wavelength or diode array detector which can be
adjusted to monitor multiple wavelengths either in sequence or simultaneously.
93
UV-VIS Detectors
UV-VIS Detectors
Variable Wavelength Detector
26
This type of UV-Vis spectrophotometer allows sequential monitoring of any

wavelength between 190 and 600 nm. A deuterium lamp emits a continuous
spectrum from 190-600 nm. The chosen wavelength of light passes through the
flow cell after being mechanically determined by the grating. Most variable
wavelength detectors are time programmable and you may also obtain a UV
spectrum of a component of interest by stopping the flow and trapping the
component in the flow cell, then rotating the grating. Some variable wavelength
detectors may also have a tungsten lamp for radiation from 340-850 nm.
94
UV-VIS Detectors
UV-VIS Detectors
Diode Array Detector
Vis
Lamp
Achromatic
Lens
Diode Array
Detector
Flow Cell
UV
Lamp
Homium
Filter
Grating
Optical
Slit
27
The diode array detector can provide detection at a single wavelength or

simultaneously at multiple wavelengths. This detector also has the ability to store
spectra for peak purity analysis, library searching, and creation of extracted
signals. This is the schematic for an HP 1100 diode array. The combined
tungsten and deuterium lamps emit radiation from 190-850 nm. The radiation is
collimated through the flow cell, then a mechanically controlled slit. The
radiation is dispersed at the holographic grating into individual wavelengths of
light. Each photodiode receives a different narrow wavelength band. A complete
spectrum is taken approximately every 12 ms and spectra and signals are created
and stored.
95
UV-VIS Detectors
UV-VIS Detectors
Diode Array Spectral Capabilities
Graphical and Numerical Results
Spectra
Purity Match Purity Match

764 999
Three dimensional data allows one to:
200
• Perform peak purity.
• Search user-created libraries.
Wavelength (nm) 400 200 Wavelength (nm) 400
• Recreate signals from stored

Signal
spectra.
impure pure
28
Three dimensional data can be very useful to the analyst. In peak purity analysis, spectra across
the peak are compared with an average spectrum from the peak. If the data correlates well, a high
purity factor is reported. If the data does not correlate well, then the peak is considered impure.
With the availability of spectra, one can also compare spectra of unknown chromatographic peaks
with those of known stored library spectra and identify the unknown. Finally, if enough spectra
are stored during a chromatographic run, a chromatogram can be produced from any signal
selection within the limits of the spectral collection. These chromatograms are called extracted
signals.
96
UV-VIS Detectors
UV-VIS Detectors
Worksheet
OCOCH3
Would UV-detection be suitable for:
a) Separation of polyvinylacetate C C
polymers.
COOR
b) Separation of phthalates.
COOR
c) Inorganic anions.
d) Separation of triazine pesticides. SCH3
N N
CH
C H NH 3
2 5 NHCH
N CH
Ametryn 3
29
97
Fluorescence Detection
Flow
Cell
Excitation
Light
Source
Emission
Variable Excitation
and
Emission
Wavelengths
Photomultiplier
Tube
30
The fluorescence detector is a highly sensitive and specific detector for HPLC. A
1000 fold increase in sensitivity over UV detection is possible. About 20% of
compounds can naturally absorb UV radiation becoming excited and subsequently
emitting radiation at a lower energy and longer wavelength than the excitation
energy. Many others can be made to fluoresce through derivatization. Radiation
from a deuterium or xenon source is focused onto the first grating. This grating is
rotated so that only the appropriate wavelength will focus upon the flow cell. The
sample fluoresces and radiation is emitted in all directions. The emission
radiation is only measured, however, 90 degrees to the incident radiation away
from any interfering stray light. The fact that both the excitation and emission
wavelengths are specific makes this detector quite suitable for trace analysis in
complex matrices.
98
Derivatizing Agents
Functional Group Reagent
-NH o-Phthalaldehyde
2
-NHR 9-Fluorenylmethylchloroformate
-COOH p-Bromophenylacylbromide
2-Naphthacylbromide
-OH Phenylisocyanate
-CHO,=C=O 2,4-Dinitrophenylhydrazine
-CO-COOH 2,4-Dinitrophenylhydrazine
31
The above is a list of common derivatizing agents and what functional groups
react with these agents. Derivatizing agents are useful when samples do not
naturally fluoresce, but excellent detection limits are desirable. This list is not
complete.
99
Pre-Column Derivatization
Pre-Column Advantages
Derivatization • Reaction conditions are freely
chosen.
Reagent Sample
• Derivatization reaction can occur
slowly.
• Derivatization can serve as a
purification step.
Mixing • Excess reagent can be removed.
Disadvantages
• Artifacts and multiple peaks can
occur.
• Reaction must be very reproducible.
Heated up to • Separation may be more difficult.
99 C
32
Derivatization may be applied pre or post-column. Pre-column derivatization

may be carried out on-line as shown or off-line. One of the advantages of pre-
column derivatization is that the reaction can occur slowly. In post-column
derivatization, the reaction must occur as the mobile phase flows through tubing
from the end of the column to the detector. The excess tubing in post-column
derivatization can lead to band broadening. An advantage of post-column
derivatization is that you do not have to separate excess reagent and other
products from the sample. An example of pre-column derivatization is amino
acid derivatization with OPA. An example of post-column derivatization is
carbamate analysis.
100
Refractive Index Detection
Refractive Index Detection
Refractive Index Detectors
Reference Sample
Beam Deflection Cell Cell
Fresnel Prism
Laser Interferometer
Photodiodes
Sample
Reference
Sample
Reference
33
The refractive index detector is one of the most universal LC detectors. Anything
that changes the refractive index of the mobile phase can be detected. It is also
one of the least sensitive LC detectors. Refractive index detectors must always be
thermostatically controlled as the refractive index will change with temperature.
The most common type of refractive index detector is the beam deflection device.
The Fresnel prism can be used for microbore work. The laser interferometer is
the most sensitive but can be the least reliable.
101
Light Scattering Detection
Light Scattering Detection
Light Scattering Detector
Detector 2
Detector 1 Detector 3
k*c = 1 + 2A2 C
R(θ) Mw P(θ)
R (θ): excess intensity of

scattered light at angle θ
c: sample concentration
Solvent Mw : weight average
molecular weight
Laser Beam A : second virial coefficient
z
K*: optical parameter
Absolute weight 4∏2 n2 (dn/dc)2 / λ4 N ,
and size of 0 A
n: refractive index
molecule may Glass Cell
be calculated
from scattered dn: refractive in increment
light as a dc
function of
angle
34
Laser light scattering detectors allow the absolute molecular weight determination
of polymers and biopolymers from MW 1000 to hundreds of millions. The
polarized laser beam passes through the flow cell. The sample scatters light at all
angles. Detectors placed around the flow cell receive the scattered light.
Absolute molecular weight data calculations are then performed by the computer
based upon the equation above.
102
Electrochemical Detection
Electrochemical Detection
Electrochemical Detectors
Reference
Electrode
Auxillary
Electrode Current
µ Amps
Flow
Cell
Limiting
Current
Mobile X Y X Y
Phase e-
Diffusion
e-
Current
-1.5 -1.0 -0.5
0.5 1.0 1.5 V

Working V
Electrode Residual
Current
Reduction Oxidation
Potentiostat
Wave Wave
35
Electrochemical detectors are sensitive devices which can detect traces of readily
oxidizable or reducible compounds. The detector flow cell has three electrodes: a
reference electrode, working electrode, and an auxiliary electrode. The potential
between the working and auxiliary electrode may be selected based upon a
voltammogram where the optimum voltage can be determined. The reference
electrode provides a stable and reproducible voltage to which the potential of the
working electrode can be referenced.
103
Conductivity Detection
Conductivity Detection
Conductivity Detectors
Schematics Applications
F
water
r fixed soap products
cell
resistor
detergents
C Ions
Balance
A ref.capacitor control
D E
Acids in
Bases } soft drinks
blood
Salts
plating baths
nuclear fuel reprocessing
streams
B
~
variable
resistances
36
Conductivity detectors are most commonly used for detection of inorganic and
organic ions usually after ion exchange chromatography. This detector measures
the conductance of the mobile phase. The sensitivity of the detector is largely
dependent upon the initial conductance of the mobile phase.
104
HPLC-MS
HPLC-MS
HPLC-Mass Spectrometry
Interfaces
Particle Beam
Thermospray
Total Ion
Continuous Flow FAB
Chromatogram Electrospray
252
Selected Ion
Full Monitoring
Scan
126
113
111
200 224
37
The mass spectrometer is a potentially powerful detector for liquid

chromatography. The most common LC-MS interfaces include the particle beam
interface, continuous flow FAB, thermospray, and electrospray. The beauty of
the mass spectrometer is its ability to provide molecular weight information and
sometimes, structural information.
105
HPLC-MS
HPLC-MS
Electrospray LC-MS
38
Electrospray combined with API and APCI is currently the most promising LC-
MS technique. Eluent is injected through a stainless steel capillary which is held
at 4 to 6 kV relative to a cylindrical electrode. The ions are desorbed from
charged droplets and transported into the mass spectrometer. The ions typically
have multiple charges. As a result, quadrupole mass spectrometers which
measure the mass to charge ratio can be used for detection of high molecular
weight compounds.
106
Radiometric Detectors
Disintegration
14 C-Methionine 12 C-Methionine
PPO
- POPOP
Scintillator
Naphthalene
Dioxane
Fluorescence
Event
End-on End-on
PMT Coincidence PMT
Electronics
Multi-Channel
Analyzer Computer
39
A radiometric detector monitors the amount of radioactivity in the mobile phase.

Prior to analysis, the analytes are labeled with radioactive isotopes such as 14C.
The radioactive isotopes undergo a disintegration to 12C along with the production
of a beta particle. The beta particle interacts with the scintillator and the
scintillator releases energy in the form of fluorescence. The fluorescence is
followed with end-on photomultiplier tubes.
107
Worksheet
Worksheet
Worksheet
1. What is the difference between a dual-piston parallel and a dual

piston series pump?
2. How does a ball valve work?
3. Describe the types of tubing used in HPLC instrumentation.
40
108
Worksheet
Worksheet
Worksheet
1. Name some of the maintenance considerations for an auto-

injector.
2. Overall, which LC detectors are known for their sensitivity?
3. Which LC detectors are known for their universal nature?
41
109
Worksheet
110
HPLC Troubleshooting
• Basic Maintenance and Troubleshooting of the Solvent

Delivery System, Injection System and Detection System
• How to Troubleshoot Baseline Performance Problems
• Causes of Peak Shape Performance Problems
112
Record Keeping
Record Keeping
Record Keeping
Tests for HPLC Columns
The Standard Chromatogram For a newly acquired column perform and record the following
under isocratic conditions:
1. Record theoretical plates, N, accompanied by:

Length and internal diameter of column
Sample compound and k’ value
Mobile and stationary phase
Mobile phase flow rate
Sample and size
The Logbook
Temperature
2. Peak Symmetry.
Date Logbook Service
3. Include phenol and amine } for testing against acids and
11/2/94 Check Valves
bases.
12/1/94 Pump Seals 4. Record column pressure.
12/4/94 New Column
Record:
Analyses Performed
Service Dates
A record of HPLC instrument maintenance should be chronicled in order to

facilitate timely repairs and maintenance. From this record, one can reliably
predict the need for such preventive maintenance as pump seal or ball-valve
replacement. Chromatographers should have at their disposal a reliable test
mixture to use when there is a need to distinguish between method problems and
instrument failures. The inclusion of a weak acid and base can test the acidity of
reversed-phase columns.
113
Proper Care of the HPLC
pH Range
■ Instrument pH range 2.3 - 9.5

■ Extended pH range 2.3 - 12.5
Corrosive to stainless steel
■ Hydrochloric acid
■ Inorganic acids and strong acids
■ Alkali halides (sodium chloride, lithium iodide)
■ Carbon tetrachloride with 2-propanol or THF
■ Complexing agents (EDTA, citric acid, acetic acid)
Attacks quartz and vespel

■ Alkaline solutions, pH>11
These substances are not recommended. If used, the pump and other parts of the HPLC should be
thoroughly flushed when analyses are completed.
HPLC instrument manufacturers will specify the permissible mobile phase pH

range of their instrument. The ranges here are for HP instrumentation. The
extended pH range kit should be acquired when basic pH’s are required. Use of
the listed mobile phase additives will necessitate more frequent maintenance.
When additives such as these or buffers are used within the HPLC, make certain
that the flow path is flushed before the instrument is shut-down.
114
Peak Retention Time and Precision
Peak Retention Time and Precision
Peak Retention Time and Area Precision
Peak retention time precision:

with oven: < 0.3%
without oven: < 0.7%
Peak area precision: <1.5%
Causes of Peak retention time and area irreproducibility:
_____________________________________________
_____________________________________________
A convenient way to assess instrument pump performance is to monitor peak

retention time and area precision. Shown above are typical relative standard
deviations for the 1090. If a known analysis begins to have larger than normal
deviations, pump maintenance may be required.
115
Common Pump Problems
Common Pump Problems
Common Pump Problems and Maintenance
High pressure
diaphragm pump
• Air Entrapment
Mixing chamber
and compliance – Degas Mobile Phase
– Prime the Pump
Switching Diaphragm
• Check Valves
valve
• Piston Seals
Check
valves
• Pistons
Pistons Piston
seals
Damping unit • Leaks
• Switching Valves
• Diaphragm
Dual syringe
metering pump
The most frequently experienced pump related problem revolves around periodic
baseline disturbances which can be attributed to air in the pump itself. This sort
of problem is quickly resolved by sparging the mobile phase and repriming the
pump. Other pump problems include leaking metering pump seals, scratched
pistons, damaged ball-valves and in-line filter blockage. A diaphragm pump may
experience a leaking diaphragm noted by the presence of a milky eluent or oil
around the pump.
116
Pressure Problems
Pressure Problems
Troubleshooting Pressure Problems
Pressure Problem
Pressure T oo High Pressure T oo L ow Pressure F luctuations
● L oosen capil lary at col umn i nlet ● Make certain it is the correct ● Degas and reprime the pump.
frit. mobi le phase. ● Check for loose or improperl y
● Check the injector to needle seat ● T ighten loose fittings or replace seated fittings.
capillary. fi ttings. ● Check metering pump seal s.
● Keep breaking the system in half ● Change metering pump seals. ● Check solvent inlet frits.
until the bl ockage is found. ● Change or clean clogged
solvent inlet frit.
When the system pressure exceeds the maximum pressure the liquid
chromatograph will shut down. System high pressure is usually a result of
particle build up on the column inlet frit, on a filter installed after the injector or
on a guard column’s inlet frit whichever is in-line first. The second most likely
blockage point is the needle seat capillary, the first restriction after injection. If
the blockage is not found then systematically break the system in half until it is
found. Lower pressure than normal is usually the result of a leak in the system,
either at a fitting or metering pump seal. A clogged solvent inlet frit may also
produce such an error as not as much solvent as usual can be pulled out of the
reservoir. Fluctuating system pressure is the result of a leak, blocked solvent inlet
frit or air in the pump.
117
Baseline Fluctuations
Pr oblem:
T he chr omatogram has baseline fluctuations.
I s problem in the detector or pump?
How would you tell?
There are many causes for baseline noise or wander. One of the first things that
you should do is isolate the cause to the detector or pumping system. This task is
easily accomplished. Monitor the chromatographic signal with the detector and
pump on. Then, turn off the pump. Does the noise remain? If the noise goes
away, the problem is in the pumping system or the problem is with your method.
If the noise remains, the problem is probably in the detector. The chromatogram
above is the result of air in the pump.
118
Noisy Baseline
Noisy Baseline
Noisy Baseline: Pump Problem
• Have you changed your mobile phase composition?
• Have you changed your acquisition wavelength?
• What mobile phase was last used in your instrument?
• Do you have a miscibility problem?
• Are your solvents dirty?
If you are experiencing a noisy baseline, ask yourself the questions above. All of
these problems are related to the method not instrument problems. If you have
just changed the mobile phase then the new solvent may contain contaminants.
Noisy baselines may also be the result of immiscible solvents.
119
Mixing Problems
Mixing Problems
Mixing Problems
Problem:
A UV-absorbing mobile phase is dynamically

mixed with a non-UV-absorbing mobile phase.
Residual pump noise is produced. A static
mixer reduces the noise level.
Drawback:
Addition of delay-volume.
Static Mixer
When a highly UV-absorbing mobile phase is mixed with a UV-transparent

mobile phase, a noisy baseline can result. The baseline noise is a result of
inadequate mixing. This same problem can occur when two mobile phases are
slightly immiscible such as the TFA and water/acetonitrile mixtures. The addition
of a static mixer can decrease noise by producing adequate mixing. The static
mixer will add an additional delay volume which should be taken into
consideration.
120
Manual Injection Valve
Manual Injector Valves - Six Port Fixed Loop
L oad I nject
Common Problems: Sample

Syringe
To
■ Improper syringe size or type Waste
■ Valve Blockage
■ Injection-port leakage
■ Sample carryover
■ Cross-port leaks
To
Column To
Column
Sample Loop
From (Fixed Volume)
Pump
There are several common problems experienced by manual injection valve users.
Blockages are common unless the operator carefully rinses the sample loop at the
end of usage. Sample carryover can occur unless the sample loop is flushed
between injections. Five times the sample loop volume should be injected into
the manual valve in order to avoid concentration gradients within the sample.
With time, the rotor seal within the injection valve will develop cross-port leaks.
When this occurs, the injection volumes, thus area and peak height will not be
consistent. At this point, the rotor seal will have to be replaced. GC syringes
(sharp tip) should not be used for lc injection, as they will scratch the rotor seal.
121
Auto-Injectors
Auto-Injectors
Auto-Injectors: Common Problems and

Maintenance
Problems: Maintenance:
Sample related Needle seat
• Blockage of replaced when
needle or necessary
tubing Rotor or seals in
when auto-sampler
samples valve replaced
are not pre- when necessary.
filtered. Regular cleaning
• Lack of of optical sensors
peak light to ensure
reproducibil alignment.
ity when a Syringe washes
density and was cycles
gradient observed.
Six-Port forms in the
Waste vial.
Switching
Needle:
Valve • Needle
blocked
from
Column Pump septum.
• Bent
Needles
Common maintenance of auto-injectors includes needle and needle seat

replacement, rotor seal replacement in valves, and syringe seal replacement.
Always filter samples in order to avoid needle and capillary blockages. Perform
individual instrument requirements regarding syringe washes.
122
Good Column Practices
• Filter solvents before use.

• Pre-treat samples which contain strongly retained components of no
interest.
• Flush column frequently with strong solvent.
• Avoid extreme column temperatures > 60 C.
• Keep the mobile phase pH between 3 and 7. If operating outside of
this pH range use a pre-column.
• Use fresh buffer solutions and aqueous mobile phases or treat them
with sodium azide.
• To prepare column for storage, purge column of buffers and leave
in appropriate solvent. Cap tightly.
• Avoid physically mishandling columns: banging, dropping or over-
tightening fittings.
Good column practices will preserve the lifetime of your column. Always filter
your solvents and samples to remove particulates. Remove strongly retained
sample components with solid phase extraction prior to injection. Store columns
tightly capped with appropriate mobile phases. Silica based columns should only
be used between the pH range of 2 to 8 and temperatures below 80 degrees C.
Try not to pressure or solvent shock your column.
123
Column Frit Replacement
Old
fr it New
F r it
1. Carefully remove
the column frit.
2. Make a slurry of 4. Place a new frit on

stationary phase. top of the mound.
3. Using a Pasteur 5. Replace column end

pipette, mound the fitting.
stationary phase on top of
the column.
The frit on the inlet side of the column may become clogged causing higher than
normal pressure readings. At that point, you may decide to replace the column or
UHSODFHWKHFROXPQLQOHWIULWDVWDLQOHVVVWHHO PSRUHVL]HGLVNZKLFKKROGVWKH
column material in place. To replace the frit, make a slurry of the same packing
material that is currently in the column. Using a Pasteur pipette, mound the
packing material at the top of the column. Place the new frit on top of the packing
material and replace the column end fitting. Realize that the column will never be
as good as it was when new, however performance should improve.
124
Column Regeneration
Column Regeneration
Column Regeneration
Problem:
After prolonged use or insufficient precautions
the column will be fouled by
build-up of adsorbed materials.
Reversed-Phase: Silica Gel:
75 mL water + 4 x 200 ml injections DMSO 75 mL THF

75 mL methanol 75 mL methanol
75 mL chloroform 75 mL aqueous 2% acetic acid
75 mL methanol 75 mL aqueous 2% pyridine
75 mL THF
75 mL methylene chloride
Wash with next mobile phase
When strongly adsorbed components have become attached to the surface of the
stationary phase you may see a degradation in peak shape and resolution. The
column may be restored through the column regeneration procedures listed above.
Procedure frequency is dependent on sample components.
125
Detector Performance
Baseline Noise Determination F or Refer ence
mAU
T ime
Record width of baseline in mAU or RI units for later comparisons.
In order to chronicle detector performance you may record the relative standard
deviation of the baseline. Excessive baseline noise may be attributable to lamp
decay, dirty flow cells or other correctable problems.
126
Detector Time Constant
Detector Time Constant
Detector: Time Constant
T ime Constant
T oo
Mor e High I nfor mation
F ilter ing lost
T oo T oo
L ess L ow much
F ilter ing infor mati on
T ime
(min.)
The detector time constant must be set properly or one of two problems may
occur. If the detector time constant is set too high, there will not be enough data
points to adequately define the chromatographic peak shape so area and retention
time data will suffer. If the detector time constant is set too low, then excessive
noise will result because signal averaging is too frequent.
127
Detector Heat Exchangers
Detector Heat Exchangers
Detectors: Heat Exchangers
Enhance detector performance by ensuring constant mobile phase

temperature in flow cell.
with heat
exchanger
without heat
exchanger
T ime
(min.)
Americas’ Technical Center
Excessive baseline noise may result when an application utilizes a high column
temperature along with a high column flow rate. The noise results when the
mobile phase has not come to temperature equilibrium before entering the flow
cell. Refractive index changes occur causing noise as the mobile phase cools. A
heat exchanger before the detector flow cell can remedy this type of noise. The
heat exchanger is simply a capillary welded into a metal block.
128
Noisy Baselines
Noisy Baselines
Noisy Baselines
T ime
(min.)
Possible Causes:
■ Dirty Flow Cell

■ Detector Lamp Failing
■ Pulses from Pump if Periodic
■ Temperature Effects on Detector
■ Air Bubbles passing through Detector
Common detector problems include poor sensitivity, drift, and high frequency
noise. Poor sensitivity can be a result of dirty solvents or flow cells, improper
detection wavelength or a failing detector lamp. Drift occurs when columns are
not yet equilibrated or when the lamp has had insufficient time to warm up. High
frequency noise can be a result of line voltage problems.
129
Drifting Baselines
Drifting Baselines
Drifting Baselines
Gradient Elution
Temperature Unstable (Refractive Index Detector)
Contamination in Mobile Phase
Mobile Phase Not in Equilibrium with Column
Contamination Bleed in System
Drifting baselines are common while performing a gradient analysis due to the
changing composition of the mobile phase. In other situations, drifting baselines
indicate that a column is still equilibrating or the detector is warming up.
Contamination problems may also be a factor.
130
Ghost Peaks
Ghost Peaks
Ghost Peaks
60 Ghost Peaks - Peaks which appear even when no sample is

injected.
15 Problem - Dirty Mobile Phase
30
15
0
3 7 15 17
20% - 100%
MeOH Gradient
No Sample Injected
If ghost peaks (peaks which do not result from your sample) appear during
gradient analyses the problem can usually be traced to unclean mobile phases,
particularly water. At the beginning of a gradient run, impurities in water may
stick to the column and the concentration of the impurity is enriched. During the
gradient, a stronger mobile phase is introduced onto the column and the impurities
begin to elute creating unwanted chromatographic peaks.
131
Extra-Column Dispersion
Increasing Extra-Column Volume
■ Use short, small internal diameter tubing between the injector and the
column and between the column and the detector.
■ Make certain all tubing connections are made with matched fittings.
■ Use a low-volume detector cell.
■ Inject small sample volumes.
Excessive extra-column dispersion will cause a loss of resolution. Extra-column

dispersion is a result of too much tubing or internal diameters which are too large.
The flow cell may also cause excessive extra-column dispersion when it has a
large volume. Large injection volumes may also cause a loss in resolution. The
maximum injection volume is dependent upon the internal diameter of the
column.
132
Peak Shape
Peak Shape
Peak Shape: Doublets
Void Volume in Column
Normal Doublet Peaks
■ Void Volume in Column

■ Partially Blocked Frit
■ Only One-Peak a Doublet- Coeluting Components
If all peaks in your chromatogram appear to have some form of doublet

appearance then the cause is usually associated with the column or instrument.
As the silica gel dissolves, the packing material may settle creating a void in the
column. The column void can produce poor peak shapes including tailing or
doublets. For small bore and microbore columns, the inlet frit may clog without a
large change in pressure resulting in the formation of doublet peaks. When just a
few or one peak in the chromatogram appears to have a doublet appearance, the
cause can be attributed to a co-eluting peak.
133
Peak Shape
Peak Shape
Peak Shape: Broad Peaks
• All Peaks Broadened:

– Loss of Column Efficiency.
– Column Void.
– Large Injection Volume.
– High Viscosity Mobile Phase.
• Some Peaks Broadened:

– Late Elution from Previous
Sample.
– High Molecular Weight.
– Sample - Protein or Polymer.
As an HPLC column ages, the chromatographic peaks will broaden. When the
resolution is no longer acceptable, the column will have to be discarded. Other
causes of broad chromatographic peaks include high viscosity mobile phases and
large injection volumes. If just one peak in the chromatogram appears broad it
may be a late eluter from an earlier injection.
134
Peak Shape
Peak Shape
Peak Shape: Tailing Peaks
Causes
Symmetry > 1.2
Some Peaks Tail:
Secondary - Retention Effects.
Residual Silanol Interactions.
Small Peak Eluting on Tail of Larger Peak.
Normal Tailing
All Peaks Tail:
Extra-Column Effects.
Build up of Contamination on Column
Inlet.
Heavy Metals.
Bad Column.
Normal Tailing
In reversed-phase liquid chromatography, interaction between weak acids and

weak bases with residual silanol groups can cause tailing. The poor peak shape
can be controlled with the proper pH or with the addition of a modifier such as
triethylamine to prevent weak base tailing. If all peaks in the chromatogram
appear tailed, the peak shape has resulted from a problem with deterioration of the
column or because of extra-column effects.
135
Peak Shape
Peak Shape
Peak Shape: Fronting Peaks
2000
1500
mAU
1000
500
0
0 5 10 15 20 25
Time (min)
Normal Fronting
Symmetry < 0.9
Causes:
Column Overload
Small Band Eluting Before Large Band
Most peaks which have a fronting appearance are the result of mass overloading.
In addition to the peak shape, an overloaded peak will also have a slight retention
time shift to an earlier retention time (usually around 10%). Coelution of
chromatographic peaks will also cause fronting when a small peak elutes just
before a large peak.
136
Peak Shape
Peak Shape
Peak Shape: Negative Peaks
Normal Negative
Causes:
■ Absorbance of sample is less than the mobile phase.
■ Equilibrium disturbance when sample solvent passes through the
column.
■ Normal with Refractive Index Detectors.
The presence of negative peaks is not usually something to be concerned about.

Negative peaks will occur in UV detection when the sample absorbs less than the
mobile phase. You may also see a negative peak when the sample solvent passes
through the detector. Negative peaks are normal with refractive index detection.
137
Worksheet
Worksheet
Worksheet
1. Suggest a possible cause for the following non-ideal

chromatogram:
mAU
Time
(min)
The presence of negative peaks is not usually something to be concerned about.

Negative peaks will occur in UV detection when the sample absorbs less than the
mobile phase. You may also see a negative peak when the sample solvent passes
through the detector. Negative peaks are normal with refractive index detection.
138
Worksheet
Worksheet
Worksheet
1. Suggest a possible cause for the following non-ideal

chromatogram:
mAU
TIME (min)
139
Worksheet
Worksheet
Worksheet
Suggest reasons for the following problems:
1. Flow rates are correct, check valves are working properly, but early
peaks in gradient elution do not have reproducible retention times:
2. Baseline is very irregular - high general noise level:
3. Baseline has systematic periodic noise:
140
Worksheet
Worksheet
Worksheet
Suggest reasons for the following problems:
1. With a UV detector, height is reproducible, but area and retention

times are not:
2. Height and area are not reproducible but retention times are:
3. Reproducibility is good, test mixture looks good but some sample

peaks are broad and tailed:
141

HPLC Theory

Încărcat de

Informații document

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

HPLC Theory

Încărcat de

Drepturi de autor:

Formate disponibile

Practical High Performance

Mass Spectrometry Data Systems

Manual Part Number H5930A-90000

Agilent Technologies, Inc

 2000 by Agilent Technologies, Inc.

INTRODUCTION TO HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ..............1

In This Section You Will Learn:

In This Section, You Will Learn

• The Historic Progression of Liquid Chromatography

• 1906 - Mikhail Semenovich Tswett Mg

• 1940’s - Partition and Paper CH

M.S. Tswett. Ber. Dtsch. Bot. Ges. 24: 384-393 (1906)

• Modern separation science began at the turn of the century with M.

Chromatography is a separation process in which the components to be separated

Typical Column Packing Support

Typical Column Packing Support

Silica gel is commonly used as a stationary phase in adsorption chromatography

Typical Column Supports – Styrene DVB

Typical Column Supports - Styrene DVB

CH = CH 2 CH = CH 2 - CH 2- CH - CH2 - CH - CH2 - CH - CH2 -

CH = CH 2 - CH 2- CH - CH2 - CH - CH2 - CH - CH2 -

Cross-linked polystyrene is made from the copolymerization of styrene and

Modes of Liquid Chromatography

Modes of Liquid Chromatography

Types of Compounds Mode Stationary Phase Mobile Phase

Neutrals Reversed Phase C-18, C-8, C-4, C-2 Water/Organic

Compounds insoluble Normal Phase Silica, Amino, Cyano Organics

High MW Compounds Size Exclusion Polystyrene Silica Gel Filtration-

Differences in Gas and Liquid Chromatography

Differences in Gas and Liquid Chromatography

Only about 20% of known organic

Solute Molecular Weight

Gas Chromatography Liquid Chromatography

The components of a high performance liquid chromatograph include: solvent

W = peak width at base

Sample components typically produce gaussian shaped peaks. Components

• Column Stationary Phase Selection

The resolution of chromatographic peaks can be controlled by the selection of

1. Name three differences in gas and liquid chromatography.

2. What is the most widely used mode of HPLC?

3. What mode may be used for separation of ions in solution?

1. Name the parts of an HPLC instrument.

2. What is the symbol for an unretained component’s elution

3. What is the difference between normal and reversed phase?

In This Section You Will Learn:

In This Section, You Will Learn

• What factors influence the resolution between sample

The Goal of Separation

The Goal of Separation: Resolution Between

The most important goal of the chromatographer is to achieve adequate resolution

Tools for Achieving a Separation

Tools for Achieving a Separation

The experimental variables available to the liquid chromatographer to achieve

0.8 1.00 1.25

For equal peak areas, R of 1.5

The graphic above illustrates the resolution values for overlapping

Calculate a Resolution Value

Calculate a Resolution Value

Calculate the resolution between the first two chromatographic

Factors Influencing Resolution

Factors Influencing Resolution

N: Total number of theoretical plates available; column efficiency.

The degree of resolution between two chromatographic peaks is dependent upon

Capacity, selectivity and efficiency are illustrated graphically in the