Nothing demonstrates the importance and maturity of High Performance Liquid Chromatography (HPLC) more than this compendium of practical wisdom about how to master the complexity of instrumentation and the problems associated with the chemical aspects of the technique.
We shall soon celebrate the centennial of the introduction of chromatography by
T.M. Tswett, who first demonstrated the concept and practice of differential migration processes which have revolutionized analytical chemistry over the past forty years. In the early fifties, gas chromatography lead the way in exploring the tremendous breadth of chromatography and thus the gas chromatograph has become the paradigm of a new era in analytical chemistry. In the late sixties it was followed by HPLC that has become, and still is, the most versatile separating tool using sophisticated instrumentation and a variety of chromatographic systems. Of course, this stems also from the dual nature of chromatography as being not only a precision microanalytical tool, but also an indispensable process for the preparative/production scale purification of biological substances in particular.
After the introduction of HPLC in the late sixties, the technique experienced a meteoric growth and established itself as the leading analytical tool in the pharmaceutical industry. Since then, HPLC has found wide application in all branches of science and technology. Today the worldwide roles of HPLC instruments and supplies amount to over two billion USD, and the market is still expanding further.
The novice may often find the instrument and the bewildering array of columns and eluents nonplussing. Indeed, the complexity is high, but not so high that at the present its use would require an operator who is a highly trained specialist. The erudite books offer little or no help in getting oriented to finding the right one among a half dozen 1/16" ferrules that look almost the same, but, if an inappropriate one is used in a given fitting, it will be ruined. Dr. Kromidas's book is a gold mine of useful tips. This practice-oriented book does not fall short of explaining the reasons underlying the problem, and what is just as important, it voices caveat from the consequences of the mistakes one can commit in trying to gain control over the instrument and the separation process.
The advent of HPLC has not only brought us elaborate instrumentation, but has also made reversed-phase chromatography the leading modality of analytical liquid chromatography. An estimated eighty to eighty-five percent of separations are carried out by using alkyl silica stationary phases. In the seventies reversed-phase chromatography set a new direction to HPLC by dwarfing the significance of ionexchange and normal phase chromatography. As a result, a new generation of chromatographers might think of normal phase as reversed-reversed-phase chromaVII
tography. It is gratifying that Practical Problem Solving in HPLC pays ample attention to the instrumentation, columns, and operation of reversed-phase chromatography
The forty-five families of tips in this book handsomely cover the present scope of HPLC and besides novices, even a seasoned chromatographer can learn a few tricks from it. The author has laid down the links to developments in HPLC which now move forcefully ahead, for instance, the increasing use of the mass spectrometer as the detector for HPLC. However, many other new problems, as well as opportunities, are coming from the employment of high voltage to bring about separations by capillary electrochromatography and by its cousin, high performance capillary electrophoresis. The new techniques require thorough familiarity with classical HPLC, that stays uncontested the chief method of chromatographic analysis, and inspiration and knowledge to master many of the practical aspects in the future ought to come from books like Practical Problem Solving in HPLC . It is concise yet rich in practical information, a combination that would be difficult to find in print elsewhere. It helps everybody to be a better practicing chromatographer and may give relief to many who have difficulties in gaining control over the instrument and the chromatographic process at large.
December 1999 Professor Csaba Horvath Department of Chemical Engineering Yale University, New Haven, CT, USA
1.
Introduction
1.1
How to use this book 1
1.2
HPLC -the development of a name 2
1.3
Frequently used abbreviations and symbols in this book 4
1.4
General tips for newcomers 5
1.5
Check list for reversed-phase HPLC 10
1.6
Some important chromatographic terms 13
2.
Simple Tests and Decision Criteria 15
Tip No.
OJ What does the name of a column material tell us? 15
02
Is this Cl8 column the right choice for my sample? 18
03
Why are polar solutes well separated with one CI8 column and hardly
at all with another? 20
04
How can I clean the RP phase quickly? 23
05
How best do I degas my mobile phase? 24
06
Methanol or acetonitrile? 26
07
The pH of the mobile phase to too high/too low -what can I do? 28
08
What is the right ionic strength of the buffer? 29
09
How to make sense of the dead volume of an isocratic apparatus? 31
10
Producing a gradient chromatogram -influence of instrumentation 34
11
Does the pump work correctly, precisely or accurately? 36
12
How to test an HPLC instrument and its modules? 39
13
Injection of solutes as aqueous solutions 41
14
What is the largest tolerable injection volume? 43
15
How critical are temperature changes? Part I 45
16
How critical are temperature changes? Part II 47
17
How to choose HPLC equipment and a supplier? 53
18
Is the current method a robust one? 57
3.
Problems and their Solutions 61
19
Sample preparation -how critical are which mistakes? 61
20
Flushing of an HPLC equipment 63
21
Dirt in the UV detection cell 65
22
The lamp is new -what happened to the peak? 67
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Tip No.
23
What are the causes of pressure changes or deviations? 69
24
Is the right or the left pump head defective? 71
25
Baseline noise and damping 72
26
The retention times increase -is it the pump or the mobile phase? 75
27
Which buffer is right for which pH? 77
28
An interesting alternative for the separation of acids and bases with
a buffer (for a detailed discussion on the separation of acids and
bases see Chapter 5) 78
29
What can be the reasons for a change in retention times? 81
30
I use up a lot of RP columns; what should I do? 83
31
Why does my normal-phase system not work any more? 85
32
Chemical tailing at the presence of metal ions 87
33
How to avoid memory effects? 91
34
How do the default values on my PC affect the resolution? 93
4.
Tips to Optimize the Separation 99
35
Which is the right injection technique to get sharper peaks? 99
36
My peaks appear too early -how can I move them in'an
RP system to later retention times? 101
37
How can I increase the plate number? 103
38
Limit of detection: how can I see more? 105
39
How can I speed up a separation? 107
40
How can I optimize a separation? 108
41
Dead volume, capacity factor, selectivity -how can I use them
in everyday life? III
42
Which flow is optimal for me? 113
43
How can I optimize a gradient elution? 115
44
Separation of ionic solutes: what works out best -endcapped phases,
inert phases, phosphate buffer or ion pairing reagents? Part I 118
45
Separation of ionic solutes: what works out best -endcapped phases,
inert phases, phosphate buffer or ion-pairing reagents? Part II
(see also Chapter 5) 120
About ionizable solutes, sun, and lectures in the afternoon 122
5.
Retention of Ionizable Components in Reversed-Phase HPLC 123
5.1
Introduction 123
5.1.1
History 123
5.1.2
Analyte Ionization 124
5.1.3
Ionization and HPLC retention in reversed-phase HPLC 124
5.1.4
Factors that should be considered prior to method development 127
5.2
Method development 127
5.2.1
General Approach to method development 127
5.2.2
Basic method development 130
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5.2.3
Method optimization 134
5.2.4
Conclusion 137
5.3
Method fine tuning 137
5.3.1
Solvation of the acids 137
5.3.2
Ionization of the acids 138
5.3.3
Chaotropic effects 139
5.4
Concluding remarks 157
5.5
References to Chapter 5 158
6.
Appendix 159
6.1
Some chromatographic and related abbreviations (selection) 160
6.2
ILTPAC recommendations for symbols in chromatography
(a selection) 163
6.3
Solvent mixtures of equal elution strength for reversed phase
chromatography (according to L. Snyder) 164
6.4
LTV absorption bands and molar extinction coefficients of some
typical chromophores 165
6.5
List of tables 166
6.6
HPLC textbooks 168
6.7
Trends in HPLC 169
6.7.1
HPLC in routine analysis 173
6.7.2
HPLC in a research environment 173
Index 177
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