Practical Problem Solving in Hplc

Quick and easy solutions to HPLC problems! For 45 typical questions of HPLC users, the answer and general conclusions are presented on no more than 4 pages each. The book covers - simple equipment tests and selection criteria for columns, buffers etc. - specific problems and how to solve them - options for optimizing separations. The book also features a special chapter on the retention of ionizable components in RP-HPLC, references, data tables and check lists. It's a first-aid kit for every HPLC user. Stavros Kromidas is managing director of NOVIA, a company offering consulting services and training courses for analytical laboratories. For five years he was sales manager for the HPLC manufacturer Waters in Germany. He is author and editor of numerous books.

Stavros Kromidas and are the authors of Practical Problem Solving in HPLC, published by Wiley.

Introduction

1

(14)

How to use this book

1

(1)

HPLC-the development of a name

2

(2)

Frequently used abbreviations and symbols in this book

4

(1)

General tips for newcomers

5

(5)

Check list for reversed-phase HPLC

10

(3)

Some important chromatographic terms

13

(2)

Simple Tests and Decision Criteria

15

(46)

What does the name of a column material tell us?

15

(3)

Is this C18 column the right choice for my sample?

18

(2)

Why are polar solutes well separated with one C18 column and hardly at all with another?

20

(3)

How can I clean the RP phase quickly?

23

(1)

How best do I degas my mobile phase?

24

(2)

Methanol or acetonitrile?

26

(2)

The pH of the mobile phase to too high/too low-what can I do?

28

(1)

What is the right ionic strength of the buffer?

29

(2)

How to make sense of the dead volume of an isocratic apparatus?

31

(3)

Producing a gradient chromatogram-influence of instrumentation

34

(2)

Does the pump work correctly, precisely or accurately?

36

(3)

How to test an HPLC instrument and its modules?

39

(2)

Injection of solutes as aqueous solutions

41

(2)

What is the largest tolerable injection volume?

43

(2)

How critical are temperature changes? Part I

45

(2)

How critical are temperature changes? Part II

47

(6)

How to choose HPLC equipment and a supplier?

53

(4)

Is the current method a robust one?

57

(4)

Problems and their Solutions

61

(38)

Sample preparation-how critical are which mistakes?

61

(2)

Flushing of an HPLC equipment

63

(2)

Dirt in the UV detection cell

65

(2)

The lamp is new-what happened to the peak?

67

(2)

What are the causes of pressure changes or deviations?

69

(2)

Is the right or the left pump head defective?

71

(1)

Baseline noise and damping

72

(3)

The retention times increase-is it the pump or the mobile phase?

75

(2)

Which buffer is right for which pH?

77

(1)

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

(3)

What can be the reasons for a change in retention times?

81

(2)

I use up a lot of RP columns; what should I do?

83

(2)

Why does my normal-phase system not work any more?

85

(2)

Chemical tailing at the presence of metal ions

87

(4)

How to avoid memory effects?

91

(2)

How do the default values on my PC affect the resolution?

93

(6)

Tips to Optimize the Separation

99

(24)

Which is the right injection technique to get sharper peaks?

99

(2)

My peaks appear too early-how can I move them in an RP system to later retention times?

101

(2)

How can I increase the plate number?

103

(2)

Limit of detection: how can I see more?

105

(2)

How can I speed up a separation?

107

(1)

How can I optimize a separation?

108

(3)

Dead volume, capacity factor, selectivity-how can I use them in everyday life?

111

(2)

Which flow is optimal for me?

113

(2)

How can I optimize a gradient elution?

115

(3)

Separation of ionic solutes: what works out best-endcapped phases, inert phases, phosphate buffer or ion pairing reagents? Part I

118

(2)

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

(3)

About ionizable solutes, sun, and lectures in the afternoon

122

(1)

Retention of Ionizable Components in Reversed-Phase HPLC

123

(36)

Introduction

123

(4)

History

123

(1)

Analyte Ionization

124

(1)

Ionization and HPLC retention in reversed-phase HPLC

124

(3)

Factors that should be considered prior to method development

127

(1)

Method development

127

(10)

General Approach to method development

127

(3)

Basic method development

130

(4)

Method optimization

134

(3)

Conclusion

137

(1)

Method fine tuning

137

(20)

Solvation of the acids

137

(1)

Ionization of the acids

138

(1)

Chaotropic effects

139

(18)

Concluding remarks

157

(1)

References to Chapter 5

158

(1)

Appendix

159

(18)

Some chromatographic and related abbreviations (selection)

160

(3)

IUPAC recommendations for symbols in chromatography (a selection)

163

(1)

Solvent mixtures of equal elution strength for reversed phase chromatography (according to L. Snyder)

164

(1)

UV absorption bands and molar extinction coefficients of some typical chromophores

165

(1)

List of tables

166

(2)

HPLC textbooks

168

(1)

Trends in HPLC

169

(8)

HPLC in routine analysis

173

(1)

HPLC in a research environment

173

(4)

Index

177

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Practical Problem Solving in Hplc

Summary

Author Biography

Table of Contents

Supplemental Materials