Gradient elution demystified

Of the various ways in which chromatography is applied today, few
have been as misunderstood as the technique of gradient elution,
which presents many challenges compared to isocratic separation.
When properly explained, however, gradient elution can be less
difficult to understand and much easier to use than often
assumed.

Written by two well-known authorities in liquid chromatography,
High-Performance Gradient Elution: The Practical Application of the
Linear-Solvent-Strength Model takes the mystery out of the practice
of gradient elution and helps remove barriers to the practical
application of this important separation technique. The book
presents a systematic approach to the current understanding of
gradient elution, describing theory, methodology, and applications
across many of the fields that use liquid chromatography as a
primary analytical tool.

This up-to-date, practical, and comprehensive treatment of gradient
elution:

* Provides specific, step-by-step recommendations for developing a
gradient separation for any sample

* Describes the best approach for troubleshooting problems with
gradient methods

* Guides the reader on the equipment used for gradient
elution

* Lists which conditions should be varied first during method
development, and explains how to interpret scouting gradients

* Explains how to avoid problems in transferring gradient
methods

With a focus on the use of linear solvent strength (LSS) theory for
predicting gradient LC behavior and separations by reversed-phase
HPLC, High-Performance Gradient Elution gives every chromatographer
access to this useful tool.

JOHN W. DOLAN, PHD, is a Principal at LC Resources. He is author of the popular " LC Troubleshooting" column in LCGC Magazine and coauthor with Lloyd Snyder of Troubleshooting LC Systems.

Of the various ways in which chromatography is applied today, few have been as misunderstood as the technique of gradient elution, which presents many challenges compared to isocratic separation. When properly explained, however, gradient elution can be less difficult to understand and much easier to use than often assumed.

Written by two well-known authorities in liquid chromatography, High-Performance Gradient Elution: The Practical Application of the Linear-Solvent-Strength Model takes the mystery out of the practice of gradient elution and helps remove barriers to the practical application of this important separation technique. The book presents a systematic approach to the current understanding of gradient elution, describing theory, methodology, and applications across many of the fields that use liquid chromatography as a primary analytical tool.

This up-to-date, practical, and comprehensive treatment of gradient elution:

• Provides specific, step-by-step recommendations for developing a gradient separation for any sample
• Describes the best approach for troubleshooting problems with gradient methods
• Guides the reader on the equipment used for gradient elution
• Lists which conditions should be varied first during method development, and explains how to interpret scouting gradients
• Explains how to avoid problems in transferring gradient methods

With a focus on the use of linear solvent strength (LSS) theory for predicting gradient LC behavior and separations by reversed-phase HPLC, High-Performance Gradient Elution gives every chromatographer access to this useful tool.

Preface xv

Glossary of Symbols and Terms xxi

1 Introduction 1

1.1 The General Elution Problem and the Need for Gradient Elution 1

1.2 Other Reasons for the Use of Gradient Elution 4

1.3 Gradient Shape 7

1.4 Similarity of Isocratic and Gradient Elution 10

1.4.1 Gradient and Isocratic Elution Compared 10

1.4.2 The Linear-Solvent-Strength Model 13

1.5 Computer Simulation 18

1.6 Sample Classification 19

1.6.1 Sample Compounds of Related Structure (Regular Samples) 19

1.6.2 Sample Compounds of Unrelated Structure (Irregular Samples) 19

2 Gradient Elution Fundamentals 23

2.1 Isocratic Separation 23

2.1.1 Retention 23

2.1.2 Peak Width and Plate Number 24

2.1.3 Resolution 25

2.1.4 Role of Separation Conditions 27

2.1.4.1 Optimizing Retention [Term a of Equation (2.7)] 27

2.1.4.2 Optimizing Selectivity a [Term b of Equation (2.7)] 28

2.1.4.3 Optimizing the Column Plate Number N [Term c of Equation (2.7)] 28

2.2 Gradient Separation 31

2.2.1 Retention 32

2.2.1.1 Gradient and Isocratic Separation Compared for Corresponding Conditions 34

2.2.2 Peak Width 38

2.2.3 Resolution 39

2.2.3.1 Resolution as a Function of Values of S for Two Adjacent Peaks (Irregular Samples) 42

2.2.3.2 Using Gradient Elution to Predict Isocratic Separation 45

2.2.4 Sample Complexity and Peak Capacity 47

2.3 Effect of Gradient Conditions on Separation 49

2.3.1 Gradient Steepness b: Change in Gradient Time 50

2.3.2 Gradient Steepness b: Change in Column Length or Diameter 51

2.3.3 Gradient Steepness b: Change in Flow Rate 55

2.3.4 Gradient Range : Change in Initial Percentage B (₀) 58

2.3.5 Gradient Range : Change in Final Percentage B (_f) 60

2.3.6 Effect of a Gradient Delay 63

2.3.6.1 Equipment Dwell Volume 66

2.3.7 Effect of Gradient Shape (Nonlinear Gradients) 67