ABSTRACT
Pavel Jandera University of Pardubice, Na´m. Cˇs. legii, Pardubice, Czech Republic
I. INTRODUCTION 3
II. THEORY OF RETENTION IN ANALYTICAL
GRADIENT-ELUTION CHROMATOGRAPHY 9
A. Calculation of retention times and of retention
volumes 9
B. Bandwidths and resolution in
gradient-elution LC 17
III. REVERSED-PHASE CHROMATOGRAPHY WITH
BINARY GRADIENTS 19
IV. NORMAL-PHASE CHROMATOGRAPHY WITH
BINARY GRADIENTS 25
V. ION-EXCHANGE GRADIENT ELUTION
CHROMATOGRAPHY 34
VI. EFFECTS OF THE INSTRUMENTATION AND
OF THE NONIDEAL RETENTION BEHAVIOR
ON THE RETENTION IN GRADIENT ELUTION 36
A. Effects of the dwell volume on retention in
gradient elution LC. Retention data in gradient
elution with an initial hold-up period. Gradient
preelution and postelution 37
B. Effects of the adsorption of strong solvents on
retention 48
VII. GRADIENT ELUTION METHOD DEVELOPMENT 55
A. Transfer of gradient methods and effects of
changing operating conditions on separation 56
1. Changing flow rate of the mobile phase in
gradient elution chromatography 58
2. Changing column diameter in gradient
elution chromatography 59
3. Changing column length in gradient elution
chromatography 61
4. Rapid prediction of the effects of changing
gradient steepness (gradient range) and
initial mobile phase composition on the
separation 62
B. Optimization of gradient elution separations 69
1. Peak capacity and fast gradients 69
2. Optimization of gradients for specific
separation problems 71
VIII. CHROMATOGRAPHYWITHTERNARY
GRADIENTS 78
IX. PECULIARITIES OF GRADIENT ELUTION
SEPARATION OF HIGH-MOLECULAR
COMPOUNDS 81
X. CONCLUSION 90
ACKNOWLEDGMENTS 90
SYMBOLS 92
REFERENCES 96
APPENDIX A. Correction of the retention volume
in normal-phase HPLC for the
column uptake of polar solvents
during gradient elution (solvent-
demixing effect) 104
APPENDIX B. Schematics of a spreadsheet program
for optimization of gradient elution 107
Many complex samples contain compounds that differ widely in
retention, so thatHPLC in isocratic elutionmode with amobile phase
of fixed composition often does not yield successful separation of the
individual solutes. To keep the time of analysis within acceptable
limits, the retention factors of the most strongly retained sample
components, k, usually should be lower than 10. Once the appropriate
chromatographic column is selected, the retention can be controlled
by setting appropriate flow rate, column temperature and-most
efficiently-the composition of the mobile phase. In the isocratic
elution mode, the working conditions are kept constant during the
separation run and in many cases satisfactory results are obtained.
However, for some complex samples weakly retained compounds
elute as poorly-if at all-separated bands close to the column hold-
up time under the conditions adjusted for adequate retention of
strongly retained solutes (Fig. 1A). On the other hand, with the
mobile phase adjusted to achieve desired resolution of weakly
retained compounds, the elution of strongly retained sample compo-
nents can be slow, their peaks are broad and their concentration in
the eluate may even fall down below the detection limits (Fig. 1B).
