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AAT Bioquest

What is the difference between an isocratic and a gradient elution method?

Posted December 16, 2021


Answer

Isocratic and gradient elution methods are two different modes of separation in high-performance liquid chromatography (HPLC). 

Basis of comparisonIsocratic elution methodGradient elution method
DefinitionIs an HPLC mode in which the concentration of the mobile phase remains constant over the full analysis time.Is an HPLC mode in which the concentration of the mobile phase is changed throughout the analysis.
Retention of componentsRetention of the components is not affected because of the unchanging concentration

Retention of the components is affected because of the varying concentration

Polarity of the mobile phasePolarity of the mobile phase remains the same throughoutPolarity of the mobile phase may gradually increase or decrease throughout the separation process
Effect of column dimensions on selectivity or order of elutionSelectivity or elution order is not dependent on column dimensions. The peaks elute in the same order.Selectivity or elution order changes with changes in column dimensions.

 

Additional resources

Thermodynamic modeling of protein retention in mixed-mode chromatography: An extended model for isocratic and dual gradient elution chromatography

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