Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1344, Issue -, Pages 66-75Publisher
ELSEVIER
DOI: 10.1016/j.chroma.2014.04.010
Keywords
Gradient chromatography; Distorted gradients; Excess adsorption isotherms; Methanol; Acetonitrile; Symmetry-C-18
Funding
- University of Tennessee
- Oak Ridge National Laboratory
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The formation of a concentration shock layer of either methanol or acetonitrile used as the modifier in steep RPLC aqueous/organic gradients was predictable from their excess adsorption isotherm and was directly observed from their elution profiles recorded in UV absorption. A discontinuity of the concentration profile of the organic modifier arises and grows along the column. Its effects on the peak shapes and the resolution levels achieved in steep gradient chromatography were measured in the recorded chromatograms of a sample mixture containing 14 different analytes uniformly distributed across the gradient retention window. The results showed severe peak shape deformation for some compounds and a significant degradation of the baseline resolution of weakly (when using acetonitrile) and moderately (when using methanol) retained analytes. Solutions to limit this loss of resolution may lie in changing the initial gradient conditions or in adjusting the curvature of the gradient profile at the column inlet. (C) 2014 Elsevier B.V. All rights reserved.
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