Isocratic vs. gradient

An excellent and comprehensive review has covered HPLC analysis of AOs and light stabilisers up to 1990 [576]. Normal vs. reversed-phase and isocratic vs. gradient-elution HPLC separation of synthetic mixtures of additives and of solvent extracts from polymers were discussed. 

Romanyshyn, L. Tiller, P. R. Alvaro, R. Pereira, A. Hop, C. E. Ultra-fast gradient vs fast isocratic chromatography in bioanalytical quantification by liquid chromatography/ tandem mass spectrometry. Rapid Commun Mass Spectrom 2001, 15, 313-319. 

Analytical methods for the determination of one antidepressant and/or its metabolite(s) were usually performed in isocratic mode, with total run times from seconds to a few minutes. However, as previously mentioned, multianalyte procedures are preferable, particularly if the method is intended for clinical or forensic analysis. Gradient separation was usually applied when the most common antidepressants were included in the methodology however, total chromatographic run times varied widely, from 5 to 40 min [57, 76], depending on column length, extraction technique (offline vs. online techniques), biological matrix or the specific application of the method. 

The median capacity factor kg is not directly related to the retention time under gradient conditions. In fact, it can be shown that under some conditions kg has the same value for all the peaks in a chromatogram obtained under programmed conditions. In deriving eqn.(4.67), the relative retention a is assumed to be independent of the composition. In other words, plots of retention (In k) vs. composition (isocratic conditions are assumed to yield parallel lines. 

For specific samples the optimum shape may deviate from this general rule. The retention and selectivity under gradient conditions may not follow the expected pattern because of anomalies in the isocratic retention vs. composition relationships. 

The approach of Jandera and Chura ek allows the optimization of the resolution of one given (arbitrary) pair of sample components and the minimization of the retention volume of another (arbitrary) solute. It requires knowledge of the isocratic retention vs. composition relationships of these three solutes. The information needed may be acquired from gradient elution experiments performed as part of the optimization procedure, or from separate isocratic experiments. The selection of the three arbitrary solutes considered during the optimization process appears to have a large effect on the result and the resolution cannot be optimized throughout the chromatogram. 

L.A. Romanyshyn, P.R. Tiller, R. Alvaro, A. Pereira, C.E.C.A. Hop, Ultra-fast gradient vs. fast isocratic LC in bioanalytical quantification by LC-MS-MS, Rapid Commun. Mass Spectrom., 15 (2001) 313... 

Romanyshyn, L. Tiller, PR. Alvaro, P. Pereira,A. Cornells,E.C.A. Ultra-fast Gradient vs. Fast Isocratic Chromatography in Bioanalytical Quantification by Liquid Chromatography/Tandem Mass Spectrometry, Rapid Commun. Mass Spectrom. 15, 313-319 (2001). 

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