Theory of Stepwise Gradient Elution in Reversed-Phase Liquid Chromatography Coupled with Flow Rate Variations:  Application to Retention Prediction and Separation Optimization of a Set of Amino Acids

Abstract

The coupling of stepwise mobile phase gradient elution and flow programming is proposed as an integrated approach to the general elution problem in reversed-phase liquid chromatography. A model is developed to describe the above separation process performed under simultaneous programming of two separation parameters by extending our previous work on the rigorous derivation of the fundamental equation governing the concentration gradient of organic modifier in the mobile phase, that is, a single gradient elution mode (Anal. Chem. 2005, 77, 5670-5677). The theory was tested in the retention prediction and separation optimization of 18 o-phthalaldehyde derivatives of amino acids in eluting systems modified by acetonitrile or methanol. The retention prediction obtained for all solutes under all dual-mode gradient conditions was excellent. In addition, it has been shown that the combination of mobile phase and flow rate programming modes is particularly favorable, whereas the separations among the analytes were considerably improved by using the acetonitrile eluting system, as compared to those obtained by the methanol system.

Multimode Gradient Elution in Reversed-Phase Liquid Chromatography: Application to Retention Prediction and Separation Optimization of a Set of Amino Acids in Gradient Runs Involving Simultaneous Variations of Mobile-Phase Composition, Flow Rate, and Temperature

“…As pointed out above, the theory of two-mode gradient elution has been developed in refs –. This theory can be readily extended to three-mode gradients involving simultaneous variations in φ, T , and F .…”

“…Equation is inapplicable for the gradients of the second category, the theory of which is still under development in our laboratory. In particular, in previous publications, − we studied two-mode gradient elution in liquid chromatography involving simultaneous changes in (a) flow rate and mobile-phase composition, , and (b) temperature and mobile-phase composition . Here, we examine the more general case of gradients with simultaneous changes in mobile-phase composition, i.e., organic modifier content, φ, flow rate, F , and temperature, T , and present a very simple stepwise method that allows for the calculation of the elution time of a sample solute under all gradient conditions.…”

Description of the Retention and Peak Profile for Chromolith Columns in Isocratic and Gradient Elution Using Mobile Phase Composition and Flow Rate as Factors

“…A change in the mobile phase flow rate accelerates or slows down peak elution, causing the chromatogram to be compressed or expanded, but without effects on the selectivity. However, the flow rate is rarely implemented as an experimental variable in RPLC, due to the narrow range of this factor that can be applied using conventional microparticulate columns, although some interesting reports on this topic have been published [7][8][9][10][11][12][13][14][15]. With conventional columns, the use of high flow rates is limited by the rapid increase in column back-pressure.…”

Comparison of interpolation polynomials with divided differences, interpolation polynomials with finite differences, and quadratic functions obtained by the least squares method in modeling of chromatographic responses

“…The modeling of the responses can be performed in two ways. The first one includes the application of an already-known theoretical or mechanistic model [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24], while the second one involves empirical modeling [4][5][6] where the experimentally obtained data is fitted (approximated) by empirical equations. DoE methodology usually fits a polynomial model to the data (most commonly a second-order polynomial model) by multiple linear regression using the least squares method of approximation.…”