Optimization of Peptide Separations in High-Performance Liquid Chromatography

Authors:
Publisher website
Google Scholar
Abstract
Optimization of peptide separations in HPLC involves more than simply optimizaing the mobile phase in terms of flow-rate and gradient-rate. The first step in optimizing the separation of a complex peptide mixutre obtained from chemical and/or proteolytic digestion is the selection of the mode or combination of modes required for the separation. For example, should a single or multimodal approach be used, and which mode(s) (SEC, IEC, RPC) should be selected? The second step is to make an asssment of the performance characteristics of the HPLC columns (selectivity, efficiency and deviations from ideal behaviour) to be used. The most logical approach to assessing column performance is to employ HPLC peptide standards designed specifically for this purpose under a set of standard chromatographic run conditions. The third step involves utilizing the information obtained with the standars and any knowledge concerning the expected size, charge and polarity of the peptide fragments to select the initial mobile phase conditions for each mode of chromatography. In SEC, we are concerned with the pH, ionic strength and whether or not denaturing or benign conditions should be used in the mobile phase. In IEC, we must consider pH which can drastically affect the net charge on the peptide fragments and, thus, the overall separation. Denaturing conditions may be required to eliminate conformational effects of large peptides. In RPC, decisions such as the ion-pairing reagent to be used and the pH of the mobile phase are important issues. The fourth step involves the optimization of the gradient-rate and flow-rate to provide maximum resolution in the minimum time period. A detailed explanation of these steps are included.

This publication has 40 references indexed in Scilit: