Tryptic fingerprinting on a poly(styrene—divinylbenzene) reversed-phase column

• Published in: Journal of Chromatography A 1990-07, Vol.512, p.315-323
• Main Author: Swadesh, Joel K.
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Proteins
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(01)89498-X

The properties of poly(styrene—divinylbenzene) (PS—DVB) in gradient reversed-phase peptide separations have not been fully explored. Comparisons of selectivity and fraction capacity with silica alkyl bonded phases remain to be established. Investigation of the effects of gradient rate and flow-rate on separation are desired. The present work examines separations of synthetic peptides and tryptic fragments of three species of cytochrome c and reduced, carboxymethylated human plasminogen. Fingerprinting is a means to localize the position of one or more variant amino acids within the sequence of a large polypeptide. Reproducibility of separation is a dominant issue, potentially affected by variability in pump performances. The susceptibility of the peptide fingerprint to changes in pump performance can be examined by systematic variation of the flow-rate and gradient rate. PS—DVB exhibited predictable separations equivalent to those of alkyl bonded phases. The selectivity parallelled that observed for bonded phases. Two observations made in the course of these studies may be of interest to the theory of reversed-phase liquid chromatography. First, the peak capacity was found to be a simple function of the gradient rate. Also, the resolution was observed to increase with increasing flow-rate in a separation of horse cytochrome c tryptic fragments at fixed gradient rate.