MECHANISM OF PEPTIDE SEPARATIONS BY SOLID-PHASE EXTRACTION CAPILLARY ELECTROPHORESIS AT LOW PH

A device for on-line extraction and concentration of peptides from a d ilute sample matrix prior to direct capillary electrophoretic analysis is described. The technique, termed solid phase extraction capillary electrophoresis (SPE-CE), can facilitate analysis of peptides in the l ow nanograms per milliliter range. Peptides from a sample matrix are a dsorbed on a reversed phase resin (C-8 or C-18) cartridge in-line with an uncoated fused-silica capillary and subsequently released for free zone electrophoresis by injection of an organic elutant. Unlike previ ous designs and commercially available packed-inlet capillaries, the d evice is easily constructed from common laboratory materials and is ap plicable to a wide range of conventional instrumentation and methods. This device and method has been developed for use in our laboratory as a stand-alone preparative technique, specifically to provide a second -dimensional orthogonal separation of biologically derived HPLC fracti ons of peptides in a single analysis. To this end, extensive effort wa s required in both device construction and method development to attai n the successful separations which are reported in this study. Extract ions of dilute peptide mixtures from sample injections exceeding, but not limited to, 20 times (48 mu L) the capillary volume with apparent recovery greater than 80% are shown, The selectivity of extraction of individual components of a very dilute peptide mixture (31 ng/mL with 280 mu L of sample injected) is presented, The ability to efficiently extract the individual peptides from the sample was found to be concen tration-dependent for the individual peptide components over a 1600-fi eld dilution of a common calibration mixture of nine model peptides, V arying the injected volume of elution buffer demonstrated the importan ce of minimizing the amount of buffer used to desorb peptides to maxim ize the resolution of individual peptides, This study highlights imple mentation for direct SPE-CE for peptide analysis and discusses the SPE tip-induced mechanism through which reversal in electroosmotic flow o ccurs.