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 dilute 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 low nanograms per milliliter range. Peptides from a sample matrix are adsorbed 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 previous designs and commercially available packed-inlet capillaries, the device is easily constructed from common laboratory materials and is applicable 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 fractions of peptides in a single analysis. To this end, extensive effort was required in both device construction and method development to attain the successful separations which are reported in this study. Extractions of dilute peptide mixtures from sample injections exceeding, but not limited to, 20 times (48 microL) 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 microL of sample injected) is presented. The ability to efficiently extract the individual peptides from the sample was found to be concentration-dependent for the individual peptide components over a 1600-field dilution of a common calibration mixture of nine model peptides. Varying the injected volume of elution buffer demonstrated the importance of minimizing the amount of buffer used to desorb peptides to maximize the resolution of individual peptides. This study highlights implementation for direct SPE-CE for peptide analysis and discusses for SPE tip-induced mechanism through which reversal in electoosmotic flow occurs.