Separation of Nanoliter Samples of Biological Amines by a Comprehensive Two-Dimensional Microcolumn Liquid Chromatography System

Abstract

A two-dimensional liquid chromatography system based on the combination of a charge separation mechanism, provided by anion exchange chromatography, with a hydrophobic separation mechanism, provided by reversed phase chromatography, is presented. A 90 cm long anion exchange microcolumn is coupled to a 3 cm long reversed phase microcolumn. Both microcolumns are interfaced by two electronically controlled valves. Effluent from the anion exchange microcolumn is collected in a sample loop and then concentrated onto the head of the reversed phase microcolumn. Analyte peaks consisting of tagged amines elute from the second column and are detected by laser-induced fluorescence. The entire operation is controlled by a personal computer. The resolving power of this system is demonstrated with a two-dimensional chromatogram of the peptides obtained from a tryptic digest of porcine thyroglobulin. The sensitivity of the system is demonstrated with a two-dimensional chromatogram of the contents of a single bovine chromaffin cell.