Negatively charged sol-gel column with stable electroosmotic flow for online preconcentration of zwitterionic biomolecules in capillary electromigration separations

Abstract

A negatively charged sol-gel coating was developed for on-line preconcentration of zwitterionic biomolecules in capillary electrophoresis (CE), using asparagine and myoglobin as representative zwitterionic bioanalytes. The sol-gel coating was created by using a solution containing three precursors: mercaptopropyltrimethoxysilane (MPTMS), tetramethoxysilane (TMOS), and n-octadecyltriethoxysilane (C18-TEOS). The resulting sol-gel coating contained chemically bonded mercaptopropyl functional groups that were further oxidized by hydrogen peroxide to the corresponding sulfonic acid moieties. Such a surface-bonded sol-gel coating can carry a negative charge over a wide range of pH due to the presence of deprotonated sulfonic acid groups. Under favorable pH conditions, the negatively charged sol-gel coating can facilitate the extraction of positively charged analytes from a zwitterionic sample through electrostatic interaction. This principle was employed to extract myoglobin and asparagine by passing aqueous samples of these zwitterionic analytes through a negatively charged sol-gel column. The extracted analytes were then desorbed and focused via local pH change and stacking. The local pH change was accomplished by passing a buffer solution with a pH above the solute p/ value, while a dynamic pH junction between the sample solution and the background electrolyte was utilized to facilitate solute focusing. The sorption/desorption phenomena could, perhaps, also be explained on the basis of ion-exchange and local pH junction effects. On-line preconcentration and analysis results obtained on sulfonated sol-gel columns were compared with those obtained on an uncoated fused silica capillary of identical dimensions using conventional sampte injections. Using UV detection, the presented sample preconcentration technique provided a sensitivity enhancement factor (SEF) on the order of 3 × 103 for myoglobin, and 7 × 103 for asparagine. © 2005 Wiley-VCH Verlag Gmbh & Co. KGaA, Weinheim.