A porous layer open-tubular capillary column with immobilized pH gradient (PLOT-IPG) for isoelectric focusing of amino acids and proteins.

A porous layer open-tubular capillary column with immobilized pH gradient (PLOT-IPG) was prepared in two steps. First, a partially filled porous layer open tubular capillary column containing active epoxy groups was synthesized by in situ polymerization of acrylamide, glycidyl methacrylate, and N,N'-methylenebisacrylamide. Then, an aqueous solution of commercial carrier ampholytes (CAs, Pharmalyte) was focused in the prepared polymer column. With the column immobilized by Pharmalyte 4.5-6.0 (narrow pH range) or Pharmalyte 3.0-10.0 (wide pH range), proteins with known pIs were separated and a good linear correlation between pI values and migration times was obtained. Meanwhile, resolution was improved compared to the traditional gradient with a wide pH range. A mixture of low molecular weight amino acids was separated with the narrow pH range PLOT-IPG column; from the obtained results, the resolution of our new column was calculated to be at most 0.09 pH unit. In addition, human serum proteins were analyzed with the newly developed wide and narrow pH range PLOT-IPG columns; its sensitivity and resolution in the narrow pH range one were better than capillary electrophoresis.