Post‐column derivatization in narrow‐bore capillaries for the analysis of amino acids and proteins by capillary electrophoresis with fluorescence detection

S. Douglass Gilman, Jeremy J. Pietron, Andrew G. Ewing

Research output: Contribution to journalArticlepeer-review

Abstract

The construction and performance of a post‐column reactor for capillary electrophoresis with laser‐induced fluorescence detection in 10 μm i.d. capillaries is described. The post‐column reactor is based on a gap design and relies primarily on diffusion for addition and mixing of derivatizing reagents. Reactors with gaps of 4 to 160 μm are easily built with minimal micromanipulation between two 10 μm i.d. capillaries. Optimal separation efficiencies and sensitivities are obtained with gaps of less than 10 μm. Separations of amino acids and proteins with o‐phthaldialdehyde/2‐mercaptoethanol post‐column derivatization and laser‐induced fluorescence detection are shown. Linear detector response is found for amino acids and proteins, and peak efficiencies as high as 230,000 theoretical plates are obtained for some analytes. The use of relatively small columns for capillary electrophoresis results in a separation and detection system that produces very low mass detection limits for biological molecules from picoliter sample injections. Mass detection limits of 130 and 5.2 attomoles are obtained for glycine and human transferrin, respectively.

Original languageEnglish (US)
Pages (from-to)373-384
Number of pages12
JournalJournal of Microcolumn Separations
Volume6
Issue number4
DOIs
StatePublished - 1994

All Science Journal Classification (ASJC) codes

  • Mechanical Engineering
  • Filtration and Separation

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