Isoelectric focusing of proteins in a microcapillary with universal concentration gradient detection

Abstract

AbstractProteins were separated by capillary isoelectric focusing (CIEF) performed in a 5‐μm i.d., 12‐cm long uncoated capillary, and detected by an on‐column concentration gradient detector based on Schlieren optics. The narrow capillary allows the application of a 15‐kV separation voltage. The high voltage resulted in a very narrow 30‐μm wide zone for carbonic anhydrase, which corresponded to a 60‐fL zone volume. All focused proteins could be detected by the universal concentration gradient detector without derivatization in less than 7 min. On‐column mass detection limits for proteins were at the 100 amol level, which was lower than that of a UV‐vis absorption spectrometric detector. The detection volume of the detector was calculated to be 20 fL, the smallest detection volumes reported to date for optical or spectroscopic detectors. This microcapillary CIEF‐concentration gradient detector system may be a useful technique for direct analysis of proteins at the single cell level.