Regulation of electroosmotic flow and electrophoretic mobility of proteins for concentration without desalting

Abstract

Proteins were concentrated and separated in 0.6% poly(ethylene oxide) (PEO) solution using a capillary filled with Tris–borate (TB) buffer prior to analysis and detected by laser-induced native fluorescence using a pulsed Nd:YAG laser. During the concentration and separation, PEO solution entered the capillary by electroosmotic flow. When proteins dissolved in high salts (phosphate-buffered saline) were separated using 0.6% PEO solution prepared in 200 m M TB buffer, pH 9.0, the limits of detection (LODs) at signal-to noise ratios=3 for carbonic anhydrase (CA) and α-lactalbumin (α-lac) were on the levels of sub μ M and μ M, respectively. The LOD values compared to those obtained in 38 m M TB buffer were relatively high, which is likely due to salt quenching, Joule heating and poor stacking. To improve sensitivity for analysis of proteins in high-conductivity media, two on-line concentration approaches without desalting were developed. When using a capillary filled with 1.5 M TB buffer, pH 10.0, and PEO solution prepared in 800 m M TB buffer, pH 9.0, the LOD values for CA and α-lac were 13.8 n M and 126.0 n M, respectively, which were about 4.7 and 11.2-fold sensitivity enhancements compared to those obtained by a conventional hydrodynamic injection (30 cm height for 10 s), respectively. The sensitivity was further improved by injecting a short plug of low pH buffer after protein injection using a capillary filled with 1.5 M TB buffer, pH 10.0, and PEO solution prepared in 400 m M TB buffer, pH 9.0. A linear relationship between the peak height and the injection volume up to 0.81 μl was obtained and the LOD values for CA and α-lac were down to 4.7 and 37.8 n M.