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

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 mM 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 μM and μM, respectively. The LOD values compared to those obtained in 38 mM 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 mM TB buffer, pH 9.0, the LOD values for CA and α-lac were 13.8 nM and 126.0 nM, 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 mM 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 nM.