On-line concentration of trace proteins by pH junctions in capillary electrophoresis with UV absorption detection

We report an on-line concentration approach based on pH junctions for the analysis of trace proteins under acidic conditions by capillary electrophoresis (CE) with UV absorption detection. Stacking is due to decreases in the electrophoretic mobilities of proteins when migrating from the sample zone to a relatively high-pH buffer filled in the capillary. Acidic buffers prepared from tris(hydroxymethyl)aminomethane (co-ions) and propanoic acid were suitable. With respect to speed, resolution, and stacking efficiency, it is appropriate to conduct the analysis of proteins under discontinuous conditions: pH 3.8 (inside the capillary), 2.8 (protein samples), and 3.3 (anodic reservoir). To minimize protein adsorption on the capillary wall, capillaries dynamically coated with single, double, and triple layers of polymers have been made and tested. Capillaries dynamically coated with three layers of neutral, cationic and neutral polymers in sequence were used to separate four proteins with good reproducibility. When using a 60-cm capillary, the peak height increased linearly with the injection volume up to 1.42-μl and peak profiles were sharp, indicating stacking of proteins. As a result, the limits of detection for lysozyme, myoglobin, carbonic anhydrase, and α-lactalbumin were 1.9, 3.2, 11.3 and 6.5 nM, respectively. Furthermore, this method has been applied to the analysis of about 1.31 and 0.66 μl of 5.00 and 0.20 μM peptic and tryptic digests of β-casein, with results of detecting 26 and 12 peaks in 21 and 14 min, respectively.