Novel poly(vinyl alcohol)-based column coating for capillary electrophoresis of proteins

A novel and simple method for the preparation of chemically bonded poly(vinyl alcohol) (PVA) coating to silica capillary inner wall was developed, and the PVA-coated capillary columns were employed for capillary electrophoresis (CE). The coating procedure included pretreatment of the capillary inner wall, silanization, aldehyde group functionalization and PVA immobilization. Electroosmotic flow of the coated capillary was almost suppressed over a wide pH range (pH 3–10). High-efficiency separations of cationic proteins (including cytochrome c, lysozyme, α-chymotrypsinogen A) at pH 3.0–5.0 and of anionic proteins (including myoglobin and trypsin inhibitor) at pH 10.0 were achieved with the PVA-coated capillary. Moreover, a “dual-opposite-injection” approach was adopted for simultaneous separations of both cationic and anionic proteins at neutral pH with the prepared column. In this CE mode, positively charged proteins migrated from one end of the column to the detector while negatively charged proteins from the other end to the detection window. Good run-to-run repeatability was obtained in all of the protein CE separations performed in this work. The PVA-coated column can also afford long-term stable uses for protein separations, as demonstrated in 100 repeated uses using a single capillary with the relative standard deviation values of the retention times less than 0.9%. Moreover, good column-to-column reproducibility was demonstrated by protein CE separation with 10 different columns. The results indicate that the present method for PVA-coated capillary preparation is promising for protein CE applications.

Research highlights▶ A novel PVA-coated column is fabricated for protein capillary electrophoresis. ▶ The column promises high efficient separation of proteins. ▶ The column affords long-term stable uses for protein separations. ▶ “Dual-opposite-injection” method can be used for protein separation at neutral pH.