Carboxymethylchitosan covalently modified capillary column for open tubular capillary electrochromatography of basic proteins and opium alkaloids

A novel open tubular (OT) column covalently modified with hydrophilic polysaccharide, carboxymethylchitosan (CMC) as stationary phase has been developed, and employed for the separations of basic proteins and opium alkaloids by capillary electrochromatography (CEC). With the procedures including the silanization of 3-aminopropyltrimethoxysilane (APTS) and the combination of glutaraldehyde with amino-silylated silica surface and CMC, CMC was covalently bonded on the capillary inner wall and exhibited a remarkable tolerance and chemical stability against 0.1 mol/L HCl, 0.1 mol/L NaOH or some organic solvents. By varying the pH values of running buffer, a cathodic or anodic EOF could be gained in CMC modified column. With anodic EOF mode (pH<4.3), favorable separations of basic proteins (trypsin, ribonuclease A, lysozyme and cytochrome C) were successfully achieved with high column efficiencies ranging from 97,000 to 182,000 plates/m, and the undesired adsorptions of basic proteins on the inter-wall of capillary could be avoided. Good repeatability was gained with RSD of the migration time less than 1.3% for run-to-run (n=5) and less than 3.2% for day-to-day (n=3), RSD of peak area was less than 5.6% for run-to-run (n=5) and less than 8.8% for day-to-day (n=3). With cathodic EOF mode (pH>4.3), four opium alkaloids were also baseline separated in phosphate buffer (50 mmol/L, pH 6.0) with column efficiencies ranging from 92,000 to 132,000 plates/m. CMC-bonded OT capillary column might be used as an alternative medium for the further analysis of basic proteins and alkaline analytes.