Continuous beds for microchromatography: detection of proteins by a blotting membrane technique

Continuous beds have been used as matrices for cation- and anion-exchange chromatography of proteins on columns with an i.d. in the range of 0.005-0.015 mm. On-tube uv detection is not feasible at low protein concentrations with these narrow-bore columns. Therefore, a more sensitive detection system has been developed based on blotting technique: as the protein zones leave the microcolumn chromatographically they become adsorbed onto a rotating polyvinylidene difluoride blotting membrane. The protein spots can then be visualized by means of Coomassie brilliant blue, immunomethods, and other standard techniques. By using an immunomethod 0.015 ng of human transferrin can easily be detected. The blotting membrane can be washed with water without loss of adsorbed protein. This is an attractive feature because the presence of salts, etc., diminishes the accuracy in the determination of molecular weights of proteins by mass spectrometry. The microcolumns are easy to prepare. A solution of appropriate monomers is sucked into a piece of fused silica tubing. The rod formed upon polymerization contains channels through which the eluent can pass. No supporting frit is required because the polymer rod is anchored by covalent bonds to the tubing wall.