Direct coupling of immobilized metal ion affinity chromatography and capillary isoelectric focusing in a single capillary

A procedure for the direct coupling of affinity chromatography and capillary isoelectric focusing in a single capillary is described. A unified capillary device was made to implement this procedure. The inner surface of a fused-silica capillary was tandemly coated with a chelating polymer, polyiminodiacetate, at the inlet side and with a neutral polymer, polydimethylacrylamide, at the outlet side. After loading a fluorescence-labeled recombinant Fab with a hexahistidine tag, a model sample, the device was rinsed with a high-salt buffer and then with a carrier ampholyte solution to fill the device. The bound Fab was eluted by filling the nickel-chelate column with an anode solution, 100 mM phosphoric acid. A positive voltage was applied at the chelate column side with a pressure at the same side, so as to slightly overwhelm the anodic electroosmosis produced in the acidified chelate column and gradually move the focused protein bands through a stationary fluorescence detector. The mixture of the labeled recombinant Fab at variable concentration and labeled bovine serum albumin at 50 nM was analyzed with the capillary device. A linear relationship between the peak area and the concentration was demonstrated for the Fab at 3.2 pM to 10 nM. The coefficients of variation for the peak area and detection time at 10 nM were 4.3% and 4.4%, respectively. The coupled procedure described here allows total transfer of the specifically adsorbed proteins from the affinity column to the capillary for isoelectric focusing without any compromise in separation efficiency. Removal of excess salts and concentration of dilute samples are also attractive features of the coupled procedure that can provide a new option for the analysis of charge variants of a protein in biological samples.