Protein determination by high-performance gel-permeation chromatography: applications to human pancreatic juice, human bile and tissue homogenate

A high-performance gel-permeation chromatography (HPGPC) method to determine the proteins of human pancreatic juice, bile, and tissue homogenate has been developed. A diol-type silica gel column (35×8 mm I.D., 5 nm average pore diameter) was used under a column temperature of 8°C. The eluent was acidic phosphate buffer with a high concentration of sodium chloride, nonionic detergent of polyoxyethylene (20) cetyl ether (Brij 58), glycerol and 2-propanol. The UV wavelength used for the protein detection was 210 nm. Analytical time was within 3.5 min. Good correlation coefficients were obtained with this HPLC method at a column temperature of 8°C and a spectrophotometric bicinchoninic acid (BCA) method. A photometric pyrogallol–red molybdate complex method was found to correlate well with this HPLC method and with the BCA method only for tissue homogenate. Since this HPGPC protein assay method is simple, convenient, rapid, reproducible, and reliable, it is expected to be generally applicable to clinical and also to biochemical research.