Azorubin in aqueous and methanolic solutions; spectrophotometric studies.

The original quantitative procedure for bilirubin of Van den Bergh (1) was based upon comparison of the color of an ether extract, first with ferric thiocyanate solutions and later with cobalt sulfate standards. This inaccuracy of standardization has been eliminated by the availability of purified bilirubin in both solid form and in comparatively stable solutions, and by the adaptation of the procedure to spectrophotometers and photoelectric colorimeters, both of which isolate narrow bands of light as compared to the entire spectrum used with visual colorimeters. The second problem was that of precipitation of the proteins by the addition of ethanol in the determination of total bilirubin, accompanied by subsequent loss of some bilirubin by adsorption, and by incomplete dissociation from the assumed protein-bilirubin complex. The solution to this problem was worked out by Malloy and Evelyn (2), who found that the serum proteins did not precipitate in 50% methanol provided the final protein concentration was below a certain level. At this concentration of methanol, full color development of the azorubin takes place. The final dilution in the original method was about 1 to 40 or 50, with a protein concentration of 300-400 mg. per 100 ml. Observations during the present study indicate that protein precipitation in methanol does not occur under the proposed test conditions, if the serum dilution is 1 to 8 or more-that is, a protein concentration of not more than 1%. Above this protein concentration precipitation almost invariably occurs.