Studies on natural bilirubin. 2. A comparative study on the azo-dyes obtained from three bilirubin fractions of dog bile fractionated by means of ion exchange resin
Igaku Kenkyu 29(8): 2643-2651
The author carried out the partition chro-matographic and spectrochemical studies on azo-dyes obtained by combining diazonium salt with three natural bilirubin fractions isolated from dog bile by means of ion exchange resin; and obtained the following results. 1. When the solvents in the combination of ethyl acetate-ethanol-n-butanol-methanol in the ratio of 3:1:1 or 4:1:1 and ethylace-tate-ethanol-n-butanol-methanol in the ratio of 4:1:1:0.5 or 4:1:1:1 are used as the developing agents, there appears a red spot having a clear cut difference in Rf value between the azo dye derived from indirect bilirubin and the azo dye derived from direct bilirubin, but in the original spot there remains a blue dye and this hardly shows any difference with respect to the partition between the two. 2. When the developing agent in the combination of methyl-ethyl-ketone glacial acetic acid-water (15:5:6) is used, between the azo dye derived from indirect bilirubin and that from direct bilirubin there is partitioned a blue spot having different Rf values, and still another yellow spot of the same Rf. 4. The azo dye derived from indirect bilirubin shows the absorption maximum at 540-550 m[mu] and when concentrated hydrochloric acid is added to it, the absorption maximum is at 580 m[mu]. Azo dye derived from basic bilirubin has its absorption maximum at 550 m[mu], and when conc. hydrochloric acid is added to it, the maximum is at 555 m[mu]; while the azo dye derived from ester-type bilirubin has the absorption maximum at 540-550 m[mu], and when conc. hydrochloric acid is added to it, the maximum at 550-555 m[mu]. 5. Between azo dyes derived from two direct bilirubin fractions there can be recognized hardly any difference of the absorption maximum both before and after the addition of conc. hydrochloric acid, but when the same are compared with the absorption maximum in the case of indirect bilirubin, distinct differences can be recognized. 6. When the aqueous solution of basic bilirubin is transferred into chloroform after addition of [image]/10 hydrochloric acid, acting directly with the diazo agent in the chloroform solution, the azo dye thus derived is purplish red and shows the absorption maximum at 550-552 m[mu]; and even after addition of conc. hydrochloric acid the tone of the color hardly changes, showing the absorption maximum at 555 m[mu]. Furthermore, in the chloroform solution there can be recognized indirect bilirubin remaining without any direct reaction, and the azo dye of this bilirubin shows the absorption maximum at (530-) 550 m[mu], and at (570-) 580 m[mu] after addition of hydrochloric acid. 7. When ester bilirubin is transferred into chloroform after saponification, its azo dye shows the absorption maximum at 552 m[mu], and when conc., hydrochloric acid is added to it, the absorption maximum is at 580 m[mu].