THE

CHEMISTRY

OF JAFFES CREATININE.

REACTION

FOR

V. THE ISOLATION BY ISIDOR

(From the

OF THE RED COMPOUND.* GREENWALD.

The Roosevelt Hospital, York.)

Harriman

Research Laboratory,
New

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(Received for publication,

September

21, 1928.)

In a recent number of this Journal (l), there was described a compound of 2 molecules of creatinine, 1 of picric acid, 3 of sodium hydroxide, and 3 of water. This was originally obtained by adding a solution of creatinine picrate in sodium hydroxide to absolute alcohol. Better yields were obtained in later experiments in which an additional molar equivalent of creatinine was used. The creatinine in this compound was found to be present in a form that does not give Jaffes reaction. The full color value of the creatinine present could be obtained only after solution of the compound, addition of acetic acid, and heating the solution to boiling or allowing it to stand at room temperature for at least an hour. It had previously (2) been found that, although only 1 mol of picric acid entered into the reaction, the maximum color in Jaffes reaction was not obtained unless at least 2 mols of picric acid were present. It was, therefore, believed to be of interest to ascertain what might be precipitated by running into alcohol an alkaline mixture containing 2 mols of picric acid for each mol of creatinine. The first preparation yielded a red precipitate which, upon analysis, appeared to be a mixture of the compound previously described with a new one containing 1 molecule of creatinine, 1 of picric acid, and 2 of sodium hydroxide. By using a little more picric acid (2.5 or 3 mols) and by avoiding the use of too great an excess of sodium hydroxide, the new compound was obtained in a nearly pure condition.
*Read before the Section of Organic Chemistry, Society, Swampscott, Masc.., September, 1928. 103 American Chemical

104

Chemistry

of Jaffes Reaction.

After drying in vacua, the substance forms a brilliant red, hygroscopic powder. When dissolved in water and diluted to contain 10 mg. of creatinine per 500 cc. and compared in a calorimeter with 0.5 N K&&O,, as in Folins original method for the determination of creatinine (3), the color obtained corresponds to only about 20 per cent creatinine instead of the calculated 26.8 per cent. But this is probably due to dissociation, for, if the solution of the new compound in 10 cc. of water is treated with a mixture of 15 cc. of
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TABLE I.

Analyses

of the New

Compound

of Creatinine, Lead) Hydroxide.

Picric

Acid,

and

Sodium

(or

Picric per

acid.

cent

Nitrogen. ~_____ per

Sodium. per

cent

cent

Calculated creatinine, Same with Preparation <

a compound of 1 molecule of 1 of picric acid, 2 of NaOH.. .. 0.5 mol HfO.. . ... ... .. .. ... ... . 12.. . . . . . . . , . . . . . . . . . . . . . . . . . . . . 13.. . . . . . . , . . . . . . . . . . . . . . . . . . . . . 20. . . . .. ... .. ... .. ... .. ... ...

for

54.2 53.1 51.6 53.5 52.5

~-

19.9 19.5 19.6 19.5 20.3

10.9 10.7 10.8 11.1 11.5

Lead.

Calculated for a compound of 1 molecule of creatinine, 1 of picric acid, 2 of lead hydroxide, 2 of HzO.. ... . . , . . . . . . . . . . From Preparation 13.. . . . . . . . . . . .. . . * 9.15 per cent when first dissolved in dilute when dissolved in hot 67 per cent H80,.

26.6 24.3 acetic

9.76 9.W acid; 9.20 per

48.1 51.7 cent

1 per cent picric acid and 5 cc. of 10 per cent sodium hydroxide and is then immediately diluted to 500 cc. the full color value is obtained. It is not necessary to wait 10 minutes for the color to develop. There can be no doubt, therefore, that it is the formation of this compound that is responsible for the red color of Jaffes reaction. This does not altogether contradict the view previously expressed (2) that it is the formation of the red tautomer of creatinine picrate that is responsible for the production of the color, for the new compound may be regarded as a compound of the red tautomer

I. Greenwald
with 2 mols of sodium hydroxide. Treatment of fairly concentrated solutions of the new compound with hydrochloric acid results in precipitation of the red tautomer. A solution of the new compound gives a red precipitate when treated with basic lead acetate solution. When filtered, washed, and dried over HzS04, the composltion of this precipitate agrees quite closely with that calculated for a compound of 1 molecule of creatinine, 1 of picrjc acid, 2 of lead hydroxide, and 2 of water. As in the case of the dicreatinine compounds, both the sodium and the lead compound contain more base than the form&e would require. With the high equivalent weight of lead, this results in a decided effect on the creatinine and picrjc acid content. That the nature of the combination between picric acid and creatinine in the new compounds is quite different from that in the dicreatinine compounds, is shown not only by the difference in the behavior of the creatinine but also by differences in the behavior of the picric acid. Analyses for nitrogen in the new lead compound give the same value when the substance is treated directly with hot 67 per cent sulfuric acid as when it is first treated with dilute acetic acid. Moreover, there is no marked destruction of the picric acid when a solution of the sodium compound is treated with dilute hydrochloric acid.
EXPERIMENTAL.

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The analytical

methods

were those previously Preparation.

employed

(1).

Details of Preparation 13.-To a solution of 17.2 gm. (0.075 mols) of picric acid in 300 cc. of boiling water, there were added 3.39 gm. (0.03 mols) of creatinine. After the solution had been cooled to 15, 30 cc. of 10 per cent NaOH were added from a pipette, the mixture being stirred constantly. The red solution was decanted from the undissolved material, which was now treated with 30 CC. of 2 per cent NaOH. Complete solution was now effected, and the two solutions were mixed and allowed to stand for 15 minutes. Some sodium picrate separated. The mixture was filtered, the filtrate being received in 3000 cc. of absolute alcohol and stirred constantly. The red precipitate obtained was filtered on a

106

Chemistry

of Jaffes Reaction.

hardened paper, with suction, washed with alcohol, and dried over sulfuric acid. The yield was 3.1 gm. or 25 per cent of the calculated. Recovery of Creatinine Picrate from the Sodium Compound.

1.49 gm. of Preparation 20 were dissolved in 35 cc. of water and the solution was than acidified with acetic acid. The precitate that formed was partly red and partly yellow. After standing overnight, it was all yellow. The crystals were filtered out and recrystallized from hot water. They melted at 222 and contained 67.4 per cent pick acid and 24.3 per cent nitrogen. The values calculated for creatinine picrate are 67.0 per cent pick acid and 24.6 per cent nitrogen. The yield was 0.89 gm. The correction for solubility (4) in the total of 85 cc. of filtrate was 0.16 gm., a total of 1.05 gm. The calculated yield was 1.18 gm. Color Development from the New Compound. 37.5 mg. of Preparation 13 were dissolved in water, diluted to 500 cc., and compared in a calorimeter with 0.5 N K2Cr207 set at 8.0 mm. The reading obtained was 11.0 mm., equivalent to 7.36 mg. of creatinine or 19.6 per cent. 37.2 mg. of the same preparation were dissolved in 10 cc. of water, a mixt,ure of 15 cc. of 1 per cent picric acid and 5 cc. of 10 per cent NaOH was added, and the mixture at once diluted to 500 cc. The reading was 9.1 mm., equivalent to 8.92 mg. of creatinine or 26.9 per cent. This is the calculated amount.
BIBLIOGRAPHY.

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1. Greenwald, I., J. Biol. Chem., 1928, lxxvii, 539. 2. Greenwald, I., and Gross, J., J. Biol. Chem., 1924, lix, 601. 3. Folin, O., 2. physiol. Chem., 1904, xli, 223. 4. Greenwald, I., Biochem. J., 1926, xx, 665.