ANALYTICAL CHEMISTRY
1372 ganic substances which failed to react with the benzidine reagent are acetic acid, aluminum sulfate, ammonium chloride, arsenic acid, barium hydroxide, barium acetate, cadmium chloride, calcium phosphate, chromium fluoride, lead nitrate, lead acetate, mercuric chloride, potassium chloride, potassium carbonate, potassium hydroxide, sodium chloride, sodium hydroxide, and stannous chloride. Reacting organic compounds are classified in Table 11. Though some of the reducing substances listed have been previously described ( 2 , 4,6 ) they are included because most of the published data are the result of “descending” paper chromatography, a method which gives slightly different R, values from the “ascending” technique employed in this investigation. The method utilized here for the calculation of the K! is also *lightly different. The benzidine reagent did not react with the following carbohydrates: adonitol, arabin, glycogen, i-inositol, inulin, mannitol, raffinose, sorbitol, starch, and sucrose. Thirty-eight amino acids and amino acid derivatives were also nonreactive. Miscellaneous compounds that were tested and did not react \yere: adenosine triphosphate (dibarium salt), atropine sulfate, betaine, betaine hydrochloride, choline chloride, acetylcholine chloride, citric acid, creatine, creatinine zinc chloride, epinephrine, folic acid, gelatin, glutathione, sodium heparin, histamine dihydrochloride, lactic acid, e o d i m malonate, nicotinamide, phlorizin, pyruvic acid, sa1cosine, sulfadiazine, sulfathiazole, tartaric acid, thiamine hydrochloride, thiourea, tyramine hydrochloride, urea, and uric acid.
bait nitrate, and niaiiganese nitrate, for example, have been aided by this procedure. At 85’ C. none of the amino acids reacts with the benzidine reagent, a factor of importance in the analysis of glycoprotein hydrolyzate~. The characteristic color and R! reactions of alloxan (Table 11) may be of value in the determination of this Pubstance in biological fluids and tissues.
DISCUS SIOk
ACKNOWLEDGMENT
Six of the inorganic compounds tested yielded R/ values a t the origin as well as a second spot in a higher R f range (Table I). It is difficult to explain these results, as well as the apparent discrepancy between the R, values of cations wch as cerium and cobalt, and anions such as nitrate and chloride. It seems important that the R f values of nianv of these compounds are close to those of a number of carbohydrates, necessitating the elimination or identification of inorganic impurities in carbohydrate samples. Many inorganic substances form benzidine-blue a t room temperature when ammonium hydroxide is added to the benzidine reagent ( 3 ) . Thus, positive identifications of cerium nitrate, co-
The authors wish to express their thanks to Jean Wright for her technical assistance and to Sam Seifter for his interest and advice concerning thie work.
I
Tahle 11. Reactions of Organic Compounds with Benzidine Reagent
a
Compound Rf Description 0.28 Brown S-Acetyl glucosamine 0.48 Violet (violet t a i l ) “ Alloxan 0.24 Brown hrabinose 0.46 Brown Ascorbic acid 0.19 Brown Dextrin 0.33 Brown Fucose 0.16 Brown Galactose 0.14 Brown Glricosamine hydrochloride 0.18 Brown Red-brown Glucose 0.38 Glucuronic acid 0.07 Brown Lactose 0.25 Brown Levulose 0.08 Brown Maltose 0.22 Brown Mannose 0.72 White Oxalic acid 0 . 8 5 Brown Phloroglucine 0.39 Brown Rhamnose 0 32 Brown Ribose 0 28 Rrow-n Xylose a,!’ea. A tail is a colored area extending f r o m inajor s p o t into Glnrose was standard for determination of all Rf valupa
LITERATURE CITED
(1) Bacon, J. S. D., and Edelman, J., Biochem. J., 48, 114 (1981). (2) Evans, E. E., and Mehl, J. W., Science, 114, 10 (1951). (3) Feigl, F., “Qualitative Analysis by Spot Testa,” New Yolk, Elsevier Publishing Co., 1946. (4) Horrocks, R. H., and Manning, G. B., Lancet, 1, 1042 (1949). (5) Partridge, S. M., Biochem. J., 42, 238 (1948). RECEIVED for review February 20, 1452.
Accepted M a y 26, 1952.
Microdetermination of Phosphorus GEORGE R. NAKAMURA, University of Caliifornia, Berkeley, Calif.
RAPID method has been developed for the estimation of
A phosphorus in biological materials and is used routinely in this laboratory. It is an adaptation of King’s colorimetric determination of phosphorus in a micro range of 1 to 10 micrograms of phosphorus (1).
To a measured amount of sample in a small borosilicate glass tube, 10 X 75 mm., which had been calibrated to a 1-ml. mark, was added 0.1 ml. of 60 to 70% perchloric acid. The tube was placed in a sand bath (as many as 20 tubes have been run simulTable I. Phosphorus Recovery from Phosphoric Esters by Micromethod P Present, Compound Disodium glycerophosphate. 5H20
Calcd.,
P P Found, Theoret.,
P Found,
Y
Y
%
5.07
5.10 5.05 5.05
10.13
10.20 10.10 10.10
%
Barium phosphoglyceric acid
4.83
4.80 4.80 4.78
9.65
9.60 9.60 9.56
Disodium phenyIphosphate2H,O
6.11
6.10 e. 10 6.05
12.21
12.20 12.20 12.10
taneously) a t about 190’ to 200” C.; excessive heating beyond this range caused undesirable splattering. The length of diges tion period depended on the nature of the sample; the phosphoric esters listed in Table I required a digestion time of only 2 minutes. If the sample contained a large amount of organic material, the content of the tube became brown and then discolored. If the discoloration did not occur within 15 minutes, a drop of 30% hydrogen peroxide was added and the tube was reheated for an additional 5 minutes. The cooled content was then diluted with about 0.5 ml. of water, and washed down the wall of the tube. To the digestion mixture, 0.1 ml. of 5% ammonium molybdate and 0.1 ml. of sulfonic acid (an aqueous solution containing 0.2% 1,2,4-aminonaphthol sulfonic acid, 12% sodium bisulfite, and 2.4y0 sodium sulfite) were added and brought to the 1-ml. mark with water. The resulting blue solution was transferred to a matched cell and compared with a standard in a Jr. Coleman spectrophotometer a t 700-mr wave length using an adapter for 10 X 75 mm. cuvets. Micropipets, such as obtained from &licrochemicalSpecialties, 1834 University Ave., Berkeley, Calif., were extremely useful in transferring 1to 100 PI. of samples. LITERATURE CITED
(1) King, E. J., Biochemical J., 26, 292 (1932). RECEIVED for review March 13, 1952.
Accepted M a y 26, 1952.
