986 chromium and 1 drop of phosphoric acid in parallel groups of experiments. S o black specks were obtained from egg albumin alone. Phosphoric acid gave occasional black specks that were removed by several nitric acid treatments and mere carbon particles encased in the ash. Egg albumin and chromium yielded occasional black specks not removed by acid or oxidizing agent,s. Egg albumin, phosphoric acid, and chromium always yielded refractory black specks in amounts visually proportional t o t,hat of the added chromium (readily seen with 1 y of chromium). These samples, when assayed for chromium employing nitric acid-hydrochloric acid solubilization of the ash as in the usual procedure, yielded low chromium recoveries. The black specks that remained after acid treatment were fused with sodium peroxide, worked up, and assayed for chromium. Amounts of chromium approximating those lost in the acid solubilization werc found in the acid insoluhle residues. The presence of acid-insoluble black specks in the ashes of dry-ashed biological materials, that cannot be removed by repeated nitric acid t,reatments, indicates the transformation of chromium present into refractory oxides or phosphates. The presence of phosphate in biological material that is dry ashed preparatory to microdetermination of chromium is always a potential source of low recoveries. This danger is largely overcome, however, in the wet-ashing procedure where a lower temperaturp is used. The complete solution of the ash obt,ainedfrom the dry- or wetashing process is an important step in the analysis. Most ashe3 obtained by dry ashing without the addition of filter paper 0 1 ’ cotton were less soluble than the ash from the same material obtained by wet ashing. It is advisable t,o heat dry-ashed samples with a few drops of nitric acid before oxidation and to time the completion of the ashing and acid treatment so that, difficultly soluble ashes may be warmed with distilled water and allowed to stand overnight. ( A 3 to 1 nitric acid-hydrochloric acid mixture is used if the residue is insoluble in nitric acid. Aqua regia is not recommended, as lower recoveries are generally obtained. ) Some materials, particularly larger quantit,ies of urine, yield ashes that give turbid solutions. Good recoveries are generally obtained in these cases; a turbid solution should not he interpreted as prima facie evidence of overheating. A t,urhidity or even a precipitate on addition of t,he hypobromite does not adversely affect the analysis and will disappear on acidification. Various oxidants (permanganat,e, persulfate, bismuthate, hypobromite) have been used t o effect the transformation of chromium(II1) to chromium(V1). Hypobromite is very effective, and the excess is readily removed by acidification and addition of phenol water. The products thus formed do not affect the reagent blanks if the excess bromine is kept low enough to avoid formation of sufficient polybromophenols t o give a turbidity. Times of heating the chromium(II1) solution with hypobromite, from bringing the solution rapidly t o a boil over it burner to heating 0.5 hour on a mater bath, have been recommended to complete the oxidation. Jarvinen (‘7) cautions against too rapid heating lest the sodium hypochromite be decomposed before oxidation is completed. Several series of experiments were run to ascertain conditions that would reliably complete t h r oxidation. The oxidation under the conditions employed is complete in 5 minutes 07 less in the absence of mineral salts. The presence of mineral salts slows down the completion of the oxidation. The time of heating recommended (10 to 20 minutes) was found t o be sufficient for all the media studied even if the solutions were slightly turbid. The color forming reaction between chromium(V1) and lj5diphenylcarbohydrazide has been known for half a century ( 3 , 11). However, the mechanism of the reaction has only been recently elucidated ( 1 ) . The red-violet color is due t o the formation of a n inner chromium complex in which the chromium is present in the bivalent state. The practical amount, of 1,5-diphenylcarboh)-drazide necessary for full color development was investigated and it was found
ANALYTICAL CHEMISTRY that a large excess was necessary. I n most m e s 0.5 ml. of the color forming reagent proved t o be sufficient. However, when more than 2 y of chromium was present in samples that yielded a large amount of mineral ash-e.g., urine-an increase in the amount of reagent increased chromium recoveries. The increaee varied from 6 to 12% in the case of urine and was less than 5% in the case of plasma. I n the present procedure the use of 1 ml. of reagent is recommended. Full color development, compared to that in distilled water, is inhibited by the presence of mineral salts. This was shown by adding 0.5 to 5.0 y of chromium to a salt solution simulating a solution of the ash derived from 5 to 50 ml. of urine and developing the color with 0.5 ml. of reagent in 10-ml. volumetric flasks. This mineral salt effect can be overcome by using more reagent ( u p to the point where a turbidity results on adding the reagent) or hy diluting the ash solution to :t larger volume. -425-ml. volumetric flask is recommended for Jnniples containing more than 5 y of chromium and/or more mineral ash than that derived from 10 t o 15 ml. of urine. Several absorbance readings are required, since the rate of intensity increase, length of maximinn intensity plateau, and subsequent fading depend on chromium and mineral salt’ concent rations. K i t h the conditions and sample type described here, color development. is usually a t a maximum in about 5 minutes and fades little at the lower a1)sorl)ances within 0.5 hour. The success of the simplified method described here for small w n p l e s of the various materials is largely due to the fact that, the mineral salt effect remains small for up to 10 t o 15 ml. of urine with normal or subnormal mineral content while the other materials contain less mineral matter. The method has been applied to volumes of urine up to 25 ml., plasma and sera up to 15 ml., and the buffer solutions up to 50 ml. with good success. Since most of the samples handled in this investigation were of smaller size, detailed recovery st,udies of the larger samples were not made. LITERATURE CITED
(1) Bose, hI., ‘Vature, 170, 213 (1952) : Scieme and Culture ( I n d i a ) , 19, 213 (1953). (2) Cahnmann, H. J., and Bisen, R., .Is.\L. CHEW.,24, 1341 (1952). (3) Caseneuve, P., Bull. SOC. chim., 23(3), 701 (1900); 25(3), 761 (1901). (4) Dingwall, A, and Beans, H. T . , Pioc. .\-atZ. d c a d . Sci. U . E., 20, 416 (1934).
(5) Ferold, H. L., “Egg Proteins,” in “Advances in Protein Chemistry,” Val. VI, D. 200, dcademic Press. New York, 11951. (6) Grogan, C. H., and Oppenheiiner. H., A r c h . Biochern. and Biop h y s . , in press. (7) Jarvinen. K. K., 2. anal. Chenr., 75, 1 (1928). (8) AIancuso, T. F., I n d . Med. and S u r g . , 20, 393 (1951). (9) hIancuso, T. F., and Hueper. W. C.. Ibid.,20, 358 (1951). (10) Middleton, G., and Stuckey, R. E., A n a l y s t , 78, 532 (1953). (11) lIoulin, A . BUZZ.SOC. chim., 31(3), 205 (1904). (12) Xess, A. T., Smith, R. E . , and Evans, €1. L., J . A m . Chem. Soc., 74, 4685 (1952). (13) Saltsman, R . E., ANAL.CHEM.,24, 1016 (1952). (14) Urone, P. F., and Anders, H . K.. I h i d . , 22, 1317 (1950). (15) U. S. Public Health Service, “Health of Workers in Chromate Producing Industry.” Publ. 192 (1953). RECEIVED for review June 9, 1954. Accepted 1:ebruary 5 , 1955. Presented in part before the Division of Biological Chemistry at the 126th Meeting of the . ~ X E R I C A S CHEMICAL SOCIETY,New York, September 19%.
Petroleum-Cor rection I n the review article on “Petroleum” [ANAL.CHEM.,27, 599 (1955)l in the fourth paragraph in the second column of page 601, the second sentence should read: XlcCabe (116) reported unpublished work of Haagen-Smit and Fou, who automatically measured the total oxidant in the atmosphere by oxidation of phenolphthalin t o phenolphthalein. HARRY LEVIN
