V O L U M E 27, NO. 6, J U N E 1 9 5 5
983
impurities in the commercial product. On the other hand, 2,4,5trichlorophenol does not have similar effects on the crystallization of 2,4,5-trichlorophenoxyacetic acid. Under the usual conditions of recrystallization from the melt a t room temperature, 2,4,5-trichlorophenoxyacetic acid will always crystallize into stable form I in the presence of as much as 5y0 2,4,5-trirhlorophenol.
The microscopic fusion data obtained in this investigation may be used in characterizing 2,4,5-trichlorophenoxyaceticacid and the following related compounds: 2,4,5-trichlorophenoxyacetic acid, 5-methoxy-2,4-dichlorophenoxyacetic acid, 2,4,5trichlorophenol, potassium salt of 2,4,5-trichlorophenoxyacetic acid, sodium salt of 2,4,5-trichlorophenoxyaceticacid, 2,3,6trichlorophenoxyacetic acid, and 2,5-dichlorophenoxyacetic acid.
CONCLUSIONS ACKNOWLEDGMENT
2,4,5-Trichlorophenoxyaceticacid is dimorphous, and in t hr The author wishes t o thank Loren Knowles for the use of pure state will usually crystallize from solution or from the melt his photomicrographic facilities at the Ethyl Research Laborainto its stable form. However, it will crystallize more easil:. tories and Leonard Siebylaki for obtaining the s-ray diffraction into its metastable form in the presence of small amounts of the data. following impurities: 2,3,6-trichlorophenoxyacetic acid, 2,sLITERATURE CITED dichlorophenoxyacetic acid, 5-methoxy-2,4-dichlorophenosyacetic acid, or the sodium salt of 2 , l , ~ - t r i c h l o r o p h e n o s ~ ~ c e t i c (1) drceneaux, C. J., ANAL.CHEM.,25, 486 (1953). (2) Kendall, D. N., Ibid., 25, 382 (1953). acid. (3) Kofler, L., and Kofler, il., “Mikrornethoden Bur Kennzeichnung The polymorphic nittiire of 2,4,5-t~richlorophenoxyacetic acid organischer Stoffe und Stoffgernische,” Universitats-Verlag Wagner, Innsbruck, 1948. and the polymorphic nature of the sis related compounds inves(4) Mecrone, W. C., ANAL.CHEM.,21, 436 (1949). tigated make it difficult t o develop any quantitative analytical (5) Mitscherlich, E., “Gesamrnelte Schriften von Eilhardt Nitschermethods for commercial 2,4,5-trichlorophenoxyaceticacid by milich. Lebensbild, Briefmeschsel und ribhandlungen,” Alexancroscopic fusion techniques. However, simple and rapid qualitader Xtscherlich, Berlin, 1896. tive fusion methods of detection have been developed for the RECEIVEDfor review J u n e 19, 1954. Accepted September 21, 1954. Presodium and pot,assiuni salts of 2,4,5-trirhlorophcnosy~~rrtir acid sented a t the Regional Conclare, .*vERICAx CHmncar. SOCIETY,New Orleans, La., December 1953. in 2,4,5-trichlorophenos~~~r~tic. acid.
Microdetermination of Chromium in Small Samples of Various Biological Media CHARLES H. GROGAN, H. J. CAHNMANN,
and
ELIZABETH LETHCO
National Cancer Institute, National lnstitutes o f Health, Bethesda
During studies of the metabolism of chromium i t became necessary to develop a rapid routine method for the microassay of chromium in various buffer solutions, protein solutions, plasma, sera, paper strips, and urine. A method has been developed based on the spectrophotometric determination of chromium as the colored reaction complex of chromium(V1) with 1,5-diphenylcarbohydrazide (svm-diphenylcarbazide). The method employs rapid ashing procedures which are applicable to small samples of several biological media relatively free of interfering materials, such as iron. All steps in the analysis are performed with only a single transfer at the point of color development. Recoveries of 95% or better were obtained in most cases under a variety of conditions on samples ranging in size from 10 1.1. to 10 ml. containing from 0.1 to 5 y of chromium. The standard deviations for the procedures were all within the range 0.02 to 0.06 y . The simplified procedures described permit the routine analysis of a large number of samples daily with a high degree of accuracy and precision in a variety of biological media.
B
ECAUSE of t h r continued interest in and investigation of
reported (8,9,15)cancerigenic hazards encountered in working with chromium, its ores and chemical compounds, a study of the metabolism of chromium, and in particular its interaction with proteins, was undertaken (6). In view of the large number of microassays of chromium involved, it was necessary t o have :t rapid and sensitive micromethod adaptable t o the routine deteimination of chromium in a variety of media such as plasma or other protein solution., buffers, paper strips, and urine.
14, Md.
A publication from this laboratory (2) describes a method of high sensitivity for the microdetermination of chromium in blood. While this method was found t o be basically applicable t o several of the media encountered in the present investigation, neither this nor other published methods ( I S , 1 4 ) were entirely suitable for the routine analysis of large numbers of samples of the type t o be handled. Therefore, modifications were worked out which considerably simplified and shortened the procedures. These modifications were largely permissible because of the absence of interfering amounts of iron and of acid-insoluble chromium compounds (chromite ore) and also because the size of the samples involved was relatively small. Liquid samples ranging from 10 1.1. t o 10 ml. and filter paper strips up t o 100 sq. em. were success fully analyzed. The present paper describes the simplified procedures which readily permit the routine assay of up t o 60 samples per person per day. The exact number depend. on the nature of the material to be analyzed and the size of the sample. These procedures proved t o be of great value in the authors’ investigations. It was felt that they will also be of assistance t o other investigators who study the role of chromium in biological processes and to workers in the field of leather chemistry. The method consists of the following steps: a wet or dry ashing of the sample, hypobromite oxidation of trivalent to sexivalent chromium, and spectrophotometric determination of the sexivalent chromium in the form of the red-violet complex which it forms on reaction with 1,5-diphenylcarbohydrazide(sym-diphenylcnrhazide) (3,11). REAGENTS
Distilled water, double-distilled IIater, 30% hvdrogen peroxide, phenol water, and sodium hvpobromite reagent TTere the same aa previously described ( 2 , 1 4 ) .
984
ANALYTICAL CHEMISTRY
Nitric acid, prepared by distilling or double distilling C.P. or reagent grade acid until 5-ml. portions gave sufficiently lo~v reagent blanks in the procedure ( 4 ) . Hydrochloric acid, prepared by saturating doub1e:distilled water, cooled in an ice bath, with gaseous hydrogen chloride after passing it through scrubbing towers of distilled water, concentrated sulfuric acid, and a dry ice trap. Sulfuric Acid. . 4 12.5 volume 7 0 solution of special reagent (low in nitrogen and arsenic) in double-distilled water was used. Traces of reducing substances were destroyed by dropwise addition of 170 potassium permanganate solution to the hot acid solution until a pale pink color persisted for 1minute. Potassium Dichromate. A stock solution of the dried powdered primary standard grade mas prepared in double-distilled water to contain 100 fig of chromium(V1) per ml. Potassium Chromate and Chromic Acetate. iipproximately 0.2M solutions were prepared from reagent grade salts. The exact chromium concentrations were determined iodometrically by comparison with a dichromate primary standard. Chromic Potassium Sulfate. A solution containing 0.1 gram per liter of reagent grade salt was prepared and the exact chromium concentration determined microanalytically by comparison with a dichromate primary standard. 1,SDiphenylcarbohydrazide Reagent. This reagent was prepared and preserved as described ( 2 ) ,except for the following modifications. The solutions of phthalic anhydride in either 95 or 99.9% ethyl alcohol Tvere added while hot t o the solid 1,5diphenylcarbohydrazide either in a volumetric or narrownecked flask marked a t the desired volume (dilution t o a n absolute volume is not critical). I n this nay, solution is more readill. effected and the keeping quality of the reagent considerably improved. This improvement appears to be due to the removal of dissolved oxygen from the solvent by boiling. The reagent, 1% hen pi epared in the cold n ith alcohol purified by distillation over silver oxide, did not keep better than a reagent similarly prepared in nonredistilled 59. or 99.97, alcohol. APPARATUS
Glassware Cleaning. .A11 glassware used in the determinations was scrupulously cleaned by scrubbing and soaking in a detergent (such as Glim), rinsed with distilled water, immersed in an acid bath (aqua regia diluted 1 to 1 with distilled water) for 1 to 2 hours, rinsed with distilled and double-distilled water, and dried. Test Tubes. All steps in the analysis prior to the color development are conveniently carried out in borosilicate glass test tubes of 25-mm. diameter. For liquid samples up to 5 ml. the 100-mm. length tube is adequate in most cases. For larger samples, or those in which there is extensive foaming, the 150or 200-mm. lengths are preferable. Instruments. All p H measurenients were made n-ith a Beckman ;\lode1 G pH meter and all spectrophotometric determinations were made with a Beckman LIodel DL- spectrophotometer using 1-cm. Corex cells. PROCEDURES
For best results, aliquots should not contain more than approximately 5 y of chromium. Larger amounts up to 11 y were determined with slightly lower recoveries. Wet Ashing. Add to the sample 0.5 ml. of nitric acid followed by 2 to 4 drops of 3Oy0 hydrogen peroxide to reduce all sexivalent chromium present t o trivalent chromium. Sexivalent chromium in the presence of chlorides and the absence of water might lead to the formation of volatile chromyl chloride and consequent low chromium recoveries. When relatively large amounts of protein or substances precipitated on addition of nitric acid are present, the reduction of the sexivalent chromium is frequently incomplete, probably because of occlusion of the chromium in the precipitate. In such cases, warm the tube gently until all the precipitate dissolves, cool, and then add the hydrogen peroxide. Mix and let stand 20 to 30 minutes. If heating is started immediately when little water is present, the hydrogen peroxide will frequently react with the nitric acid and decompose suddenly nith spattering. Remove excess mater from mixtures that foam considerably in a water bath before completion of the ashing with nitric acid. Evaporate the nitric acid slowly over an open flame, allowing it t o reflux and wash down the sides of the tube. The tubes are held nearly horizontally with wooden clothespin-type clamps to avoid losses by spattering and entrainment. Samples containing little organic matter are ashed in an average time of 3 to 5 minutes. If large amounts of organic matter are present, repeat the nitric acid treatment one or more times until a white or slightly yellowish ash is obtained. Most
of the oxidation takes place just before the test tube becomes dr . A nearly dry residue ca? be obtajned a t the bottom of the t u i e while several drops of acid are still present about halfway up the tube wall. By returning these drops several times to the residue, a very efficient oxidation can be achieved. Deflagration may occur a t this point unless care is taken to heat the sample gently. Dry Ashing. Place t,he test tubes containing the samples, supported in metal racks, in a muffle oven and cover light,ly with aluminum foil to prevent contamination. Evaporate liquid samples t o dryness in a water bath before placing in the oven. Permit the temperature to rise to 420" to 460" C. within 2 to 4 hours and maintain it overnight. hshing is generally complete the following morning. If not, as evidenced by carbon particles, complete the ashing with nitric acid as in t'he wet ashing procedure. Solution of Ash and Oxidation. Dissolve the ash in each tube by washing down with 4 to 5 ml. of double-distilled water with warming if necessary. St,rongly heated ashes may be baked on the glass and not readily soluble. Then add several drops of nitric acid (or a 3 to 1 mixture of nitric and hydrochloric acids) and evaporate the acid gently, allon-ing it to reflux and dissolve the residue. The removal of acid at this point must be fairly complete or the subsequent addition of empirically established amounts of sodium hypobromite solution and sulfuric acid (as described below) will not bring the pH of the final solution within the range for color development. Add 0.5 ml. of sodium hypobromite solution to the aqueous Polution of the ash. Heat for 10 to 20 minut'es in a gently boiling ir-ater bath. pH Adjustment and Color Development. Cool and add a sufficient, number of drops of 12.5y0 sulfuric acid (10 to 20) to adjust the final volume to the optimal pH (1.3 to 1.7) for color development. The exact, number of drops required is determined by calibrating the procedure empirically with droppers so that it is not necessary to measure the pH in each case. The yellowbrown color of free bromine appears on acidification. If it does not, incomplete ashing (orgmic reducing materials) or deteriorated hypobromite is indicated. Transfer the content of the test tube to a 10- or 25-ml. volumetric flask (see discussion). Rinse twice with 2 to 3 ml. of double-distilled water. Add 0.5 ml. of phenol water to decolorize the bromine. Add 1.0 ml. of 1,s-diphenylcarbohydrazidereagent, dilute to volume, mix and read the absorbance a t 543 mw in a spectrophotometer. (With the Beckman Model DU, a slit width of 0.025 to 0.030 was used.) Make several readings betxeen 2 and 10 minutes to obtain the maximum absorbance. Calculation. For the determination of chromium in a variety of media, recoveries are based on control samples that contain known amounts of chromium in double-distilled water that are carried through the entire procedure. For the routine determination of chromium in a single medium, such as plasma, a reference curve that reflects the absolute losses is established for this medium. $11 recoveries are ultimately computed on the basis of a standard reference curve established by developing the color directly in volumetric flasks (no transfer) with added amounts of a standard dichromate solution. RESULTS
The chromium recovery values listed in Tables I, 11, and I11 represent averages of two to six determinations. The standard deviations were computed from the values of all the individual determinations and not from the average values. Unless otherwise noted chromium(II1) was added as potassium chromium(111) sulfate and chromium(F'1) as potassium dichromate. Table I shows that the recoveries were always 95% or better when chromium(II1) or chromium(V1) was added t o either distilled water or buffer solutions. The chromium recoveries from human plasma are shown in Table 11. Part A gives data obtained on adding a constant amount of chromium t o varying amounts of plasma and Part B shows data obtained on adding varying amounts of chromium to a given amount of plasma. The recoveries were between 94 and 101%. The recoveries of chromium from urine, paper strips, and egg albumin are shown in Table 111. The wet-ashing procedure was used for urine, the dry-ashing procedure for filter paper, and both procedures for egg albumin (see discussion). Table I V illustrates an application of the method in a series of dialysis experiments in which the binding of chromium to human
V O L U M E 27, NO. 6, J U N E 1 9 5 5 Table I.
985
Recoveries of Chromium(II1) and Chromium(VI), Wet-Ashing Procedure Cr(VI), y
Cr[III), y
Added
Found
Added
Found-
A. Added t o Double-Distilled Water 0,974 0.97s 1.13 1.14C 2.97 2.90 2.26 2.24 5.78 5,S6 4.52 4.47 0 = 0 0 4 u=OO3
1.00 3.00 6.00
B.
Cr Added, 0.42 0.82 1.61 3.22
Added to Buffersd Cr r o u n d ,
Y
Crc"1T
CrcIII)
y
through entire procedure. * Carried Starting with addition of phenol water. Starting with oxidation step with sodium hypobromite.
d Added t o 500 pl. of acetate buffer p H 5.30 or phosphate buffer pH 7.35, of ionic strength 0.1.
Table 11. Recoveries of Chromium Added to Human Plasma A.
2.00 7 of Cr(VI) Added to Varying Amounts of Plasma, Wet Ashing Plasma,
C r Found,
pl.
B. Varying Amounts of CrWIJ and
Cr(VI1 Added
Cr Found, C r Added,
7
Wet Ashing CrCIII) crcv1,
y
favorable conditions for obtaining a soluble ash must be arrived a t empirically for each material. The choice of wet or dry ashing depends on the type of material to be analyzed. Plasma or eerum can be ashed with equal facility by either procedure. The wet ashing of filter paper is tedious, while the dry ashing proceeds very smoothly. Urine gives better results when wet ashed, since dry ashing yields a difficultly soluble ash. Erratic results were also obtained when egg albumin was dry ashed. The addition of chromium-free filter paper or cotton, sufficient to soak up all liquid before drying and dry ashing, permits the successful dry ashing of egg albumin and other biological materials that give erratic results when dry ashed alone. (It is necessary to check the paper and cotton to find a type or lot that does not contain detectable amounts of chromium in the amount used in the dry ashing.) The paper or cotton acts as a support and generally a fluffy readily soluble ash is obtained. Black specks were occasionally noted in the ash obtained from egg albumin on dry ashing. They occurred more frequently in ashes obtained without supporting media during ashing, in which cases the ash was dense and concentrated. These black specks were insoluble in nitric acid, sulfuric acid, sulfuric acid and hydrogen peroxide, and aqua regia, and were not carbon. Recoveries of chromium from materials moistened with nitric, sulfuric, or phosphoric acid prior to dry ashing decreased in that order. Insoluble black specks were always present when phosphoric acid was used, occasionally with sulfuric acid, but not with nitric or organic acids as maleic or oxalic. Recoveries were better if no acid was used a t all.
to 200 p l . of Plasma y
Dry Ashing CrWI) Cr(V1)
Table IV.
Recoveries of Chromium(II1) and Chromium(VI) from Dialysis Experiments Cr
Added,
plasma and egg albumin was studied. The total chromium as distributed in the system could be accounted for in most cases to 100 f 5%. DISCUSSION 0
The various methods of dry and wet ashing of biological materials in preparation for trace metal determination are surveyed in a recent review (IO) and possible sources of error discussed. No data pertaining to chromium are presented. In the authors' experience the most serious errors in the microdetermination of chromium are introduced during the ashing process. These are sometimes due to mechanical losses from spattering, creepage. deflagration, and incomplete ashing. In most rases, however. they are due to baking on or fusion of the ash resulting in it; subsequent incomplete dissolution. In the viet ashing procedure baking-on or fusion can be controlled manually. Thc most
uHa
Mg.
4.14 4.14 5.30 5.30 6.40 6.40 7.35 7.35 7.35
2.31 2.31 2.31 2.31 2.31 2.31 2.31 2.31 2.42
Valence of Added Crb 6 3 6 3 6 3 6 3 3 and 6
Total Cr Found, Mg.c 2.18d 2.38 2.24 2.16 2.40 2.45 2.40 2.27 2.38'
Recovery,
%
94.5 103 97 93.5 104 106 104 98.7 96-100
pH's 4.14 a n d 5.30 in sodium acetate-acetic acid buffers of ionic strength
0.1.
pH's 6.40 and 7.35 in sodium and potassium phosphate buffers of ionic strength 0.1. Chromium(V1) added a s potassium chromate and chromium(II1) a s chromium(II1) acetate. C Each value of total chromium found is pummation of separate amounts of chromium found inside and outside dialpsis bag a i end of each expenment. d f O . O 1 mg. 8 Mean value of seven separate dialysis experiments: maximum deviation -0.06 and +0.04 mg. -
Investigation of the cause and nature of the black specks was made. Chromium(II1) nitrate and sulfate can decompose when strongly heated to yield chromium(II1) oxide which when ignited is highly refractory to -~ chemical attack. Ness and coTable 111. Recoveries of Chromium Added to Urine, Paper Strips, and Egg Albumin workers ( l a ) showed that chroCr Found, y I n Egg Albuminc mium(II1) phosphate when I n Urinen I n Paper Strips b Wet Ashing Dry Ashingd heated over 300' C. yielded Cr Added, y CrIII CrVI CrIII CrV' CrIII CrV' Cr"' Crh" black amorphous m a t e r i a l s 0.50 0.52 0.49 0 0.46 0.47 0.47 0.51 that required fusion for solu1.00 00 .. 95 74 0 .. 4 97 6 1.86 1.82 1:sl 1.92 I:96 1:92 tion. Egg albumin [ovalalbu2.00 1:95 1:87 5.00 4.71 4.90 4.79 4.72 4.60 4.88 4.85 4.88 min cont,ains 0.10 t o 0.13% u = 0.05 u 0.04 u = 0.04 phosphorus ( 6 ) ]was dry ashed a Added t o volumes of urine u p t o 10 ml., wet ashing only. b Added t o Eections of Whatman 3 M M paper from 0.5 cm. X 1 inch u p t o 1 X 2 inches, dry ashing only. per se, with 1 drop of 10% C Added t o amounts u p i o 500 p l . of a 10% solution. phosphoric acid, with 1t o 6 Y Of d Dry-ashinc: d a t a for egg albumin obtained b y addition of filter paper or acid washed cotton prior to dry ashinp. chromium, and with 1 to 6 y of _ _ _ _ ~ ~ _ _
-
~~
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,ained from 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 slons 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 an 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 wnples 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
