V O L U M E 24, NO. 8, A U G U S T 1 9 5 2 the wave nunibers a t nhich the is0 and normal sapogenin band systems should occur. It can be seen that none of them possess the characteristic system of four sapogenin absorption bands, although some contain one or t n o bands a t nearly the same frequencies. Curve c is the spectiurn of a triterpenoid sapogenin. Such a compound could not be mistaken for a steroidal sapogenin because it does not have the entire system of four bands. However, if a substantial amount of such substance occurs mixed with a small amount of qteioidal sapogenin in a plant extract, it can lead t o an erroneouslv high estimate of the amount of steroidal sapogenins because of its absorption a t 982 cni.-l Certain other substances found in plant materials also have xeak absorption bands near 982-987 cm.-’ but do not have the complete system of four sapogenin bands. Therefore, although the infiared method appears to give positive evidence of the presence or absence of steroidal sapogenins, the estimatc of percentayc of sapogenin is only semiquantitative if considerable amount. of nonsteroidal material are present. Severtheless, it is the ouly method now available y a crude material. which will give even an approximate a ~ a on Two worhers, onp czrr?ing out the heiiioly,is and crude sapo-
1341 genin isolation steps and the other the infrared procedure can handle 40 t o 50 samples in a &hour n eek. ACKNOW LEDG.\IEYT
The authors wish to thank A. E. Jones for assistance in various phases of this research, and T. D. Fontaine for the sample of dihydrodiosgenin. LITERATURE CITED
(1) KoAer, L., “Die Saponine,” Vienna, J. Springer. 1927. (2) Kofler, L., and Raum, H., Hiochem. Z.. 219, 335 (1930). (3) Marker, R . E.. and Applerweig. S . , C‘liem. Eng. S e w s , 27, 3348 (1949). (4) Narker, R. I-:., Wagner, R . R.. L-lshafer. P. K., JVittbecker, E. L., Goldsmith, D. P. J., a n d Iluof, C. H., J . A m . C’hem. Soc.. 69, 2167 (194ij. (5) Roserikranz. C;., Pataki, J., and Djcrassi, C., Ibid., 73, 4055 (1951). (6) Wall, hI. E., Krider, RI. hl., liothman, E. S., and E d d y , C . R.. S.Bio2. Chem., in press.
R E C ~ I \for T Dreview Febriiary 10. 1052. Accepted I I a y 23, 1952. in a series on steroidal rapugenins: for paper I see (6).
.Second
Microdetermination of Chromium in Blood H. J. CAHNMA” AND RUTH BISEN Cancerigenic Research Laboratories, .Yational Cancer Institute, 4ictional Institutes of Health. Rethesda, .\Id. In the course of investigations of lung cancers among chromium workers it became necessary to hav e a method for the quantitativ e deterinination of traces of chromium in blood. As the chromium coiicentration of most blood saniples which had to be analyzed w-a8 in the order of magnitude of 0.01 to 0.1 p.p.m., a method with a high degree of sensiti\ity and precision was required. A spectrophotometric micromethod is described, based o n the w-ell-known reaction of hexavalent chromiuni with s-diphenylcarbazide, which results i n the formation of a pink color. This reaction is sensitive to 0.005 p.p.m. if the color is read in a Beckman spectrophotometer, Model DU. The standard deviation was determined for chromium concentrations ranging from 0.02 to 0.32 p.p.m. and found to be 0.005 p.p.m., when 10-1111. blood samples were used.
A
LTIIOUGH a great, numbtxr of toxic maiiifestntions of chromium have heen described in t81ieliterat,ure (1, 7 , 20, 39), very litt,le is known about the nirtabolism of chromium in the human body. The authors became interested in chromiumnietabolisin because of the high incidenw of cancer anioiig chromate -1s the producand chromite workers (3-6, 18, 20, 21. Z+,2;). tion of chromium iii the United Statw hns incrcvtsed t,rcmendousl>during the past tn-o decades, thv study of certain biochemical aspect.s of chroniium cancer beconies n i u r c ~arid niore iniport,ant,. In the course of the investigations it 1)ecanit~necessary t’ohave a method for the quantitative determination of very small aniounts of chroniium in blood. The method must be sensitive and it must have a high degree of reprodueihilit~-for such low blood chroniiuni concentrations as 0.1 p.p.ni. mid Iew. I t must also permit the determination of chroniium prewnt as soluble chromium salts or as organic chroniiuni componnds, as well as chromium in chromite which might be present in the blood of chromite workers in the form of very fine particles (cf. 2 3 ) . Among the various possible methoda (including spectrophotometric, spectrographic, polarographic, and radioisot’ope proce-
dures) a spectrophotometric method was first investigated, n-hich is based on the format,ion of an int,ensely colored compound when s-diphenylearbaxide is added to a dichromate solution. [The nature of this colored substance is still unknown. Therefore, t,he authors prefer to call it a compound rather than a complex, following a suggestion made by Feigl (Is)]. Many ot,her color reactions have been suggested (cf. I), but none of them is so sensitive and so specific as the diphenylcarbazide reaction. The principle of this method has been known since the beginning of this century (9,SO), and a great number of publicat,ions have dealt with its application to the detection and niicrodeterminat,ion of chromium in minerals, steels, soils, and water (29, s3, 34). Relat,ively lit,t,lework has been done on biological material and only a fen- methods have dealt, with the colorimetric niicrodetermination of chromium in blood ( 1 , 7 , 28, 38). Akatsuka and Fairhall ( 1 ) stat(, that t,heir method permits only a rough estiniat,ion of t h e chloiiiium content of blood and tissues. Brard ( 7 ) uses large blood samples in his determination. Mitchell and Gray’s niethod (28) cannot be used if the sample contains less than 3 micrograms of chromium. rill three methods are r:rt,her tedious and not easily adaptable to the analysis of a large number of blood samples containing minute amounts of chroniium. Urone and Andere’ method (38)is by far superior to both Braid’s and Mitchell and Gray’s methods. Vsing this procedure, sat,&factory results were obtained when blood samples cont’aining chromium in concentrations great,ert,han 0.1 p,p.ni. were analyzed. However, insufficient precision x-as obtained for the wthors’ purposes a t chromium concent,rations below 0.1 p.p,m. In view of the fact that the chromium concentrations of most samples t o be analyzed in this laboratory ranged from about 0.01 to 0.1 p.p.m. it appeared desirable to develop an alternative procedure which gives an increased precision and can therefore be used advantageously for these extremely lo\\- chromium concentrations. [Determinations carried out in this laborat,ory and in the 1aborat.ories of the Division of Industrial Hygiene, Ohio Department of Health ($6), shon- that blood of chromiuni workers contains about 0.01 to 0.16 p.p.m. of chromium, whereas normal human blood usually contains less than 0.01 p.p.ni. Somewhat similar results are re-
A N A L Y T I C A L CHEMISTRY
1342 ported in two publications which came to the authors' attention after completion of the present work ( 2 ,I.)% The method presented in this paper consists of a dry or wet ashing of the blood, followed by an oxidative alkaline fusion which serves to transform trivalent to hexavalent chromium and also to dissolve any chromite particles that may be present. Diphenylcarbazide is added t o an aqueous solution of the melt after removal of the iron m ferric oxide and proper adjustment of the acidity. The intensity of the chromium-diphenylcarbazide color, n.hich follows Beer's law (Figure 11, is then read in a spectrophotometer. REAGENTS
Distilled Water is prepared in an all-glass distilling apparatus. Double-Distilled Water. A small quantity of alkaline permanganate solution is added to distilled water. The solution is boiled until about one fourth has evaporated, and the remainder is distilled. Precautions should be taken to avoid the carrying over of any spray, and all-glass equipment should be used (cf. 58: 40). Dichromate Stock Solution. Powdered and dried potassium dichromate (2.823 grams of primary standard, analytical reagent, Rlallinckrodt) is dissolved in double-distilled Kvater and the total volume is brought to 1 liter. This solution is diluted 1 to 100 wit,h double-distilled water. Each milliliter of this solution corresponds to 10 micrograms of chromium. Nitric Acid. Reagent grade nitric acid (specific gravity 1.4) is redistilled using a distilling head that prevents entrainment of the acid. (Most reagent grade nitric acid contains chromium.) A slow stream of filtered air or nitrogen is passed over the surface of the acid in order to facilitate removal of the vapor. -4middle cut of the distillate is used (12). Hydrochloric Acid. Distilled water is saturated with hydrogen chloride gas. ( X o s t reagent grade hydrochloric acid contains chromium.) The receiving flask is cooled in an ice bath. Hydrogen Peroxide, 30a/0, reagent grade. Ammonia Water. One volume of reagent grade ammonia water (28 to 297,) is diluted with 4 volumes of distilled water. Carbonate Mixture. A molar mixture of reagent grade sodium and potassium carbonates is used. Both salts are predrird. The mixture is kept in a tightly closed container. Potassium Chlorate, KC103,analytical reagent. Sodium Chloride Solution, 0.37, sodium chloride, reagent grade, in double-distilled water. Sulfuric Acid. A 25% solution (v./v.) is prepared from reagent grade concentrated sulfuric arid and double-distilled water. In order to destroy traces of reducing substances, a few drops of a dilute potassium permanganate solution are added to the hot' aqueous aci 1 until a very faint pink color persists for 1 minute (33). Diphenylcarbazide Solution. One gram of phthalic anhydride, analytical reagent, is dissolved in hot 957, ethyl alcohol (L7.S.P.). After being cooled to room temperature, the solution is added t,o 62.5 mg. of reagent grade s-diphenylcarbazidc 111 a 25-nil. volumetric flask and brought to the mark with 95% alcohol. The flask is closed tightly by means of a suitable sealing material such as Parafilm and kept in the freezing compartrncnt of a refrigerator. The solid tiiphenylcarbazide is also kept in a tightly closed container in the refrigerator. CLEANING OF G L l S S W A R E i N D PL.ATINU.MWARE
The glassware is cleaned I)>- wccessive washings with a detrrgent, a concentrated nitric acid-concentrated hydrochloric arid-3 4 volumes), and distilled water. The water mixture (1 glassware should remain immersed in the acid mixture for 15 minutes or longer. Kjeldahl flasks and glass beads which have become etched through frequrnt use should eit'her remain immersed in t,he acid solution for several hours or be treated with hot acid solution for several minutes. Although the use of etched flasks and beads never led to erratic results, it is recommended that badly etched flask.%anzi heads he replaced by new ones a8 a matter of precaution. Platinum crucibles and covcrs arc heated in concentrated hydrochloric aci? and rinsed ivit,h x-ater. Then they are gently rubbed with a moistened mixture of fin finall>- rinsed with ivatt>r ansl rli
+ +
PROCEDURE
Ashing. A dry-ashiny procedure is recommended only for small blood samplcs of u p t.o 2 ml. A vet-ttshing procedure can be used for blood samples rtinging from a fraction of 1 ml. to 30
ml. or possibly more (10-ml. samples were used for most of the determinations carried out in this laboratory). DRYASHING. One milliliter of blood is placed in a platinum crucible of about 15-ml. capacity. The blood is evaporated t o dryness on a steam bath. The drying process is completed by placing the crucible in a n oven a t 100" C . and raising the temperature t o 110" C. The heating is continued in a muffle oven from 250" to 52.5' C. [If the heating elements of the oven are exposed] the crucible should be protected against possible contamination (cf. IS) by placing it in a porcelain evaporating dish and covering it with a beaker. The beaker ha5 two small holes on opposite sides in order to permit the accumulated fumes to escape.] Between 380" and 410' C. swelling and frothing occur. h temperature of 525' to 540" C. is maintained for 40 minutes. If 2 ml. of blood are used, swelling and frothing take place somewhat above 400" C. and the temperature of 525' to 540' C. must be maintained longer than 40 minutes for complete ashing.
0.400;-
+ t-
I 0.300
w n -I
3
E o.200:I 0
0.IOOt
1
2
3
4
5
6
7
CONCENTRATION (MICROGRAMS CHROMIUM ADDED) Figure 1, Chromium Concen tration-Optical Density C w \ e s \\-ET ASHISG. Three milliliters of nitric acid are added to the blood in a Kjeldahl flask. (Flasks of 30-ml. capacity are used for blood samples up to 10 to 15 ml. For larger samples correspondingly larger flasks should be used.) A4glass bead is added to prevent bumping. After thorough mixing, the flask is allowed t,o stand for several minutes, then 0.5 ml. of hydrogen peroxide is added. The flask is again shaken, alloned to stand for a few minutes, then heated gently over an open flame with frequent shaking until the foaming subsides. It is then placed on a Iijeldahl rack and the contents of t,he flask are boiled. After some t,ime the solution becomes clear. As soon as faint brown vapors d a r t to form, a small funnel (diameter about 18 to 20 length of stem about 50 mm.) ie placed in the neck of the and the flame is lowered as much a-5possible. At this point t.he react,ion should be watched carefully. An exothermic reaction start,s soon with the evolution of h e a w brown vapors. After this reaction subsides, the flame may be raieed slightly. Heating is continued until the reaction mixture turns black and the residue most dry. When the flask has cooled somewhat, 1 ml. cid is added through t,hc funnel and the mixture is evaporat,ed to complete dryness. Then the flask is placed in a muffle oven overnight a t about 500" C. (If many samples are to he worked up simultaneously, it is convenient to place all the Iijeltlahl flasks in a sheet metal basket Ivhich can be easily moved in and out of the oven. If necessary, t.he flasks can be protected against possible contamination by sliding sleeves made of glass tubing over their necks. The use of t,eet tubes for this purpose is not recommended, because they prevent free circulation of the air.) The next morning the flask is allowed to cool and 1 ml. of nitric acid is added through the funnel, The mixture is evaporated to dryness, then the flask is heated in a large yellow flame until
V O L U M E 2 4 , NO. 8, A U G U S T 1 9 5 2 heavy brown vapors appear. This operation is repeated once more. After cooling, 6 ml. of hydrochloric acid and 3 drops of hydrogen peroxide are added to the residue. The contents of the Hask are mixed and then boiled. Before dryness is reached, the vapors should be tested n i t h potassium iodide-starch paper for chlorine and nitrosyl chloride. As soon as this test becomes negative, t,he solution can be evaporated completely. ( I n some rare cases it is necessary to add 1 ml. more of hydrochloric acid before completion of the evaporation.) Then 1 ml. of hydrochloric acid is added to t h r residue and the flask is heated until the walls are \vel1 washed down by the condensed acid vapors. The funnel is rinsed with 2 ml. of distilled water and then removed. (The use of a q i n g e and needle is recommended for all risings.) The contents of the flask are heated to incipient boiling and then transferred with a hand pipet to a platinum crucible of about 15-ml. capacity. The flask is rinsed three times with 1 ml. of distilled water containing 3 drops of hydrochloric acid. The rinses are heat'ed to incipient boiling before each transfer. The combined liquids are evaporated to dryness on a steam bath. Fusion. If the blood sample was dry ashed, about 1 ml. of distilled water and then 200 mg. of carbonate mixture and 20 nig. of potassium chIoriLte are added t'o the residue in the platinum rrucible. If the blood sample was \vet ashed, 1 ml. of ammonia water is used instead of the distilled water. The crucible is heated on a steam bath to dissolve the salts and then tilted in such a manner that all parts of the crucible covered by the ash are wetted. The solution is evaporated to dryness. The drying process is completed by placing the covered crucible in an oven a t 100" C. and raising the temperature to a t least 120' C. Heating is then continued in a muffle oven from 400" to 750' C. (If necessary, the crucible can he prot,ected by placing it in a porcelain evaporating dish and covering it with another evaporating dish of the same size.) If no chromite is suspected in the blood sample, the crucible is removed from t'he oven. If the presence of chromite is considered possible, the crucible is kept a t i 5 0 " C. for 10 minutes. Color Development. One milliliter of double-distilled water is added to the cold melt and the covered crucible is heatc.d on a steam bath for a few- minutes to disintegrate the salt cake. Then the cover is rinsed down with 1 ml. of double-distilled water. The solution containing suspended ferric oxide is transferred to a 15ml. graduated centrifuge tube and the crucible is rinsed three times with 1 ml. of double-distilled water. After each addition of rinse water, the crucible is heated for a short time on a steam bath. The suspension is centrifuged for 4 minutes Tith a centrifugal force of about 2000 t,o 2500 gravity a t the tip of the centrifuge tube, The supernatant liquid is decanted into a 10-nil. volumetric flask. The residue is washed once with 1 ml. of doubledistilled water and once with 2 inl. of sodium chloride solution hy centrifugation. The combined liquids are acidified with 0.5 ml. of 28% sulfuric acid. The volumetric flask is swirled around to liberate carbon dioxide from the supersaturated solut'ion. One half milliliter of diphenylcarliazide reagent is added and the solution is brought t o a total volume of 10 ml. with double-(listilled water. The optical density of the solution is read in a spectrophotometer a t a wave length of 543 nip against a blank containing the same amounts of reagent anti sulfuric acid as the test solution. [A Beckman spectrophotometer., Model DU, and 1-em. cells were used. Cell corrections ~ v e r emade according to Caster (8). The slit v\.idth was srAt a t 0.03 mni.] Readings are made between 5 a n d 15 minutes ktfter the addition of the diphenylcarbazitle. STANDARDIZATIO&
The chromium concentration of t,he analyzed blood sample can be derived from the optical demit- reading by comparison with a previously prepared standard reference curve. The standard reference curve is established in the following manner. The optical density of a blood blank is determined with normal blood using t,he procedure outlined above. Increasing amounts of dichromate stock solution are added by means of an ultramicroburet to othei, samples of the same blood. ( A Gilniont buret, \vas used.) Thew chromium-containing blood samples are also worked up in the iianie manncr. From the optical densities obtained, the optical density for the blood blank is deducted. The t,hus correct.ed optical densities are plotted against. the amount of chromium added (Figure 1, curve 11). The opt.ical density of a reagent, blank is determined using the same procedure as for blood, except that the blood is replaced by distilled water. In the analysis of a blood sample of unknown chromium content, t.he optical density of the reagent blank is
1343 deducted from the observed optical densit,\.. The chroniium content can then be determined from the correct,ed optical density value h y comparison with the standard reference curve. .I new standard reference curve should he est,ablished about every 2 years or whenever a new batch of polid diphenylcarhazide is used (cf. IO). DISCUSSION
The sensitivity of the color reaction uwd is very high. -is little as 0.005 microgram of chroniium pc.r milliliter of final soluLion can he detect,c>dwith a Beckman spectrophotometer. The main prohleni encountered in applying this reaction to the determination of extremely small amounts of chromium in blood consists in eliminating numerous factors that, interfere with the precision of the method. Among such factors are incomplete osidation of trivalent to hexavalent chromium, presence of minute :mounts of reduring agents, lose of chroniium t,hrough volatilization, and interference of heavy metals. In the dry-ashing procedure B Huffy reddish ash is obt,ained. Occasionally black charwal spots arc visihle. In this case heating must be continued, as charco:rl prevents the complete oxidation of t8hcchromium t o t,he hexavalent state in the subsequent fusion. Overheat,ing must be avoided. If the temperature ex~ n complete d ashing becomes ceeds ,550"C., the fluffy ash sirit difficult. The use of Kjeldahl flasks in the wet-ashing procedure prevents mechanical losses through .spattering. Perchloric acid is not a suit,able oxidizing agent because its use leads to the formation of volatile chromyl chloride (19, 22, 38). Oxidation mixtures containing concentrated sulfuric acid are not practical when Kjeldahl flasks arc used for the ashing, bec*auscthe subsequent evaporation of the d f u r i c acid is difficult,. C'oncentrated nitric acid is an excellent oxidizing agent. The cvaporntion of the nitric acid, however, must be carefully roritrollrd. If it is carricd out too fast,, the reaction tends t,o heconir violent, Complete ashing can he achieved by successive additions and evaporations of nitric acid. However, time is savcd : ~ n dl(w nit,ric acid is uiied if the inconipletely ashed residue is heated in a muffle oven overnight. Hydrogen peroxide is added twice in t,he rourse of the wet ashing because it. reduces instantaneously any hexavalent chromium present and thus prevents the formation of chromyl chloride. It has been said that chromic chloride is volatile a t lon- temperatures ( 1 7 , 28). Experiments carried out i n this lahoratory show that no chromium is lost when chromic (ahloride is heated to several of hundred degrees centigrade (cf. 1 4 ) . Excessive s~perhcat~ing the Kjeldahl flasks should be avoided whenever an evaporation to dryness is carried out. S u p e h ~ i t i n g( ~ I S C S the flasks to kiecwme etched within a short time. The addition of hydrochloric. wid t ( J t,he fully ashed sample and its subsequent evaporation sc'rve to eliminate all nitrates as nitrosyl chloride: HNO, 3HC1 +2tI,O SOCI CIlr
+
+
+
.iny riit,rates left would he partly reduced to nitrites during the following alkaline fusion. The nitrous acid liberated upon the acidification of the aqueous solutio11 of the melt, prior to the color developinent, would reduce the hexavalent chroniium. Several fusion niixt,ures were investigated and a carhonato chlorate mixture was finally cmhosen as the most, satisfactory. LIixtures containing riit,rates as an oxidizing agent cannot he used for t,he reason outlined above. Sodium peroxide is an escellent solvent for chromite, but i t strongly attacks porcelain, iron, nickel, and platinum crucibles. The carbonate-chlorate niixture used in this procedure also attacks porcelain, nickel, and iron crucibles considerably, but platinum crucibles only very slightly. Dingwall, Crosen, and Beans ( I S ) used a mixture of sodium bicarbonate and potassium chlorate. Because of the high fusion temperature of this mixture (ea. 800" C.) and the high chlorate concentration, t,he platirium crucible is Considerably at'tacked.
ANALYTICAL CHEMISTRY
1344 The use of a molar misture of sodium and potassium carbonate serves to lower the fusion temperature to about 700" C. Neither potassium chlorate nor potaesiuni chloride which is formed during the fusion interferes with the intensity of the chromium-diphenylcarbazide color. Finely powdered chromite, up to many times the amount which would be suspected in blood, is readily dissolved under t,he experimental conditions of the procedure presented in this paper. A standard reference curve was established by adding known amounts of chromium in the form of chromite powder to blood. The curve obtained can be superimposed on the standard reference curve of Figure 1. The chromium content of the chromite powder used was determined iodometrically ($7). The diameter of the chromite particles was 4 microns or less. The chromite was intimately mixed with powdered lactose in order to permit accurate weighing.
0.08C
I
I
I
I
I
I
> 0.07C L= v)
z W
0 -I
3 0.06C I-
8 5 2 MICROGRAMS IRON
PER ML.
0.050
WAVELENGTH (mp) Figure 2. Absorption Spectrum of the Irons-Diphenylcarbazide Compound Ferric ions should not be present in the final solution in which the chromium-diphenylcarbazide color is developed. They form a brown color with diphenylcarbazide which interferes with the determination of chromium (10, 31, 33). hlthough the interference of iron is generally recognized, indications In the literature concerning the extent to which iron interferes are contradictory. Although Dingvall et al. (11, 13) deny any interference of iron, provided that measurement8 are made a t longer TTave lengths than 500 mp, Davis and Bacon (10) find a strong interference of even small amounts of iron. Sandell ( 3 3 ) states that chromium may be determined in the presence of a limited amount of iron. In experiments carried out in this laboratory the findings of Davip and Bacon could be confirmed. Figure 2 shon-s the absorption curve of the iron-diphenylcarbazide compound betv-een 516 and 570 mp. .4 solution of ferric chloride containing 520 micrograms of iron (the amount normally present in 1 ml. of human blood) n-as added to a volumetric flask. -4fter adjustment of the pH to the value used in the determination of chromium, an excess of diphenylcarbazide reagent was added and the volume brought to 10 ml. A comparison of the curves shown in Figure 1 and Figure 2 shows that the iron-diphenylcarbazide compound absorbs enough light a t 543 mp to simulate the prescnce of about 1 microgram of chromium in 1 ml. of blood Since preliminary attempts to remove ferric ions by complexing them with phosphoric acid (7, 10, 91) led to low chromium recoveries, no further experiments were done in this direction. If
the melt froin the carbonate-chlorate fusion is dissolved in TTater, a fine powder of red ferric oside remains undissolved and can be removed by centrifuugabion. This residue retains only a negligibly small amount of chron~iumthrough adsorption or occlusion. The use of a very dilute sodium chloride solution for the second rinsing of the ferric oside precipitate serves to prevent peptization. After completion of the fusion, the inside of the platinum crucible is covered 1rit.h a yellow film containing iron and traces of platinum. This film is insoluble in water and therefore the h a 1 solution does not conta,in platinum. This is important because platinum reacts n-ith diphenylcarbazide to form a compound that absorbs light a t 543 nip. The diphenylcarbazide reagent used is the one proposed by Ege and Silverman (15). If kept in a tightly closed cont.ainer in the freezing compartment' of a refrigerator, it is stable for about 1 mont,h, after which time it assumes a brownish tint. Although a slight discolorat,ion does not, interfere with the determination, a fresh reagent was prepared as soon as a discoloration became apparent. The solid diphenylcarba,zide is also kept in a refrigerator in order to retard deterioration (10). -4s shown in Figure 3, the concentration of the reagent in the h a 1 solution has a definite influence upon the t,iiiie needed for the development of the maximal color. The addition of sulfuric acid to the reaction mix%urebefore the color is developed brings the pH of the solution to the optimal value of 1.5 to 1.6. d t a pR below 1 t,he color intensity is d e creased considerably and the color fades rapidly. .4t a p H above 2, full color development is vcry slow (cf. 30,33). The molar extinction coefficient of the chromium-diphenylcarbazide conipound at the wave length of its maximal absorption (543 mp) was found to be 3.11 X lo4 (based on chromium). This is in good agreement' with t,he value of 3.14 X lo4 reported by Rowland (32) and Ege and Silverman (16). Somewhat higher values wcre found by Crone and .Inders (38)and Dingwall, Crosen, and Beans (13). Differences in the quality of the diphenylcarbazide used (cf. 1 0 ) arc probably responsible for these discrepancies. A great number of determinations were made in which known amount,s of chromium were added to blood as p o M u m dichromate, chromium potas.6mi sulfate, or finely powdered chromit,e.
0~050L---
1i
0.040
UJ
J
1/
0.020
0.0I ott'
IO
1-0.08 MICROGRAMS CHROMIUM B 0.04 ML. DIPMNYLCARBAZIDE REAGENT PER ML.
P-0.08 MICROGRAMS CHROMIUM B 0.01 ML DIPHENYLCARBAZIOE REAGENT
PER )r
20
ML
543mp
SLIT W I D T H ' 0 0 3 m m
30 40
50 60
3
TIME (MIN.) Figure 3.
Time-Opticai Density Curves for Different Diphenylcarbazide Concentrations
1345
V O L U M E 24, N O . 8, A U G U S T 1 9 5 2 The mean value t'or tlie chromium recovery is 93 to 94%) n o matter in which form the chromium is added. The st:indard reference curves established with 1 nil. of blood (dry-ashing procedure) and with 10 1111. of blood (x-et-ashing procedure) TWIT identical. The standard deviation for the clcxtcrmination of chromium was calculated on the b&s of a great numher of data obtained \Then amounts of chroniium ranging from 0.2 to 3.2 micrograms were added to blood samples of 1 to 10 ml. If the dry-ashing procedure is wed, the standard deviation is 1 0 . 0 6 micrograni of chromium; if the !vet-ashing procedure is used, the standard deviation is 1 0 . 0 5 microgram of chromium. REFERENCES
-Lkatsuka, h.,and Fairhall. L. T., J . Iiid. fl!iu., 16, 1 (1934). -Uwens, TV., Ber. l ' l l l l n t e r i ~Korbg?'. Cnfa.llrned. u . Berujskrankh., Frankfurt/M., September 1938, p. 97'3, Leipzig, Georg Thieme, 1939. Baetjer, 1.31.. A / d f . I n d . Huu. 0 c c i i i ) u t l o u u l .Wed., 2 , 487 (1950). Ihid., p. 505. Ridstrip, L., i l r c h . bclaes 7ithd. . s w i , i / ~( t hyg., 8, 500 (1950). Bourne, H. G., Jr., and Tee, H. T., Iiid. Med. and Surg., 19, 563 (1950). Arard, Daniel, ";lctualitbs Scientifiqur.: et Industriellcs," S o . 225, Paris, Hernmn et Cie., 1935. Caster, W. O., .Is.u..CHEAT., 23, 1229 (1051). Caeeneuve, P., Bull. suc. chiin., [ 3 ] 23, 701 (1900); [3] 25, 761 (1901). Davis. 1%C., and Bacon, d.,J . SOC.C ' h i m . Itid., 67, 316 (1948). Dingwall, .L.,and Heans, H. T., A m , J . Cariwr. 16, 1490 (1932). P , T., Proc. .\-r!tl. .Icad. Sci., 20, 416 Dingwall, A , , antl U ~ N I I [I. (1934). Dingwall, 9.. ('ioaeii. K.G., arid Beans, H. T., -4m. J . C ' U J I C W , 21, 606 (1934). Doerner, H. -4., L-. ,S.Dt,pt. Interior, liur. Mines, Ttcii. P a p e r 577 (1937). Ege, J. F., Jr., and bSilrernian.L., AA-.LL. CHEAL,19, 693 (1047). Feigl, F., personal comniunication. Gray, S. J., and Sterling, I
