Spectrochemical Determination of Trace Quantities of Cobalt in Animal Tissues ROBERT
G. KEENAN and JOHN
F. KOPP
Occupational Health Field Headquarters, Public Health Service, U. S. Department of Health, Education, and Welfare, Cincinnati, Ohio
The accurate chemical determination of submicrogram quantities of cobalt in animal tissues has been rendered difficult in the past by the presence of varying amounts of interfering elements, notably iron, as well as by the use of methods of insufficient sensitivity. These difficulties have been overcome in this laboratory by the development and application of two highly sensitive spectrochemical methods. One method is applied directly to the ash of tissues of exposed animals containing at least 0.025 y of cobalt per gram of fresh tissue, in excess of that present in normal tissue. The other method was developed for the determination of cobalt in normal tissues. It employs the principle of preliminary chemical concentration of the cobalt with an organic complexing reagent, followed by collection in a mixture of aluminum oxide, lithium chloride, and graphite as constant base material for direct current arc spectrography. The latter method has been used for the determination of millimicrogram quantities of cobalt in the tissues of small normal animals.
a high concentration of phosphate. The efficiency of thiq extraction has been found to he very high and this method of isolating cobalt was applied succe-sfully to all animal tissues encountered in this investigation. Mitchell and Scott had reported aluminum to be a satisfactorv carrier element for their group precipitation procedure and had found the aluminum oxide residue from the ashed precipitate to be a suitable spectroscopic matrix. Therefore, in the present method, aluminum nitrate is added to the chloroform solution containing the extracted cobalt rompleu. After ashing, ho\\ ever, a mixture of lithium chloride and graphite is added as a spectroscopic buffer to the aluminum oxide residue. This buffer ?\as found by Keenan and White ( 5 )to be effective in the suppression of cyanogen bands and background and also in the enhancement of line spectra. By the use of this buffer in both methods of the present paper, a sensitivity of 0.005 y of cobalt on the electrode has been attained. Cobalt present in the tissues of three species of normal animals has been determined by the second method. DIRECT SPECTROGRWHIC \IETHOD FOR C O B i L T IN TISSUES O F EXPOSED ANIMALS
I
N T H E course of an experimental investigation of t,he toxicity of cobalt, the need arose for the det'ermination of submicrogram quantities of this element in the tissues of animals exposed to cobalt metal fume a t an air concentration approximating 1 mg. of cobalt per cubic meter. The purpose of the present paper is to present the two spectrochemical met'hods developed for the determination of cobalt (1) retained by the soft tissues of animals exposed to known concentrations of cobalt metal fume for specific periods of time or (2) deposited in the hones of exposed animals or present normally in the bone and soft tissues of unexposed animals. These methods have shown a high degree of sensitivity and accuracy on analyses of more than 1000 tissur, samples from dogs, rabbits, guinea pigs, rats, and mice. The first method is a direct spectrographic procedure which i p applied to the ash of soft tissues from exposed animals only. The tissues should contain a t least 0.025 y of cobalt per gram of fresh tissue in excess of the quantity present in normal tissue. Under the conditions of this method, normal tissue aPh does not yield a detectable line of cobalt. The second method includes a cobalt concentration procedure and is designed for the determination of cobalt in the soft tissues of normal animals and in the bone samples of both exposed and normal animals. This method involves the extraction of cobalt with an organic complexing reagent and addition of a carrier element to provide hulk, followed by ashing and spectrographic analysis of the resulting oxide mixture. This procedure is based upon the principles set forth by other investigators (3, T , 8, 1 0 ) for the determination of trace elements in plants, soils, and biological materials. Mitchell and Scott ( 7 , 8, 1 0 ) employ a mixture of 8-quinolinol, tannic acid, and thionalide for the quantitative precipitation of a group of trace elements including cobalt, from a solution buffered a t a p H of 5.2 and containing the unwanted alkali and alkaline earth elements and phosphate. However, the extremely large amount of phosphate in bone ash precluded the use of Mitchell and Scott's group separation procedure. Instead, it was decided to extract the cobalt with l nitroso-2-naphthol, in accordance ~5ith Saltzman's preliminary extract'ion procedure ( 9 ) ,to circumvent the difficulties imposed
Quantitative analysis by the direct spectrographic method may be applied to the tissue samples (other than hone) of cobalt-exposed animals. These tissues should contain a t least 0.005 e, of cobalt per 2 mg. of tissue ash in excess of the amount of this element in the normal tissue ash used as base material for the preparation of the analytical curves. The 2-mg. quantity of ash, introduced in dilute hydrochloric acid solution to the electrode crater, has been found to be the optimal quantityforspectrographic exposure, as repoited by others who have analyzed these materials ( 2 ) . Bone tissue vields too large a quantity of ash to be amenable to analvsis by this procedure and should he analyzed either chemically (9) or b r the alternative spectrographic method, if the cobalt content is too low for the successful application of the chemical method. REAGENTS
.411 reagents are anal) tical ieagent grade, except where othern ise indicated. Double distilled water, from an all-glass borcsilicate glass still, is used in the preparation of all reagent solutions. Electrode Waterproofing Solution. Dissolve 20 grams of paraffin wax in C.P. benzene and dilute to 100 ml. Standard Cobalt Solutions. Dissolve 0.1247 gram of cobalt oxalate [a specially purified salt containing 0.0005% nickel prepared by a procedure t o be published ( I ) ] or equivalent quantity of a soluble cobalt salt in 15 ml. of 6-V hydrochloric acid and dilute to I liter with double distilled water. One milliliter of this solution contains 50 y of cobalt. Make successive tenfold dilutions of this stock solution as needed to prepare the individual standard solutions. Palladium Internal Standard Solution. Extensive experimentation with several metals led to the choice of palladium as internal standard. The volatilization rates of cobalt and palladium are similar, as shown by Vallee and Peattie ( l a ) . Rloreover, palladium has not been detected in the animal tissues and the intensity of the 3460.8 A. line of palladium is not affected by a variation in the cobalt concentration. With an electrode content of 0.5 y of palladium, the 3460 A. line falls in the middle portion of the densitometric range, as is desirable for the internal standard line. Dissolve 20.0 mg. of palladium chloride dihydrate in water and dilute to 100 ml. Dilute 1 part of this stock solution u ith 9
185
ANALYTICAL CHEMISTRY
186 parts of water to obtain an internal standard solution containing 0.5 y of palladium per 0.05 ml. Spectroscopic Buffer Mixture. Mix and grind thoroughly 1.000 gram of spectroscopically pure graphite and 0.400 gram of C.P. lithium chloride, using a mullite or agate mortar reserved for this purpose. Store over concentrated sulfuric acid in a desiccator. (Store ingredients of this mixture in same desiccator to facilitate weighing and grinding procedures.)
cent transmittance values of the cobalt 3453.5 and palladium 3460.8 A. lines and of the background adjacent to the cobalt line are determined with a nonrecording microphotometer. The intensity ratios of Co 3453.5/Yd 3460.8 (with background correction) are established by reference t o an emulsion calibration curve obtained by the conventional step sector method, and the concentration of cobalt is estimated from the appropriate analytical curve. An average concentration value is calculated from the results of the triplicate determinations.
APPARATUS
Spectrograph. A Bausch & Lomb large Littrow spectrograph complete with quartz optics, a condensing lens system, and a 10-micron fixed slit. Yo sector was used during sample or standard exposures. Excitation Source. A 220-volt direct current arc, operating at 9 amperes with a gap maintained a t 10 mm. Plates. Eastman 111-0 spectroscopic plates with antihalation backing. Developing Equipment. Glass trays supported in a specially constructed galvanized metal tray maintained a t 68" F. by means of water circulated from a constant temperature bath. Densitometer. Jarrell-Ash Model 200 nonrecording microphotometer. Micropipet Assembly. A micropipet assembly, constructed in the laboratory machine shop, was used to facilitate precise control over the addition of standard and sample solutions to the electrodes.
ANALYTICAL CURVES
Preparation. Separate analytical curves are usually prepared for each type of tissue t o be analyzed. The tissues of normal animals provide the ash used as base material for each standard tissue solution, Each standard solution is prepared to contain 2 mg. of a particular tissue ash per 0.05 nil. These solutions are considered to be cobalt-free for the immediate purpose, since none of them contains a detectable quantity of cobalt by the direct spectrographic method as applied to their 0.05-ml. portions.
LEGEND: LIVER
PROCEDURE
The tissue is transferred to 6, pmcelain evapoiatmg dish 01 crucible, dried overnight on a hot plate, and dry-ashed a t 500" C. in a muffle furnace. The ash is extracted Wi€h 5 mi. af 6N hydrochloric acid and about 10 ml. of hot distilled water. The solution is filtered through Whatman KO.42 paper into a 12,j-m1, Phillips beaker (borosilicate glass). The filter is washed three times with hot distilled water and the washings are combined n-ith the filtrate. The filter is transferred to the original dish, dried, and re-ashed in the furnace. The ash is re-extracted and washed BP described previously, and the filtrate and washings are combined with the first filtrate. The resulting solution is evaporated to akput 5 ml. and then transferred, with rinsing, to a 15-ml. cent& fuge tQbe, graduated t o 0.1 mI. The tube is placed in the drying oven a t 105' C. and the sample solution is evaporated to a predetermined volume which will contain approximately 2 mg of tissue ash per 0.05 ml. This volume is determined from the known fresh weight of the original tissue sample (weighed after surface blood is removed by rinsing with distilled water and blotting with filter paper) and the previousIy established ash percentage of each type of tissue. Tissue portions which weigh less than 0.6 gram are ashed in porcelain crucibles. If ashing appears t o proceed too slondr, a few drops of distilled wateT are added t o the coded crucible, the mixture is dried and ashing continued U R ~ I aI satisfactorv white ash is obtained. Depending upon the estimated ash J! eight, 1, 2, or 3 drops of 6-V hydrochloric acid are added t o the cooled ash. The mixture is allowed t o stand until solution is effected. The tissue ash solutions thus prepared are now ready for electrode loading. Spectroscopically pure graphite electrodes, by 12 inches, are cut into 1.5-inch lengths. Uniform craters, 4.5 mm. wide and 3 mm. deep (including the cone produced by the tapered end of the drill), are drilled into each, using an electrode shaper. A\fterthe craters are waterproofed with the paraffin wax solution, the cone-shaped bottoms of the craters are slightly more than filled ~ i t habout 10 mg. of the ground and des'lccated lithium chloride spectroscopic buffer mixture. -411samples are run in triplicate A hen sufficient solution is available .4volume of 0.05 ml. of the sample solution is added to the electrode crater containing the buffer mixture, using a PickardPierce blood pipet mounted in the micropipet assembly. The electrodes are dried a t 85" C. for 15 to 20 minutes; after cooling, 0.05 ml. of the internal standard solution is added with a micropipet and the electrodes are dried a t 105" C. for 1 hour, after which they are ready for spectrographic exposure. The electrodes containing the samples are burned as anodes in a 220-volt direct current arc operating a t 9 amperes, n i t h the gap maintained a t 10 mm. throughout each exposure. Clem, freshly pointed, spectroscopically pure graphite electrodes serve a- the cathodes. The exposures are continued (usually 25 to 35 seconds) until burnout, as evidenced by increased wandering of the arc accompanied by the disappearance of the reddish radiation due t o lithium in the arc. The central portion of the arc is focused on the slit of the spectrograph. The plates are developed and fixed a t 68' F. After the plates have been air-dried, the per
A PA N C R EA S 0 LUNG I
I .06
.I
-
I
a
1-J
I 8
KIDNEY
I
I
L
I
2
3
INTENSITY RATIO
Figierum 1. Analytical curves for cobalt added
t6
normal tissue ash
I n establish&@ each curve, t h e electrodes are loaded with the designated quangidies of spectroscoplc buffer and the appropriate tissue ash solution, then dried a t 105" C. AfteF drying, 0-,' 0,005-, 0.010-, O.O%-, 0.050-, 0,125-, O.2N0-,0.500-, grid 1.25--, quantities of cobalt we added to triplkste sets of t%ctrodes. .Ifter drying for 20 mim&e,. 0.5 y of palladium is added a l a , after additional drying for T hoiiv, the spectro@aphic exposui%%are conducted and the intensity mtlos of Co 3&%.B/Pd 3460.6 are determined as indicated in Dhe procedure. f i e intensity r a t h thus obtained are plotted & dually for ea& correspondin% electrode content of added c o t d t and an average f w v e is drawn The curves for cobalt added to normal liver, lung; kidney, and pancreas ash are shown in Figure 10. Discussion. The curves shown ih Rgure 1 cover r~@%of the umal working range of this method, which extends from @',0@5 or 0.010 to 1.25 y of cobalt in 2 mg. of &sua ash; these coraetnarations are in excess of the quantities present in normal tissue a&. I n practice, the lung and pancreas curves have been extended' to include increased quantities of cobalt up t o 50' y as the need arm@ for the analysis of larger quantities of this element. At t h e w higher concentrations, the 3455.2 rl. line of o o b d t was used a4 the analysis line. The variation of these curves viith respec6 t o each other is believed by the authors to b e due to the varyitlg inorganic composition of the ash of the difTe?-ent tissues. This effect could be minimized by the use of a l a r g a quantity of buffer. However, it was feared that this might r e m b in a sacrifice of sensitivity, as previous work with 20-mg. charges of this buffer limited the
V O L U M E 28, NO. 2, F E B R U A R Y 1 9 5 6
187
sensitivity of lead, vanadium, titanium, and molybdenum determinations to 0.1 y in the matrix ( 5 ) . As maximal sensitivity was desired, the amount of buffer was maintained a t 10 mg., a quantity which was sufficient to suppress background and act as an enhancing agent in the production of the cohalt spectrum.
Table I. Recovery of Cobalt and Deviation of Recovery from Expected Values for Indicated Amounts of Cobalt Added to 2 M g . Portions of Liver .4sh CO
NO.
Added, Y
; 3
0,005 0.010
4
0.050
20.4 21A 11D 13D 20.4 21.4 13D 27D 20.4 21.4 11D 13D 27 D
0,025
0.10 0.10 0.10 0.10 0.25
0.25 0.25
0.25 0.35 0.35
0.35 0 35 0.35
co Recovered, Y 0.0048a 0.00805
Deviation, Y
-0.0017 0.0072
0.110 0.107
+0.010
0.098
0.250
0.252 0.243 0.231 0.322 0.383 0.335 0.363 0.335
R
- 0,0002 - 0.0020
0.0233" 0.0428a
0,100
Kecoyery
-
retention of cobalt in the soft tissues, in excess of the quantities present normally.
CONCENTRATION METHOD FOR DETERMINATION OF COBALT IN BONE AND NORMAL ANIMAL TISSUES Prior to the application of this method, separate tissues are prepared as for the direct spectrographic method except that the acid solutions of the ashed samples are not adjusted to definite volumes. Instead, these solutions are transferred to individual separatory funnels and subjected to the cobalt separation procedure.
96 0 80.0 Mean
93.2 65 6 88 7 110.0
+0.007 -0.002 0.000 0.000 +0.002 -0,007 -0.019 -0.028 f0.033 -0.016 +0.013 -0.015
107.0
96 0 100.0 100.0 100.8 97.2 92 4 92.0 109.4 95.7 103.7
Mean
9.5 7 100 1
Results of individual analyses.
After the concentration method described below was developed and applied to the determination of cobalt in normal animal tissues, the analytical curves of the direct method were replotted, using as ordinates the total electrode content of cobalt-i.e., normal plus added cobalt. The same variation of the curves shown in Figure 1 resulted; the individual curves were displaced slightly but still exhibited their independent character. Hence, it appears to the authors that this independent nature of the separate curves is not due to the residual cobalt in the tissue ash, but to a variation in the composition of the ash. ACCURACY
To test the accuracy of the method, a series of analyses was carried out to determine the per cent recovery. Normal liver and kidney tissues were used as the base materials to test the lower end of the working range, extending from 0.005 to 0.05 y of added cobalt per 2 mg. of tissue ash. The working range from 0.1 to 0.4 7 of cobalt per 2 mg. of ash was tested by using previously analyzed liver and kidney samples as base materials. I n these recovery determinations, known quantities of cobalt were added to electrodes containing the quantities of spectroscopic buffer and tissue ash designated in the quantitative procedure. -411 solutions, including the internal standard, were added as described previously. Finally, the spectrographic exposures and cobalt estimations were performed in triplicate, except where indicated otherwise, in conformance with the procedure. An indication of the accuracy of the procedure may be obtained from the results of the recovery of cobalt added to liver tissueq, presented in Table I. As reported in Table I, the average recovery of 0.005- to 0.050-7 quantities of cobalt added to liver ash was 89%, as determined by individual determinations. The mean recovery of 0.10- to 0.35-r amounts of cobalt was 100% as a result of conducting triplicate determinations on 13 ashed samples. The error of these latter determinations lies within *loyo, showing the advantage of triplicate determinations. Similarly, recovery determinations using 11 ashed kidney samples as base material gave a mean value of 102%. Consequently, the degree of accuracy of the direct spectrographic method, indicated by these recovery data, was considered satisfactory for the purpose of determining the
RE4GEYTS
-411 reagents are analytical reagent grade. Double distilled water, from an all-borosilicate glass still, is used in the preparation of all solutions. In addition to those listed under the direct method, the following are required: Nitric Acid, Concentrated. Redistill the C.P. reagent in an allborosilicate glass still. Nitric Acid, 1 to 1, and Hydrochloric Acid, 1 to 99. Dilute the redistilled reagents with water. Phosphoric Acid, 1 to 49. Dilute the C.P. reagent with water. Methyl Orange Indicator Solution, 0.1% in water. 1-Nitroso-2-naphthol. Dissolve 2.5 grams of the reagent in 125 ml. of glacial acetic acid and dilute to 250 ml. with water. Sodium Citrate Solution. Dissolve 423 grams of the dihydrate salt in almost a liter of water in a separatory funnel, adjust the p H to 9 with sodium hydroxide, and extract metallic impurities with a solution of 100 mg. per liter of dithizone in chloroform until a green colored extract is obtained. Adjust the aqueous solution to p H 7 with citric acid and remove excess dithizone by washing with several portions of chloroform. Separate the aqueous layer and dilute to 1liter with ~ a t e r . Aluminum Nitrate Solution. Dissolve 1.472 grams of the nonahydrate salt in 20 ml. of redistilled 95% ethyl alcohol. Two milliliters of this solution contains the equivalent of 20 mg. of aluminum oxide. PROCEDURE
Separation of Cobalt. The separation of cobalt from the other inorganic constituents of tissue ash is conducted in accordance with Saltzman's detailed procedure (9) for the preliniinary separation of cobalt, using 1-nitroso-2-naphthol and including the removal of impurities by shaking the extract with 25 ml. of 1 to 99 hydrochloric acid. The chloroform solution of the extract is then separated and transferred t o a porcelain crucible containing 2 ml. of the alcoholic solution of aluminum nitrate. The mixture is evaporated to dryness on the steam bath and is ready for ashing. Ashing. About 5 ml. of concentrated nitric acid is added to the crucible containing the l-nitroso-2-na.phthol residue and t h e mixture is evaporated to dryness on- the steam bath. T h e residual material is then subjected to alternate dry ashing in a muffle furnace a t 450' C. and wet ashing with concentrated nitric acid on a hot plate. During the latter process the crucible is covered with a watch glass, placed so that a smal open space permits the slow escape of vapor. When the cyce has been repeated twice, the residue is ashed R third time in the furnaccb; a t this point there is produced an easily crushed, grayish white powder and ashing is considered to be complete. Preparation of Electrode Charge. The residue of aluminuiii and cobalt oxides from the ashing procedure is crushed with a clean glass nail, then weighed on glazed paper, and three or four separate 5.0-mg. portions are weighed out for analysis. (The absolute amount is not critical but must be known to calculate the aliquot portion on the electrode.) T o each weighed portion is added approximately 5 mg. of the lithium chloride-graphite spectroscopic buffer mixture. The mixtures are ground separately in an agate mortar until thoroughly mixed and then they are added to the waterproofed craters of spectroscopically pure electrodes. The crater is the same size as that used in the direct s ectrographic method. A4fterthe addition of 0.5 y?f alladium, tge electrodes are dried for 20 minutes a t 80" to 85 $and then for 1.5 hours a t 105" C. Spectrographic and Densitometric Procedures. The exposure conditions, plates, and plate-processing procedure are the same as those of the direct method. The intensity ratios of Co 3453.5 to Pd 3460.8 are determined as described previously; the cobalt concentrations in the aliquot portions on the separate electrodes ~
1
ANALYTICAL CHEMISTRY
188 are estimated from an analytical rurve obtained from a series of standards. PREPARATION OF CURVE
A series of known quantities of cobalt, ranging from 0.03 to 1.00 T, is added to porcelain crucibles containing 2 ml. of the aluminum nitrate and 5 ml. of the 1-nitroso-2-naphthol solutions. The ashing, electrode loading, spectrographic and densitometrir procedures are conducted as described above. The plot of I co rasa,s/I Pd 31b0.8 us. the quantity of cobalt on the electrode is prepared as the analytical curve and is shown in Figure 2. The values plotted in Figure 2 resulted from the densitometric treatment of the individual spectrograms prepared from 5.0-mg. portions of the ashed standard residues. The present working range of the method is from 0.006 to 0.23 y of cobalt on the electrode. ACCURACY
Known amounts of cobalt, varying from 0.03 to 0.4 y, were carried through the entire extrartion, ashing, and spectrographic procedure. The results of this experiment, obtained by averaging the quadruplicate determinations of cobalt concentrated in the aluminum oxide residue, are presented in Table 11. The recovery data presented in Table I1 indicate the accuracy of the method to lie between 90 and 98%, with a mean value of 93.7% obtained with this set of determinations. These recov-
c
,005L
--
I
G. 3 4 5 3 . 5 P i 3460.8
,05
.5
I
1.0
INTENSITY RATIO
Figure 2. Analytical curve for cobalt in aluminum oxide, using lithium chloride graphite buffer eries are considered adequate for a working range extending from 0.006 to 0.09 Y cobalt. DETERMIXATION OF COBALT CONTENT OF NORMAL TISSUES
The cobalt content of tissues from normal dogs, rabbits, and rats was determined in individual samples by the cobalt concentration method. The results are reported in Table 111. The data reported in Table I11 show cobalt to be widely disTable TI. Recovery of Cobalt by Chemical Concentration tributed throughout the animal organism. I n the dog, the small Method glandular tiswes contain the highest concentrations of thielement, whereas the liver, spleen, muscle, and bone contain coCo Taken Co Detd. Total for E x Blg. On Co Av.Co Recovbalt a t the lowest and a t almost a constant concentration. On Electrode, Recovered, Recovery, traction, ery. Y total electrode 7 7 % I The small glandular tissues, with the exception of rabbit adrenals, were not taken from the rabbit and rat for cobalt anal0.03 26.4 5.3 0.0059 0 029 5.6 0.0057 0 027 ysis a t the time these control animals were sacrificed. How0,0055 0 028 5.2 0.0066 0 032 0.029 96.7 5.5 ever, as in the case of the glandular tissues of the dog, the rabbit adrenals show the highest cobalt concentration. I n the remain0.04 27.3 5.4 0.0080 0.041 0 031 6.2 0.0071 ing tissues of both rabbits and rats, cobalt appears to be con5.9 0.0084 0 039 0.0069 0.033 0.036 90.0 5.7 centrated to a greater estent in the spleen, pancreas, and kidney than in the other tissues, although this relationship does not 0.10 28.0 6.5 0.0213 0.092 6.1 0.0208 0.095 hold for the absolute quantities of cobalt in the entire organs. ... 4.9 0.0147= 5.8 0 0163" ... 0.9904 90.4 Upon investigating the latter relationship, repeated statements were noted in the literature that cobalt had been found concen0.20 23.1 5,3 0.0415 0,189 6.0 0.0485 0 193 trated in the liver, pancreas, and spleen. The present stud\. 4.0 0.0248n 5.6 0.0410 o:iij 0.186 93.0 bears this out partially on a total organ basis only; however, most data in the literature Tere obtained from ruminants whose 0.40 26.0 5.1 0.0800 0.408 6.4 0 0870 0.354 metabolism of this element may differ from that of dogs, rabbits, 5.1 0.0893 0.456 5.3 0.0725 0 356 0.394 98 5 and rats. The only data on any of the latter species appear to be Mean 93 7 those of Josland and McNaught ( 4 ) , who analyzed certain tissues, composited from seven rats, by a chemical method. The a Intensity ratio, Co 3453/Pd 3160 not in agreement with others in respective sets. mesent data were found to agree well with McNaught'e corrected values ( 6 ) . A coniTable 111. Cobalt Content of Kormal Animal Tissues Analyzed Individually parison of both sets of determinations is presented in Table (Expressed in micrograms of cobalt per gram of fresh tissue) IV. Dog Rabbit Rat .4s reported in Table IV, No. of Concn. Av. S o . of Concn. -4v. S o . of Concn. Ar. anirange, concn., anirange, concn., anirange, concn.. there are some differences bey Co/g. 7 Co/g. rnals y Co/g. y Co/g. mals y Co/g. y Co/g. Tissue mala tween the results obtained by 2 Lung 0 008-0 015 0.012 8 0.010-0.063 0.032 0.030-0.071 0 050 the two methods. However, 0 007-0 008 0.008 2 9 0.027-0.086 0.054 0.017-0.030 0 , 0 2 6 Liver 2 9 0.047-0.163 0 010-0 014 0.012 0.03&0.088 0 069 Kidney 0.103 the agreement is believed to be 2 0 005-0 006 0.006 7 0.046-0.602 0 . 1 3 1 0.044-0.189 0.116 Spleen 2 0.052-0.246 0.109 Pancreas 0 012-0 022 0 017 0.071-0.134 0.101 3 satisfactory after consideration 2 Muscle 0 006-0 007 0 007 0.015-0.024 0 . 0 2 1 0.015-0.104 0.049 4 of the different sources of ani2 0.007-0.048 0,030 0.028-0.103 0.069 0 002-0 007 0 , 0 0 5 Bone 2 l: 0.069-0.316 0.170 0 074-0 167 0.121 Adrenals mals and the probable differ2 0 047-0 125 0.086 Pul. lymph nodes 0 156-0 360 0.258 2 Thyroid ences in their diets. 1 0 112 Pituitary Brain 2 Of perhaps greater signifi0 O l e 0 013 0 . 0 1 2 cance is the report in the present
189
V O L U M E 2 8 , NO. 2, F E B R U A R Y 1 9 5 6 paper of the quantity of cobalt occurring in bone and muscle tissues, a fact which may have escaped earlier detection because of the lack of a method as specific and sensitive as the one reported here. SUMMARY Two spectrochemical methods have been developed for the determination of trace quantities of cobalt in animal tissues. One, applied directly t o the ash of tissues from exposed animals, is used for those samples containing 0.023 y of cobalt per gram of fresh tissue in excess of that present in normal tissue. The analysis error is within +lo%, as shown by triplicate recovery determinations of cobalt. The other method, referred to as the cobalt concentration method and developed for the analysis of cobalt in normal tissues, employs the principle of preliminary chemical concentration of the cobalt. hluminum is added as the carrier element to the isolated cobalt and provides (as the oxide aft'er aahing) with lithium chloride and graphite the constant base material for spectrographic analysis. This method possesses a degree of sensitivity of about 0.001 p.p.m. for a 30-gram sample of fresh tissue. The average analysis error over the 0.006- to 0.1-y portion of the working range is about -6%. This method provides greater accuracy in individual sample analysis than does the direct method. The cobalt present in normal tissues and undetectable by the direct method has been determined in the tissues of the dog, rabbit, and rat by the concentration method. These data coniprise the first published report of highly accurate individual determinations of the millimicrogram quantities of cobalt present in the organs of small normal animals. The high degree of sensitivity realized Ivith both spectrochemical methods is due in part t,o the lithium chloride-graphite buffer system which suppresses background and exerts an enhancing effect on the line spectra of trace elements.
Table IV. Comparative Cobalt Content of Normal Rat Tissues Determined Chemically and Spectrochemically KeenanKeenanMcKaught, Kopp, 3IcXauglit. Kopp Tissue y Co/G. y Co G y CofOrgan y Co/Organ 0.11 0,050 Lung 0.30 0,02(i 0:iB 0 : 040 Liver 0.14 0.13 0.116 0.128 Spleen 0.11 0.10 0.043 0,069 Kidney0 22 0 13 0.066 0.109 Pancreasu 6.97 0.049 ... hluscleb 2.42 ,.. O.OCi9 Boneb .Issuming a n organ weight of 2 wanis for pancreas. b Estimated weights of these tissGes in individual animals are based on percentage weight d a t a of rat tissue3 in relation t o body weight given by Skelton ( 2 2 ) .
The sensitivity and accuracy of either procedure may prove t o be useful to those engaged in the determination of cobalt in plants, soils, rocks, or other materials. ACKNOWLEDGMENT The authors are indebted to Herbert E. Stokinger, under whose direction this work was performed, for his constructive criticim and guidance in the preparation of this paper. LITERATURE CITED
Birmingham, D. J., and Keenan, R. G.. unpublished results. Daniel, E. P., Hewston, E. &I., and Kies, XI. W.,ISD. ENG. CHEM.,ANAL.ED. 14, 921 (19421. Heggen, G. E., and Strock, L. W., .%NAL. CHEM. 25, 8 5 9 (1953). Josland, S.W., and McNaught, K. J . , .Yeu' Zealand J . Sci. and Technol. 19, 536 (1938).
Keenan, R. G., and White, C. E., ..~N.%L.C H E Y .25, 887 (1953). McNaught, K. J., S e w Zealand J . Sci. and Technol. 30A, 109 (1948).
Mitchell, R. L., and Scott, R. O., J . SOC.C'hem. Ind. 66, 330 11947).
Mitchell, R. L., and Scott, R. O., Spectrochim. Acta 3 , 367 OTHER 4PPLICATIONS The principles of the cohalt spectrochemical methods described in this paper are being applied to similar problems in this laboratory. Thepe include determination of vanadium in animal tissues, trace element5 in animal diets, and lead in hair.
(19481.
Saltaman, B. E., i l ~ aCHEM. ~ . 27, 284 (1955). Scott, R. O., and Mitchell, R. L.. J . SOC.Chem. Ind. 62, 4 (1943). Skelton, H.. Arch. Internal M e d . 40, 140 (1927). Vallee, B. L., and Peattie, R. W., ANAL.CHEM.24, 4 3 4 (1952). R E C E I Y Efor D review April 15, 1955, .Iccepted October 31, 1955
Spectrophotometric Determination of Total Hemoglobin in Plasma KEITH B. MCCALL Division o f Laboratories, Michigan Department o f Health, Lansing, M i c h .
A simple method has been developed for the spectrophotometric determination of total hemoglobin in plasma. All hemoglobin is first converted to methemoglobin; the absorbance of this solution is then evaluated before and after a small amount of cyanide is added to convert all methemoglobin to cyanmethemoglobin. The change in absorbance observed is directly proportional to the total hemoglobin and is calculated directly. The method offers the obvious advantages of simplicity, stable reagents, and the production of stable colors with an acceptable degree of precision and accuracy, which are not affected by varied dilutions of the test plasma.
T
HE determination of hemoglobin in irradiated liquid plasma has been a difficult problem because of the inherent color of d l lots of plasma and the fact that glucose is added t o stabilize
the proteins which are present. Of the many methods which have been proposed, such as that of Karr and Chornock (5)and Creditor ( I ) , none have offered the combined advantages of stable reagents, stable colors, and high accuracy and precision. The purpose of this investigation was to develop an analytical procedure that has these characteristics. THEORY
Since solutions of methemoglobin and cyanmethemoglobin follow Beer's law very well, the total hemoglobin concentration in plasma may be evaluated directly by the difference between the absorbance, A , of cyanmethemoglobin, AcJfHb,and the methemoglobin, AMHb,in the plasma a t the same wave length (540t o 550-mp range) using a constant cell thickness (Figure 1). This principle has been applied by LIichel and Harris ( 4 ) t o the determination of methemoglobin in whole blood at 635 mp. The absorptivity, aMHb and aCMHb, for methemoglobin and for
