604
A N A L Y T I C A L CHEMISTRY
the first water wmh of the extract if the mixture is shaken a t all. (2) The solution containing the vitamin A extract may be made up in hexane and an aliquot placed directly on the chromatographic column, thus eliminating the evaporation step necessary if ether is used. It has been noted that there is a definite breakdown of vitamin -4alcohol if a solution of it is taken to dryness under reduced pressure, in accordance with the statements of Hjarde (6). Evaporation should, therefore, be avoided. The method of deactivating the adsorbent under controlled conditions is similar to that employed by Datta, Overell, and Stack-Dunne ( S ) , and gives a means by which the degree of activation of the adsorbent may be varied over a wide range to suit a particular chromatographic separation problem. In addition, it provides a means by which the adsorbent is msintained a t the desired level of activation for an indefinite period.
LITERATURE CITED
(1) Cama, H. R., Collins, F. D., and Morton, R. A , Biochem. J . , 50, 48 (1951). (2) Chilcote, M. E., Guerrant, S . B., and Ellenberger, H. A , , B N ~ L . C H E M 21, . , 1180 (1949). (3) Datta, S. P., Overell, B. G., and Stack-Dunne, hl., ,Vatwe, 164, 673 (1949). (4) Dowler, M. W., and Laughland, D. H., ABAL. CHEM., 24, 1047 (1952). ( 5 ) Fox, S. H., and Mueller, A , J . Am. Pharm. Assoc., Sci. Ed., 39, 621 (1950). (6) Hjarde, A., Acta. Chim. Scand., 4 , 628 (1950). (7) Hoffmann-La Roche and Co., .I.-G., Swiss Patent 256,699 (March 16, 1949). (8) Morton, R. A., and Stubbs, A. L., d n a l y s f , 71, 348 (1946); Biochem. J . , 41, 525 (1947). RECEIVED for review September 4, 1932. Accepted December 29, 1952.
Colorimetric Determination of Vanadium with 8-Quinolinol Application to Biological Materials K. A. TALVITIE Division of Occupational Health, Public Health Sercice, Federal Security Agency, Cincinnati, Ohio During toxicological studies of vanadium, a specific and sensitive method for the determination of this element in biological materials was required. A colorimetric method was developed based on the measurement of the intensity of the magenta-black color of the compound of vanadium and 8-quinolinol(8-hydroxyquinoline) in chloroform medium. When the color is read photometrically, the method permits the determination of vanadium in the range of 1 to 50 micrograms with an average error of 3~0.32microgram. The method is applicable to studies of the distribution and the rate of excretion of vanadium in experimental animals and is useful in evaluating industrial exposure to vanadium compounds by analysis of urine specimens.
D
URING laboratory and environmental studies of the toxicity of vanadium, a method for the determination of small amounts of this element in biological materials was required. Only a few methods of sufficient sensitivity for these determinations were available. A colorimetric cupferron method ( 2 ) sensitive to 2 micrograms of vanadium per 100 grams of plant tissue had been published but was found to be unreliable in other hands (6). Although a spectrographic method (3)and the phosphotungstate, colorimetric method (9, 11) were found suitable for some samples, neither of these had the necessary sensitivity when applied directly to samples of high ash content. Efforts a t improving the sensitivity by extraction of the vanadium from the ash as the 8-quinolinol derivative, using chloroform as the extracting medium, led to the conclusion that the resulting colored extract aould, in itself, have the required photometric sensitivity and specificity. Montequi and Gallego ( 7 ) proposed the use of the color of the chelate in the chloroform extract as a sensitive method for the detection of vanadium; hoxever, they were unable to develop a quantitative colorimetric method because of the variations in shades of color obtained. Subsequent investigators similarly failed to find the color of the chelate in chloroform medium satisfactory for quantitative use. Sandell (10) was unable to prevent interference by iron and used the chloroform extraction only for the separation of vanadium prior to determination by the phosphotungstate method. Bach and Trelles ( 1 ) reported nonconformity of the color with Beer’s law and substituted isoamyl alcohol as the extracting medium, obtaining, thereby, a linear relationship between absorbancy and concentration. The red color in isoamyl alcohol medium, however, is more subject to
interference by other metals than is the magenta-black color in chloroform medium. I n the course of the present study a stable solution of the vanadium derivative of 8-quinolinol in chloroform was produced. This solution was found to follow Beer’s law and to possess an absorption maximum a t a wave length relatively free of interferences and was, therefore, suitable for use in the quantitative determination of vanadium. Conditions for the complete extraction of vanadium and separation from interferences including iron were developed and suitable ashing procedures for biological samples containing vanadium were found. The experimental data obtained and the procedures adopted for the analysis of samples are presented below. REAGENTS
Methyl Orange Indicator, 0.1%. Dissolve 0.1 gram of C.P. methyl orange in 100 ml. of distilled water. Sdfuric acid, 4 N . Nitric Acid, 4 N . Bubble filtered air through concentrated nitric acid until the acid is water-white, standardize by any convenient means, and dilute the appropriate volume to 1-liter. Ammonium Hydroxide, 4 N . Standardize ammonium hydroxide by titrating with the 4 N nitric acid using methyl orange indicator. Dilute the appropriate volume to 1 liter. Ammoniacal BufIer Solution, pH 9.4. Dilute 200 ml. of .4N ammonium hydroxide and 100 ml. of 4 N nitric acid to 2 liters with distilled water. It is not necessary to check the pH of this solution if the ammonium hydroxide and nitric acid used are exactly equivalent in strength. Phthalate Bllffer Solution, pH 4.0. Dissolve 12.77 grams of C.P. potassium biphthalate in water and dilute to 250 ml. Alcohol-Free Chloroform. Extract the alcohol from 2 liters of reagent-grade chloroform by shaking vigorously with six sepa-
605
V O L U M E 25, NO. 4, A P R I L 1 9 5 3 rate portions of distilled water, each portion being one fourth the volume of the chloroform. Cover with a layer of water and store in a refrigerator if possible. 8-Quinolinol Solution, 0.57,. Dissolve 5 grams of reagentgrade 8-quinolinol in 1 liter of alcohol-free chloroform. 8-Quinolinol Solution, 0.1%. Dilute 100 ml. of 0.5% 8-quinolinol solution to 500 ml. with alcohol-free chloroform. Stock Vanadium Standard. One milliliter contains 100 micrograms of vanadium. Dissolve 0.2296 gram of C.P. ammonium metavanadate in 25 ml. of 4 N sulfuric acid and dilute to 1 liter with distilled water. Make tenfold dilutions of the stock solution as needed.
Table I.
Effect of pH on Extraction of Vanadium Absorbancy 0.326 0.491 0.688 0.795 0.816 0.816 0.810 0.780 0.710 0.528 0.281
PH 2.42 2.60 2.88 3.26 3.58 4.07 4.51 4.82 5.24
5.53 5.89
EQUIPMEhT
Measurements of absorbancy ir-ere made with a Beckman quartz spectrophotometer, Lfodel DU. All measurements were made in 22-nim. Kimble brand test tubes matched optically within 0.57, and having an effective light path of 20.0 mm. An accessory test-tube adapt01 n as used. hleasurements of p H were made with a Beckman Laboratory Model G p H meter and glass electrode. Wet ashing of samples was conducted in 125-ml. and 250-ml. borosilicate glass, conical Phillips beakers with spout (Corning Glass Works). Extractions were made in 125-ml. borosilicate glass, glass-stoppered, pear-shaped, Squibb separatory funnels (Corning Glass Works). Sample solutions were filtered through 20-ml. medium porosity, porcelain Selas filtering crucibles (Fisher Scientific Co.). EXPERI M ElTA L
Absorption Spectrum. The color of the chelate of pentavalent vanadium with 8-quinolinol in chloroform solution has been reported to undergo variations in hue (‘7). These variations probably were due to the small amount of alcohol added to chloroform as a preservative. Ordinary chloroform was found to yield a solution having a color of more or less reddish hue depending upon the alcohol content; furthermore, the hue became more reddish on standing. Chloroform which was free of alcohol, on the other hand, yielded a stable solution which had absorption maxima a t 365 and 550 mp and which followed Beer’s law throughout the useful range of concentrations. Visually. the solution had a magenta color a t low concentrations of vanadium and a black color a t high concentrations. The absorption spectrum of the pentavalent vanadium derivative of 8-quinolinol in alcohol-free chloroform is presented in Figure 1. 1.2
0
0 t
z
a
4
5
6
Extracted from 60 ml. of solution at pH 4.0 with two 5-ml. portions of 0.1708-quinolinol in chloroform Extracted from 60 ml. of solution at pH 4.0 with three 5-ml. portions of 0.570 8-quinolinol in chloroform, removed from chloroform with 50 ml. of buffer at pH 9.4, and re-extracted at pH 4.0 with two 5-ml. portions of 0.170 8-quinolinol
The solution for the absorbancy measurements was prepared by diluting standard vanadium solution to 50 ml., adjusting the pH to 4.0, and extracting with 0.057, 8-quinolinol solution. The reference solution was a blank obtained in the same manner.
0.8
m
3
MICROGRAMS OF V P E R M I L L I L I T E R
1.
a
2
Figure 2. Absorbancl- cs. Concentration of 8-QuinolinolVanadium Complex in Chloroform 2.
1.0
I
Inasmuch as the maximum a t 365 mfi has no analytical usefulness in the present application because of the high absorption of other metallic derivatives and of 8-quinolinol itself in this region ( 5 ) ,the 550-mp wave length was selected for determinations of concentrations. Curve 1 of Figure 2 illustrates the straightline relationship between concentration and absorbancy obtained when pure solutions of vanadium are extracted Tr-ith a chloroform solution of 8-quinolinol. Curve 2 of Figure 2 represents the actual working curve, some of the vanadium having been lost as a result of two additional extraction steps which are necessary to prevent interference by iron.
0.6
0
u)
m
a
0.4
0.2
0.0
400
500
600
700
BOO
WAVE LENGTH, M I L L I M I C R O N S
Figure 1. Absorption Spectrum of 8-Quinolinol\-anadium Complex in Chloroform
Extraction pH. To determine the proper pH for the most efficient extraction of vanadium, 50-ml. portions of a solution containing 1.25 micrograms of vanadium per milliliter were adjusted to varying pH values and extracted TTith single 10-ml. portions of 0.05y0 8-quinolinol. Measurements of pH were made on the aqueous layers after extraction and measurements
606
ANALYTICAL CHEMISTRY
of the absorbancy a t 550 nip were made on the extracts. These results, which are presented in Table 1, indicate efficient extraction in the pH interval 3.5 to 4.5 and verify the observation of Sandell (IO) that vanadium extracts readily a t the pH corresponding to the intermediate color of methyl orange indicator. Subsequently, samples were adjusted to p H 4.0 before extraction, Potassium biphthalate was found not to interfere significantly with the extraction and was adopted as a buffer, Stability. The absorbancies of two solutions of the chelate containing the equivalent of 10 micrograms of vanadium per milliliter were found to remain unchanged after a week of exposure of the solutions to diffuse light from a north ~ i n d o w . One solution was prepared with new, alcohol-free chloroform and the other with reclaimed chloroform. KO evidence of deterioration was detectable in alcohol-free chloroform or in 8-quinolinol solutions which had been kept for 3 months in a refrigerator when not in actual use. Interfering Metals. Of prime importance is the removal of iron since the black ferric derivative also extracts a t pH 4.0 and absorbs strongly a t 550 mp. Separation of iron is accomplished by shaking the chloroform extract with an ammoniacal buffer solution having a pH of 9.4. The iron remains quantitatively in the chloroform layer while the vanadium transfers to the aqueous phase from which it may again be extracted after adjustment of the pH to 4.0. The derivatives of other metals (4,5 ) are relatively transparent a t 550 mp and, in addition, they are either eliminated completely or their concentration in the final extract is considerably reduced by virtue of the three successive transfers of the vanadium between aqueous and chloroform phases. In order to test the effectiveness of the separation from estraneous metals, a series of samples, each containing 30.0 micrograms of vanadium and 0.55 gram of sodium dihydrogen phosphate as well as the metal i n question, was analyzed according to the procedure described herein. These samples were evaporated to dryness with 2 ml. of nitric acid and the residue was dissolved in 25 ml. of water acidified with 1 nil. of nitric acid. The results, which are presented in Table 11, s h o that ~ satisfactory analyses may be obtained in the presence of a t least 0.5 mg. of these metals. A low result was obtained in the presence of 0.5 mg. of tungsten, but not in the presence of 0.1 mg. of tungsten. Aluminum, iron, bismuth, and titanium gave turbid solutions but did not interfere seriously with the extraction of the vanadium. Higher amounts of aluminum and iron caused eniulsification of the chloroform layer. With aluminum it was found helpful to increase the acidity slightly by the addition of a drop of 4 iV acid and with iron the emulsion could usually be broken by increasing the volume of the chloroform extracting solution. In the presence of several milligrams of iron it was found necessary to extract with a stronger gquinolinol solution and to shake for a longer time in order to convert the ferric phosphate to the quinolinolate. PREPARATION OF SAMPLES
For biological samples ION in interfering substances and having vanadium in an easily soluble form, a wet method of ashing is preferable. Ashing of urine is accomplished readily with nitric acid alone, whereas the addition of potassium sulfate is desirable in the ashing of samples having low inorganic content such as blood and soft tissue. The bisulfate which is formed by the action of nitric aeid reduces the tendency of the organic residue to ignite spontaneously, while the nitrate formed speeds destruction of the organic matter. In order to avoid a violent action in the wet method, sufficient nitric acid should be present during the initial evaporation to give a light-colored solution as the sample approaches dryness. Polyphosphates which are produced in the ashing process are hydrolyzed by slow evaporation of a solution of the ash in the presence of an excess of nitric acid in the manner described by Saltzman (a), the evaporation serving a180 to eliminate nitrites. If not hydrolyzed to the orthophosphate, the polyphosphates,
Table IJ,
Recovery of \'anadium 3Ietals Metal Added, hlg
None
0.5 0.5 0.5 0.5 0.5
A1
Bi Co
Cr Cu
0.5 Fe 0.5 M n 0.5 M o 0 . 5 Ni 0 . 5 Sn 0 . 5 Ti 0.5 U 0.5 W 0.1 w 5 . 0 Zn
in Presence of Other
Vanadium. Micrograms Added Found 30.0 30.9 30.0 30.6 30.0 30.2 30.0 29.2 30.0 30.7 30.0 30.6 30.0 30 7 30.0 30.9 30.0 30.2 30.0 30.1 30.0 30.3 30.0 30.8 30.0 30.5 30.0 21.7 30.0 30.1 30.0 31.0
through complex formation, would prevent complete estraction of the vanadium. Nitrites have a destructive effect on both the 8-quinolinol and the methyl orange indicator and must, therefore, be absent from the final solution. Samples high in iron and silica are preferably ashed by ignition and the ash is fused with sodium carbonate as prescribed by Sandell (IO) for silicate rocks, without the addition of nitrates either in the ignition or the fusion. The fusion removes all but a trace of iron, depolymerizes polyphosphates, and oxidizes the vanadium to the pentavalent state. Specific procedures for preparation of different types of biological specimens and appropriate sizes of samples are presented below.
Human Urine. Transfer 50 to 150 ml. of urine to a 250-ml. Phillips beaker and add concentrated nitric acid equivalent t o 20y0 of its volume. Evaporate to dryness on a hot plate just below the boiling point. Cool, add sufficient nitric acid to wet the residue, and return the beaker. to the hot plate. When the residue is dry, cover the beaker with a borosilicate watch glass and place on a second hot plate a t high heat (350' to 400' C.) for a few minutes or long enough to fuse and partially decompose the inorganic nitrates, as evidenced by the evolution of brown nitrogen dioxide fumes. Cool and repeat the process of wetting the residue with nitric acid, evaporating to dryness with gentle heat, and fusing a t high heat until a pure white residue is obtained. From three to six such treatments are necessary. To the ash add 2 ml. of nitric acid and 25 ml. of water and evaporate just to dryness on a steam bath (not a hot plate) to hydrolyze polyphosphates. Add 1 ml. of nitric acid and 50 ml. of water and evaporate to 25 ml. on a steam bath. Cover the beaker with a watch glass and allow to cool. If only a small amount of precipitated silica is present, the solution need not be filtered. Rabbit Urine. Add nitric acid to the sample until a clear solution is obtained using a drop of capryl alcohol to reduce foaming. Transfer an aliquot equivalent to 20 ml. of urine to a 250-ml. Phillips beaker, add 5 ml.. of nitric acid, and proceed as for human urine. Complete ashmg is difficult to judge because of the yellow color of the residue. Blood and Soft Tissue. Transfer not more than 5 grams of blood or up to 15 grams of tissue to a 125-ml. Phillips beaker and add 0.1 gram of potassium sulfate for each gram of sample and 1 ml. of concentrated nitric acid for each gram of sample. Heat gently until the foaming subsides and then proceed as for urine. Bone, Char the sample in a porcelain dish in a muffle furnace a t 400' C. and ignite to a carbon-free ash by increasing the temperature to 700" C. over a period of several hours. Crush the ash to a powder with a pestle and return to the furnace if necessary. Add 2 ml. of nitric acid to not more than 1 gram of the ash contained in a 250-ml. Phillips beaker. Add 25 ml. of water and evaporate just to dryness on a steam bath. Add 1 ml. of nitric acid and 50 ml. of water and evaporate to 25 ml. on a steam bath. The wet method also may be used for the ashing of bone. Feed and Feces. To a tared porcelain dish add 10 rams or more of the sample and dry to constant weight a t 110' Char in a muffle furnace a t 400" C. and ignite to a carbon-free ash by increasing the temperature to 700"'C. over a period of several hours. Crush the ash with a porcelain pestle and return to the muffle furnace if necessary. Mix 1 gram of the ash with 5 grams
8.
607
V O L U M E 2 5 , NO. 4, A P R I L 1 9 5 3 of anhydrous sodium carbonate and fuse in a platinum crucible over a blast lamp until a quiet melt is obtained. Immediately upon removing from the flame, rotate the crucible in such a manner aFi to solidify the melt in the form of a shell and then cool the crucible rapidly bv partial immersion in a tray of cold water. Remove the cooled melt by tapping the inverted crucible sharply on a hard surface covered with a clean sheet of paper. Transfer the melt to a beaker and digest with 50 ml. of water on a steam bath. Hasten disintegration of the melt by crushing with a flattened stirring rod. Reduce manganate, when present, by the addition of a single drop of 500, ethyl alcohol. Transfer the Polution and residue to a 100-ml. volumetric flask, dilute to 100 ml., mi.; and allow the residue to settle. Siphon off at least 50 ml. of thP rlear, supernatant solution and, if necessary, filter tiy suction through a Selas crucible. 4 Y A LYTIC 4 L PROCEDURE
RESULTS
In the case of saniplas prepared by wet ashing, transfer 25 ml of the sample solution to a 125-nil. separatory funnel using enough rinse water to make a total volume of about 50 ml. Add a drop of methyl orange indicator and neutralize to the intermediate color of the indicator with 4 N ammonium hydroxide or 4 K nitric acid and 1 to 50 dilutions of these reagents. I n the case of samples fused with sodium carbonate, transfer 50 ml. of the clear e.;tract from the fusion to a 125-ml. separatory funnel, neutralize with 4 h ‘ sulfuric acid to the intermediate color of methyl orange, and remove carbon dioxide by alternately shaking and releasing the gas through the stopcock. Clean the stem of the funnel with a cotton swab. Extract the vanadium by shaking for 2.5 minutes with each of three successive 5-ml. portions of 0.5y08-quinolinol in chloroform, drawing off the chloroform layers into a second separatory funnel containing 50 ml. of the ammoniacal buffer solution. Shake the combined chloroform extracts with the ammoniacal buffer solution for 5 minutes, then draw off and discard the chloroform layer. T a s h the aqueous phase by shaking for a minute with 5 nil. of chloroform. If the chloroform layer has a distinct color, repeat washing with second 5-ml. portion of chloroform. Add a drop of methyl orange indicator to the aqueous phase and neutralize t o the intermediate color of the indicator with 4 S nitric acid Before final adjustment of the pH with the diluted reagents, stopper and invert the funnel, and open the stopcock to allow any chloroform or ammoniacal buffer in the bore to drain back into the funnel. Clean the stem of the funnel with a cotton swab, add 2 ml. of phthalate buffer and then re-extract the vanadium by shaking for 2.5 minutes R-ith each of two successive 5.00-nd. portions of 0. lCc 8-quinolinol in chloroform.
Table 111. Recovery of I‘atiadium Added to Samples Sample Human urine, 100 ml.
Human urine, 150 inl.
Rabbit urine, 20 ml.
Vanadium. hficrograms Added Recovered Error 10.0 10.0 0.0, 20 0 10.3 -0., 30 0 30.2 +0.2 40.0 39.4 -0.6 50.0 49.6 -0.4 9. n -0.1 10 0 20 o 19 7 -n 0 19.7 - 0 . 32 30 30 1 +i, +0 i1 3 0 .00 30.1 400 .00 39 4 -0 4 39.4 - 0 . 66 50 50 0 5 0 .00 50.0 0 .0 5.0 5.0 0.0 10 0 10.5 + 0 . ,3 20.0
40.0 00.0
Bone ash, 0.5 gram
2 5 5 0 in o 20 n 40 0
Muscle. 5 grains
5 0 10 0
1.5.0
20.0 25.0
Liver, 3 grams Blood, 5 grams Rabbit feed, 5 grams Rabbit fecea, 5 grama
.
.j 0
10 0 5 0
20.4
41.0 59.6 2 8 5 2 9 9 19 7 39 8 5.2 10.6
-0.4
T1.O
-0.4 +0 3 to 2 -0 1 -0 3 -0 2
+0.2
15.3 20.3 25.2
t 0 .j +0.3 +0.3 +0.2
5 0 9 8
0 0 -0.2
+0.2 +0.5 25 Q n 26.8Q +0.9 Average i o ,32 a Values reported were corrected by subtracting normal vanadium cont e n t obtained by means of blank detprminations. Normal vanadlurn content of other Eamples was below detection limit of method, 14,Sa
5.2
I5.0a
Draw off these extracts into a glass-stoppered, graduated cylinder, note t.he total volume, and invert gently to mix. The cylinder will retain water droplets when the chloroform extract is transferred to a photomet’er cellLF Measure the absorbancy a t so0 nik using 0.1% &quinolinol solution in the reference cell. Determine the vanadium content from a calibration curve obtained by treating a series of known solutions of vanadium in the same manner as the prepared samples. Each of the known solutions may be prepared by adding 4 ml. of 4 S nitric acid and the appropriate volume of standard vanadium solution to 50 ml. of water contained in a 125-ml. separatory funnel. Because of the large amount of iron, the first extraction of the vanadium from blood samples should be carried out with 10, 5, and 5 ml. of 1% 8-quinolinol solution and the shaking time extended to 5 minutes for the 10-ml. portion. Table I11 presents results of analyses of samples t o which known amounts of vanadium were added and which a-ere then analyzed according to the described procedure. The results of these analyses show that vanadium can be determined in the range of 1 t o 50 micrograms m-ith an average error of &0.32 microgram. Vanadium was added as a solution t o these sam les before they m-ere ashed except in the case of the bone sampEs to which the vanadium was added after ashing. In a later test of the recovery of vanadium from bone, the vanadium was added as a solution in 50% ethyl alcohol to bone meal sieved to ass 40 mesh. Because of an unusually high contamination of t f e bone meal with iron, the ash which was obtained by ignition was fused with sodium carbonate. The average of four analyses of the ash was 12.85 & 0.35 micrograms of vanadium with 12.85 micrograms computed to be present in each 0.5 gram of ash. DlSCUSSION
Although the procedure as given calls for initial extraction of vanadium with three 5-ml. portions of 8-quinolinol solution and final extraction with two 5-ml. portions, the volumes and number of these portions may be varied to suit the range or sensitivity desired. Somewhat better adherence to Beer’s law may be obtained by the use of three 5-ml. portions of 8-quinolinol solution in the final extraction. I t is not essential to accuracy, however, that all of the vanadium be extracted if the standard curve is derived by the same procedure used for the samples. The author has found that for efficient extraction of the vanadium, the chloroform solution should contain an excess of 30 or more times the theoretical amount of 8-quinolinol. Thus, if much over 50 micrograms of vanadium are to be extracted, either the concentration or the volume of the extracting solution should be increased. Also, the complex does not form instantaneously; therefore, if the shaking time is decreased, it is well to extend the contact time of the chloroform and aqueous layers t o ensure complete reaction of vanadium with 8-quinolinol. The method has been most useful for samples of high ash content-such as bone, feed, urine, and feces-for iyhich samples it has a sensitivity several times as great as the dirert spectrographic method. The color system has a sensitivity twice that of the phosphotungstate method. ACKNOWLEDGMENT
The author is indebted to D. H. Byers and H. E. Stokingrr, under whose direction this vr-ork m s done, for many helpful criticisms. LITERATURE CITED
(1) Bach. .J. M., and Trelles. R. d.,d n a i t ~ sosoc. q v f m . n r g c n t i n n , 28, 111 (1940). ( 2 ) B e r t r a n d , D., BuU. soc. c h ’ n . , 9, 128 (1942). (3) Daniel, E. P., Hewston, E. Xf., and Kies, hf. IT,,IND.ENG. CHEM.,- 1 x . a ~ .ED.,14, 921 (1942). (4) G e n t r y . C . H. R., a n d Sherrington, L. G., A n n l y s t , 75, 17 (1950). ( 5 ) >\loel!er, T., IND. ENG.CHEJI., ANAL. E D . , 15, 346 (1943). (6) Rfonier-Williams, G. W., “Trace Elements in Food,” pp. 478-9, London, C h a p m a n & Hall, L t d . , 1949. (7) Xlontequi, R., a n d Gallego, &I., A n d e s SOC. espafi. fis. 1/ guZm., 3 2 , 1 3 4 (1934). (8) Saltaman, B. E., AXAL.CHmf., 2 4 , 1 0 1 6 (1952). (9) Sande!!. E. B., “Colorimetric Determination of Traces of Metals,” pp. 440-3, A-ew York, Interscience Publishers, 1944. ENG.CHEY , A N A L ED., . 8, 336 (1936). (10) Sandell, E. B., IND. (11) Snell, F. D., a n d Snell, C. T., “Colorimetric Methods of Analysis,” 3rd e d . , Vol. 11, pp. 455-6, K e w York. D. V a n Kost r a n d Co., 1949. RECEIVEDfor review -4ugust 28, 1952. Accepted January 23. 19.53.
