Determination of Vitamin A in the Presence of Tocopherols ~~~
D. T. EWING AND L. H. SHARPE Kedzie Chemical Laboratory, Michigan State College, East Lansing, Mich.
0. D. BIRD Research Laboratories, Parke, Dazis & Co., Detroit 32, Mich. A esters and a-tocopherol, and distilled natural vitamin A esters and Type IV mixed tocopherols. The separation is highly successful for mixtures containing a-tocopherol, but is not satisfactory for mixtures containing Type IV mixed tocopherols. The method is simple and accurate, even for mixtures containing very large percentages of a-tocopherol, and does not depend on destruction of the vitamin A and analysis by difference. Rather, the two vitamins are separated without destruction of either, and spectrophotometric measurements are made on the quantitatively recoi-ered vitamin A.
The direct application of the Morton-Stubbs correction to vitamin A preparations containing tocopherols in appreciable quantity yields high results for the vitamin A potency. A solution to this problem is the separation of the two vitamins and subsequent quantitative recovery of vitamin A, to which the Morton-Stubbs correction can then be applied. The separation is carried out by chromatographing the vitamin mixture on activated alumina; this procedure gives quantitative recovery of yitamin .4. Data are presented for mixtures of pure vitamin .4 and pure a-tocopherol, distilled natural vitamin
I
Morton-Stubbs correction was applied to a mixture containing vitamin A alcohol and mixed tocopherols, was circumvented by destroying the vitamin A in the mixture by means of sulfuric acid, and comparing the absorbancy of the treated and untreated mixtures. The amount of vitamin A present was then determined by difference. The authors mention that attempts t o find a technique suitable for the separation of tocopherols from vitamin .4 in the sample have not been successful.
S RECEXT years there has been a great deal of interest in the
application of chromatography to the separation of vitamin A (both as esters and as the alcohol) from other substances(2,Q,6 , V . Various adsorbents have been employed, among them alumina, bone meal, and calcium diphosphate. Many different solvents such as benzene, acetone, ethyl ether, petroleum ether, and mixtures of these solvents have been employed for preparing, developing, and eluting the chromatogram. The success of Hjarde (6) in separating vitamin A, by chromatography, from mixtures containing other substances which interfere with its spectrophotometric determination, and the success of workers in this laboratory in using a similar technique for mixtures containing other vitamins, prompted the investigation which is the basis of this paper. Chromatographic separation has been investigated as an expedient for making_possible the determination of vitamin .4 in . the presence of tocopherols. Data are presented for mixtures of pure vitamin A and pure a-tocopherol, distilled natural vitamin A esters and pure a-tmopherol, and distilled natural vitamin -4 P 15 CM esters and Type I V mixed tocopherols. The method was as follows: The vitamin A-tocopherol mixture wassaponified and the two vitamins n-ere separated by chromatographing on alumina. 7 MM The Morton-Stubbs correcA I 2 CM. ID tion (8)was used in the spectrophotometric estimation of the vitamin A. The influence of tocopherols on the U.S.P. vitamin -4 ?. assay has been studied by Fox and Mueller (5). The considerable overcorrective influence of tocopherols, Figure 1. Chromatographic which resulted when the Column
I
APPARATUS AND MATERIALS
Ether. Mallinckrodt's C.P. anhydrous ethyl ether was distilled from anhydrous sodium sulfite and potassium hydroxide to remove peroxides and water. The material w m used not later than 3 hours after distillation. Isopropyl Alcohol. Isopropyl alcohol, 9 9 7 , obtained from the Shell Oil Co. was distilled before use. Hexane. Skellysolve B was chromatographed on a column of activated silica gel, 3.75 cm. in diameter and about 75 cm. in length. The first 1200 ml. of liquid (transparent down to 214 mp) passing through the column was used. Vitamin A Acetate. Merck's pure crystalline synthetic vitamin A acetate, with a claimed potency of 2,906,000 U.S.P. units per gram, was stored a t -20' C. under carbon dioxide until use. This material had an a t 325 mp (in isopropyl alcohol) of 1480. The value given by Cama, Collins, and Morton (1) for the pure synthetic acetate is 1530. Distilled Natural Vitamin A Esters. S o . 1, with a potency of 467,000 U.S.P. units per gram, and KO.2, with a potency of 438,300 U.S.P. units per gram, were furnished by Parke, Davis & co. a-Tocopherol. Pure a-tocopherol was obtained from Distillation Products Industries as a sealed glass ampoule containing 1 gram. The material was stored a t -20" C. until use. Mixed Tocopherols. Type IV mixed tocopherols, with a potency of 40.25y0, were furnished by Parke, Davis & Co. Spectrophotometric Instruments. The Beckman Model DU spectrophotometer and 1-cm. quartz cells were used for all absorbancy measurements. Fraction Collector. Automatic collection of fractions was accomplished by means of a Technicon automatic fraction collector.
I1
7?-
CONSTRUCTION OF CHROMATOGRAPHIC COLUMNS
The column proper was made of borosilicate glass tubing, 7-mm. inside diameter, and its construction is illustrated in Figure 1. SectionA is the solvent reservoir, B thecolumnproper, andCthe column outlet. The constriction between B and C provides a 599
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ANALYTICAL CHEMISTRY
neck in which a cotton wad may be placed to support the adsorbent ill the column above. No particular criteria were used to determine the dimensions of the column, except that the size happened t o be convenient. The important point is that B should be of the same inside diameter in all columns that are t o be used for a series of experiments with a given adsorbent, in order that rate of flow, length of column of adsorbent, threshold volume of materials being separated, etc., be the same for all columns.
Preparation of Tocopherol Solution. This solution was prepared by dissolving a known weight of tocopherol in a known volume of hexane. Charging Column with Adsorbent. A small wad of cotton about the size of a pea was tamped lightly into place at the constriction between B and C, Figure 1, and a small cork was placed in the column outlet. The column was filled with hexane to a point about 0.25 inch above the A-B constriction, the cork was removed from C, the air bled from C, and the cork replaced. The column was then refilled with hexane to the original level and lightly tapped t o remove air bubbles trapped by the cotton PROCEDURE wad. A 2.5-gram portion of the adsorbent was allowed t o run Preparation of Vitamin A Alcohol Solution. SAPONIFICITIOX.down into the column of hexane and any adsorbent adhering to The sample of vitamin A containing not more than 0.66 gram of the walls of A was washed down with a small portion of hexane oil was saponified by boiling for 0.5 hour with 20 ml. of 95% The adsorbent was settled into place by gently tapping column ethyl alcohol and 2 ml. of a 50-50 (weight-volume) aqueous soluB with a glass rod which was covered with rubber tubing. The tion of potassium hydroxide. The saponification was carried column was then ready for use. out in an all-glass apparatus consisting of a 50-ml. round-bottomed The objection may be raised that this method of preparation flask with ground-glass neck, and a small water-cooled reflux of the column results in differential settling of the particles of condenser. The temperature of the mantle used for heating was adsorbent. Results showed, however, that the method was so adjusted that boiling was just sustained. After 0.5 hour the highly satisfactory, inasmuch as columns prepared in this manner heat was removed and the flask and its contents were cooled by gave reproducible results. directing a stream of cold water over the outside of the flask. The method of charging a column with dry adsorbent and then placing a solvent like ether or hexane on the column as a prewash sometimes leads to breaking apart of the column of adsorbent by vapor bubbles of solvent resulting from the heat of adsorption generated when the solvent comes in contact with the dry adsorbent. OS The method of charging the column with a slurry of adsorbent in such a low-density solvent as hexane leads to settling out of the adsorbent before the slurry can be poured into the column. OE I n whatever manner the column is charged, it is important that its characteristics be reproducible. Chromatography. The prepared column was subjected to suction until the hexane was about 1 cm. from the surface of the 0.7 adsorbent, and the desired aliquot of solution t o be chromatographed was introduced. The material was washed down onto the adsorbent with three OE 5-ml. portions of hexane: the level of the liquid was never allowed to go below that of the adsorbent. The suction was then removed and the quantity of solvent (as determined by the development 2 01 analysis given in a following section) required t o develop the I chromatogram was added, care being taken not to stir the adsorb-
? 4
04
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0.2
0.S
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07
01
0.c
2
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6 7 8 9 1011 12 FRACTION NUMBER (EACH FRACTION = 4 ML.)
5
0.6
1314
Figure 2. Development Analysis of Pure a-Tocopherol and Pure Vitamin A Alcohol
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a-Tocopherol Vitamin A alcohol
EXTR.4CTION. A 40-ml. portion of cool distilled water was added t o the mixture in the flask, and the resulting solution transferred to a separatory funnel. The saponification flask was rinsed with 30 ml. of hexane, this rinse added to the contents of the separatory funnel, and the first extraction carried out. Three subsequent extractions of the saponification mixture with 30ml. portions of hexane &'ere performed, and the hexane extracts combined. The combined extracts were then washed with six 50-ml. portions of distilled water, in the separatory funnel, and transferred t o a 125-ml. flask, a 5-ml. hexane rinse of the separatory funnel also being transferred to the flask, and dried with from 5 to 10 grams of anhydrous sodium sulfate. The dried hexane extract was transferred t o a 200-ml. volumetric flask by means of a funnel with cotton-plug filter. The sodium sulfate remaining in the drying flask was extracted with six 10-ml. portions of hexane t o ensure complete removal of the vitamin A alcohol and these portions were added to the volumetric flask. The volume was then adjusted t o 200 ml. with hexane, This solution was used for chromatographing.
2 0.e I
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0.3
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0.0 FRACTION (EACH
NUMBER
FRACTION = 4 ML.)
Figure 3. Development Analysis of Type IV Mixed Tocopherols and Natural Vitamin A Esters 1
IV mixed tocopherols - - - - Type Distilled natural vitamin A esters 1
601
V O L U M E 25, NO. 4, A P R I L 1 9 5 3
Collection of Fractions. Columns were prepared in the standard manner and chromatograms of pure synthetic vitamin A alcohol, vitamin -4 alcohol from the natural products, pure a-tocopherol, and Type IV mixed tocopherols were run separately, using a 1 to 2 (ether-hexane) mixture to develop the Chroniatograms. Fractions of 4.0 ml. each were collected, using a Technicon automatic fraction collector, while the columns were in the process of development. This was done in order that absorbancy measurements of the fractions might be made, and the course of development of each chromatogram plotted. For vitamin A alcohol the absorbancy of each of the fractions was obtained a t 325 mp, using a 1 to 2 (ether-hexane) mixture as the reference solution. The same procedure was adopted for the tocopherols, the measurements, however, being made a t 290 mp.
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e4
n
The plots of these chromatograms are given in Figures 2 and 3.
' h4
I .o
0
0.9
0 3 290
300
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320
330
340 0.8
WAVELENGTH
(MILLIMICRONS)
Figure 4. Analysis of Mixtures of Natural Vitamin -4Esters and a-Tocopherol
-- --.-.- -
Solution of natural vitamin A esters 1 and pure a-tocopherol before chromatographing Solution after chromatographing Natural vitamin A esters 1 Mixture contained equivalent of 687 of vitamin A alcohol and 956y of a-tocopherol
ent a t the top of the column. The chromatogram was allowed to develop, and was eluted with 99% isopropyl alcohol, the eluate being collected in a 25-ml. volumetric flask, and made up to volume with isopropyl alcohol and pure hexane according to the procedure described under Treatment of Solutions. These eluate solutions were such as to facilitate direct absorbancy measurements by the Beckman Model DU spectrophotometer, using 1-em. cells.
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WAVELENGTH
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(MILLIMICRONS)
Figure 6. Analysis of Mixtures of Natural Vitamin A Esters and a-Tocopherol
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Solution of natural vitamin A esters 1 and Type IV mixed tocopherols before chromatographing Solution after chromatographing Natural vitamin A esters 1 ,Mixture contained equivalent of 64v of vitamin A alcohol and 8917 of a-tocopherol
10
0 9
Treatment of Solutions on Which Absorbancy Measurements Were Made. Because all solutions of vitamin A and tocopherols were made up in hexane, while isopropyl alcohol was to be used as the solvent for absorbancy measurement.?, and because it was desired to eliminate having to evaporate solutions containing vitamin A alcohol (owing to its instability), the follon-ing procedure \vas adopted:
0 8 n
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07
06
The final volume of all solutions which were to be examined spectroscopically was set up to be 25 ml., and whenever a hexane solution aliquot was to be made up with isopropyl alcohol, enough pure hexane was added t'o bring the volume of hexane up to 5 ml., the remainder of the 25 ml. being isopropyl alcohol. The solution used in the reference cell was, accordingly, a 1 to 4 (volume-volume) mixture of hexane and isopropyl alcohol.
0 5
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0 3
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300
310
WAVELENGTH
320
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340
(MILLIMICRONS)
Figure 5. Analysis of Mixtures of Natural Vitamin A Esters and a-Tocopherol
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Solution of natural vitamin A esters 2 and pure atocopherol before chromatographing Solution after chromatographing Natural vitamin A ester8 2 Mixture contained equivalent of 687 of vitamin A alcohol and 9567 of rr-tocopherol
Recovery. An average recovery of vitamin A, from the column, n-as found to be 94.4% on the basis of four experiments in which the individual recoveries were 95.7, 94.8, 93.5, and 93.8%. Mixtures of Distilled Natural Vitamin A Esters and Tocopherols. Four mixtures-distilled natural vitamin A esters 1 and 2 and a-tocopherol, distilled natural vitamin A esters 1 and 2 and Type IV mixed tocopherols-were made up and carried through the entire procedure in the recommended manner. The cor-
ANALYTICAL CHEMISTRY
602 Table I. Applicability of Method to Mixture of Natural Distilled Vitamin A Esters and a-Tocopherol 310 Sample No. 1 2 3 4
Av 1 2 3
4
Av.
Ware Length, mp 325 334 , after Chromatographing
Vitamin A Mixture 69 6 81 3 68 1 80 3 69 3 81 5 83 1 71 4 69 6 81 6 Vitamin A Esters 2
la
63.5 62.9 60.5 62.4 62 3
65.2 63.5 63.1 65.9 64 4
74.6 73.0 72.8 74.8 73 8
72 70 72 72 71
3 4 5 5
9
at 325 mp, Morton-Stubbs corrected = 73.9. Observed potency of mixture = 73.9 X 1900 = 140,400 U.S.P. units of vitamin A per gram. Value corrected for column loss = 148 700 U.S.P. units of vitamin .I per gram. Known potency = 150,500 U.S.P.’unitsof vitamin A per grain. b at 325 mp, Morton-Stubbs corrected = 71.4. Ohserved potency of mixture = 71.4 X 1900 = 136,600 U.S.P. units of vitamin A per gram. Value corrected for column loss = 143,600 U.S.P.units of vitamin A per gram. Known potency = 142,000 U.S.P. units of vitamin A per gram. Correction constants used were those for isopropyl alcohol as solvent, inasmuch as presence of 20% of hexane in solvent doea not change vitamin .I curve enough to warrant calculation of new constants. Q
FRACTION NUMBER (EACH FRACTION : 4 ML.)
Figure 8. Development Analysis of a-Tocopherol and Vitamin A Alcohol on Controllably Deactivated Alumina -.-.-. Tocopherol (65 % sulfuric acid-35 % water)
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Tocopherol (63% sulfuric acid-37ojc water) Tocopherol (60% sulfuric acid40o/c water) Vitamin A alcohol (60 % sulfuric a c i d 4 % water)
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WAVELENGTH
I !
(MILLIMICRONS)
Figure 7. Analysis of Mixtures of Natural Vitamin A Esters and a-Tocopherol
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Solution of natural vitamin A esters 2 and Type IV
m i x e d tocopherols before chromatographing
Solution after chromatographing Natural vitamin A esters 2 Mixture contained equivalent of 58y of vitamin A alcohol and 8917 of a-tocopherol
responding curves are given in Figures 4, 5, 6, and 7. The datzi as to obtained potency, etc., are given in Table I. ADSORBENT
The alumina which was on hand in this laboratory proved to be too highly activated for direct use in this work-that is, both components of the mixture were so strongly adsorbed that a good separation was impossible. In order to deactivate the alumina, it was left, spread in a thin layer, open to the atmosphere of the laboratory (at the time the relative humidity was
15%) for about 10 hours. This gave an alumina (alumina X ) of suitable degree of activation for the work. It was desired, however, to be able to control the degree of activation more closely than the above permitted. Because the amount of water vapor adsorbed on the surface of the adsorbent can control the degree of activation of the adsorbent, and the amount of water vapor adsorption is a function of the water vapor pressure, the adsorbent may be deactivated controllably by exposure to an atmosphere of controlled humidity. Accordingly, 10-gram samples of alumina were spread in thin layers on Petri dishes. These were placed in desiccators over approximately 300 ml. of sulfuric acid-water mixtures of varying sulfuric acid content. The samples were then allowed to come to equilibrium with the water vapor of the produced atmospheres, a process which takes about 48 hours. Information as to the course of development of chromatograms of a-tocopherol on 2.5-gram columns of aluminas of these various strengths was obtained by collecting 4.0-ml. fractions from these columns in the standard manner. The data are presented as a plot in Figure 8. The ordinate is the ratio between the absorbancy a t 290 r n p of any given fraction to that of the fraction containing the largest amount of tocopherol. Development plots of a-tocopherol are given for several samples of controllably deactivated alumina, each sample being deactivated to a different extent. Included in Figure 8 is a similar development plot of a chromatogram of pure vitamin A alcohol obtained from one of the above aluminas-that which had been deactivated by exposure to a 60 to 40% (by weight) sulfuric acid-water mixture (alumina Y). This particular chromatogram of vitamin A alcohol and 01tocopherol on alumina Y may be compared with that in Figure 2 which was obtained for the alumina used in all of the actual sep-
V O L U M E 25, NO. 4, A P R I L 1 9 5 3
603
aration work (alumina X ) for the pure materials. It becomes evident that as good a separation is obtainable with the one alumina as with the other. It appears, therefore, t8hatthe alumina to be used for the separation of vitamin A alcohol and a-tocopherol may possess an activity in the range between that of alumina X and the alumina I-,and still give good separation. CHOICE OF SOLVENT SYSTEM
A good solvent system t o be used for developing a chromatogram (1) must give good banding of the components to be separated, and (2) must give good separation. The actual developer rn hich is used is most often a compromise between (1) and (2)that is, a solvent system is employed which gives the best separation consistent with sharp handing. for the particular adsorbent being used.
were carried through the procedure. The observed values for the potency of the mixtures differ from the known values by about 1yo. It will be noted from the ratio curves corresponding to mixed tocopherols-natural vitamin A mixtures that a highly unsatisfactory degree of separation has been obtained. The curves of the development analysis performed on these materials (Figure 3) show that the mixed tocopherols have been resolved into a t least two components, as two peaks appear in the curve. The first peak may be attributed to a-tocopherol, while the second peak and rather long tail may be attributed to nonalpha forms of the tocopherol. These two peaks arise because of the difference in behavior of the different isomers of tocopherol on alumina-one or more of the other isomers being more strongly adsorbed than the alphaisomer. This results in overlapping of the bands of tocopherol and vitamin A in the chromatogram, and leads to incomplete separation. Type IV mixed tocopherols such as that used in this portion of the work are a mixture of the natural tocopherols, and are relatively rich in nonalpha forms of tocopherol. The method, therefore, breaks down. If, however, one were dealing with such a preparation of mixed tocopherols as Type VI, where the percentage of nonalpha forms was relatively low, then the method here presented would probably be applicable-that is, the separation of tocopherol from vitamin A would be relatively good.
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WAVELENGTH
320
3x)
340
(MILLIMICRONS)
Figure 9. Typical Curves for Mixture of Tocopherol and Vitamin A Alcohol
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Solution of pure vitamin A and pure a-tocopherol before chromatographing Solution after chromatographing -.-*-a Pure vitamin A alcohol Mixture contained 747 of vitamin A alcohol and 8267 of a-tocopherol
In thie work the optimum solvent system was found to be A less polar mixture of ether and hexane resulted in unsharp banding and tailing of the tocopherol. A more polar mixture resulted in incomplete separation of the components.
a 1 to 2 (volume-volume) mixture of ether and hexane.
RESULTS AND DISCUSSION
The degree of separation of vitamin A alcohol from a-tocopherol is probably best illustrated by a comparison of the ratio curves ( A / & & ) of pure vitamin A alcohol, and those of the vitamin A alcohol-a-tocopherol mixture before and after chromatographing. A typical set of curves is given in Figure 9 for a weight mixture of approximately 12 to 1 (tocopherol-vitamin A alcohol). These curves show essentially complete separation of the two components. If, however, this weight ratio is increased t o approximately 24 to 1, the separation is less complete, as shovn by Figure 10. The data illustrating the applicability of the method to mixtures of natural distilled vitamin il esters (1 and 2) and a-tocopherol are given in Table I. Synthetic mixtures of the above
WAVELENGTH (MILLIMICRONS)
Figure 10. Typical Curves for iMixture of Tocopherol and Vitamin A Alcohol -Solution of pure vitamin A and pure a-tocopherol before chromatographing Solution after chromatographing Pure vitamin A alcohol Mixture contained 67r of vitamin A alcohol and 1 6 5 2 ~of a-tocopherol
----.-.-e
The saponification procedure has been altered somewhat from that recommended in the U.S.P. XIV vitamin A assay. Hexane has been substituted for ether in the extraction of the saponification mixture. The use of hexane has a twofold advantage. (1) There is no tendency of the mixture to form an emulsion with hexane during either extraction or washing. In the case of ether extraction, however, a persistent emulsion will form during
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) (7)
Hjarde, A., Acta. Chim. Scand., 4 , 628 (1950). 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-
