20 ml. of hot concentrated nitric acid and add this liquid to the beaker. Decompose the precipitate with nitric acid, and evaporate t o dryness on a hot plate. Carry out a t least three evaporations with 5 ml. of concentrated hydrochloric acid each time, to ensure complete removal of nitric acid. Dissolve the residue in 50 ml. of a 5M sodium chloride solution and add 7 ml. of glacial acetic acid. Transfer the solution to a 100-ml. volumetric flask by a water wash and dilute to volume (the resulting DH is 1.8 to 1.9). Transfer a 35-ml. aliquot into the cell. Before reading and adding the titrating solution, deaerate the solution by passing nitrogen through it for 15 minutes. Continue this bubbling for 3 minutes after each addition of the paramolybdate solution, prior to turning on the current. “
240
i
I
i
f
/
2 160
w LL Lz
3 0
80
I
The readings were carried a t each 0.1 ml. a t the beginning of the titration, then for each 1 ml. around the equivalence point, and next around each 0.2 ml. until various points in line were obtained. After the dilution correction is made ( l a ) , the equivalence point is graphically determined in the usual way. Figure 2 shows a typical curve. LITERATURE CITED
(1) Ranks, C. V., Iliehl, H., ANAL. CHEM.19,222 (1947). (2) Blatt, A. H., ed., “Organic Syntheses,” Coil. Vol. 11, p. 414, Wiley, New York, 1944.
0
Figure
0.8
1.6
2.4 3.2 4.0 MOLYBDATE SOLUTION, ML.
4.8
5.6
6.4
2. Amperometric determination of thorium in a monazite sample
(3) Britton, H. T. S., German, W. L., J. Chem. SOC.1931, 1429. (4) Deshmukh, G. S., Bokil, I., Bull. Chem. SOC.Japan 29,449 (1956). (5) Gordon, L., Stine, C. R., ANAL. CHEM.25,192 (1953). (6) Gordon, L., Vanselow, C. H., Willard, H. H., Zbzd., 21, 1323 (1949). (7) GuibB, L., Souchay, P., J . chim. phys. 54,684 (1957).
(8) Hillebrand, W. F., Lundell, G. E. F., Bright, H. A., Hoffman, J. I., “Applied Inorganic Analysis,” 2nd ed., p. 542, TViley, New York, 1953. (9) Kaufman, L. E., Trav. inst. &ut radium (U.S.S.R.) 4 , 313 (1938). (10) Kawahata, M., Mochizuki, H., Kajiyama, R., Bunseki Kagaku 8 , 2 5 (1959). (11) Kolthoff, I. M., Lingane, J. J., “Polarography,” 2nd ed., Val. 11, p. 457, Interscience, New York, 1952. (12) Zbzd., p. 890. (13) Lingane, J. J., Laitinen, H. A., IND. ENG.CHEM.,ANAL. ED. 1 1 , 504 (1939). (14) Manning, D. L., Ball, R. G., Menis,
O., AXAL.CHEM.32,1247 (1960). (15) Metzger, F. J., Zons, F. W., J. Znd. Eng. Chem. 4,493 (1912). (16) Moeller, T., Schweitzer, G. K., Starr, D. D., Chem. Reus. 42, 63 (1948). (17) Pecsok, R. L., Parkhurst, R. M., ANAL.CHEM.27,1920 (1955). (18) Rodden, C. J., ed., “Analytical Chemistry of the Manhattan Project,” p 169, McGraw-Hill, New York, 1950. (19) Zbid., 171. (20) SchoeEer, W. R., Powell, A. R., “Analysis of Minyals and Ores of the Rarer Elements, 3rd ed., p. 108, Griffin, London, 1955. (21) Zbid., p. 262. (22) Smales, A. A,, Airey, L., At. Energy
Research Establishment, Harwell, England, C/M 131. (23) Tung, S. C., Kang, E. K., Hua
Hsueh Hsueh Pao 25, 33 (1959) (24) Willard, H. H., Gordon, L , ANAL. CHEY.20,165 (1948).
RECEIVED for review February 10, 1961. Accepted Kovember 28, 1961.
Differentiation of Vitamins D2 and DB by Infrared Spectrophotometry W. W. MORRIS, Jr., J. B. WILKIE, S. W. JONES, and LEO FRIEDMAN Food and Drug Administration, U. S. Department o f Health, Education, and Welfare, Washington 25, D . C.
b Infrared spectrophotometry can b e used to determine the form of vitamin D present, either b y visual examination o f the spectrum between 10 and 1 1 microns or b y spectrophotometric neutralization techniques when nonuniform background i s present. The amounts of vitamin Dz or D1 can also b e estimated b y spectrophotometric neutralization. A technique i s described that i s potentially useful for the determination o f the proportion of each form of vitamin D present in mixtures of th= two by means of a reference curve relating the ratio of a b sorbance differences (A10.3p - Ala.5 p ) /
A10.4 p - A10.5 p) to the per cent composition of the mixture. The amount of each form could then b e calculated from the total vitamin D content of the sample. The accuracy o f these procedures i s within f 15%.
U
there has been no means of differentiating between vitamin D2and vitamin DB,except by bioassay procedures. Both forms are equally potent as measured by the rat bioassay ( I S ) , while vitamin D2 has only 1/100th the potency of vitamin D3 as measured by the rhick bioassay (I). Horn-erer. bioassay procedures are P TO THE PRESENT TIME
costly and too time-consuming for routine control work, and the need exists for a more rapid procedure for the differentiation of the two forms of vitamin D. Infrared spectrophotometry appeared to offer a rapid and sensitive means of differentiating between vitamins D2 and DB. Jones (7) found that the infrared spectrum of vitamin Dz exhibits a strong band a t 10.3 microns (970 cni.-l) due to the carbon-hydrogen bending vibration of the -C-H group in the unsaturated side chain; the saturated side chain of vitamin D3 does not exhibit this strong band. VOL. 34, NO. 3, M A R C H 1962
381
OD2
f
go1
SI
< 02 10
pqpqpqpq 25
0
I
I1 10
1W
15
50
10 3
11 10
11
11 10
11 10 WAVELENGTH
Figure 1. Infrared spectra, between 10.0 and 1 1 .O microns, of pure vitamin Dz, pure vitamin D3, and mixtures of vitamins Dz and D3
Absorption in this region of the infrared spectrum due to isolated trans double bonds has been applied to the determination of the trans fatty acids (9),the detection of hydrogenated fats in butter (d), and to the differentiation of Pennsylvania-type oils from other oils (3) and of stigmasterol from sitosterols (6). This report presents a method for utilizing differences in the infrared spectra of vitamins Dzand D3 for the identification of the form present. When used with the SbCla color inhibition method for total vitamin D ( 1 4 , this procedure permits the determination of both vibmins D2 and Da with an accuracy of *15% (the accuracy of the SbC13 color inhibition method) and can be applied to mixtures of the two compounds.
IO3:
REFERENCE MATERIALS
Vitamins D2 and D3 were purchased from the California Corp. for Biochemical Research, Los Angeles, Calif. Both the melting points and the ultraviolet spectra were used as criteria of purity. Stock solutions in carbon disulfide were prepared fresh daily. PROCEDURE
Preparation of Sample. A sample containing approximately 5 mg. (200,000 Units) of total vitamin D is saponified and extracted by a modification of the method of Wilkie et al. ( 1 4 ) ; the modifications include extension of the saponification time to 30 minutes and the use of petroleum ether (b.p. 30" to 60" C.) instead of diethyl ether for the extraction of vitamin D. I n addition, during the first extraction, 5
382
ANALYTICAL CHEMISTRY
75
The infrared spectra of vitamin DZ and vitamin D3 between 10 and 11 microns, and of mixtures of the two a t a total vitamin D concentration of 5 mg. per ml. are shown in Figure 1. The same cells and slit widths are used for each determination. The spectrum of vitamin D3 exhibits a singlet a t 10.4 microns, while that of vitamin Dz exhibits a strong absorption maximum a t 10.3 microns with a subsidiary peak a t 10.4 microns. Both forms
50
0
25
Figure 2. Relation of ratio of absorbance differences to per cent composition of mixtures of vitamin Dz and vitamin
D3
APPARATUS
Spectral measurements were made with a Perkin-Elmer 221 infrared spectrophotometer equipped with sodium chloride optics. Haenni-type cells (4) with a light path of 1 mm. and a capacity of 0.25 ml. were used; the cell windows were of sodium chloride. Carbon disulfide was used as the solvent. The qualitative scans were obtained with a slit width of 216 microns, while the quantitative scans were obtained m-ith a slit width of 82 microns.
loo
RESULTS AND DISCUSSION
grams of S a 2 S 0 4 10HzO . is added to facilitate separation of the two phases. The final extract is concentrated to a volume of about 5 ml. and purified by chromatographic procedures. Chromatographic Purification. From two to four chromatographic columns are used depending upon the nature of the impurities present. I n materials which contain xanthophylls and/or provitamin D, these impurities are removed by a magnesia-Hiflo Supercel column (14), after which vitamin -4 is removed by a column of Celite 545polyethylene glycol 600-iso-octane (11, 15); the latter step was routinely used for all samples. If decomposition products of vitamin A are suspected to be present, they are removed by passage through a Florex XXS column (10) Carotene, if present, is removed by the subsequent use of an alumina column (14). The purifted extract is evaporated to dryness in vacuo and dissolved in carbon disulfide to produce a concentration of 2 to 5 mg. per ml. Spectrophotometric
Vitamin
D2
Capsule
I r r a d i a t e d Yeast
Procedures.
The infrared spectrum of the carbon disulfide solution of the purified material is recorded. I n addition, the total vitamin D content of the original sample is determined by the official U.S.P. method of assay for total vitamin
D (1.9).
exhibit low absorption at 10.5 microns. I n mixtures of the two, the absorption a t 10.3 microns progressively increases with the amount of Dz. Figure 2 is a plot of the ratio of absorbI.C ance differences( A 0 . 3 p - A10.5 IL)/(AIO.~ p ) of these mixtures against the per cent composition of the mixture. The calculated ratios of absorbance
WAVELENGTH
Figure 3. Infrared spectra, between 10.0 and 10.5 microns, of samples in which the form of vitamin D present can b e determined b y visual examination of the spectrum
Table I.
Comparison of Infrared Spectrophotometry with Other Assay Procedures for Vitamin D
Form of Vitamin D Present SpectroVisual photoexaminametric Label Claim tion neut. 50,000 U D?/capsule Dz, no D3 ... 50 ,000 U Ds/capsule D? no Da ... 40,000 U D?/gram DP,no D3 ...
Sample Vitamin capsule Vitamin capsule Irradiated yeast Poultry feed concentrate 200,000 ICU/gram 200,000 ICU/gram 3,000 ICU/gram 0
D3
Dt
(?)
D3 (?)
Spec. Keut.O
.. .
...
DB,no Dz
200 ;0oo U/gram .. .
D,, no D?
3,000 U/gram
...
Chemical assay 53,800 U/capsule 69,000 U/capsule 47,000 U/gram
Vitamin D Content Bioassay Rat 60,000 U/capsule 70 ,000 U/capsule
...
200,000 U/gram 306,000 U/gramb
200,000 U/gram 162,000 U/gramb
3,000 U/gram
3,300 U/gram
Chick
...
...
200,000
ICU/gram
>200,000
ICU/gram 3,000 ICU/gram
Approximate content.
* Different samples of the same lot.
differences are independent of the concentration of total vitamin D; the ratio for 1007, vitamin Ds is approxirnately -0.25, while that for 100% vitamin D2 is about 1.40. This relationship is potentially useful for the determination of the per cent of each form of vitamin D present in materials containing both forms. While only the results of a typical experiment are shown, these ratios are reproducible. I n the application of this relationship to the analysis of mixtures, it may be advisable t o repeat this calibration curve each time a sample is run. The relation of the absorbance difference p - A l 0 . 5 p) to the concentration of vitamin Dz and vitamin D3is linear for each form up to a concentration of 20 mg. per ml., the highest used. The use of the absorbance difference automatically corrects for nonspecific horizontal background. In some cases, the form of vitamin D present in a sample can be determined by inspection of the infrared spectrum of the extract. The infrared spectra of two commercial preparations claimed to contain vitamin D? are shown in Figure 3. The spectrum on the left is that of a vitamin Dz capsule preparation (50,000 Units per capsule). Visual examination indicates that the vitamin D present is almost entirely vitamin D,, as confirmed by the calculated ratio of absorbance differences, 1.6. Impurities present which absorb a t 10.3 to 10.4 microns may have caused this ratio t o be slightly higher than that found for pure vitamin Dz. The spectrum on the right is that of an irradiated yeast product; the peak a t 10.3 microns and the almost complete lack of a peak a t 10.4 microns indicate that vitamin D2 is the predominant, if not the only, form present. Use of the ratio of absorbance differences, in this case, is not feasible since the subsidiary peak a t 10.2 microns indicates the presence of provitamin D2 (ergosterol) which also has an absorption
1’
10.4 10.0
10.5
WAVELENGTH
10.0
10.5
WAVELENGTH
Figure 4. Application of spectrophotometric neutralization in the infrared region to determination of form of vitamin D present Left. Poultry feed concentrate (3000 Units vitamin Da/gram) 1. Sample vs. solvent 2. Sample vs. 1 mg.vitamin Ds/ml. 3. Sample VI. 2 mg. of vitamin Da/ml. Right. Poultry feed concentrate (200,000 Units vitamin Da/gram) 1. Sample VS. solvent 2. Sample vs. 5 mg. of vitamin Da/ml.
maximum a t 10.3 microns with a lesser peak a t 10.4 microns. In samples with nonuniform background, spectrophotometric neutralization (6, 8) is used to determine the form of vitamin D present. I n this technique vitamin Dz or D3 is used in the reference cell, and the concentration is increased by known amounts until the specific peak attributable to vitamin D in the sample cell disappears into the recorded curve. At this point the concentration of Dz or D3 in the reference cell is approximately equal to that in the sample cell. This procedure can be used for an estimation of the amount of vitamin D present in the sample with an accuracy of =k15%. This process is illustrated in Figure 4. The spectrum on the left is that of a poultry feed concentrate in a cornmeal
filler claimed to contain vitamin D3 (3000 Units per gram). I n curve 1, the peak a t 10.4 microns and the lack of a peak a t 10.3 microns suggest that this is vitamin D3; however, in the presence of nonuniform background absorbance, this is not conclusive identification. I n curves 2 and 3, 1 and 2 mg. per ml., respectively, of vitamin D3 are used in the reference cell. The disappearance of the peak a t 10.4 microns into the recorded curve when the concentration of vitamin D3 in the reference cell is 2 mg. per ml. shows that the vitamin D of the sample is almost entirely D3 and indicates the approximate amount present. On the right of Figure 4 is the infrared spectrum of another poultry feed concentrate claimed to contain vitamin D3 (200,000 Units per gram). VOL. 34, NO. 3, MARCH 1962
383
The lack of a peak absorption at 10.3 microns and the singlet at 10.4 microns are indicative of vitamin D3. The peak at 10.4 microns is neutralized by the addition of 5 mg. per ml. of vitamin D3 to the reference cell. Here, too, the form and the approximate amount of vitamin D is elucidated by the technique of spectrophotometric neutralization. The application of these procedures to the determination of the form and the approximate amount of vitamin D present in some commercial preparations is summarized in Table I. In some cases, inspection of the infrared spectrum was sufficient to identify the form of vitamin D present; in other cases, spectrophotometric neutralization was necessary. Thus, the finding by infrared spectrophotometry that in poultry feed concentrates the vitamin D present in the D3 form is confirmed by the chick bioassays. Despite the relative insensitivity of spectrophotometric neutralization in the infrared as compared to that in the ultraviolet,
the results obtained by this procedure are comparable to those obtained by the chemical assay for total vitamin D by the method of U.S.P. XVI (13). ACKNOWLEDGMENT
The authors are indebted to Ronald Yates, Division of Cosmetics, Alma Hayden and Jonas Carol, Division of Pharmaceutical Chemistry, and Daniel Banes, Bureau of Biological and Physical Sciences, all in the Food and Drug Administration, for their interest and suggestions during the course of this investigation. LITERATURE CITED
(1) Assoc. Official Agr. Chemists, Washington 4, D. C., “Official Methods of Analysis,” 8th ed., 1955. (2) Bartlett, J. C., Chapman, D. G., J . Agr. Food Chem. 9, 50 (1961). (3) Fred, M., Putscher, R., ANAL.CHEM. 21, 900 (1949). (4) Haenni, E. O., J . Assoc. Oj%. Agr. Chemists 42, 215 (1959).
(5) Johnson, J. L., Grostic, M. F Jensen, A. O., ANAL.CHEM.29,468 (1957). ( 6 ) Jones, J. H., Clark, G. R., Harrow, L. S., J . Assoc. Ofic. Agr. Chemists 34, 136 (1951). ( 7 ) Jones, R. K., Chem. in Can. 2, 94 (1950). (8) Powell, H., J . A p p l . Chem. 1956, 488. (9) Report of the Spectroscopy Committee, J.A.O.C.S. 36,629 (1959). (10) Schmall, M., Senboroski, B., Colarusso, R., Woolisch, E. G., Schaefer, E. G. E., J . Am. Pharm. Assoc., Sci. Ed. 47, 839 (1958). (11) Theivogt, J. G., Campbell, D. J., AXAL. CHEM.31, 1375 (1959). (12) United States Pharmacopeia XV, 1955. (13) United States Pharmacopeia XVI, 1960. (14) Wilkie, J. B., Jones, S. W.,Kline, 0. L., J. Am. Pharm. ASSOC., Sci. Ed. 47, 185 (1958). (15) Wilkie, J. B., Jones, S. W.,Morris, W.W.,J. Assoc. O j i c . A g r . Chemzsts 42, 422 (1959). RECEIVEDfor review September 6, 1961. Accepted December 19, 1961. In part: 5th International Congress on Kutrition, September 1960, and 74th Annual Meeting of the Association of Official Agricultural Chemists, October 11, 1960.
Spectrographic Solution Procedures for the Determination of Some Less Familiar Elements in Iron and Nickel Base Alloys J. P. McKAVENEY and G. L. VASSILAROS Crucible Steel Co. of America Research laborafory, Pitfsburgh 7 3, Pa.
b Spectrochemical analysis by the solution-rotating disk technique i s proposed for the determination of less familiar elements in typical iron and nickel base alloys to relieve the wet chemist from the long and difficult task of developing a separation scheme for each new alloy composition. Chemical procedures required for the sample solution preparation are outlined for beryllium, niobium, magnesium, silver, yttrium, and zirconium. Chemical aspects of the solution are empirically examined both from the view of solvent effect on spectrographic response and chemical interaction between solvent and metallic ion.
T
HE TECHNIQUE of direct spectrographic analysis of solutions is fairly well known for most of the common alloying elements present in iron and nickel base alloys, but little information is available for the less familiar elements. This has not been because of lack of investigation of the physical
384
ANALYTICAL CHEMISTRY
aspects of the problem, as seen from the excellent work of Feldman (3) m-ith the porous-cup technique, Pagliassotti and Porsche (8, 9) with the rotating disk, and Zink ( 2 1 ) with the vacuum-cup technique. Also on the physical side, Margoshes ( 5 ) has worked on improved source techniques to increase sensitivity. However, on the chemical side, only the work of Baer and Hodge ( 2 ) has considered one aspect of the problem, namely, the effect of the solvent composition on spectral response. It is the authors’ conviction that the physical and chemical aspects of the problem should go hand in hand for successful application of the spectrographic solution technique. Too often in the American metals industry there is complete separation of chemical and spectrographic laboratories. The spectrographer who has been trained only in the solid specimen technique cannot solve analytical problems for less familiar elements by the solution technique because of lack of experience with chemical solutions. On the other hand, while use of a met chemist as a consult-
ant might be of advantage, this approach often fails because he does not understand the spectrographic technique. Success, therefore, will lie in the hands of the spectrographer with the chemical background. The spectrographic solution technique can be a porerful tool because of the advantages for minimizing segregation and metallurgical history as well as eliminating the need for preanalyzed chemical standards. Also, another point often orerlooked is the nearly complete elimination of interelement effects, e.g., carbon on niobium because of niobium carbide formation, or sulfur on manganese through formation of manganese sulfide in the solid solution of the metal. The proper chemical solvent will decompose completely the intermetallics, and total niobium or manganese can be determined easily. The specialty steel analyst is often challanged when analyzing research heats for less familiar elements because of the lack of solid specimen spectrographic standards or a chemical procedure free of interferences. I n this labora-
