Determination of Vitamin A in Whale liver Oils Activated Glycerol Dichlorohydrin OLAF K. BKWKKAE' I n s t i t u t e of !Vutrition, University of Oslo, Oslo, Norway Activated glycerol dichlorohydrin has been applied to the determination of vitamin A in whale liver oils. The absorption curves of the color reaction were found to be identical in shape to those developed by pure vitamin A with the same reagent, which indicates a high degree of specificity of the reaction. This has been demonstrated by analyses of mixtures of vitamin .-i and purified kitol.
S
EVERAL investigators have shown that whale liver oils give ultraviolet absorption curves with the maximum displaced toward wave lengths shorter than 325 mp, which is characteristic of pure vitamin A ( 1 , d , 6, 7 , 10, 11, IS). It has been demonstrated that this is caused by the presence of kitol (6, 8): which has an absorption maximum a t approximately 285 mp. Therefore, vitamin A cannot be determined directly in these oils by the commonly used ultraviolet spectrophotometric method (3, 9). Sobel and Werbin ( 1 2 ) have applied glycerol dichlorohydrin to the determination of vitamin A . Their results were completely verified by the author. Studies are reported here in which activated glycerol dichlorohydrin (activated GDH, J. B. Shohan Laboratories, Newark, N. J.) has been applied t'o the determination of vitamin A in whale liver oils. rill investigated samples gave colors with absorption curves identical with those developed by pure vitamin A and the same reagent (Figure l). This indicates that activated glycerol dichlorohydrin reacts sprcificallj- with the vitamin A in these oils.
test on noninterference with the color reaction, mixtures of vitamin A and kitol were analyzed. Table I shows that kitol does not interfere with the color reaction. I t has been confirmed that cholesterol, which is present it1 fairly large amounts in whale liver oils, does not interfere wit1 the color reaction (IO). .inalyses were carried out on eight samples of whale liver 011,. some of them concentratw. Weighed portions of the ails ww
Table I.
Analyses of Mixtures of Vitamin A and Kitol with Activated Glycerol Dichlorohydrin
llixture No.
Vitamin
A
6.45 6.45 6.45 6.45
1
2 3 4
Vitamin A
Kitol
.\f'acrograms 0 13.95 27.90 oa.80
Total
Theorrti % uf
Found
cal
6.45 6.40 6.45 6.42
100.0 99.3 1OO.h 99 6
per m1.-
6.45 20.40 84.35 62.25
For this study a standard curve for vitamin A was made using vitamin A natural ester concentrates, Control YOS. PC-3 and PC-4, from Distillation Products, Inc. Vitamin A content is expressed in micrograms, calculated on the following basis: pure vitamin A alcohol (Distillation Products, Inc., Control KO. K-842) gave an average Et?&. (325 mp) value of 1756 in absolutcx ethanol. I n the same solvent PC-3 and PC-4 gave, respectively, 100.75 and 158.4, which confirmed data supplied by the producers. Csing the assumption that the absorption measured is entirely attributed to vitamin A, this gives a content of 5.7474 vitamin A in PC-3 and 9.07% in PC-4. Figure 2 shows the standard curve' of the color reaction. Six different weighings were made and diluted in chloroform (U.S.P. grade), and measurements were taken on the Beckman spectrophotometer a t wave length 555 mh. All dilutions were made, and all colors developrd and measured at 25" c;. As kitol causes the principal interference in the ultraviolet absorption curves of whale liver oils, special studies were carried out on purified kitol (Distillation Products, Inc., Control No. B641). It gave an absorption curve in the ultraviolet (Figure 3 ) with absorption maximum at approsimately 285 mp, and corresponding E:?&, value of 465 in absolute ethanol, and 469 in isopropyl alcohol. I n cyclohexane the absorption maximum w a ~ found a t 287.5 mp, and an E:?&, value of 461. (Each value is the average of three different weighings.) As pure kitol is reported (2') to have an (290 mp) value of 707, it indicates approximately 60% purity of the analyzed sample. Only in strong concentration could a color be obtained with activated glycerol dichlorohydrin, givingan E:?&, (555 nip) value of approximately 3 . That no consistent curve could be plotted between 400 and 700 mp suggests that the developed color was caused by small amounts of impurities, rather than by kitol itself. For further Present address, Vitamin Division. Norwegian Fisheries Research Institute. Bergen, Norway. 1
400
500
555 600 WAVELENGTH IN
Figure 1. AbsorptiQn Curves 1. Pure vitamin A 11. Whale liver oil
1530
mp
7(
1531
' V O L U M E 21, N O . 1 2 , D E C E M B E R 1 9 4 9
caused b j kitol, but this irrelevant absorption in the ultraviolet spectrum does not seem to interfere with the color reaction. This should justify the conclusion that the determinations by the use of activated glycerol dichlorohydrin give the true values of the vitamin A content of whale liver oils. The results will be further studied by biological experiments.
0.4
0.:
SUMMARY
The use of activated glycerol dichlorohydrin provides a valid method for the determination of vitamin A in whale liver oils. Kitol and some closely related compounds do not interfere with the reaction.
4-
u
0
-I
ACKNOa LEDGiMEhT
0.2
The author wishes to express his thanks to J. G. Baxter for the samples of kitol provided for this work, and to E. M. Nelson, Vitamin Division, Food and Drug Administration, for allowing checking analyses to be carried out in his laboratories.
0.1
'0.C
Figure 2. Standard Curve for Vitamin ..i I
Table 11.
Determination of Vitamin -4 in Whale Liver
Oils Actirstrd glycerol dichlorohydrin coinpared with ultraviolet nieasurements~ Spectrophotometric Drtnu. AbsorpOil tiqn lorn. Color So rnaxims (325 mp'! Detns. Differences mii .wg./y. .Mg./g. MQ. % 56.74 315 99.b 49.93 6.81 11.7 315 48.84 8.39 14.7 100.5 57.23 40.92 24.2 169.13 310 128.21 297. (I 315 81.76 16.76 17.1 173.0 98,52 315 84,96 12.5 170.5 97.10 16.14 308 11.56 29.9 16.52 29.0,4.96 20.3 314 28.02 35.17 7.15 61.13 36.65 14.7 42 99 6.34 315 7.5.5 8
lissolved in ether, two equal portions were pipetted O U T . the ether was evaporated in vacuum, and suitable dilutions were made up in absolute ethanol and chloroform. Dilutions containing be'ween 5 and 8 micrograms were used, allowing easy checking of the absorption curves of the developed colors. All samples were run in duplicate. Table I1 summarizes the results of ultraviolet s n d color readings from thr invtstigations Table I1 shows that the valuea obtained by usiiig activated glycerol dichlorohydrin are 10 to 30% lower than those obtained by the spectrophotometric method based on E:& (325mp). I n the same table are also shown the absorption maxima of the different samples of n-hale liver oils. The displacements range from 10 to 17 mp below 325 mp, which is the characteristic wave length for the absorption maximum of vitamin A. If kitol were the only irrelevant absorbing substance, the displacements should give good criteria for the correction of the ultraviolet absorption readings a t 325 mp. The relation between absorption maximum and correction could be worked out either experimentally bl analyzing mixtures of pure vitainin -4and kitol, or theoretically hased on the known absorption curves and constants for both mbstances. The differences obtained between the methods are higher in the present analyses than expected theoreticall\-. This indicates: a considerable irrelevant absorption in addition to that
WAVELENGTH I N
mp
Figure 3. Absorption Curve of Purified Kitol LITERATURE CITE11
Brlckkan, 0. R., Hvalradetv Skrijttei (Oslo), KO.32 (1948). (2) Clough, F. B., Kascher, H . M..Roheson, C. D., and Baxter, J . (1)
G., Science, 105, 436 (1947). 113) Edisbury, J. R . , A,naZgst.65, 484 (1940). (4) Embree, N. D., and Shantz. E:. XI.. .I. Am. Chem. Soc., 65, 908 (1943). 65, 910 (1943).
1,s) Ibid.,
(6) Haines, R. T. M., and Druriiinond, J . C., A d y s t , 63, 335 (1838). ( 7 ) Kringstad. A,, and Lie, J., T i d u . Kjpmi. Rergcesen M e t . , I, 83 (1941). 8.8, Ibid.. 2, 14 (1942j.
Morton, R. A . , and Stubba, A. L., Biochem. J . , 42, 195 (1948). Nakamiya, Z . , Kvieumi, K., and Kawakanii, I.. Bull. I n a t . Phgs. Chem. Research (Tokyo),20, 576 (1941). 1~11) Pritchard, H., Wilkinson, H., and Morton, R . A., Biochem. J . . (9) (10)
31, 268 (1937). 112)
Sobel, A. E., and Werbin, H., ISD. ESG. CHEM.,ANAL.ED.,18,
570 (1946). (13) Willstaedt. H., and Jensen, J . B., Satme, 143, 474 (19393. RECZIVEDFebruary 14. 1919.
