INDUSTRIAL .4ND ENGINEERING CHEMISTRY
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VOL. 12, NO. 11
B y observing these essential factors, a long series of tests was made on each of seven fish liver oils having a wide range in vitamin A potency. Analysis of the spectrophotometric Calculated Conversion E:?mm. Value a t 328 mF data shows that the mean of as fex as two deterFactor Bioassay SpectroSpectrominations gave a satisfactory result ( ~ per 2 T y p e of Fish Liver Oil value photometer Vitameter photometer Vitameter cent) . L’. s. P. unitslg I n the study with the vitameter, reproducibility was determined by making a series of tests 2066 2019 31.46 32.20 Halibut (3898) 65,000 1806 1796 60.39 Halibut (16,519) 50,500 50.12 on the same fish liver oils mentioned in connec2355 2321 78.68 79.85 Mixed (4288) 185,300 2295 2225 hlixed (16,319) 295,100 128.60 132.60 tion with the spectrophotometer. Analysis of Halibut (18,869) 31,900 15 05 15.12 2120 2110 the data established that a satisfactory 2191 40 07 2203 hlixed (16,949) 39.85 87,800 2222 2150 1.37 Cod “nonsap.” (9758) 1.35 3,000 value can be obtained from a minimum number Mean value 2152 2122 of six tests. The vitameter mean values on the lone: series were in close agreement with those of t h i spectrophotometer. ” By correlating the spectrophotometer and vitameter data oil, 2222. For the vitameter, the mean conversion factor is with those from the biological procedure, conversion factors 2122, also but 3 per cent different from that derived from the have been calculated. The several values are in good agreeunsaponifiable fraction of the reference liver oil, 2190. The ment, the mean value being, in this instance, 2137. This is, average of the mean conversion factors in Table XI, 2137, is therefore, a close approximation between the potency of a in close agreement with that obtained from routine bioassay fish liver oil in U. S. P. units per gram and the spectrophotoand spectrophotometric results on fish liver oils during the past several years. It is also very close to Barthen’s factor metric Et?m. value. 2064 referred to by Wilkie (IO). It is thus apparent that i t is possible, under the conditions of Conclusion technique as carried out (1) to obtain a quantitative evaluaThe E: value can be accurately determined spectrotion of vitamin A by measuring the extinction coefficient a t phot,ometrically under a carefully controlled procedure. It 328mp; and (2) by applying a conversion factor such as 2137, is therefore possible, b y employing the proper conversion to express the potency in U. S. P. or International units per factor, to evaluate satisfactorily the vitamin A potency of gram. fish liver oils in units per gram. Discussion and Summary Literature Cited I n this critical study of the applicability of the spectrophotometric method to the determination of vitamin A in fish (1) Carr, F. H., and Price, E. -I., Biochem. J . , 20, 497 (192G). liver oils, the characteristics of the operative procedures have (2) Drummond, J. C., and Coward, K. H., Ibid., 14, 734 (1920). (3) Drummond, J. C., and Morton, R. A., I b i d . , 23, 785 (1929). been carefully investigated with respect to the Bausch & (4) Morton, R. A . , “Absorption Spectra of Vitamins and HorLomb spectrophotometer and the more specialized instrumones”, London, Adam Hilger, 1935. ment, the Hilger vitameter. By means of an elaborate series (5) Morton, R. A,, and Heilbron, I. 31., Biochem. J . , 22, 087 (1928). of determinations on several oils, reproducibility has been (6) Munsell, H. E., “The Vitamins, A Symposium”, Chap. I V , pp. 87-109, Chicago, American Medical Association, 1938. demonstrated mith both, and a comparison of the results (7) Rosenheim, Otto, and Drummond, J. C., Biochem. J., 19, 7 5 3 shows good agreement. (1925). The investigation has established the follov ing funda(8) Takahashi, K., Kakamiya, Z., Kamakami, K., and Kitssato, T., mentals with respect to the spectrophotometric technique: Inst. Phys. Chem. Res. (Tokyo), Sci. Papers, 3, 81 (1926). (9) U. S.Pharmacopeia XI (Second Supplement), p. 1 3 4 , 1939. (10) Wilkie, J. B., J . Assoc. Oficial Agr. Chem., 23, 336 (1940). Since vitamin A in solution may lose considerable of its absorptive power after a time, the test reading should preferably be made within an hour after preparation of the solution. PREBESTED before the Division of Biological Chemistry a t the 9Sth MeetThe vitamin is equally absorptive in isopropyl and absolute ing of the American Chemical Society, Boston, .\Iass. ethyl alcohols. The cells should be carefully paired and their surfaces kept scruDulouslv clean. EsDecial care should be taken that no oil residues remain on the cell surfaces. The values of a fish Temperature Correction in F o r m u l a liver oil determined through a range of 50 times difference in concentration were found to be in agreement, showing that Beer’s for Viscosity of Latex law holds. This is also true when one concentration is examined in cells of Is THE authors’ paper entitled “Examination of Rubber Latex different length, showing that Lambert’s law holds within the exand Rubber Latex Compounds. I. Physical Testing hIethods” perimental error of the method. [IND.ENG. CHEY., Anal. Ed., 9, 182-9 (1937)], Formulas 7 If a condensed spark is used as source of light, its position with and 8 on page 188 should read respect to the optical axis of the system must be maintained with the highest degree of precision. This is ordinarily very difficult 72.5 = 711 - 0.02 (25 - T)] (7) to accomplish since erosion of the electrodes is unavoidable during the course of sparking. On the other hand, if a Hilger hydrogen 7 ’ 2 6 = ~ ’ [-l 0.02 (25 - T)] (8) tube is employed, its constancy is definite -with respect to the optical axis, and one is able to obtain satisfactory results in reThese corrected formulas agree with the Crude Rubber Comgard to reproducibility and applicability to routine assays. mittee “Tentative Procedures for Testing the Variability of For visual com arison, optimum results are obtained if the Xormal and Concentrated Latex” [ I N D . ENG. CHEX., Anal. photographic emufsion has been processed to a density of 0.6. Ed., 11, 593 (1939)l. A few substances may interfere with t,he determination of vitamin A in fish liver oils. Among these the most important are: The authors’ attention has been drawn t o this error in a private (a)the unsaturated long-chain fatty acids (particularly in lorn communication from W. S. Davey of the Rubber Research Instipotency oils) and ( b ) the metallic salts such as copper oleate and tute of Malaya. ferric oleate, both of -which can be detected analytically if present H. F. JORDAX, P. D. BRASS,AND c. P. ROE in amounts having significant absorption.
TABLE XI.
CORRELATION OF E:?m. AT 328 M,LCWITH BIOLOGICAL POTENCIES OF FISH LIVEROILS
Fm,
