Physicochemical Assay of Vitamin A J
J
NORRIS DEAN E>IBREE, Distillation Products, Inc., Rochester, N. Y.
T
HE vitamin A potencies of various materials are determined in many laboratories by one or both of two wellknown physicochemical methods. The method which is often considered to be the more exact depends upon the measurement of the absorption of ultraviolet light a t the maximum (328 mp) of the vitamin A absorption band of an alcohol solution of the material or a purified extract (1, 2, 4, 8 ) . The vitamin A concentration is proportional to the value of F1% ~1 cm. (328 m ~ ) . =
a1 log -II 2
where c = concentration in grams per 100 ml., d = depth of optical cell in centimeters, IO = intensity of the light incident on the solution, and Z = intensity of the light transmitted by the solution.
Several solutions of vitamin A which were being assayed in the regular work were split up into two parts. One part was put into the amber test tube, and the other part into a clear test tube of the same dimensions. The tightly stoppered test tubes stood in a glass beaker on the laboratory bench 12 feet from the window of the room. After a few hours of this exposure to daylight, the solutions were again tested and the potencies of the vitamin A concentrates calculated. Cloudy days during which no sun shone were chosen for these tests, in order to help compensate for the higher actinic power of summer daylight. The solutions contained 20 to 25 units of vitamin A per ml.
The other method, which is almost as exact, depends upon the measurement of the absorption of light a t the maximum (620 mp) of the absorption band of the blue colored reaction product formed by mixing a chloroform solution of the material with ten times its volume of a saturated chloroform solution of antimony trichloride (3, 5, 6). The vitamin A concentration is proportional to the value of (620 mp). If a spectrophotometer is not available, this color measurement may be made with a photoelectric colorimeter using a sharp-cut filter. I n this latter case a calibration curve is made relating the vitamin A potency of the solution being tested to the depth of the blue color.
Table I1 gives the results of these tests, which show that serious losses of vitamin A potency result from exposure of solutions of vitamin A in clear glass test tubes, while only small losses are found if amber test tubes are used. Contrary to prevalent opinions, solutions in chloroform are about as stable as solutions in alcohol, and solutions of vitamin A in the alcohol form are about as stable towards light as those in the ester form, This last fact should be borne in mind because, owing to the greater resistance of vitamin A esters to autoxidation, solutions of vitamin A esters are sometimes not handled so carefully as those of vitamin A alcohol.
TABLEI. STABILITY OF SOLCTIOKS OF VITAMIK A KEPT IK BROWSGLASSBOTTLES [Halibut liver oil distillate diluted with cottonseed (Wesson)oil] Solvent Eth lalcohol Cycrohexane Hexane Ether Chloroform Benzene
Location of Maximum Absorption 328 327 328 328 333
333
Concentration
0.0822
0.0863 0.0844 0,0848 0.0846 0.0846
E
had been diluted with cottonseed (Wesson oil) oil to a potency of slightly over 30,000 U. S. P. units per gram. Since solutions of vitamin A are so stable in the absence of ultraviolet light, it seems desirable that they be handled entirely in apparatus which will absorb these harmful radiations. The apparatus, in general, must be transparent, so that the operator may separate layers, look for emulsion formation, etc. Test tubes made of an amber glass which seemed suitable for the manufacture of such apparatus were furnished to the author by a glass manufacturer. Some tests of the usefulness of this glass were made.
(lrax.)
1 hour 24 hours 7 days 50 days
15.1 14.6 16.1 16.6 13.2 14.4
15.1 15.0 15.8 16.3 13.6 14.3
15.1 14.6 15.6 15.8 14.4 14.3
TO LIGHTON SOLUTIONS OF TABLE11. EFFECTOF EXPOSURE VITAMINA
13.1 14.5
16.0
11.9 13.2 14.7
Material
When most fish liver oils and concentrates are tested by either of these methods the assays are precise and reproducible, especially in commercial laboratories doing the work as a routine procedure. The vitamin A solutions are prepared and tested within a half hour or, a t most, an hour. However, the assays of low-potency fish liver oils, food products, and certain pharmaceutical preparations are not so satisfactory. These assays often involve extractions, saponification, etc., and take from 2 to 6 hours to complete. The potencies determined by these assays are often slightly, and many times surprisingly, low. The poor results are attributed to the instability of vitamin A in dilute solution. The degeneration of the vitamin is often assumed to be due to oxidation, but handling the solutions under inert gas does not improve the results to any great extent. It has been shown (7) that vitamin A is destroyed by ultraviolet light, but it is not generally realized that the annoying instability of dilute solutions of vitamin A is almost entirely due to this effect, I n Table I are given data which show the constancy for several days of the ultraviolet absorption of solutions of vitamin A stored in 50-ml. brown-glass-stoppered bottles. The solvents are of the ordinary c. P. grade, and the source of the vitamin A is a halibut liver oil distillate which
Vitamin A alcohol concentrate A 8-1-40 Vitamin A alcohol concentrate B 9-4-40 Vitimin A alcohol concentrate B 8-28-40 F i s i liver oil C, 8-23-40 Fish liver oil C, 8-23-40 Fish liver oil distillate D , 827-40 Fish liver oil distillate D, 8-
Exposure to Daylight
Original Potency0 Solvent Final Potency
Hour8 5.6 3,260,000 EtOH Cloudy day
72 93
5.0 2,989,000 EtOH Cloudy day
92 100
.. .. .. ..
4.0 Cloudy and rain 5.0 Cloudy day 4.0 Cloudy day 5.0 Cloudyday
2,980,000
91 104
93 105
45
....
4.0 Cloudyday
216,000
CHCla
30,000 EtOH 30,000 CHCls 216,000 EtOH
CHCL
Color of Test Tube
Clear Amber Clear Amber Clear Amber
96 59 101 73 98
63 101
....
Clear Amber Clear Amber Clear Amber
80 101
85 101
Clear Amber
27-40 (328 mp) in alcohol. Taken to be 2000 times Measured with Evelyn photoelectric colorimeter using Rubicon 620 filter. 0
b
Unfortunately, no amber laboratory glassware is regularly made by the apparatus manufacturers. The author believes that every laboratory performing assays for vitamin A on food, medicinal, and physiological preparations needs at least the following types of amber glassware: separatory funnels, conical flasks, volumetric flasks, and boiling flasks for Soxhlet extractors. 144
March 15, 1941
ANALYTICAL EDITION
Tliere are undoubtedly other substances which could well Le handled in amber glassware. The Kimble Glass Company, Vineland, K. J., has available the glass used in the work reported here, and is compiling a list of items which ‘le used in sufficient quantity to permit stocking* The coefficient of expansion is 64 x lo-’ per ’ c.9 and apparatus made of it can be heated with reasonable care. The company will welcome suggestions concerning possible listings.
Acknowledgment T l ~ eauthor thanks George H. IVait for the experimental data given in Table 11 and the Eastman Kodak Re-
145
search Laboratories for the optical measurements made for Table I.
Literature Cited (1) Barthen and Leonard, J . Am. Phalm. Assoc.,26, 515 (1937). (2) Coward, Dyer, and Morton, Biochem. J., 26, 1593 (1933). (3) Dann and Evelyn, Ibid., 32, 1008 (1938). (4) Ewing, D. T., Vandenbelt, J. M., Emmett, A. D., and Bird, 0. D., IND.ENG.CHEM.,Anal. Ed., 12, 639 (1940). (5) Koehn and Sherman, J . Bid. Chem., 132, 527 (1940). (6) Notevarp and Weedon, Biochem. J., 32, 1668 (1938). (7) Smith, Robinson, Stern, and Young, Ibid., 33, 207 (1939). (8) Wilkie, J . Assoc. Oficial Agr. Chem., 23, 336 (1940). COMMCNICATION
20 from the laboratories of Distillation Products, Inc
Determination of Small Amounts of Zinc in Plant Materials A Photometric Dithizone Method HALE COWLING’ AND E . J. MILLER, Michigan Agricultural Experiment Station. East Lansing, Rlich.
D
IPHENYLTHIOCARBAZONE, commonly referred to as “dithizone”, has been used by several investigators (2-4, 8) as a colorimetric reagent for the determination of small amounts of zinc. The reaction between dithizone and zinc in slightly alkaline solution results in the formation of a red complex which is quantitatively extractable with chloroform or carbon tetrachloride. The reaction is extremely sensitive and the color sufficiently stable to form the basis of an excellent colorimetric method. However, more than a dozen other metals also react with dithizone to form extractable colored complexes. This lack of specificity has been the chief difficulty in the use of the reagent. It is possible to eliminate interferences to some extent by careful regulation of the pH a t which the extractions are carried out and by the use of the selective complexing action of certain inorganic anions, but neither of these methods is entirely satisfactory. Holland and Ritchie (5) reported the important observation, for which they give credit to R. H. Caughey, that in 0.02 N ammonium hydroxide solution sodium diethyldithiocarbamate, the copper reagent usually referred to as “carbamate”, inhibits the reaction of all metals with dithizone except zinc. These workers proposed a colorimetric method for the determination of zinc in foods in which carbamate is used to eliminate interferences by other metals which form dithizone complexes. This method, although it represents an important step toward the solution of the problem, did not give highly reproducible results in the authors’ hands when color intensities were measured with a photoelectric colorimeter. It was hoped by an investigation of the action of carbamate in the determination of zinc to develop a method free of interferences and capable of an accuracy comparable with that obtainable in measuring color intensities with modern photoelectric colorimeters.
dithizone complex and the colorless carbamate complex. The result of this effect is a reduction in the color intensity of the dithizone extract, as is shon-n by a comparison of the curves on Figure 1. Curve 1 represents the relationship between the amount of zinc present and the per cent light transmission of the carbon tetrachloride extract obtained when no carbamate is present, and curve 2 represents the same relationship when 12.5 mg. of carbamate are present. A comparison of the two curves shows that the presence of the carbamate appreciably reduces the color intensity of the carbon tetrachloride extract obtained for all amounts of zinc. The effect of varying the amount of carbamate present
Effect of Carbamate on Extraction of Zinc as Dithizonate Zinc can be quantitatively extracted as dithizonate from aqueous solution a t a pH between 8 and 9 with carbon tetrachloride containing excess dithizone. I n the presence of carbamate, however, complete extraction of the zinc as dithizonate does not occur. The zinc is distributed between the red 1
Present address, American Viscose Company, Marcus Hook, Penna.
FIGURE 1. EFFECT OF CARBAMATE ON RELATION BETWEEN COLORINTENSITY OF DITHIZONE EXTRACT AND AMOUNT OF ZINC PRESENT 1. No carbamate resent 2. 12.5 mg. of o a r t a m a t e present
Filter. Sextant green (3.52 mm.). Cell thickness. 1 cm.
