Antimony Trichloride Reaction of Vitamin EDGAR M. SHANTZ, Distillation
Products, Inc., Rochester,
D
N. Y.
IN
RECENT years, many investigators have reported on methods for the physicochemical estimation of vitamin D in fish liver oil. Many of these methods (1-4,7,8) depend upon the measurement of the yellow color developed when antimony trichloride or some modification (6, 6,9)of this reagent is added to the vitamin D-containing fraction which is usually freed from vitamin A, sterols, and other color-producing substances by a chromatographic procedure. Some years ago when these laboratories were actively investigating this problem, a reference calibration curve of the antimony trichloride yellow color was made up using crystalline calciferol (British Drug House) as a standard. It was found that variations in the conditions of concentration, time, light, and temperature all had a marked effect in the development of the yellow color a t 500 mp. Some of these effects have been overlooked by other investigators. These observations are published here in the hope that they may enable others to obtain closer agreement in their results.
MICROGRAMS OF CPLUFEROL PER ML 4
0.4
EXPERIMENTAL PROCEDURE
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One milliliter of a chloroform solution of calciferol was measured into a colorimeter tube, and 10 ml. of a chloroform solution of antimony trichloride (saturated a t 20" C.) were added rapidly from an automatic pipet. The intensity of the orange color was determined in an Evelyn photoelectric colorimeter, using a 500 mp filter. (The filter was made up by E. E. Richardson of the Eastman Kodak Research Laboratories by adding some components to the 500 mp filter supplied by the Rubicon Company to a sharper band.) From the galvanometer reading function , approximately proportional to the optical density, was determined. Using this basic procedure, the conditions of concentration of calciferol, time, light, and temperature were varied.
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EFFECT OF VARYING CONDITIONS
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The intensity of the yellow color of the CONCENTRATION. antimony trichloride-calciferol reaction product was not proportional to the amount of calciferol in the aliquot tested, except a t very low concentration (5 micrograms or less per ml.). With larger amounts the color development was much less than would be expected from the intensity of color a t lower concentrations (Figure 1, upper). These observations were confirmed by measurements made a t 500 mp on a recording spectrophotometer. Thus a calibration curve is recommended. If a conversion factor is used, it must be limited to a small range of optical density. TIME. Calciferol-antimony trichloride colors must be measured after an exact interval of time. At 30" C. the color intensity reached a maximum after 4 minutes, then slowly and steadily faded (Figure 1, center). LIGHT. The maximum color development was reached when the reaction was allowed to take place in the dark. When the color was developed in a shaded corner of the room on a bright day, the results were about 10% low, and when allowed to stand near the window, the results were about 15% low. TEMPERATURE. Changes in temperature had a profound effect on the color development (Figure 1, lower). The intensity increased with temperature to a maximum a t 42' C., beyond which it decreased. Between normal ranges of room temperature (19' to 33" C.) there was a difference of 40% in the color intensity of the same solution.
4
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8
12
16
TEMPERATURE *C-.
Figure 1.
Intensity
OF CalciFerol-Antimony Trichloride Color, f, at 500 ma
Upper. Rotted against concentration of calciferol Center. 0,0014% solution plotted against time Lower. 0.0008% solution plotted against temperature at which color was allowed to develop
closely if reproducible results are t o be obtained. I n this laboratory satisfactory checks were obtained (*2%) on samples of calciferol by the following procedure:
DISCUSSION
The author is not attempting to set up strict conditions under which vitamin D determinations with antimony trichloride should be run. This is left to the individual investigator, but attention is called t o the variables which must be controlled
One cubic centimeter of chloroform solution, calculated to contain about 0.07 to 0.25 mg. of calciferol, was measured into a colorimeter tube which had been allowed to come to constant temperature by inserting in n rack of steel tubes immersed in a
179
I N D U S T R I A L A N D E N G I N E: E R I N G C H E M I S T R Y
180
LITERATURE CITED
controlled temper3ture bath at 30" C. Ten cubic centimeters of antimony trichloride solution (saturated in chloroform at 20" C.) were then added. The reagent had also been previously brought to 30' C. by immersing the container in the constanttemperature bath. The steel tubes were covered to exclude light and the color -a as allowed to develop for exactly 4 minutes. The colorimeter tubes were removed and immediately read on an Evelyn photoelectric colorimeter, usin a 500 mp filter. The amount of vitamin D was calculated from a calibration curve prepared from crystalline calciferol.
Brockmann, H., and Chen, Y., 2.phy8iOl. chem.. 241, 129 (1936). Emmerie, A., and Eekelen, M. van, Acta Brevia Needand. Physiol. Pharnacol. M'icrabiol., 6, 133 (1936). (3) Ewing, D. T., Kingsley, G. V.,Brown, R. A,, and Emmett, A . D., IND. ENG.CHEX.., ANAL.ED.,15, 301 (1943). (4) Milas, N., Heggie, R., and Raynolds, J., Zbid., 13, 227 (1941). (5) Nield, C. H., Russell, W. C., and Zimmerli, A., J . Biol. Chem.. 136, 73 (1940). Raoul, Y., and Meunier, P., C m p t . rend., 209, 546 (1939). Ritsert, K. E., Merck's JahreJber., 52, 27 (1938). Wolff, L., Z . Vitaminforsch., 7, 277 (1938). Zimmerli, A., Nield, C, H.. and Russell, W. C., J . Biol. C h r m . 148, 245-6 (19431.
In the estimation of vitamin D by the antimony trichloride procedure, the conditions of concentration, time, light, arid temperature must be rigidly controlled to obtain reproducible results.
A
Vol. 16, No. 3
Versatile Liquid-Liquid Extractor the extractant, an out,let,should be provided a t the bottom of the flask, 9.
W. D. LONG Horton & Converse,
Los Angeler, Calif.
Figure 2 illustrates the extractors used in conjunction with a boiling flask and condenser for continuous operation. Extraction time is much reduced by this arrangement. Figure 3 is a design of an apparatus utilizing this principle for extracting or percolating solid materials or for conducting adsorptions or catalytic reactions. The cylinder, 1,may be packed. or a thimble containing the material to be extracted may be inserted into the chamber. The unit is operated in the same manner as the liquid-liquid extractor.
MAKY
liquid-liquid extractors have been described. A large number of these are patterned after the device described by Marshall (1) which, although labor-saving, is slow a n d t h e r e f o r e expensive to operate. In addition certain materials are heat-labile to the extent that they are destroyed in the boiling flask. T h e a p p a r a t u s herein described utilizes the well-known gas lift principle to overcome these objections and presents many additional possibilities for extraction procedures, catalytic reactions, adsorptions, etc. It is vompact, simple to build, and very economical to operate. Figure 1 Low vacuum (0.5 inch) or a slight air or gas pressure serves equally well to operate the unit whether it be used as a small laboratory unit or for large-scale extractions. The extractant may be either the heavier or the lighter liquid. The basic apparatus, which may be used as a batch extractor and which has been used to advantage in procedures normally carried out with separatory funnels, is shown in Figure 1. In operation, vacuum a t 1 or pessure at 2 causes the lighter liquid in 3 to drop to 4, where a plug of air enters tube 5 and pushes the liquid in 5 into reservoir 6. Increased height of the liquid at this point causes a downflow through tube 7, where it bubbles out through a fritted-glass bubbler and rises t o the liquid interface, 8, thence to point 4, momentarily sealing opening to tube 5 . Gas entering through tube 3 again forces liquid into reservoir 6. A low vacuum or gas pressure causes a continuous, rapid bubbling action. Then the heavier liquid is to be
LITERATURE CITED
(1) Marshall, F, C.B., Chem. Ne~os.143, 235-6 (1931).
x
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c c
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f Figure 2
Figure 3
