V O L U M E 27, NO. 3, M A R C H 1 9 5 5
443
Table 111. Absorbance of Color Produced by Various Samples of Digitonin (Anthrone Method) Absorbance, 0.2 RIg. Digitonin 0.193 0.198 0,199 0,200 0,199
Sample
Table IT. Sample 3 5 6
7
Standard Deviation 0,010 0.011 0,005 0,019
0.010
Precipitation of Cholesterol by Digitonin (Liebermann-Burchard Reaction) Cholesterol Recovery, Mg. 201 199 200 202
Theory, l l g . 200 200 200 200
Three recrystallizations were made of poorly crystalline sample 3 (Table 11). Rapid recrystallization from 95% ethyl alcohol produced no change in the pattern obtained. mhen the same alcohol solution was allowed to crystallize more slowly, the pattern observed began t o take on the appearance of type C. The third recrystallization, from an ethyl alcohol-benzene mixture controlled so as to proceed a t a slow rate, produced a type C diffraction pattern sharper than the previous ones, but not nearly so distinct as that of samples 1 and 8. Although no chemical determinations of the structures of the samples before and after recrystallization were made, an examination of the infrared curves in Figure 2 indicates no chemical change occurred during crystallization. Infrared absorption spectra of five of the eight samples are presented in Figure 2. Below 9.2 and above 9.7 microns the spectra are virtually identical. Differences between 9.2 and 9.7 microns appear to be due to the relative intensity of absorption a t about 9.3 and 9.6. Sample 8, a t the
top of Figure 2, s h o w the sharpest “peak” a t 9.32. This was the sample which also gave the most clearly defined x-ray diffraction pattern of type C. Sample 7, which gave a crystalline type B pattern, sample 6 from the same supplier but noncrystalline in type, and the noncrystalline sample 3 from another manufacturer, differed very little in absorption characteristics. Absorptions a t 9.3 and 9.6 were more nearly equal in these samples than in sample 8. When sample 3 was recrystallized, the infrared absorption shown a t the bottom of Figure 2, became similar to that of sample 8. That the peak a t 9.32 microns became sharper m s actually due to diminished absorption a t 9.6. Sample 3, during recrystallization, approached sample 8 with respect both to infrared absorption characteristics and type of x-ray diffraction pattern. Data on the intensity of color production with anthrone are presented in Table 111. Samples 3 and 6 were of the noncrystalline type C. Absorbance a t 750 mp was essentially the same for all samples. Recovery by precipitation of cholesterol as the digitonide was equally good when digitonin samples of various types were used, as indicated by the data in Table IV. ACKNOWLEDGMENT
The samples of digitonin used in this study were kindly supplied by Fisher Scientific Co., Hoffmann-La Roche, Inc., s. B. Penick &: Co., and Merck & Co., Inc. LITERATURE CITED
(1) Beher, W.T., and ilnthony, W. L., J . A 7 ? ~ t ~ i t t o52, n , 519 (1954). ( 2 ) Beu, K. E., Reu. Scz. Instr., 22, 62 (1951). (3) Beu, K. E., and Claauben, H. H., Ibid., 19, 179 (1945). (4) Schoenheimer, R., and Sperry, W. M., J . B i d . Chem., 106, 746 (1934).
Sobel, A. E., and Mayer, A. AI., Ibid., 157, 255 (1945). (6) Sperry, TI’. M.,and Webb, 11 , Ibid., 187, 97 (1950). (5)
RECEIVEDfor reiTiew June 25, 1964.
Accepted October 11, 1954.
Phenol-lndo-2,6-Dichlorophenol as a Spray Reagent J. BARNABAS, Ahmednagar College, Ahmednagar, G. V. JOSHI, Wilson College, Bombay-7, lndia Indophenol dye may be used as a spray reagent, in the paper chromatography of organic acids. This dye was better than the usual indicator sprays because of its ease of preparation and its usefulness in differentiating certain acids. Separation of maleic and malonic acids is also reported.
S
EVERAL spraying materials have been introduced by Buch and coworkers ( 2 ) for the identification of organic acids on paper chromatograms. Similarly, mercurochrome has also been used to locate the positions of organic acids on filter paper disks (1). Indophenol dye may also be used as a spray reagent in the paper chromatography of organic acids. The organic acids were separated on paper strips, as well as on circular disks, using a refluxed mixture of 1-butanol, formic acid, and 11-ater-a solvent that has been used by Wiggins and Williams (3)for the separation of amino acids and sugars. Khen a chromatogram was treated with an alcoholic solution of the indophenol dye, organic acids usually developed dark pink spots immediately after spraying against a blue background, thus making the distinction between the spots and the background well pronounced; certain acids bleached the dye, thereby aiding in differentiating these acids from the rest. The spray reagent was
lndia
prepared in ethyl alcohol, and it did not require the adjustment of p H as is usually necessary for indicator sprays. MATERIALS AVD REAGENTS
Paper. Whatman filter paper S o . 1 strips (32 X 28 em.) and circular disks (36 cm. in diameter). Solvent. A solvent mixture of 1-butanol, formic acid, and n ater was prepared by refluxing for an hour a mixture of 10 ml. of 85% formic acid, 120 ml. of 1-butanol, and 10 ml. of water. A further 60 ml. of &-aterwere added in small quantities, the mixture being shaken from time to time during cooling. After having been allowed to stand for 24 hours, the upper layer was used. Spray reagent. Phenol-indo-2,6-dichlorophenol (0.1 gram) in neutral ethyl alcohol (100 ml.). METHOD AND RESULTS
rln ascending strip chromatogram (32 X 28 cm.) containing 25 y of test acid per spot, was developed in a cylinder (height 36 em., radius 6.5 cm.) in the usual manner. After development, the chromatogram was dried in air for about 3 hours, until it waa free from formic acid fumee, and it was then sprayed with the dye solution. The organic acids developed dark pink spota against a blue background, except for ascorbic acid and gallic
444
ANALYTICAL CHEMISTRY matograms, as well as by applying the color tests proposed by Buch and coworkers (d). A circular paper chromatogram (36 om. in diameter) containing 50 y of test acid per spot was prepared in the manner described [Airan and coworkers ( f ) ] . After being air-dried, the chromatogram was treated with the spray reagent, and was photographed (Figure . ._ 1). Maleic and malonic acids. which have an identical R, value in a pentanol-formic acid system, were separated. CONCLUSION
Indophenol dye is a better spray reagent than the usual indicator sprays because of its ease of preparation and use and its usefulness in differentiating certain organic acids. ACKNOWLEDGMENT
The authors' thanks are due t o F. R. Bharucha, Institute of Science, far his keen interest in this work
c.
citric F. Fumaric GLu. Gluconic MLn. Malonic
MLe. Maleic M. Malic
s.
sucoinio
T. Tartaric
acid, which bleached the dye immedirttely. With time, lactic acid, maleic acid, and malonic acid showed a tendency to bleach,
ANAL.C n m ~ .25,659 . (1953). (2) Buch, M. L..Montgomery, R.. and Porter. W. L.,Ibid., 24,489
,.~~"~
11WOZ).
(3) Wiggins, L. F..and Williams. J. A,, Nature, 170, 279 (1952). RECEIVED for review May 13, 1954.
Accepted October 28, 1954.
.."
,InriAn Inn via Golorimetric uetermination 01 Ch,,,
Inn Frchanan .VI. ~..IIIu116v
JACK L. LAMBERT and STANLEY K. YASUDA Department of Chemistry, Kansas State College, Manhattan, Kan.
Chloride i o n is d e t e r m i n e d colorimetrically over the range 0 to 180 p.p.rn. after exchanging for i o d a t e ion with g r a n u l a r silver iodate in a column. The released i o d a t e i o n reacts with o a d m i u m i o d i d e l i n e a r staroh r e a g e n t to f o r m rhe blue linear staroh-triiodide ion complex, the absorbancy of which at 615 mw is proportional t o the conoentration of chloride ion. No serious interferences w-ere f o u n d a m o n g ions commonly f o u n d in n a t u r a l waters, within the limits of their u s u a l concentrations. Bromide and iodide ions maet in the same manner as chloride i o n but are n o t commonly present.
c...
OLORIMETRIC procedures for the determination of chlo. . ride ion involving ion exchange with a solid phase reagent, such as silver chromate or silver ferrocyanide, have been described (5). Chloride ion exchanges for chromate or ferrocyanide ions, and is determined indirectly by the colorimetric determination of the released ion or the reaction products of the released ion. I n the method described here, chloride ion exchanges for iodate ion with granular silver iodate, and the concentration of chloride ion is determined by the absorbancy of the blue linear starchtriiodide ion complex formed by the reaction of the released iodate ion with cadmium iodide-linear Starch reagent in acid solution. The relatively low melting point of silver iodate permits its preparation in massive form, from which particles of uniform size suitahle for column reactions are obtained by grinding and sieving. Its n6e in a column ensures the attainment of equilibrium with chloride ion in solution.
REAGENTS AND EQUIPMENT USED
Silver iodate, granular, 100- to ZOO-mesh. Cadmium iodidelinear starch reagent (f), 11.00 grams of cadmium iodide and 2.50 grams of tu,icP-recryrtallized linear potato starch fraction per liter of solution. Hydrochloric acid,, LOA'. Standard chloride ion solution, 200 p.p.m., 0.330 gram of sodium chloride ~ e liter r of solution.
