Analyses of Catalysts Containing Metal Fluorides. Samples of silicaalumina catalyst impregnated and/or mixed with different metal fluorides have been analyzed b y t h e steaming method for different steaming times a t 760" C. (Table 111). These d a t a show t h a t both sodium and aluminum fluorides release fluorine rapidly and quantitatively. However, calcium fluoride releases its fluorine very slon ly, for one half being evolved in 2 hours. Analysis of Other Inorganic MaTKO terials Containing Fluoride. minerals, topaz and cryolite, containing fluorine, and a sample of opal glass were analyzed b y both t h e chemical a n d the steaming methods. T h e d a t a in Table I V reveal t h a t the fluorine is not so readily released from these materials as from aluminum and sodium fluorides. T h e fluorine retention is similar t o t h a t of calcium fluoride. Fluorides in general are divided into two classes: rapidly hydrolyzable and slowly hydrolyzable. However, U308 acts as an accelerator and slowly hydrolyzable fluorides may be analyzed more rapidly by use of this compound (10). Recently Silverman and Bowen
(8) were able to pyrohydrolyze cryolite
at 1200" C. using an all-nickel reactor. A 3 to 1 ratio of alumina to cryolite was necessary for complete fluoride recovery. The steaming method is a rapid and valuable tool for the analysis of some materials, but the longer chemical method should be used for unknown or unusual samples. ACCURACY A N D PRECISION
I n Table V are given data from a program in which four laboratories tested both chemical and steaming methods on a synthetic sample and on an unknown sample. The t-wo methods are equally accurate but the steaming method is more precise. ACKNOWLEDGMENT
The authors gratefully acknowledge the assistance of E. J. Kewchurch, J. S. McIlhenny, and Dorothy Webb, and thank the following organizations, for participation in the accuracy and precision tests of Table V: Technical and Research Division, Humble Oil 8: Refining Co., and Products Research and
Process Research Divisions, Esso Research and Engineering Co. LITERATURE CITED
(1) Chu, C.-C., Shafer, J. L., -1s.4~.CHEU. 27, 1429-31 (1955). ( 2 ) Cline, IT. D., Tevebaugh, R; D., Warf, J. C., Method Abstract, Analytical Chemistry of the Manhattan Project," ed. by C. J. Rodden, National Kuclear Energy Series, Vol. VIII-I, p. 239, McGraw-Hill, Ken. York, 1950. (3) Gahler, 8.R., Porter, Galen, ASAL. CHEX 29,296-8 (195i). ( 4 ) Haff, L. V., Butler, C. P., Bisso, J. I).,
Ibid., 30,984 (1958). (5) Hoffman, J. I., Lundell, G. E. F., Bur. Standards J . Research 3, 581 (1929). (6) Lee, J. E., Edgerton, J. H., Kelley, 11,T., A S A L . CHEJI. 28, 1441 (1986). ( 7 ) Powell, R. H., Menis, O., Ibid.,30, 1546 (1958). (8) Silverman, H. P., B o ~ e n F. , J., Ibid., 31, 1960 (1959). (9) Susano, C. D., Khite, J. C., Lee, J. E., Jr., Ibid.,27, 453 (1955). (10) Warf, J. C., Cline, TI-. D., Tevebaugh, R. D., Ibid., 26,312 (1954). ( l l ) W i l l a r d , H. H., Kinter, 0. B., IND.EXG. CHEX., ASAL. ED. 5 , 7 (1933). RECEIVEDfor review May 27, 1959. Acceoted Kovember 2. 1959. Division of Ana&tical Chemistri, 131st AIeeting, .1CP, Miami, Fla., April 1957,
Analytical Assay of Diosgenin STEPHEN KAUFMANN, J. C. MEDINA, and CLAUD10 ZAPATA Syntex, S. A., Mexico City, Mexico
b
Fractional sublimation is applied to the assay of crude diosgenin. The results obtained by this technique are evaluated by comparison with those obtained using two other well-known analytical methods: column chromatography and countercurrent distribution. A good agreement was obtained between results, indicating a similar accuracy for the three methods. An estimate of the reproducibility of the sublimation method gives statistical control limits within f0.34%. Because of its accuracy, precision, and simplicity, the fractional sublimation technique seems promising for use in the ndustrial quality control of diosgenin.
D
is the starting material for many steroid hormones. The yields and quality of the steroids derived from diosgenin depend to a considerable extent on the purity of the raw material. Thus, analytical procedures for assaying technical grade diosgenin are important in the production control of the steroid industry based on this sapogenin. 192
IOSGESIN
ANALYTICAL CHEMISTRY
A seemingly simple spectrophotometric method (6) for the identification and estimation of steroidal sapogenins is based on the absorption curves of the sulfuric acid chromogens originally reported by Diaz, Zaffaroni, Rosenkranz, and Djerassi ( 1 ) . This method was unsatisfactory because the impurities in technical grade diosgenin react with sulfuric acid to produce a color which interferes in the determination. Also this procedure (6) is time-consuning and difficult to apply to crude materials. Sublimation has long been recognized as a valuable technique for the isolation of pure organic compounds. I n some instances it has definite advantages over other purification methods ( 4 ) . The excellent general reviery of the topic by Tipsoii ( 5 ) provides some striking examples of its application. V h e n applied to sapogenins, sublimation of glycosides easily permits the isolation of the aglycon, as the sublimate (9). This technique for assaying technical grade diosgenin has been explored in this laboratory and compared n-ith the chromatographic and countercurrent distribution methods. The results are described below.
EXPERIMENTAL
Chromatography. Columns filled with activated alumina oxide are used for adsorption; diosgenin is eluted n i t h several portions of benzene, benzene-ether (8:2, 6:4, 2 : 8 ) , and ether and collected in fractions, nhich are evaporated to dryness. The fractions containing only diosgenin (melting point over 200" C.) are dissolved in chloroform, mixed together, evaporated t o dryness, washed with hexane, and dried to constant weight. This weight is considered as pure diosgenin for calculations. Countercurrent Distribution. A continuous extraction Craig apparatus (manufactured by E d m u n d Buhler, Tubingen, catalog Xo. 30-01-01) consisting of 35 glass tubes is used. The solvent system is composed of 99% heptane and methanol (1 to l ) , heptane being the upper moving phase. The sample is submitted to 45 transfers. Under these conditions, diosgenin has a partition coefficient of 0.667, from which a theoretical distribution curve is calculated. .A comparison of this curve n i t h the experimental curves obtained for each determination shows that all the diosgenin, almost pure, is contained in tubes 10 to 27, whereas the impurities are retained in the other tubes. Thus,
Table I. Comparative Results of Diosgenin ChromatograPhY, Sublimation, and Countercurrent Distribu-
ChromaSample tography 1 90 2 2 91 0 3 90 7 4 90 3 5 90 6
diosgenin
the purity of the sample can be expressed by the relation of the sum of the n-eights of material in tubes 10 t o 27 t o the total sum of iveights in the experimental curve. Sublimation. -4 sample of t h e crude diosgenin is subjected t o selective sublimation under high vacuum. T h e diosgenin a n d t h e more volatile impurities are sublimed a t a given temperature under high vacuum, leaving a nonsublimable d a r k residue. Resublimation at a temperature lower by about 20’ C. removes certain impurities from the original sublimate and leaves essentially purl= diosgenin in the latter. Procedure. Approximately 500 nig. of crude diosgenin are weighed evactly a n d introduced into a 10-ml. volumetric flask. T h e solution is diluted t o volume with pure chloroform. An aliquot of 1 nil. of t h e solution is carefully introduced a t t h e bottom of a glass tube 1 em. in diameter and 25 em. long, which is closed a t one end. T h e chloroform is evaporated b y passing a slow stream of nitrogen or air over t h e sample. After the solvent has completely evaporatrd, the open end of the sublimation tube is connected with the source of high vacuum (0.1 mm.), and the closed end containing the sample is immersed to approximately 4 em. in a silicon oil bath heated t o 180’ C. Diosgenin and the volatile impurity start t o sublime and deposit just above the contact level of the bath. After 30 minutes, no more material sublimes, and a dark residue is left a t the bottom of the tube. The temperature of the bath is decreased to 160’ C. and the tube is lowered further into the bath so t h a t the sublimed material stays about 3 em. below the level of the bath. The volatile impurity sublimes at this temperature and at 0.1 mm. of vacuum in 10 minutes, leaving the diosgenin behind. The sample is fractionated into three zones: -4, B, and C (Figure 1). A is the nonsublimed residue B, the diosgenin, and C, the volatile impurites. The three zones can be separated b y cutting the tube carefully into three segments
limation 87 5 91 9 90 7 92 5 89 2
Table II.
Sample -4 B C
Sub-
current Distribution 89 7 89 5 88 9 88 9 89 9
are recorded and plotted, after ordinary statistical preparation, in the form of control -charts for average (2)aiid range iR). The data beloT$ cor7
from the value obtained for the average range (I?), using the correqpondiig factors (31, UCLx
-? =
=
R
87 5 9 5
LCLx
98 7
=
87.2
CCLR = 1 049 =
0 46“; LCLR = 0
Test of Efficiency of Three Analytical Procedures
Chromatography 97.7 94.5 92.1
Diosgenin Found. % Countercurrent Sublimation distribution 98.6 99.9 94.9 95.7 93.0 93.4
containing the three different materials. Each segment is neighed exactly and subsequently washed free of organic material with pure chloroform. After drying, the segments arc Iveighed again and the weight of the three fractions is determined b y difference. The percentage of fraction B in relation to the weight of the aliquot of the original sample determines the per c m t of diosgenin in the sample. RESULTS
The results of the three different assay methods are compared in Table I. Five different samples of technical grade diosgenin were analyzed by chromatography, sublimation, and countercurrent extraction. Each determination was carried out three times and the average result reported. T o check the efficiency of the three different analytical procedures described above, three samples (A, B, and C) of diosgenin of known purity lvere submitted to these procedures. Sample A n-as prepared by repeated crystallization from acetone t o a constant melting point and subsequent chromatographic purification. This bonafide sample had a melting point of 208-8.5” C. [ a ] , = -126.5’ (chloroform) and was considered 1 0 0 ~ opure for calculation purposes. Sample B was a mixture of 50y0 of Sample A and 50% of sample 5 in Table I (the latter was considered 90% pure diosgenin for calculation purposes). Sample C contained 25% of sample A and 75% of Sample 5 in Table I. The results obtained in these assays are reported in Table 11. T o calibrate the sublimation technique during its use as a routine control method, results of consecutive determinations on a particular sample
Diosgenin calculated, % 100 95 92.5
From the above data, a fairly good reproducibility may be inferred. Subsequently modification of limits is to be expected as more subgroups are ohtained. DISCUSSION
The three analytical procedures dem i b e d for assaying technical grade dioqgenin can be carried out with similar accuracy and reproducibility. The chromatographic method gives very exact results, hut it is time-consuming and needs an experienced operator. Countfrcurrent distribution is a v-orkable analytical tool for assaying diosgenin and gives a very reliable picture of the compoqition of the sample, especially \\-ith regard to accompanying impurities. Unfortunately, it requires special equipment and each determination is time-consuming; thus, its use is limited to research problems of sapogenins. Sublimation is the quickest and simplest and requires less apparatus than the other two procedures. It can be recommended for routine industrial assay of diosgenin. LITERATURE CITED
(1) Diaz, G., Zaffaroni, A, Rosenkranz, G., Djerassi, C., J. Org. Chem. 17, 747 (1952). ( 2 ) Fischer, R., Arch. Pharm. 275, 516 (1937). (3) Grant, E. L., “Statistical Quality Control,” 2nd ed., pp. 137, 302, McGraw-Hill, Sew York, 1952. (4) Hubacher, M. H., IND.ESG. CHEM., -4v.4~.ED. 11, 50 (1939). (5) Tipson, R. S., “Sublimation,” Chay; VII, “Technique of Organic Chemistry, Vol. IV, A. Weissberner, ed.,. Interscience, New York, 1951. (6) Walens, H. A., Turner, -4., Jr., Wall, 11.E., ANAL.CHEW26,325 (1954). RECEIVEDfor review February 25, 1959. Accepted November 18, 1959. ’
VOL. 32,
NO. 2, FEBRUARY 1960
193
