increased care needed in handling a strong acid during the sampling period. I n sampling for formaldehyde in combustion gases a pair of impingers in series containing an aqueous collecting medium may be the sampling procedure of choice. Passing 30 liters of gas containing 0.5 p.p.m. of formaldehyde through chromotropic arid solution results in a n absorbance of approximately 1.0 (Table IV). If an absorbance of 0.2 is considered satisfactory for quantitative analysis, this intensity can be obtained by collecting the formaldehyde from 60 liters of gas containing 0.5 p.p.m. of formaldehyde into an aqueous medium. Below the 0.5 p.1i.m. by volume level of formaldehyde it is necessary t o use a very large gas volume and long sampling periods to obtain sufficient formaldehyde for analj-siq. blternatively, if 3 ml. of aqueous solution is added to 27 ml. of chromotropic acid reagent solution, enough solution volume is available to use a spectrophotometer cell of eithw d- or 10-cm. optical path length. It is in the analysis of combustion gascls for formaldehyde by direct collection irito chromotropic acid that the interference problem is the most critical. This is particularly the situation in automobile exhaust. The total olefin contcnt of automobile exhaust may exceed the formaldehyde concentration by a factor of 8 or 10. However, the resultb of the present investigation indicate that this olefin content need not constitute a serious interference. The aromatic hydrocarbons in automobile exhaust likewise may exceed the formaldehyde content by a factor of 5 or 10. This quantity of aromatic hydrocarbons will constitute a serious interference. Consequently, sampling into aqueous solution is the preferred
procedure. Any olefin or aromatic hydrocarbon collected in this way will still interfere when the chromotropic acid solution is added, but the collection efficiency of water for hydrocarbon is much lower than t h a t of sulfuric acid. Phenols and higher molecular weight alcohols if present in excess of the formaldehyde concentration will interfere somewhat in both aqueousand sulfuric acid-type collecting media. However, the available data indicate that both of these classes of compounds occur in much lower concentrations than formaldehyde in most combustion sources and in the atmosphere. It is in the atmospheric sampling situation that sampling directly into chromotropic acid is most advantageous. Formaldehyde is unlikely to exceed a few tenths of a part per million by volume in the atmosphere. I n smoggy atmospheres the small interference from olefins is reduced further, since the formaldehyde present is formed in part by the consumption of olefins by photooxidation reactions. Fern data are available to estimate the ratio of aromatic hydrocarbon t o formaldehyde in smoggy atmospheres. I n situations similar to the irradiation conditions resulting in the formaldehyde concentrations reported in Table IV, t h a t ratio is unlikely t o exceed 2 to 1, even without further reduction in the aromatic hydrocarbon content by photo-oxidation reactions. The small amount of laboratory data available indicates that xylenes may disappear by photo-okidation reactions about as rapidly as olefins such as 1-butene or I-pentene ( I S ) . The considerations discussed above suggest that only a small interference by aromatic hydrocarbons and olefins nil1 occur in the analysis for formalde-
hyde in smoggy atmospheres by the direct collection in chromotropic acid solution. I n other gas mixtures, and particularly those Y,ith large amounts of aromatic hydrocarbons, collection into aqueous solution is recommended. LITERATURE CITED
(1) Altshuller, A. P., Cohen, I. R., ANAL.
CHEM.32, 801 (1960). (2) Arundale, E., hlikeska, L. X.,Cheva. Revs. 51, 505 (1952). (3) Baker, R . A., Doerr, R. C., Intern. J . A i r Poll. 2, 142 (1959). (4)Beyer, G., J . Assoc. Ofic. d g r . Chemists 34, 745 (1951). (5) BOOS,R. x., -4N.kL. CHEN. 20, 964 (1958). (6) Boyd, J. &I., Logan, 11. A,, J . Biol Chem. 146, 279 (1942). (7) Bricker, C. E., Johnson, H. R.,IND. ENG.CHEM., ANAL.ED. 17, 400 (1945). (8) Bricker, C. E., Roberts, K. H., A S A L . CHEM.21, 1331 (1949). ( 9 1 Bricker, C. E., Vail, -4.H., Ibid., 22, (20 (1050). (10) Eegriwe, 2. anal. Chem. 110, 22 (1937). (11) Ellis, C., Burn, R. W., “Oxygenated Compounds in Automobile Exhaust Gases as Shown by Chemical Analysis,” Bureau of Mines Rept., 1961. (12) Rose, A. H., Brandt, C. S., J . A i r Poll. Control Assoc. 10, 331 (1960). (13) Schuck, E. A., Doyle, G. J., “Photooxidation of Hydrocarbons in Mixtures Containing Oxides of Sitrogen and Sulfur Dioxide,” Rept. No. 29, Air Pollution Foundation, San Marino, Calif., October 1959. (14) Smith, R. G. MacEwen, J. D., Barrow, R. E., A m . Ind. Hyg. Assoc. J . 20, 149 (1959). (15) West, P. W., Sen, B., 2. anal. Chena. 153, 177 (1956). (16) Yocom, J. E., Hein, G. >I., Selson, H. W., J . A i r Poll. Control Assoc. 6 , 84 (1956). RECEIVED for review September 21, 1960. Accepted December 12, 1960. Division of R7ater and Waste Chemistry, 138th hleeting, ACS, Kew York, S. Y., September 1960.
Colorimetric Assay for Diosgenin and Related a Lompounds I
S. C. SLACK and W. J. MADER Research Deparfment, Ciba Pharmaceutical Products Inc., Summit,
b A rapid assay for the diosgenin content of crude diosgenin is based upon the chromogen produced when the sapogenin is treated with perchloric acid. The specificity of the reaction and its application to structurally related compounds are discussed. The assay of crude diosgenin by the colorimetric method and by a lengthy chromatographic procedure are compared.
S
N. J.
have a prominent role in the synthesis of steroidal hormones. I n this respect, diosgenin is used as the starting material in the preparation of testosterone and methyl testosterone. Rhizomes of the plant genus, Dioscorea, yield diosgenin and its stereoisomer, yamogenin, the concentration being dependent upon the species of the plant. BPOGENINS
Crude diosgenin is obtained by extracting the ground rhizomes after acid hydrolysis. The extract is purified and the material obtained is composed of diosgenin, yamogenin, trace amounts of gentrogenin and correlogenin (31, and resinous material of unknown structure. Steroidal sapogenins h a r e been identified by the absorption spectra of their sulfuric acid chromogens (1, 4). VOL. 33, NO. 4, APRIL 1961
0
625
I.'
I. w
0
z 4
m
n
u) 0
W
4
0
)
400 425 450 475 WAVELENGTH IN m u
375
500
e
Figure 1. Absorption spectrum of diosgenin in perchloric acid
sapogenin by column chromatography was necessary prior to assay. Application of the method to a number of samples of crude diosgenin indicated that impurities in crude diosgenin react with sulfuric acid to produce a color. A more promising assay (2) based upon the treatment of sapogenins having A6 unsaturation, with iron(II1) chloride in a mixture of acetic and sulfuric acids, was studied. Absorption maxima were obtained a t 485 m9; however, sapogenins exhibiting no A5 unsaturation produced enough color to render the procedure unsatisfactory. After further study i t was found that when diosgenin is treated with 70 to 72% perchloric acid, a yellow chromogen having an absorption maximum a t 410 mp is produced. The specificity of the reaction and its applications are discussed. EXPERIMENTAL
Sapogenins dissolved in 94% sulfuric acid have characteristic ultraviolet absorption spectra which can be used for the estimation of these substances (4). Evaluation of the procedure indicated that purification of the crude
A sample of diosgenin, purified by multiple recrystallizations from acetone and whose purity was determined by phase solubility analysis, was used as a reference standard. A number of sapogenins were obtained to determine the
~~
Table I.
Color Reactions of Sapogenins with Perchloric Acid L +3
A"
i
I
specificity of the reaction. The structures of the compounds studied and their reactions to perchloric acid are given in Table I.
A solution of the compound to be studied was prepared in chloroform and an aliquot containing 125 fig. was transferred to a stoppered test tube and evaporated to dryness in a mater bath with the aid of a stream of air. Ten milliliters of 70 to 72% perchloric acid were then added and the tube shaken to dissolve the residue. After 10 minutes the visible absorption spectrum of the developed color was obtained on a Cary Model 11 spectrophotometer. A visible absorption curre of diosgenin treated nith perchloric acid is shonn in Figure 1. Concentrations of diosgenin ranging from 25 to 200 fig., treated by the recommended procedure gave linear plots of absorbance us. concentration. To assess the value of the method for the determination of the diosgenin content of crude diosgenin, five samples were assayed for monohydroxysapogenin content by chromatography on Florisil columns. The chromatographic procedure used is a modification of that proposed by Walens, Turner, and Wall (4). The method consists of dissolving the crude diosgenin sample in benzene and placing the resulting solution on a column of Florisil (60- to 100-mesh). Elution of the column with benzene and 5y0chloroform in benzene removed the monohydrosysapogenins. Further elution with chloroform removed the more polar sapogenins including any keto sapogenins and, finally, the resinous materials present were eluted with methanol. Various elution patterns were employed until the monohydroxysapogenin recovery was complete. The most favorable ratio of absorbent to sapogenin was 40 to 1. Duplicate samples mere assayed by treatment with perchloric acid using the recrystallized diosgenin sample as a standard. The absorbance of the sample and standard were measured at 410 mp using perchloric acid as the reference liquid. The per cent diosgenin in the crude samples was calculated by the following expression : A
zs
x
w s -x P
w
= Per cent disogenin
where
at 410 mp -4s = Absorbance of standard at 410 m9 Ws = Weight of reference standard W = Weight of sample P = Per cent purity of diosgenin reference standard
-4
H I OH CHLOROCENIN (YELLOW ON STANDING)
626
HECOGENIX (NONE)
ANALYTICAL CHEMISTRY
KRYPTOG'ENIN ACETATE ( Y E L L O W )
= Absorbance of sample
A standard sample should be run with each series of samples assayed. Ten determinations on a single sample run against the reference standard show
Table II. Comparison of Chromatographic and Colorimetric Assays for Diosgenin
Per Cent Monohydroxysapogenin by
Sample 1 2 3 4 5
Chromatography 87 90 87 82 93
Per Cent Diosgenin by
Colorimetric Assay 88 5 90 8 88 8 84 0 9% 3
the assay to have a standard dcvi t‘ion of 1.1%. The color produced reaches a maximum intensity after 5 minutes and is stable for an additional 30 minutes. A comparison of the results obtained by the two procedures is given in Table 11. DISCUSSION
Treatment of diosgenin. kryptogenin acetate, and yamogenin with perchloric acid yields a yellow color immediately. These sapogenins all have A 5 unsaturation. Chlorogmin which has an H and OH in the 5 and 6 positions, re-
spectively, is presumably dehydrated, giving rise to A 5 unsaturation. The dehydrated compound then reacts to give the yellow chromogen observed upon standing. Kammogenin differs from diosgenin in that there are OH groups in both the 2 and 3 positions and a keto group in the 12 position. The visible absorption spectrum of kammogenin, treated with perchloric acid, s h o w maximurn absorption a t 450 and 565 mp with minimum absorption a t 505 mp. A compound such as gentrogenin was not arailable to resolve the question of the influence of the OH group in the 2 position of a compound such as kammogenin in giving rise to the additional absorption maximum observed with this compound. The colorimetric procedure described has also been used for the determination of dehydroisoandrosterone. Plots of absorbance us. concentration are linear for concentrations of 25 t o 250 pg. The crude diosgenin samples assayed by reaction with perchloric acid yielded results generally higher than those obtained by the chromatographic procedure. The chromatographic assay has a standard deviation of 2%. The observed deviations between the two assays are reasonable as the colorimetric
assay is a measure of all As unsaturated compounds while the chromatographic assay is a measure of the monohydroxysapogenin content of the sample. The time of assay for the colorimetric procedure is approximately 30 minutes compared to 8 hours for the chromatographic assay which makes the perchloric acid procedure valuable for routine use. ACKNOWLEDGMENT
Acknowledgment is made t o Joseph O’Connor who carried out the chromatographic assay for the monohydroxysapogenin content of the samples reported in Table I1 and to Monroe Wall n-ho Pupplied some of t h e sapogenins studied. LITERATURE CITED
(1) Diaz, G., Zaffaroni, A,, Rosenkranz, G.. G., D’ieraSei, Dierasei, C., J.’ J . Org. Chent. 17; 17, 1 747-9 (1952). (2) Moss, N., Boyle, A. J., Zlatkis, A., A N A L . CHEM. 26, 776 (1954). Walens, H. A., Serota. Serota, S., Wall, M. (3) Walens. $55). E., J.Am. Chem. SOC.77,5196-7 (1955). (4) Walens, H. A., Turner. A., Jr., Wall, M. R., ANAL.CHEM.26,325-9 (1954). \
,
RECEIVED for review September 23, 1960. Accepted January 9, 1961.
Microdetermination of Formaldehyde in Air A. C. RAYNER and C. M. JEPHCOTT Air Pollufion Control Branch, Ontario Department o f Health, Toronto, Canada
b A procedure is described for determining the concentration of formaldehyde in air. Formaldehyde is collected b y drawing air a t a rate of 1 c.f.m. through an impinger containing O.OO5N hydrochloric acid. The collecting efficiency of this solution is 72% with a standard deviation of 5%. A magenta color is produced b y adding Schiff’s reagent to the formaldehyde solution; acetone increases the depth and stability of the color. The absorbance is measured in a spectrophotometer a t 560 mp. The method is sensitive to 0.1 pg. of formaldehyde per ml. of collecting solution. With a sampling period of 1 hour, a concentration as low as 5 p.p.b. in air can be determined. Typical results for urban air are presented. Diurnal variations in formaldehyde concentration are found, with peaks occurring during the rnorning and late afternoon.
A
concentrations ranging up to 1 p.p.m. by volume have been found in the air of a few large cities. I n most urban areas which LDEHYDE
have been surveyed, the average level is less than 0.1 p.p.m. The greater part of the aldehydes present in the atmosphere come from the incomplete combustion of fuels, gasoline, and refuse material. I n Los Angeles air, formaldehyde constitutes somewhat less than half of the total aldehydes and ketones present (11). No limits for the emission of formaldehyde to the outdoor air have been found in air pollution control legislation, but in a recent Russian paper, a maximum allowable concentration of 0.035 mg. per cubic meter in ambient air has been suggested (1%). Several methods for determining formaldehyde in air have been developed. Goldman and Yagoda (6) used 1% sodium bisulfite solution as an absorbing agent, and determined the formaldehyde by an iodine titration procedure. Phenylhydrazine hydrochloride solution has been employed as a collecting medium (IO),but owing to discoloration of this reagent, its use is limited t o air samples of not more than 40 liters. Barnes and Speicher ( 3 ) bubbled air through a 1.25% potassium hydroxide solution, and used
Schryver’s method (10) to determine the absorbed formaldehyde. Jacobs, Eastman, and Shepard (9) modified this method and were able to determine a minimum of 10 ,ug. of formaldehyde in 12 ml. of test solution. West and Sen (16) investigated the factors influencing the reaction of chromotropic acid with formaldehyde, and devised a spectrophotometric method for trace amounts. The present paper describes a method which is sufficiently sensitive t o determine low concentrations of formaldehyde in air. The formaldehyde is collected in slightly acidified water and determined by a colorimetric procedure, using acetone (8) and Schiff’s reagent (4, 6, 16). This reagent gives a favorable ratio of volume of sample taken for analysis to volume of reagents added. Water has been suggested (1) as a n absorbing medium, and was investigated further, as sodium bisulfite and potassium hydroxide interfere with color development. Formaldehyde was found to be more stable in weak hydrochloric acid than in water. The efficiency of the weak acid solution to collect formaldehyde was determined VOL. 33, NO. 4, APRIL 1961
627
