V O L U M E 2 4 , N O . 6, J U N E 1 9 5 2
1049
removed more irrelevant absorption than has saponification. This ratio cannot be interpreted in a etrictly absolute sense because i t is based on absorption ratios and these indicate only the relative shape of the absorption curve. Table V. Relative Efficiency of Chromatography and Saponification i n Removing Irrelevant Absorption Irrelevant Absorption, % Removed Removed b y saponifib y chromaSample I n whole oil cation tography NO. fraction 1 62.4 46.9 49.8 2 35.2 33.2 46.6 3 37.3 7.2 54.4 4 36.8 12.2 46.2 5 24.1 1.2 10.4 35.7 6 28.0 28.6 7 65.4 32.3 43.3 a Ratio expressed by ‘32 removed by chromatography. Yo removed h y saponification
,Chrom. Sap. ratio 1.06 1.40 7.55 3.79 8.66 1.25 1.34
some insight into various aspects of this problem. The work deRcribed herein indicates that a chromatographic technique may be employed to advantage as an efficient means for the removal of irrelevant absorption. Moreover. the use of different adsorbents and solvents might make it possible to obtain a greater degree of fractionation of the irrelevant absorbing material than by saponi-. fication. The chromatographic technique which has been described is simpler and more rapid than the usual saponification procedures m d the extracts obtained on chromatography cont,ain less irrelevant abjorption than do the nonsaponifiable extracts of the same oil. Because fewer manipulations are required in the chromatographic procedure than in saponification and the former method is the more rapid, it should be possible to improve the precision of vitamin A determinations both by decreasing manipulative errors and by affording an increased opportunity for replication. LITERATURE CITED
DISCUSSION
In view of the magnitude of the correction obtained in thP application of the Llorton-Stubbs procedure to the nonsaponifiable fraction of lowpotency oils, it appeared advantageous to study the effect of chromatography on such samples. The “difference curve’” v hich is ohtained by subtracting absorption values for the nonsaponifiablc fraction from the corresponding values for the whole oil does not necessarily give any indication of the nature of the irrel~vantniaterial remaining in the sample. Chromatography is onc technique that might be expected to givp
(1) Chilcote, XI. E., Guerrant, S . B.. and Ellenberger, H. A . , AXAI.. CHEM.,20,1180-8 (1949). (2) Eden, E., Biochem. J . , 46, 259-61 (1950). (3) Glover, J., Goodwin, T. IT., and Morton, R. A., Ibid., 41, 94-6 (1947).
(4) Gridgeman. Tu’. T.. Gibson, G. P., and Savage, J. P., Annlysl. 73, 662-8 (1948). 15) Hjarde, W., Acta Cheni. Stand., 4, 628-40 (1950). (6) Reed, G., Wise, E. C., and Frundt, R. J. L., ISD.ESG. CHEM., .&SAL. ED., 16, 509-10 (1944). RECEIVEDfor review M a y 16, 1931. Accepted November 5 , 1951. Contribution 201,Dirision of Chemistry. Science Service, Department of Agriculture, Ottawa. Ont., Canada.
Absorption Characteristics of Cortisone Acetate and Other Ketosteroids in Alkali JOHN M. CROSS, HENRY EISEN,’ AND RICHARD G . KEDEKSHA Rutgers University College of Pharmacy, Newark 4 , 1’. J .
HE Zimmerman reaction, as modified by Callow, Calloa-, Emmens ( I ) , has been used extensively in the analysis of ketosteroids. A modification of this procedure, which consists essentially of treating a 3-, 17-, or 20-ketosteroid n-ith 3,5-dinit8robenzoic acid and an organic alkali such as benzyltrimethylammonium hydroxide, has been reported (4). This modified procedure was invest,ignted for possible use in the analysis of cortisone acetate. I t has been reported ( 2 , 3 ) that steroids containing a A‘-unsaturated %keto configuration generally exhibit an absorption maximum in the area of 380 mp when the Zimmernian reartion is employed. Because cortisone acetate conforms to this structure, it was expected that a similar absorpt,ion maximum csould be obtained. KOsatisfactory maximum, however, was obtained when cortisone acetate was subjected to the modified Zimmerman reaction (41, In the course of these experiments it was noted that when cortisone acetate was heated with either inorganic or organic alkali, an absorption maximum wns observed a t 373 mp. PROCEDURE
One milliliter of a solution of cortisone acetate in methanol (50 micrograms per ml.) is placed in a small flask and t’he solvent is evaporat,ed on a water bath. To the dried sample 5 ml. of a 10% solution of tetraet,hylammonium hydroxide are added and the flask is sto pered and agitated gently. The stopper is then removed ancf the flask is heated on a water bath at 70” for 35 minutes. A blank is prepared by placing 5 ml. of 10% tetraethylammonium hydroxide in a similar flask and heating in a similar manner. The sample and blank are then cooled to room ~~
1 Present address, University of Connecticut College of Pharmacy, Storrs. (‘onn.
Table I.
EKect of .Alkali on Ketosteroids
Position of Absorption Compound Keto Group Ring A Maximum, -\IF Cortisone acetate 3 , 1 1 , 20 A*-Unsaturated 373 Estrone 17 Aromatic a Progesterone 3 , 20 A‘.Unsaturated 375 Testosterone 3 A&-Unsaturated 375 Dehydrocholicacid 3 , 7 , 12 Saturated Theelol Aromatic u-Estradiol Aromatic Ethinyl estradiol Aromatic a Although some absorption was noted, no significant maximum was observed in range of 350 t o 400 mp.
temperature. The absorption spectrum is then determined with a Beckman DU spectrophotometer using Cores cell8 of 1-em. light path. RESULTS AND DISCUSSION
Khen treated with alkali in this manner, cortisone acetate exhibits an absorption maximum a t 373 mp. Full development of this maximum occurs in 30 minutes. Heating for a longer period does not change the maximum reading. Readings taken over a period of 12 hours indicate that the absorption value remains constant for this time. Using this method, a concentration-optical density curve at 373 mp showed adherence to Beer’s law over a range of 10 to 80 micrograms of cortisone acetate. Benzyltrimethylammoniuni hydroxide and tetramethylammonium hydroxide gave similar results. When sodium hydroxide in concentrations of 1.0 to 6.0 was used, a similar absorption maximum mas obtained. However, the results were not so readily reproducible with sodium hydroxide as TTith the organic alkalies.
ANALYTICAL CHEMISTRY
1050 Results obtained when a number of steroids were heated separately with 10% tetraethylammonium hydroxide are shown in Table I. These indicate that ~ ~ ~3-ketosteroids ~ ~when ~ heated with alkali show a characteristic absorption maximum in the area 373 to 375 mw. ACKNOWLEDGMENT
The authors wish to thank Merck & Co., Inc., for generosity in supplying the cortisone acetate used in this work.
LITERATURE CITED
(1) Callow, N. H., Callow, R. K., and Emmens, C. W., Biochem. J., 32, 1312 (1938). (2) tFiefler,~L. F.,~and Fieser, M.3 Products Related to ~ t “Natural ~ d
Phenanthrene,” 3rd ed., p. 101, New York, Reinhold Publishing Corp., 1949. (3) Langstroth, G. O., and Talbot, S . B., J . Bid. C h a , 128, 759 (1939). (4) Tansey, R. P., and Cross, J. M., J . Am. P h a r m . Assoc., 39, 660 (1950). RECEIYED for review October 15, 1951. Accepted February 7, 1952.
Determination of Vanadium in Fuel-Oil Ash G. L. HOPPS AND A. A. BERK U . S . Bureau of Mines, College Park, M d .
TILIZATION of residual fuel oils in power boilers has, in some instances, resulted in a special type of corrosion. The trouble appears most likely to occur where combustion conditions and design cause oil ash from high-vanadium oils to pass over metal surfaces a t comparatively high temperatures (6). The ash content of fuel oil is generally much less than 0.1% ( S ) , and only a small proportion of the ash would be deposited on a probe placed in the hot-gas stream; a laboratory study of this problem therefore would require a method for determining vanadium in very small samples. Previously described analytical methods include one by Karchmer ( 2 ) for determining vanadium in petroleum ash by fusing the ash with carbonate to separate the vanadium from the interfering metals and subsequently determining vanadium colorimetrically by the color formed with hydrogen peroxide. Wrightson (8) employed a bisulfate fusion and determined traces of vanadium in petroleum ash by the color formed with diphenylbenzidine reagent. Sandell ( 4 ) has determined vanadium in silicate rocks colorimetrically by using phosphotungstic acid, after fusion and separation of the vanadium with 8-quinolinol. An extenuive spectrophotometric study was made of this phosphotungstvte method by Wright and Mellon ( 7 ) , and it was adapted to the determination of vanadium in alloy steels. .ifter attempts to adapt several methods for a colorimetric procedure in this laboratory, the phosphotungstate method was chosen as offering the best possibilities, because it was sensitive enough to be used on very small samples and was subject to a minimum of interference from other constituents of fuel-oil ash. The procedure described herein is relatively rapid, a single determination requiring approximately 20 minutes.
fore attempted by the usual fusion procedure in which the ash is fused with a mixture of sodium carbonate and potassium nitrate. Vanadium recovery was usually low by this method, and results were inconsistent, possibly because of volatilization of the vanadium salts a t the high fusion temperatures used (800’ to 900” ‘2.). When sodium peroxide was substituted aa the fusion medium, results were much better, as fusion temperature could then be dropped to 500’ C.; however, further study showed that iron caused no interference and a tedious fusion separation waa therefore unnecessary. The acid-extraction procedure described herein has been shown epectrographically to bring the vanadium content of the ash into solution. REAGENTS AND APPARATUS
Sulfuric acid, concentrated, C.P. Nitric acid, concentrated, C.P. Phosphoric acid, 85% C.P. Sodium Tungstate Solution, 0.5 M . Dissolve 16.5 grams of Folin’s sodium tunestat.e dihydrate in distilled water t o make 100 mI. of solution. Standard Vanadium Solution. Dissolve 0.535 gram of purified vanadium pentoxide in 50 ml. of 5% sodium hydroxide. Acidify
-
SAMPLING
iish deposits used in this investigation were obtained as s c r a p ings from the gas side of boiler tubes, superheaters, air preheaters, and stacks in installations firing No. 6 fuel oil. All deposits were ground to -50 mesh. Spectroscopic examination of the ash samples showed that major constituents were iron, sodium, nickel, and vanadium, with lesser amounts of magnesium, cop er, chromium, silica, calcium, and other elements normally founx in trace concentrations in heavy fuel oil. The most abundant molecular combination was the sulfate, especially in the lowertemperature deposits. The cold-water solubility of the vanadiumcontaining components of these ash samples ranged from zero in screen-tube deposits t o 100% in air-preheater scrapings. The most recent card files contained no lines that would aid in identifying such x-ray diffraction patterns as were obtained for these vanadium compounds.
IO
EXPERIMENTAL
It w a first thought that the heavy iron concentrations in the ash samples would cause interference with the vanadium determination, and separation of the vanadium from the iron was there-
MQ. VANADIUM per 25 ml. Solution
Figure 1.
Standardization Curves
