V O L U M E 2 6 , NO. 3, M A R C H 1 9 5 4 reductions. It is interesting that a value of Z’L/C = 12.5 milliamperes per millimole per liter was obtained with a cathode area of 42 square em. I n a regular polarogram of 0.001M cadmium(I1) with m = 2 mg. per second and t = 3.5 seconds, t d would be 7.0 pa The average surface area of the mercury drops is given by
&,“.
= 0.0051m2’3t2/3 = 0.0187 sq. cm.
Therefore, the current per unit electrode area in large scale experiments n a s 0.30 ma. per square cm. while in polarographic runs it would be 0.38 ma. per square cm. This means that the flux of reducible species is about equal in both cases and that the concentration gradients near the surface are similar. Similar experiments were run with nitromethane, e t h j 1 nitrate, and maleic acid in the presence of excess electrolyte. While there nas an indication of a limiting current a t very low concentrations in all cases, Z L was not too well defined and the current increased continuously. Figure 6 shows the nitromethane curves. i~ disappeared a t about l O - * X for nitromethane and a t even lower concentrations for ethyl nitrate and maleic acid. This may be due to irreversibility and low rate of the electrode reaction, accumulation of reduction products near the electrode, and contamination of the mercury surface. The low concentration runs are described here to emphasize the sensitivity and versatility of the instrument. With total currents as low as 10 ma. and correspondingly small changes in amplifier input and grid voltage, the i versus time graphs ‘sere entirely smooth and reproducible.
519 K i t h a line voltage of 220 volts, it is possible to have a large potential drop across the cell and this may become important in work with nonaqueous solutions. With cathode-calomel potentials as high as 8 volts in aqueous solutions containing only a trace of electrolyte, cell potentials of 150 volts a t currents of 3 to 4 amperes were obtainable. These applications will be expanded in future work. LITERATURE CITED
illlen. 11.J., ANAL.CHEM.,21, 178 (1949). Caldmell, C. W., Parker, R. C., and Diehl, H., IND.ENG.CHEM., ANAL. ED.,16, 5% (1944). Greenough, AI. L., Williams, W. E., Jr., and Taylor, J. K., Rev. Sei. Instr., 22,444 (19.51). Hickling, A , , Trans. Faraday SOC.,38,27 (1942). Kaufman. F., Cook, H. J. and Davis, S. >I., J . Am. Chem. SOC., 74,4997 (1952).
Lamphere, R. W., ANAL.CHEM.,23, 253 (1951). Lamphere, R. W. and Rogers, L. B., Ibid.. 22,453 (1950). Lingane, J. J., IND.ENG.CHEM.,ANAL.ED.,17, 332 (1945). Lingane, J. J., and Jones, S. L., A N ~ LCHEX., . 22, 1169 (1950). Miher, 0.W. C., and Whitten, R. K’.,Analyst, 77, 11 (1952). Penther. C. J. and Pompeo, D. J.. IND.ESG. CHEX..ASAL. ED.. 21,178 (1940).
“Vacuum Tube Amplifiers,” edited by G. E. Valley, Jr., and H. Wallman, p. 485, Sew York, AZcGraw-Hill Book Co.. 1948. Wehner, P., and Hindman, J. C., J . Am. Chem. SOC.,72, 3911 (1950). RECEIVED for review U a y 26, 1953.
Accepted January 4, 1954.
Determination of Adrenocortical Steroids in Mixtures ERICH HEFTMANN and DAVID F. JOHNSON National Institute o f Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda 74, Md.
Partition chromatography on paper is limited to relatively small amounts of substances. For conventional methods of identification and assay and for the determination of one compound in the presence of a large excess of others, column chromatography is preferred. A method for the separation of all six active adrenocortical hormones by partition chromatography on silicic acid columns is presented. Water is used as the stationary phase and a mixture of petroleum ether and progressively increasing amounts of dichloromethane is the mobile phase. Eluates are collected in an automatic fraction collector and assayed by ultraviolet spectroscopy. The sulfuric acid test serves as a method for identifying the fractions. Modifications of this method may be applied to biological extracts.
W
H I L E several methods for the separation of adrenocort,ical steroids by partition chromatography on paper are now available, there is still a need for a relatively simple procedure of quantitative analysis of hormone mixtures. The separation of adrenocortical steroids by column chromatography offers the advantage that relatively large amounts of mixtures can be handled, allowing the determination of one steroid in the presence of a large excess of another and making sufficient amounts of individual steroids available for conventional methods of identification and assay. Morris and TVilliams ( 5 ) have described the determination of individual adrenocortical steroids in blood by use of partition chromatography on Hyfio Super-cel columns with the solvent
systems toluene-ethyl alcohol-water and petroleum ethertoluene-ethylene glycol and subsequent polarographic estimation. Two methods of separating adrenocortical hormones by partition chromatography on silica gel columns have been reported. Katzenellenbogen el al. ( 4 ) have separated the acetates of the steroids, using dilute methanol or ethyl alcohol as the stationary phase and various mixtures of dichloromethane and petroleum ether a s the mobile phase. Haines ( 2 ) reported the separation of 10 mg. of each of various adrenocortical hormones on a column of 30 grams of silica gel impregnated with ethylene glycol. A series of solvent mixtures, containing increasing proportions of dichloromethane in cyclohexane, was passed through the column by means of an ingenious device, known as the “mechanical chemist.” I t occurred to the present authors that the same eflect could be produced by progressively increasing the dichloromethane concentration in cyclohexane, using an adaptation of the automatic dispenser of Donaldson et al. ( 1 ) . Using the solvent system of Haines, the two isomers A4-pregnene-llp,21-diol-3,20-dione (cor-
Table 1. Steroid
s S
B
E F
Recovery of Adrenocortical Steroids Amount Taken,
Bmount Recovered,
Y
Y
32 1 59 4 247.5 128 5 600 2 976 8
35 0 57 243 130 533 896
5 5 7 7 3
Recovery, % 109 96 98 102 89 91
8 4 0 8
ANALYTICAL CHEMISTRY
520 ticosterone, B ) and A4-pregnene-1ia,21-diol-3,20-dione ( 8 )could not be separated. Also, contamination of the eluate with ethylene glycol makes it unsuitable for assay by periodate oxidation. Using water as the stationary phase and dichloromethane in progressively increasing concentration in petroleum ether as the mobile phase, success was had in separating the six active hormones of the adrenal cortex. In work with the p a e hormones presented in this paper, the ultraviolet absorption for quantitative estimation and the sulfuric acid test ( 7 ) for identification of the steroids have been used. Other tests map be more useful in R orking with biological extracts. APPARATUS
Chromatographic Apparatus. I'wo cylindrical 1-liter separatory funnels lvith standard-taper joints are connected top to bottom. The stem of the top funnel, holding the dichloromethane, has been constricted to an opening of about 1 mm. in diameter. The bottom funnel, holding the petroleum ether, is joined to a 24/40 T outer joint, which has been attached to the top of a chromatographic tube. The latter is of the commercially available type, consisting of a tube, 22 mm. in outside diameter and 200 mm. long, with an outer joint on the bottom and a matching inner member provided with a fritted glass disk on top. All joints are lubricated with starch-glycerol paste ( 3 ) and the two stopcocks are held in place by stopcock tension clips. Fraction Collector. Automatic fraction collector (Technicon Chromatography Corp., Xew York, Pi. Y.) with drop counter adjustable to 400 drops per tube. Ultraviolet Spectrophotometer. Beckman Model D U spectrophotometer. RE4GENTS
Silicic acid was C.P. Baker analyzed, powder. It was continuously extracted with redistilled trichloromethane for 4 hours, dried a t 90' C. for 1 hour, and kept in a stoppered bottle. Sea mnd (Ilerck) was continuously extracted with ether. Petroleum ether (Skellysolve F) was washed with concentrated sulfuric acid, then with water, 3'47 sodium hydroxide, and again with water, dried over calcium chloride, and distilled. Fraction boiling a t 30" to 60" C. was collected. It was saturated with water by ehaking in a separatory funnel and allowing to stand overnight. Dichloromethane was redistilled, boiling point 40" to 41 ' C., and saturated with water as above. Ethyl alcohol (20y0), 200 ml. of ethyl alcohol (absolute, redistilled) made to 1 liter with distilled water. Sulfuric acid (Merck) was reagent grade. PROCEDURE
Stock solutions of 1 mg. per 100 ml. 20% ethyl alcohol areprepared from the following steroids: A'-pregnen-21-01-3,2O-dione (deoxycorticosterone, Q), A4-pregnen-21-ol-3,11,20-trione(dehydrocorticosterone, A ) , S,B, A4-pregnene-17a,21-diol-3, 11,20trion (cortisone, E ) , and A '-pregnene- 1l p , 17a ,2 l-triol-3,2O-dionc (hydrocortisone, F ) . The absorption maximum of all six solutions is near 248 mp. Calibration of the spectrophotometer with known amounts a t this wave length gives linear absorbance versus concentration plots of identical slope for all six compounds. Appropriate amounts of stock solutions are mixed and evapo-
rated to dryness in vacuo. The residue is quantitatively transferred with ethyl alcohol to 5 disks of filter paper, 18 mm. in diameter, which are held on a needle. .After adding 20 ml. of water dropwise to 30 grams of silicic acid while grinding the mixture in a mortar, a mobile slurry with water-saturated petroleum ether is prepared and poured into the column. Trapped gas bubbles are liberated by slowly working a tamping rod up and down the column and the silicic acid is compressed by periodically applying a pressure of 5 pounds per square inch to the top from a nitrogen tank. The dried filter paper disks are pressed to the top of the column with the tamping rod before all of the silicic acid has been transferred to the column, the remaining silicic acid is then placed on top of the paper, and finally some ether-extracted sand is added. The final column consists of, in descending order, a 10-mm. layer of sand, 20 mm. of silicic acid, the paper disks, and a 190-mm. column of silicic acid. The solvent dispenser is attached to the top of the column and both stopcocks are opened. Dichloromethane from the top funnel enters the bottom funnel and, being of greater density, immediately mixes with the petroleum ether. Some of the water dissolved in dichloromethane comes out of solution, but stays on the walls of the bottom funnel. As the mixture leaves the bottom funnel, an equal volume of dichloromethane automatically enters from the top funnel. Two hundred eluate fractions are collected a t a rate of 400 drops (approximately 6.5 ml.) per tube Der 4.5 minutes. Eauilibration of the solvents and chromatographic development' are carried out a t constant temoerature (27" (3.). ' The fractions 'are evaporated to dryness under a stream of nitrogen while the tubes are immersed in a water bath a t 60" C. Five milliliters of 20% alcohol are added to each tube and all tubes are stoppered and allowed to stand overnight to ensure complete solution of the residue. Then the absorbance a t 248 mM is determined. The contents of all tubes belonging to the same chromatographic band are pooled and evaporated to dryness. The residues are used for identification of the steroids by the sulfuric acid method. RESULTS
A representative chromatogram is shown in Figure 1 where tube numbers are plotted against absorbance a t 248 mp. The absorption peaks occurring in tubes 31, 48, 54, 63, 106, and 138 are due to Q, A , S, B, E, and F , respectively, as shown by the sulfuric acid test. There is a slight overlapping of the curves for A , S, and B. The amounts of steroids taken for analysis and recovered are given in Table I. The calculations are corrected for an average absorbance of 0.013 for the background and bascd on the absorhance of fractions having an absorption maximum near 248 mp: tuhes 30 to 33 Q, 43 to 50 A , 51 to 59 S, 60 to 72 B, 93 to 122 E, and 126 to 192 F. .Approaimatel\; 30 y of Q is quantitatively recovered in the presence of a 30-fold excess of F . In other experiments recoveries have been as good or better. With reasonable care the position of the peaks is reproducible. Constant temperature during equilibration of solvents ana chromatography is essential, but similar results have been obtained a t other temperatures between 21' and 27' C. Changes in the amounts chromatographed have no influence on the position of the pcaks, but a broadening of the chromatographic bands occurs as the concentration is increased. A , S, and B are completely separated
w .4000
a 0 cn
-
.200-
m
-
a .loo1
I
80
100
120
140
160
TUBE NUMBER Figure 1. Partition Chromatogram of a Mixture of Adrenocortical Steroids
180
200
V O L U M E 2 6 , NO. 3, M A R C H 1 9 5 4
521
when 100 y or less of each compound is used. When more than 100 y of these three compounds is present, separation may be achieved using 2 liters of petroleum ether instead of 1 liter. In that case more than 200 fractions must be collected or the numher of drops per tube must be raised. As the commercial fraction collector does not allow for the collection of more than 400 drops per tuhe, this is accomplished by a “doubling” attachment. B y use of this attachment the number of drops collected per tube may be increased u p to 800.
by partition chromatography, identification of the eluates by the sulfuric acid test, and ultraviolet assay of the fractions. The separation of steroids is carried out automatically, using a silicic acid column impregnated with water and a mobile phase of petroleum ether-dirhloromethane of continuously increasing polarity. Small amounts of adrenocortical steroids can be determined in the presence of a large excess of other adrenal steroidq ACKNOWLEDGRl ENT
DISCUSSIOX
The separation of adrenocortical steroids described is based largely on partition chromatography, as evidenced by the fact that the position of peaks is pract8icallyindependent of concentration. Partitioning takes place between the stationary water phase and a mobile petroleum ether-dichloromethane phase of gradually increasing pnlarity, The adrenocortical steroids are eluted in the order of increasing polarity. B is apparently more polar than its isomer. S. This agrees with the data on partition coefficic.iits present.ed by Pfrffer et al. (6). They observed that when various adrenocort.ira1 steroids are distributed betn.een equnl volumes of petrolclum ct,her and water, i5yoof compound S and only 2 0 5 of compound B are found in tmhepetroleum ether ph:ise. I ~ c of k material has prevented the determination of the load limitations of the columns used in this work, but Haines ( 2 ) has rcport,ed the separation of 40 mg. of hormone mixture on 30 grams of silica gel.
The authors gratefully acknowledge the advice and encouragement of Erich Mosettig of this institute. They are indebted to G. C. Riggle of the Instrument Section, National Institutes of Health. for the construction of a doubling attachment. LITERATURE CITED
(1) Donaldson, K. O., Tulane, Y. J., and 3Iarshal1, L. 3I., A N i L . CHEM., 24, 185 (1952). ( 2 ) Haines, W. J., “Recent Progress in Hormone Research,” Vol. 7 , p. 255, Sew York, -4cademic Press, 1952. (3) Herrington, B. L., and S t a r r , 31. P., IND.ENG. CHEX.,A 4 x . ~ ~ , ED.,14, 62 (1942). (4) Kataenellenbogen, E. R., Kritchevsky, T. H., and Dobriner, K.. Federation Proc., 11, 238 (1952). (5) 3Iorris, C. J. 0. R., and Williams, D. C., Biochem. J., 54, 470 (1953).
(6) Pfeffer, K. H., Ruppel, W., Staudinger, Hj., and Weissbecker. L., Naunyn-Schmiedeberg’s Arch. exptl. Pathol. Pharmakol., 214, 165 (1952).
SUMMARY
(7) Zaffaroni, A.,
The determination of the six active adrenocortical steroids in a mixture is based on preliminary separation of individual steroids
J. Am. Chem. Soc., 72, 3828
(1950).
RECEIVED for review July 18, 1953. Accepted December 11, 1 9 3 .
Colorimetric Determination of Strontium with Chloranilic Acid PETER J. LUCCHESI, S. Z. LEWIN,
and
JOHN E. VANCE N. Y.
Department of Chemistry, N e w York University, N e w York,
Strontium is determined by means of the diminution in absorbancy of a chloranilic acid solution acconipanying precipitation of strontium chloranilate. Large excesses of mineral acids were successfully removed prior to analysis by the use of Amberlite IRA-410. Filtration of chloranilic acid solutions diminishes the absorbancy and shodd be avoided.
I
ions, including strontium ( I , 3, .5, 8),and a nmn1)er of investigations have shoivn its applicability as a colorimetric reagent for calcium ( 1 , 3, /i, 6-8). These methods are all base1 on the decrease in light absorption of the chloranilic acid solutions accompanying the precipitation of the calcium snlt. In the present work, this approach has been adapted to thri determination of strontium. PROCEDURE
S C O S S E C T I O S \\-it11 311 iiivrstigation of the rate of dissolu-.
tion of stront’ium sulfate iii several aqueous media, the need
arose for an anal?;tical niethorl for strontium(I1) t h a t u ould be: rqiid and convenient, suitable for small samples of the order of 2 to 15 nil. containing strontium in milligram amounts, and
athptahle t o samples coiit.aiiiing high concentrations of hj-drochloric acid, nitric arid, or perchloric acid. Preliminary esperimerits showed that sodium rhotiiaonate, which has been employed for the detection of strontium ( J ) , is not suitalde for quantitative colorimetry olving to thc instnliility of the reagent.; turbidimetry involving strontium carbonate \vas investigated and proved to lie insufficiently rpproducible for satisfactory results. Chloranilic :ic.itl was found to fill the requirements satisfact,orily, and 1)erarise of the convenience of the colorimet,ric technique, ot,her possible approaches were not, investigated. Chloranilic acid (2,5-dichloro-3,6-dihydroxy-p-quinone) is prcripitated as the salt from aqueous solution by a variety of cat-
Neutral Solutions. For solutions having a pH hettveen 5 and i and containing no appreciable concentrations of cations other than st’rontium(11))the following procedure was used.
I n a centrifuge tube, 5.00 ml. of a stock 0.05% solution of chloranilic acid are mised with 5.00 nil. of the solut,ion to be analyzed, and the tube is cooled in ice for a minimum of 3 hours, to ensure complete precipitation, but not for more than 12 hours, as longer periods may cause precipitation of some of the unreacted chloranilic acid. The misture is centrifuged at about 1100 r.p.m. for 5 to 10 minutes, after which some of the supernatant liquid is removed for spectrophotometric measurement. .k control is employed in each determination, consisting of 5.00 ml. of the stock reagent and 5.00 ml. of distilled water; this misture is cooled and centrifuged in exactly the same manner as the other samples. -4bsorbances are measured a t 530 mp b y means of a Beckman Model DU spectrophotometer. The control is used as the spectrophotometric reference-i.e., the instrument is adjusted to read 100% transmittance v i t h the unknown in the path of the light beam and the per cent transmittance of the control is measured relative to this setting. Hence, the
