in this system and one can affect an excellent separation of protactinium from thorium. Also, by using intermediate hydrochloric acid concentrations, protactinium can be separated from zirconium and all other elements that do not form anions under those conditions. GENERAL APPLICATIONS
Using the anionic liquid-liquid extraction technique described above, it is possible to develop rapid separations of zinc from cobalt, manganese, nickel, iron, and separations of zinc, cobalt, and iron from other elements which do not form anionic chloride complexes (4). The method has been used to demonstrate the first separation of niobium and tantalum by liquid-liquid extraction (2, 8. Uranium may be extracted readily from many dilute acids such as sulfuric, hydrochloric, nitric, phosphoric, acetic, oxalic, hydrofluoric, formic, or maleic n-ith a solution of methyl-di-n-octylamine in xylene ( 5 ) . The fact that uranium can be extracted from dilute sulfate solution was verified by Brown, Crouse, and Arnold (1). These workers utilized the extraction of anionic uranyl sulfate and thorium sulfate with longchain amines dissolved in inert diluents to develop processes for the recovery and purification of these metals. A 5% solution of methyl-di-n-octylamine in trichloroethylene extracts protactinium efficiently from dilute phosphoric acid solution (6). This fact may prove valuable in the recovery of protactinium from phosphoric acid solution, because the common extract-
ants now used will not extract phosphate species of protactinium. When considering the numerous combinations of long-chain amines (primary, secondary, tertiary), the wide array of diluents available, and the many anionic aqueous systems that can be produced readily, it is seen that this technique offers great possibilities for chemical separations. Thus, for a given separation, a suitable aqueous system is produced such that the desired component is in anionic form and, therefore, extractable with a long-chain amine dissolved in a suitable diluent. By use of this technique the general field of liquid-liquid extraction may be greatly increased. For instance, while isopropyl ether does not extract cobalt from strong hydrochloric acid solution, the addition of a long-chain tertiary amine to the isopropyl ether results in excellent extraction of the anionic cobalt. Liquid-Liquid Extraction with LongChain Amines us. Anion Exchange Resins. There appears to be a strong analogy between anion exchange resins and liquid-liquid extraction with amines. I n the several extraction systems studied to date, the aqueous conditions from which an anion adsorbs on an anion exchange resin are quite similar to those required for the liquid-liquid extraction of that particular anion with a solution of a longchain amine dissolved in a suitable diluent. There exists a vast amount of work in which anion exchange resins have been used as a separations tool. However, the newer anionic liquidliquid extraction technique is fast and inexpensive, and it has higher capacities.
Also, the extraction technique has the inherent advantage of greater ease of operation of a liquid-liquid system over a solid-liquid system in continuous countercurrent work. Already, in several applications, the long-chain amines are becoming successors to anion exchange resins. While the two long-chain amines described in this paper are rather expensive, a new tertiary amine, tri(iso-octyl)amine, has recently become available from Union Carbide Chemicals Co. Because this reagent is cheap and possesses the desirable properties of low aqueous solubility and high organic solubility, it should find wide application in chemical separations. LITERATURE CITED
(1) Brown, K. B., Crouse, D. J., Arnold,
W. D., Oak Ridge National Laboratory Unclassified Report, ORNL-2173 (1956). 121 Ellenbure. J. Y.. Leddicotte. G. W.. Moore:' F. L.; ANAL. CHEW 26; \
,
mi.;
( i ~ i ) . \ - - - - I
(3) Leddiiotte, G. IT., %loore, F. L., J . Am. Chem. SOC.74, 1618 (1952). (4) Mahlman. H. 8..Leddicotte. G. W., Moore,' F. L.;ANAL. CHEM.26,
1939 (1954). (5) Moore, F. L., Oak Ridge National Laboratory Secret Report, ORNL1314 (1952). (6) Moore, F. L., Reynolds, S. A., ANAL. CHEM.,29, 1596-9 (1957). (7) Smith, E. L., Page, J. E., J . SOC.Chem. Ind. (London)67,48 (1948).
RECEIVEDMarch 20, 1987. Accepted June 13, 1957. Oak Ridge National Laboratory is operated for the U. S. Atomic Energy Commission by Union Carbide X'uclear Co.
Colorimetric Assay for Cortisone, Hydrocortisone, and Related Steroids E. P. SCHULZ
D. NEUSS Merck & Co., Inc.,
and J.
Chemical Division,
Rahway, N. 1.
b A colorimetric method for the assay of cortisone, hydrocortisone, and related steroids is based on a chromogen produced by the reaction of the steroid with 2,6-di-fert-butyl-p-cresol in alkaline solution. Cortisone produces a yellow-brown color with an absorption maximum at 471 mp, while hydrocortisone develops a blue color absorbing at 625 mp. Reproducible results are obtained by careful control of reflux time and temperature during the color development. This method is applicable to various formulations, including ointments, tablets, and sus-
1662
ANALYTICAL CHEMISTRY
pensions, although in some cases a preliminary extraction of the steroid with chloroform may be necessary.
C
employed in the determination of corticosteroids are based upon ultraviolet absorption by the conjugated 3-keto group, reduction by the 20,21-ketol group of tetrazolium reagent @),or on color development with acidified phenylhydrazine (6). All of these methods fail to distinguish between hydrocortisone and cortisone. A reagent consisting of 60% sulfuric acid in glacial acetic acid (7) URRENT METHODS
produces a yellow color (Amax, = 470 mp) with hydrocortisone. Although this method is suitable for the quantitative det,ermination of hydrocortisone, it ia not applicable to the assay of cortisone. A more versatile assay method ( I ) , involving the use of diphenylamine as reagent, produces a violet color (Amx, = 530 mp) with cortisone and a green color (Amax. = about 630 mp) with hydrocortisone. The interaction of cyclic ketones with phenols ( 2 ) and the tendency of cyclic ketones to form colored complexes with phenols (3) suggested the possibility of
developing a colorimetric assay based on the formation of a steroid-phenol chromogen. Alkaline solutions of certain steroids, when refluxed with 2,B-di-tert-butyl-pcresol, were observed to form two distinct colors, the visible spectra of n-hich are shonn in Figure 1. A blue color is produced by all steroids with the following basic molecular structure :
i/g
Pipets, 5.0 ml. (three), 10.0 ml. (one). Thermometer, 100" C. range. Magnetic stirring bar, glass covered, inch. Magnetic stirring unit. Electric hot plate. Stirrer, air driven. Filter, sintered glass, medium porosity.
still attached and immerse it in an 800ml. beaker containing 600 ml. of water a t 10' to 12' C. for 10 minutes with magnetic stirring. Add 5.0 ml. of 95% alcohol, mix, and filter through a medium porosity, sintered-glass filter. Avoid loss by evaporation throughout this procedure. Alternately, before adding the 5.0 ml. of 9570 alcohol, the solution could be filtered, transferred quantitatively to a 25-ml. volumetric flask, and diluted to 25 ml. with alcohol. Determine the absorbance a t 625 mp (blue color) or a t 471 mp (yellowbrown color), using distilled water as a reference. Correct the observed reading for the absorbance obtained by treating a reagent blank as above. This blank consists of 5.0 ml. of 95% alcohol, 5.0 nil. of DTBPC reagent, and 10.0 nil. of 5% sodium hydroxide solution. Formulations. Suspensions, lotions, and ointments containing none of the interfering substances listed below can be assayed by the following procedure. Treat a measured weight or volume of the formulation, equivalent t o 8 to 10 mg. of the steroid, with about 75 ml. of 95% ethyl alcohol in a 100-ml. volumetric flask. Shake and heat, if necessary, to ensure complete solution of the steroid, cool to room temperature, and dilute to 100 ml. with 95% alcohol. Filter through a medium porosity, sintered-glass filter. Develop the color on a 5.0-ml. aliquot of the filtrate and measure the color intensity as described above. Correct the observed absorbance values for the absorbance obtained by treating a placebo formulation in the same way. The corrected absorbance, when compared t o standard absorbance values for the same steroid, is a quantitative measure of the steroid present in the formulation. Formulations which contain interfering substances require a preliminary chloroform extraction of the steroid. Shake a measured weight or volume of the formulation, equivalent to 8 to 10 mg. of steroid, with 100 ml. of chloroform in a 250-ml. separatory funnel. Separate the chloroform phase and filter through a medium porosity, sintered-glass filter. Pipet 5.0 ml. of the filtrate into the 5O-ml., round-bottomed flask used for color development.
REAGENTS
Ethyl alcohol, 9570, either USP grade or denatured formula 2B. DTBPC reagent, 2,6-di-tert-butyl-pcresol (Eastman practical grade or Koppers technical or food grades), 0.6% solution in alcohol. Sodium hydroxide (Merck reagent), 5y0 freshly prepared aqueous solution. Steroid standard, 400 to 500 -!per 5.0 ml. of 957, alcohol. PROCEDURE
Steroids with this structure but having a ketone at the 11 position develop a yellow-brown color. The formation of either color seems to be independent of substitution a t the 17 position. The presence of fluorine a t the 9 position does not inhibit the formation of a blue color in the case of Sa-fluorohydrocortisone. The introduction of a second double bond a t positions 1 and 2, typified by the AI-dehydro derivatives of cortisone or hydrocortisone, prevents the formation of either the blue or yellow-brom color. APPARATUS
Spectrophotometer, Beckman Model DU, with 1-em. cells. Round-bottomed flask, 50 ml., 24/40 standard taper joint. Beakers, 800 ml. (two).
Set up a water bath with an S00-ml. borosilicate glass beaker containing 600 nil. of water, heated by an electric hot plate and stirred with a mechanical stirrer. Uaintain the temperature a t 100" c. Weigh t o the nearest 0.1 mg. 20 to 25 mg. of steroid standard into a 250-ml. volumetric flask and dissolve in 250 ml. of 95% alcohol. Pipet 5.0 ml. of this solution into the round-bottomed, 50ml. flask containing a glass-covered magnetic stirring bar. Add 5.0 ml. of DTBPC reagent, followed by 10.0 ml. of 5% aqueous sodium hydroxide. Lmmediately attach the flask t o a watercooled reflux condenser and immerse the flask up t o its neck in the 100' C. bath. Adjust the position of the flask so that its contents may be stirred efficiently with a magnetic stirring unit placed close to the side of the 800-ml. beaker, as shown in Figure 2. Reflux for 30 minutes after immersion. Remove the flask with the condenser
CORTI SONE
8 z U m
THERMOMETER
m
MECHANICAL, STIRRER
5 Q
REPSENT BLANK
0
I
\ --
471 WAVE LENGTH, mp
Figure 1. Visible spectra of steroid-2,6-di-terf-butyl-pcresol chromogens
\\,
H ELECTRIC O T PLATE'-
-
s2u2L
STIRRING BAR
625
\
Figure 2.
Apparatus for color development
VOL. 29, NO. 1 1 , NOVEMBER 1957
1663
Evaporate the contents to dryness on a steam bath, cool to room temperature, and add 5.0 ml. of 95% alcohol to the residue. Develop the color and measure the absorbance as described above.
A comparison of results, obtained in determining various formulations by the DTBPC method and an independent method, is shown in Tahle I. EXPERIMENTAL
The above procedure was adopted after a study of effect of alkali concentration, temperature, and reflux time on the reaction for hydrocortisone and 29 other compounds. The visible spectrum of each reaction product was recorded on a Cary Model 11 spectrophotometer.
Tabie I.
The following compounds produced a blue color exhibiting a single absorption maximum at 625 mp: hydrocortisone, hydrocortisone acetate, hydrocortisone tert-butyl acetate, hydrocortisone-21-aldehyde, Sa-fluorohydrocortisone, 9a-fluorohydrocortisone acetate, corticosterone, deoxycorticosterone acetate, progesterone, methyltestosterone, and testosterone propionate. On the other hand, cortisone, cortisone acetate, and cortisone-21-aldehyde developed a yellow-brown color with a single absorption maximum a t 471 mp. The folloring 16 compounds developed no absorption maxima in the visible spectrum under reaction conditions: prednisone, prednisolone, 21hydroxypregnane-3,l 1,20-trione, 4,5dihydrocortisone, 4,5-dihydrocortisone
Assay of Steroid Formulations by 2,6-DTBPC Method
Concentration Found Expected DTBPC Ultraviolet 25.0 mg./tablet 22 8 mg./tablet 23.7 mg./tablet
Steroid Formulation Cortisone ace- Tableta tate Cortisone ace- Saline suspensiona 25 0 mg./ml. tate Hydrocortisone Infusion solutione 0.20 mg./ml. Cortisone and Ophthalmic hydrocortipension sone acetates
Table II.
Cortisone acetate Prednisone Hydrocortisone acetate Hydrocortisone Sa-Fluorohydrocortisone
25 9 mg./ml.
0.20 mg./ml.
0 21 mg./ml.
(by tetrazolium assay) sus- 1.25y0 of each 1.28% corti- 90independent sone acetate; method available 1.22%. hydrocortisone acetate
Sa-Fluorohydro- Opthalmic SUE- 0.25% cortisone acepension tate Sa-Fluorohydro- Lotionb 0.10% cortisone Hydrocortisone Ointment 0.50% tert-butyl acetate a Preliminary extraction vith chloroform. * Preliminary extraction with cyclohexane.
Compound Cortisone
25 0 mg./ml.
0.26%
0.25%
0.10%
0.10%
0.497,
0.5070
Absorbance Data
A , Dm.a 471 mp 625 mp 0.537, 0.524, 0.523, 0.532 0 . 0 5 i , 0.069, 0.Oi9, 0.059 Av. 0,529 f 0.005 Av. 0.066 f 0.008 0.478, 0.492 0.048, 0.064 Av. 0.485 f 0.007 Av. 0.056 f 0.008 0.000 0.000 0.039, 0.029 0.232, 0.240 Av. 0.034 f 0.005 Av. 0.236 ;t: 0.004 0.031, 0.048, 0.046, 0.035 0.267, 0.270, 0,259, 0.268 Av. 0.040 =k 0.007 Av. 0.266 =t 0.003 0.038, 0.034 0.252, 0.240 -1v.0.036 f 0.002 Av. 0.246 & 0.006
9 mu-Fluorohydrocortisone
acetate
0.027, 0.037
Av. 0,032 f 0.005
H y d r o cor tis on e tertbutyl acetate
0.218, 0.234
.Sv.0.226 =t 0.008
0.025, 0.035 0.228, 0.220 Av. 0.030 f 0.005 Av. 0.224 f 0.004 Prednisolone 0.000 0.000 a Absorbance values (corrected for reagent blank) are for 400 y of steroid.
1664
ANALYTICAL CHEMISTRY
acetate, 3,1i-dihydroxypregnane-11,20dione, 4-bromo-17-hydroxy-2l-acetoxypregnane-3,l l,2O-trione, cholic acid, deoxycholic acid, apocholic acid, lithocholic acid, estrone, estradiol, ergosterol, cholesterol, and Vitamin A acetate. The largest absorbance value (1-cm. path) a t 471 mp was 0.016, obtained by treating 200 y of 4,5dihydroxypregnane-ll,2O-dione under reaction conditions. I n all instances the absorbance (1 cm.) at 625 mp was not greater than 0.002 for 200 y of steroid. DISCUSSION
The 1-cm. absorbance values, corrected for reagent blank, shown in Table I1 represent the color intensity for 400 y of steroid treated under conditions described in the procedure. Steroids of purity greater than 98%, as determined by phase solubility analysis were used. The absorbance for cortisone, hydrocortisone, and their acetates a t their respective absorbance maxima is reproducible to within 2%. dbsorbance studies indicate (Table 111) that the intensity of chromogens a t 471 and 625 mp is proportional to the concentration of steroid in the range from 200 to 500 y. A t the 100-7 level readings were about 4% low, while at the 600-7 level the readings were found to be 3Y@low. The use of 4% alkali, as compared to 5% alkali, decreased color intensity by 6%, while the use of 8% alkali had no significant effect. Several samples of 2,6-di-tert-butyl-pcresol from tn-o independent sources were used in this investigation. The color of these samples ranged from pure white to yellow, but their ultraviolet and visible absorption spectra mere identical. Reagent blanks with different samples were all of the order of 0.014 absorbance unit a t 471 mp and 0.003 at 625 mp. A 10% variation in the concentration of the DTBPC reagent had no significant effect on the intensity of color produced. The blue or yellow chromogens were stable for at least 0.5 hour. On long standing, the colors fade. Variation of the reflux time between 20 to 40 minutes produced identical absorbance readings within 1%. Careful control of temperature was essential for adequate reproducibility; this was obtained only by stirring both the 100" C. bath and the refluxing solution. Color Development with Other Phenols. Under the reaction conditions the following compounds, when used in place of the DTBPC reagent, failed t o develop colors characteristic of the hydrocortisone or cortisone steroids: o-hydroxybenzoic acid, pmethylaminophenol, 4,6-di-tert-butyl-mcresol, phenol, 0-, m-, and p-cresol,
(e),
2,6-, 3,4-, and 3,5-dimethylphenol, 1,2,3and 1,3,5-trihydroxybenzene, and mdihydroxybenzene. Absorbance readings a t 471 and 625 mp were equal to that of the reagent blank.
Table 111.
Steroid, Compound Mg. A&:?. Hydrocortisone 0,100 0.064
INTERFERENCES
For formulations containing no interfering substances, the absorbance obtained with the placebo was of the same order of magnitude as the reagent blank. Formulations containing sodium carboxymethylcellulose, dextrose, or other carbohydrates which darken on heating with alkali require a preliminary separation of the steroid with chloroform. The presence of excipients as white wax, glycerol, antifoam, sodium methyl p-hydroxybenzoate, sodium lauryl sulfate, liquid petrolatum, bacitracin, or neomycin does not interfere in color development. Diglycol stearate in small amounts is tolerated. However, large
Color Intensity vs. Concentration
Cortisone
0.200 0.300 0.400 0.500 0.600 0.100 0.200 0.300 0.400 0.500 0.600
0,134 0.199 0.266 0.337 0.390 0.127 0.266 0.402 0.529 0.670 0.7i5
formulation by extracting the cetyl alcohol with cyclohexane.
A%?/
Mg. 0.640 0.670 0.663 0.665 0.674 0.650 1.27 1.33 1.34 1.32 1.34 1.29
LITERATURE CITED
( 1 ) Clark, I., N u t w e 175, 123-4 (1955). (2) Dane, E., Schmitt, J., Ann. 536, 198-9 (1938). (3) Granick,‘ S., hlichaelis, L., J . Am. Chem. SOC.66, 1023-30 (1944). ( 3 ) Alader, W. J., in “Organic Analysis,” Vol. 2, pp. 253-75, Mitchell, John, Jr., &hem, eds., Interscience, Yew York, 1954. ( 5 ) Mader. W. J.. Buck. R. R.. AKAL. C H E 24. ~ 666 (1952). (6) Porter, C. C., Siiher, R. H., J . Biol. Chem. 185,201 (1950).
( 7 ) Szalkowski, C. R., O’Brien, 31. G., Mader, K, J., AXAL.CHEM.27, 944
amounts of diglycol stearate or other alkali-consuming substances interfere. Cetyl alcohol caused low results when present in formulations. This interfering component was quantitatively separated from a Sa-fluorohydrocortisone
(1965).
RECEIVED for review September 14, 1956. Accepted June 11, 1957. Symposium on -4naly~isof Fine Chemicals and Pharmaceuticals, Division of Bnalytical Chemistry, 130th Meeting, ACS, -4tlantic City, N. J., September 1956.
Rapid Determination of Phosphine in Air J. P. NELSON and A. J. MllUN Chemical laboratories, General Mills, Inc., Minneapolis 7 3, Minn.
b A simple, rapid method for determining phosphine in air at the parts per billion level consists of metering a known volume of. air through a tube packed with silica gel impregnated with silver nitrate. Phosphine is determined by measuring the length of black color formed in the tube and relating this to a calibration curve obtained with known mixtures. Recovery data are given for synthetic mixtures of phosphine in air.
M
METHODS for determining small amounts of phosphine in air are too involved for routine use. Beyer’s method (1) is too lengthy for routine analysis, but is satisfactory for calibration purposes. The method proposed by Kitagawa and Ogawa (2) involves passing air through a tube packed with silica gel impregnated with mercuric salts and measuring the length of coloration in the tube. This method appeared to be suitable for routine work. However, preliminary experiments showed that tubes containing silica gel and mercuric iodide formed ill-defined orange color bands that were not easily discernible with mixtures of air and phosphine. On the other hand, silica gel tubes impregnated with silver nitrate showed well-defined color bands proportional in length to the concentration of phosphine passed through them. OST
REAGENTS A N D APPARATUS
Phosphine. Generate by reacting aqueous alkali with phosphonium iodide in an atmosphere of carbon dioxide (3, 4). Collect over 50% aqueous potassium hydroxide. Silica Gel Tubes. To a solution containing 1.5 grams of silver nitrate in 100 ml. of distilled mater, add 76 grams of 16- to 28-mesh silica gel and stir the mixture for 15 minutes. Decant the excess silver nitrate solution, dry the silica gel a t 90” C. for 3 hours, and sieve t o a 30- to 50-mesh size. Heat the end of a 7-inch length of 5-mm. outside diameter glass tubing until the opening is approximately 2 to 3 mm. in diameter. Close the constricted end with a small wad of glass wool and pour in a 5-inch column of the treated silica gel. Push another small wad of glass wool down on top of the column and store the tubes in a brown glass bottle. Pump. Any pump which \vi11 force 1.3 to 1.4 liters per minute of air through the silica gel tubes is satisfactory. An A. S. Aloe Co., Catalog Yo. 70572, pump was used along with a metric scale precision met test meter for sampling and volume measurement. PROCEDURE
Set up an apparatus with rubber connections so that air flows from the pump through a vertical silica gel tube into a wet test meter. After adjusting the pump to a flow rate of 1.3 t o 1.4
liters per minute (rate chosen for convenience), replace the silica tube with a fresh one. Pass a measured volume of air through the tube so that a color band of a t least 0.8 cm. is formed. Measure the length of the color band and use a calibration curve to determine the milligrams of phosphine in the air passed through the tube. Calculate parts per billion of phosphine in the air from the following equation:
-u.u.b. _ -phosphine -
=
mg. of PHS X 100,000 1.52 x litere of air sa.mpled (1)
\\-here 1.52 is the density phosphine - of . in mg. per ml. Calibration Curve. The Duritv of the phosphine used t o prepare” the calibration curve was measured by the method of Beyer (1) and was checked each day the gas was used. Calibration was accomplished by flushing measured volumes of phosphine through silica gel tubes with nitrogen for 10 minutes. The length of each color band mas measured and plotted against milligrams of phosphine. The calibration curve v-as linear over phosphine concentrations from 0.02 to 0.21 mg. In order to check whether diluted samples gave the same results as those obtained in preparing the calibration curve, known volumes of phosphine mere metered into a partially evacuated %liter flask prior to flushing through the silica gel tubes. The results of these VOL. 2 9 , NO. 1 1 , NOVEMBER 1957
1665
