ANALYTICAL CHEMISTRY
162
I
Table 11. Analysis of a Sample of Crude Pinic Acid Acid Unidentified Pinonio Unidentified Pinic Unidentified % recovery from column
Peak Effluent Volume 30
80 240 300 445 95.2
W
2.00 2 .00 -
'1
\
z
A
Found, % 1.6 11.9 9.8
70.9 1.0
EXPERIMENTAL RESULTS AND APPLICATION
The peak effluent volumes of pure acids that would be expected as products of the oxidation of commercial a-pinene using the three methods described above are tabulated in Table I. Unless i t is so noted in the footnotes, the samples were dissolved in water-saturated chloroform or benzene, depending on the type of column. The recovery of pure acids was 99 to 100% for all columns. Recovery on samples of pilot plant batches of crude pinic acid was from 95 to 100%.
W
2.00
v)
a
e
0;
1.50
2u
$ 1.00 aL I-\ v,
0.50
0
IO0
200
300
Chromatogram of Acid Mixture by Benzene Method
Homoterpenylic, terpenylic, and terebic acids would be likely to occur together in some samples produced under acidic oxidation conditions. The terebic acid is so insoluble in benzene or chloroform that it can be filtered out of the sample solutions and d e veloped separately. Homoterpenylic and terpenylic acids are separated to the same degree with either the modified MarvelRands procedure or the benzene method. Terpenylic acid is developed as two peaks with the Marvel-Rands method. This benzene method has been successfully applied to the analysis of crude samples obtained by the oxidation of a-pinene. A sample of crude pinic acid (prepared in a pilot plant by oxidation of commercial a-pinene with potassium permanganate to pinonic acid followed by the oxidation of pinonic acid with a deficiency of calcium hypochlorite to pink acid) gave the results tabulated in Table 11.
400
ml. E L U A T E Figure 2.
ml. ELUATE Figure 3.
Chromatogram of Acid Mixture by Modified Marvel-Rands Method
Figures 1,2, and 3 show the separation of a synthetic mixture of pure acids using each of the three methods. The Marvel-Rands and modified Marvel-Rands procedures do not separate this mixture of the monocarboxylic acids (pinonic, pinononic, and nopinic). However, in the absence of pinononic acid, pinonic and nopinic acids are almost completely separated. The only real advantage of the modified Marvel-Rands technique is the complete separation of the dicarboxylic acids present (pinic and norpinic). The good separation of both the monocarboxylic and the dicarboxylic acids using benzene ax the mobile phase is illustrated in Figure 3.
ACKNOWLEDGMENT
The authors wish to thank Richard N. Moore of the Naval Stores Research Division for providing some of the pure samples of acids used in this work. LITERATURE CITED
(1) Delepine, M., Bull. SOC. chim., [5], 3,1369-82 (1936).
(2) Higuchi, T.,Hill, N. C., and Corcoran, G. B., ANAL. CHEW, 24,491-3 (1952). (3) Marvel, C.S.,and Fbnds, R. D., J . Am. Chem. SOC.,72,2642-6 (1950). (4) Mirphy, C. M.,O'Rear, J. G., and Zisman, W. A.,Ind. Eng. Chem., 45,119-30 (1953). (5) Ramsey, L. L.,and Patterson, W. I., J . Assoc. Oflc. Agr. Chemists, 28,644-56 (1945). (6) Ibid., 31,139-50 (1948). RECEIVED for review October 7, 1953. Accepted November 27, 1953.
Improved Colorimetric Determination of Urinary 17-Ketosteroids HAROLD WERBIN and SlEW ONG Argonne Cancer Research Hospital, The University o f Chicago, Chicago,
A
RECENT paper by hlasuda and Thuline (6) describing a method for estimating 17-ketosteroids in urine prompts a report on a similar modification which has proved to be of value in this laboratory for some time. Several investigators (3, 6, 7, 8) have attempted to improve the colorimetric determination of 17-ketosteroids by employing organic solvents to extract the pink chromogens formed in the Zimmermann reaction. Aqueous potassium hydroxide was used to develop the color of the steroid with dinitrobenzene and chloroform, ether or amyl acetate to
111.
extract it from 60% ( 3 ) or 68y0(5, 7 , 8) alcohol. The procedure described here differs in that: the absolute alcohol technique of Callow et al. ( 4 ) was adopted for the color formation, the alcohol concentration of the pink solution was made to 37% before extraction, and a novel apparatus was introduced to extract the steroid chromogens. These modifications practically eliminated the interfering substances that absorbed strongly a t 400 mp and permitted an average recovery of 96% of the crystalline steroid which was added to urine extracts. The apparatus described
763
V O L U M E 26, NO, 4, A P R I L 1 9 5 4 Table I.
Per Cent Transmittance of Pink Chromogens at 515 mp
DEAA, y 20
5 80.0 40 61.8 60 50.9 Timing began after addition of
Time", Minutes 10 15 20 80.7 79.8 80.1 62.5 61.8 62.0 51.0 50.6 50.7
30 80.8 62.5 51.3 1 ml. of absolute alcohol t o ether.
permits the method to be carried out routinely with a minimum of manipulation. APPARATUS
The 12-ml. test tube for the Zimmermann reaction and the 65ml. separatory funnel (available from Delmar Scientific Laboratories, Chicago 51, Ill.) employed for the extraction of the pink reaction product are presented schematically in Figure 1. The absorption spectra were measured in a Beckman Model B spectrophotometer with test tube adapter.
of the 1 hour allowed for the color formation, 8.0 ml. of distilled anhydrous ether were added from a buret to each of the separatory funnels (see Figure l),one for each test tube. Then, the subsequent steps were performed in diffuse light. Exactly 1 hour after the addition of the 2 . W alcoholic potassium hydroxide, 1 ml. of water was added to each test tube and the contents were mixed. The test tube was inserted into the mouth of the separatory funnel while it was held in an almost horizontal position. The contents were vigorously shaken in order to extract the pink chromogens. The inverted test tube was withdraarn, and after the two phases separated (2 to 3 minutes) the aqueous layer was removed. Then, 1 ml. of absolute alcohol was added to the ether solution in the separatory funnel. The pink solutions were poured into round cuvettes and read against the reagent blank (prepared in the same manner) a t 515 mp. Crystalline dehydroepiandrosterone acetate (DEAA) was used to establish the standard curve. The Girard separations were carried out according t o the procedure of Beher and Gaebler (1). RESULTS AND DISCUSSION
Table I demonstrates that the pink color of the ether solution was stable for approximately 15 t o 20 minutes.
PROCEDURE
I
The directions of Robbie and Gibson ( 6 ) or Buehler et al. ( 2 ) were followed for the hydrolysis and extraction of the urine. The color was developed by the method of Callow et al. ( 4 )in the test tubes shown in Figure 1. A reagent blank was run with each set of determinations. About 3 minutes before the end
400
450
500 I
550
000 1
I
WAVELENGTH, m p Figure 3. Comparison of Absorption Spectra of Pink Chromogens Given by Urinary 17-Ketosteroids by Callow Technique (C) and by Present Method ( P )
.---.---.After C before Girard separation (53 y) --------separation (35 y) .-.-. P beforeGirard Girard separation (ai y)
iuI, Figure 1. Test Tube and Separatory Funnel for Extraction of Zimmermann Reaction Product
4do
450
500
550
WAVELENGTH,
660
mp
Figure 2. Absorption Spectra of Ether-Extracted Pink Chromogens Stabilized with Alcohol
-0-0
Crystalline DEAA (80
y)
Crystalline estrone (102 --------- Pregnanolone from urine Etiooholan-3-a-01-li-one from urine y)
.---.---a
For comparison the pink color of DEAA (80 y ) developed by method of Callow *-.-e
et al. ( 4 )
-
After Girard separation (35
y)
The transmittance spectra resulting from the application of the new method to several steroids are presented in Figure 2. (When the ether extraction method was applied to crystalline androsterone, etiocholane-3-ol-ol-17-one, epiandrosterone, and dehydroepiandrosterone the spectra showed maxima a t 515 to 520, 515, 520. and 515 mp, respectively. These measurements were made with a Beckman DU spectrophotometer.) All showed more than 90% transmittance a t 400 mp. This is in contrast to the other procedures (3,7 , 8 ) in ivhich the pink chromogens after extraction absorbed as much as 60% a t this wave length. Thus, even with crystalline steroids the present procedure appears t o be superior in the elimination of the chromogenic material which exhibits strong absorption a t 400 mp. Estrone interfered to a small extent since the absorbance of 102 -/ a t 515 mp was that given by 20 -/ of dehydroepiandrosterone acetate. The ether extractable chromogens from 80 y or less of dehydroepiandrosterone acetate were found to obey Beer's law. Higher concentrations were not investigated. The absorption spectra of the chromogenic material obtained from a neutral urine extract by the application of both the Callow technique and the new procedure are presented in Figure 3. Khen the former was employed, the Girard separation eliminated many of the substances which caused high absorption at 400 mp. However, with the present method there was very little change observed in the shape of the spectra after the Girard
ANALYTICAL CHEMISTRY
764
separation This demonstrates that for crude urine evtracts it probably gives a measure of those steroids remaining after the Girard separation, thereby making the latter unnecessary M hen the total 17-ketosteroid content of the urine is desired. Most of the spectra from urine evtracts exhibited a minimum of 90% transmittance (Figure 3 ) and closely approvimated in shape those in Figure 2 A determination of the 17-ketosteroids of several neutral urine extracts was made The data are summarized in Table I1 together with the higher values found by the absolute alcohol technique (urines E and F are evceptions). The quantities of 17-ketosteroids obtained b\- the e\traction method before and after the Girard separations (urine samples A and B) agreed ne11 with those found b> the Callow procedure following the Girard separations. This again indicates that the extraction method measures the ketonic steroid. which are separated by the Girard reagent.
Table 11. Application of Extraction Rlethod to Neutral Urine Extracts
Urine Sample Pooled male 1 After Girard separation Pooled male 1 After Girardseparation Pooled male 2 Pooled male 2
A A B B C D
Hydrolysis and Extraction Procedure, Refeience (5)
(2)U (6)
( 2 ,h
Mg. 17-KS. Colorimetric Methods Present Callow et al. (4) 4.67 6.70 4.48 4.43 4.67 6.19 4 54 4 80 5 78 6.24 6 33 6.20
Per 24 Hours N a l e arthritic (0') 12 5 11 5 Female virilisni during cortisone therapy (8) 12.3 12.1 G Female virilism (8) 15 7 17.4 48-hour ether extraction. 6 72-hour ether extraction. Urines E , F, and G were made available through the courtesy of D. Bergenstal, Department of Medicine. L-nirersity of Chicago.
E F
(1
Table 111. Recovery of Crystalline Dehydroepiandrosterone Acetate Added to Neutral Urine Extracts Urine Sample C E
F
c
Urine Extract, y 17-KS 22 5 45 0 26 7 53 4 22 3 44 5
23 7 23 7 47 4
DEAA Added, y 20 20 20 20 20 20 20 40 20
DE.A.1 Reco\ercd 21 0 18 8 18 8 17 6 19 3 16 1 20 3 40 8 20 9
Y
Recovery, % 105 94
94 88
97 81 102 102 105 AT 96
ACKNOW LEDGMEbT
The authors are inlebted to the Schering Corp. for crystalline dehydroepiandrosterone acetate and to F. A Travers of the Ciba Pharmaceutical Products, Inc. for the crystalline epiandrosterone. EdlTard Davis, Department of Obstetrics Thanks are due to ?*I. and Gynecology, Cniversity of Chicago, for the crystalline estrone. urinary pregnanolone, and etiocholan-3-or-ol-l7-one David Fukushima kindly supplied crystalline androsterone and etiocholan-3-ol-ol-l7-one. Discussions with George V. LeRoy have been very helpful. LITERATURE CITED
(1) Beher. IT,T.. and Gaebler. 0. H.. AN-\L. CHEM..23. 118 (.1 9 5 1,) . (2j Buehler, H. J., Kataman, P. . i , , ' a n d Doisy, E. a:,Proc. SOC. Esptl. Biol. 'Wed., 78, 3 (1961). (3) Cahen, R. L.. and Salter, W. T., J . Biol. Chem.. 152, 1 8 9 (1944). (4) Callow, X . H., Callow, R. K., and Emmens, C . IT.,Biochem. J . , 32, 1312 (1938). (6)
Jlasuda, 31., and Thuline, H. C., J . Clin. E~tclooinol.and Meteb-
oli'rrn, 13, 581 (1953). (6) Robbie, IT7. d.,and Gibson, R. B., J . Clin. Endocrinol., 3, 200
The average recovery of dehydroepiandrosterone acetate which as added to diffeient amounts of urine evtract was 96% (Table 111) The recovery from the larger evtract tended to be lot\ er than that from the snialler aliquot. This was also noted by Masuda and Thuline ( 5 ) .although no quantitative data were presented
(1943).
(7) Ruppert, d.,Z . g e s . e.rpt2. M e d . , 119, 229 ( 1 9 5 2 ) . ( 8 ) Zimmermann, W,, Anton, H. V.,and Pontius, D., H o p p e - S e y l e r ' s Z. phU8iol. Chem., 289, 91 (1952). RECEIVED for reviem August 17, 1953.
Accepted .Janiiary 26. 1954.
Improved Fractional Melting Apparatus S. V. R. MASTRANGELO' and J. G. ASTON Pennsylvania State University, State College, Pa.
I
S VIEW of the utility of fractional melting as a means of purification (1), an improved fractional melting apparatus
with compressible conduction vanes has been developed which is sturdier than the others described previously (1). This apparatus is referred to as fractional melting apparatus 5. The reader is referred to the earlier publication for background and experimental detail. A test of performance of the apparatus with and without using the vanes to press out the melted substance is described. APPARATUS
A scale drawing of the apparatus is shown in Figure 1. The apparatus consists of four sections: the melting chamber, 18, and vane system, 27, the shield supplied with a heater, 17, the 1 Present address, Barrett Division, Allied Chemical and Dye Corp., Glenolden, Pa.
shield or temperature equalizer, 16 (which is not supplied with a heater), and the external cryostat envelope, 15. The melting chamber is provided with a compressible spiral vane system 27, which is the basis of the high efficiency of the apparatus. This type of vane system proved to be mechanically superior t o and simpler in design than the one used in the fractional melting apparatus 3 ( 1 ) . The vanes are made by cutting disks from S o . 36 gage sheet steel, perforating them a t random, and drilling a central and a radial hole for the vane guide tube, 28, and the transfer tube, 30, respectively. The disks are cut along one radius (opposite the radial hole) and twisted approvimately 15'. The twisted disks are then hardqoldered end t o end t o form a continuous spiral. The pitch of the spiral is about 0.25 inch when the spiral is expanded the full length of the melting chamber. The lower end of the spiral vane is soft-soldered t o the bottom of the melting chamber, and the top is hard-soldered t o the main activator vane, 20. The main activator vane, 20, consists of a copper disk l / 1 6 inch thick. This vane is hard-soldered t o the plunger tube, 19, which has a perforated cap, 12. The plunger tube, 19, is partially slotted along one side and the slot is made t o ride on a key mounted in the cryostat pumping tube, 14. This device pre-
