arrangement gives two to three times t h e intemity of the flat crystal. The x-ray tube was opcrated at 50 kv., 45 ma. The test sample consisted of a series of 1 x 1 cm. squares of various metals of various thicknesses in the following order: 1/8 inch ( 4 . 3 mm.) Pb, 0.0025 inch (-0.06 inm.) Ni, '/s2 inch (-0.8 mm.) Cu, 0.001 inch (0.025 mm.) Au, and another 1/8-inch layer of Pb. The squares were securely bolted together between steel end plates inch thick, and one surface was given a metallographc finish. The test sample WELSscanned with the spectrometer set :tt 28-angles for PbLal, NiKa, CuKa and AuLal using a slit aperture 0.025 mm. wide and a pinhole aperture 0.1 mm. in diameter. Figure 3 shows recordings of such scans made by motor-driving the sample continuously and recording the x-ray intensity with the rrhemeter recorder. Figure 4 shows plots of such scans made by advancing the sample manually by increments and measuring the
(2) Bertin, E. P., Longobucco, R. J., Advan. X-ray -4nal. 5 , 447-56 (1961). (3) Bertin, E. P., Longobucco, R. J., Advan. X-ray Anal. 7, in press (1963). (4) General Electric Co., X-ray Dept., 4855 Electric Ave., Milwaukee 1, Wis., Bull. 88-3862, 1962. (5) Heinrich, K. F. J., Advan. X-ray Anal. 5 , 516-26 (1961). (6) Lico, Inc., 625 McGrath Hwy., Sornerville, Mass., unpublished data. (7) Raag, V., Bertin, E. P., Longobucco, R. J., Advan. Electron Tube Techniques 2 , 249-59(1963). (8) Siemens-America, Inc., 350 5th Ave., New York 1, X. Y., General Equipment Catalog, 1962. (9) Sloan, R. D., Advan. X-ray Anal. 5 , 512-15 (19611. (10) Togei, K.; Siemens-2. 36, 497-501 (1962). (11) Weyl, R., 2. Angew. Phys. 13, 283-8 (1961). (12) Wittig, W. J., Develop. A p p l . Spectry. 3, in press (1963). (13) Zimmerman, R. H., Advan. X-ray Anal. 4,335-50 (1960). (14) Zirnmerman, R. H., Iron Age 186, NO. 15, 84-7 (1960). (15) Zimmerman, R. H., Metal Finishing 59, NO. 5, 67-73 (1961).
x-ray intensity with the scaler-timer. Table I lists net line and background intensities for the four lines measured with a 0.025-mm. slit and with 0.1-, 0.05, and 0.025-mm. pinholes. The data are self-explanatory and require no discussion other than to note that the 0.025-mm. slit gives intensities four or five times as high as the 0.1-mm. pinhole, and with better resolution. However, the slit resolution is not as good as might be predicted because the slit blocks cannot be brought as close to the sample surface as a pinhole without seriously shielding the primary x-ray beam. Moreover, the resolution varies from bottom to top of the slit because of varying distance from the sample. The resolution could be improved by extending the slit blocks in the direction away from the sample surface.
LITERATURE CITED
(1) ANAL. CHEM.32, No. 9, 30A, 32A-
36A, 38A (1960).
A N e w Method for Elution of Conjugated Steroids from Paper Strips Ludwig Kornel, Steroid Section, Division of Endocrinology and Metabolism, Department of Medicine, University of AlabamaMedical Center, Birmingham, Alabama INTEREST in the conjugated steroids, parallded by certain speculations as to a possible physiological role of some of thi? conjugates (1, 7 , IS), stimulated work :timed at the isolation and identification of various steroid conjugates. Method:; for their extraction (5, 6, 8 ) have been worked out and systems have been devised for the separation of various conjugated steroids using column (4) and paper chromatography (1I ) , and low (3) and high voltage (9) paper electrophoresis. Elution of the separated conjugates from paper presents a serious problem in view of their high polarity. The technique almost universally used is extraction from paper by soaking pap€r cuts in a volume of 70% aqueous methanol or, less frequently, 50% aqueous ethanol, with or without simultaneous shaking. The main disadvantage of this technique resides in the fact thak pieces of paper fibers are also eluted ,n large quantity, and their separation from the extracts is tedious and time consuming and sometimes connected with serious losses of the eluted conjugates. This paper describes a method for the elution of conjugated steroids froin paper, which easily removes even most polar conjugated CP1steroid metabolites with a relatively small volume of solvent without eluting any of the paper fibers. Thus the eluate can be realily concentrated by evaporation to dryness and used directly for further treatment.
EXPERIMENTAL
AGROWING
Areas of paper chromatograms or electrophoretograms containing the separated conjugated steroids are cut out so as t o give 1.5-2-cm. X 7-8-cm. strips with one end cut into a wideangle (approximately 120') V-shape. Two to four of such paper cuts are then superimposed on each other and clipped together between the lower edges of two microscopic slide-glasses attached to a 10- to 20-cc. syringe (from which the piston has been removed) with an adhesive transparent tape (12); the two slide-glasses are held together bv means of-a rubber band o r a small stainless-steel clamp (see Figure 1A). The syringe has a stainless-steel needle, 22-gauge.- The elution is carried out
Table 1.
with a 1 : 3 mixture of n-butanol and 70% aqueous methanol, with which the syringe is filled; 10 to 25 ml. of this solvent are used for the elution, depending on the number of superimposed strips. The eluate is collected into a test tube or B flask fixed below the elutor. It was advantageous to mount a battery of such elutors in a wooden box with a transparent plastic sliding door, to minimize the degree of eva; oration of the solvent from the paper .tr ps during the eluting process (Figure 1B). RESULTS
Table I shows the recoverv of pure conjugated CIS and CPI steroids after their s e p a r ~ t i o nby paper chromatog
Recovery of Pure Conjugated Steroids Eluted from Paper by Method Described
Elution after: Paper chromatography Paper electrophoresis
Whatman paper No. 2
3 MM
DHEA-Sa
FSb
98 97
99 96
Recovery, yo THFdiSc P-diol Gd THEGe 97 94
100 96
99 97
DHEA-S, dehgdroepiandrosterone sulfate : 17-oxo-5-androstene-3p-yl-sulfate. cortisol sulfate: 11~,17a-dihydroxy-4-pregnene-3,20-dione-2l-yl-sulfate. THFdiS, tetrahydrocortisol disulfate : 1I@,17a-dihydroxy-5p-pregnane-20-one-3a,21did-disulfate. "4 P-diol G, pregnanediol glucuronide : 20a-hydroxy-pregnane-3a-y~-p-~-glucopyranosiduronic acid. e THEG, tetrahydrocortisone glucuronide: 17a,21-dihydroxy-5p-pregnane-ll,20-dione3a-yl-p-~-glucopyranosiduronicacid. a
* FS,
VOL. 36. NO. 2, FEBRUARY 1964
443
Elution of Radioactive Conjugated Metabolites of Cortisol-4-C14 from Paper Chromatograms
Table II.
Counts/min. per cm.*eluted Radioactivity eluted, % Method of elution In I1 111 I I1 111 Soaking in 707, hIeOH 126 714 5653 95 80 93 Present method 130 840 5710 98 95 94 a I, 11, 111-paper strips with various amounts of radioactivity.
raphy (11 ) and high voltage paper electrophoresis (9). Furthermore, urinary extracts of conjugated radioactive corticosteroid metabolites of cortisol-4-CI4 administered I.V. as a tracer, were subjected to consecutive paper electrophoresis and paper chromatography of the conjugated steroids (10 ) . The chromatograms were scanned in a n automatic radioactive scanner ( 2 ) ,and the areas corresponding to radioactive peaks aere eluted, using the method described in this communication. The efficiency of the elution was compared with that of the technique of extracting the conjugates by soaking the paper in i O % aqueous methanol for 24 hours; the latter method included a double wash of the eluted paper-fiber particles, separated by centrifugation. The results are shown in Table 11. Furthermore, following the elution by either of these methods, strips were reconstituted from the paper cuts to their original size, and were rescanned for the
presence of residual radioactivity: none could be detected. The eluates obtained with the butan01-70% methanol mixture gave very little residue after evaporation to complete dryness. They could be readily used for further studies. DISCUSSION
The butanol-iO% methanol mixture used in this technique is polar enough to elute quantitatively, using relatively small volume of solvent, even most polar conjugated corticosteroids like THFdiS. Yet, the high viscosity of this mixture, and the method of elution, prevent pieces of paper fibers from being extracted from the paper together with the conjugates. An initial difficulty with this mixture resided in the fact that when a single. paper strip was used for the elution, various urinary “pigments” tended to diffuse to the edges of the paper, carry94-y
c
m
.
V
ing with them a considerable amount of conjugated radioactivity, which was virtually impossible to get off the paper even by repeated elutions with very large amounts of solvent. This was overcome by superimposing two or more paper strips and eluting them together: the power of capillary diffusion due to creation of multiple capillary spaces between the two wet strips apparently counteracted the diffusion of the pigmented material to the edges of paper, as had happened when a single strip was being eluted. Furthermore, when a syringe with a hooked needle was used, on which paper strips to be eluted were suspended, the eluting solvent, because of its high viscosity, ran mainly as a streak in the middle of the paper area. Moreover, because of the thickness of the paper used for the chromatography and electrophoresis of the conjugates (Whatman, Yo. 3MM) the solvent ran mainly along the front strip, so that only 20% of the radioactivity was eluted from the”back strip. This difficulty was overcome by using the two-slide system, whereby a lit,tle reservoir of the dripping solvent is formed between the slides, a t the upper edge of the paper strips. and an equal solvent distribution over the entire area of both strips is accomplished. The size of the needle is critical, as the speed of the elution depends on itLe., the rate of the movement of solvent along the paper strip. When a S o . 24 needle was used, \Tith twice the amount of solvent, only 6070 of the conjugated radioactivity could bc eluted. LITERATURE CITED
(1) Adams, J. B., Biochim. Biophys. Acta 71, 243 (1963).
(Piston removed )
(2) Berliner, D. L., Dominguez, 0. V., West’enskow,G., ANAL.CHEW29, 1797
(1957).
( 3 ) Cavina, G., Tentori, L., Clin. Chim. Acta 3, 160 (1958). ( 4 ) CrBpy, O., Jayle, M. F., Meslin, F., Acta Endocrinol. 24, 233 (1957). (5) Edwards, R. W. H., Kellie, A . E., Wade, A . P., Mem. Soc. Endocrinol. 2,
Sc0t;h tape
53 (1953). (6) Jayle, 31. F., “Techniques de Laboratoire,” Vol. 2 , hlasson &: Co., Parie,
22 Go nee
1954.
Rubber band Microscopic sllde glass
Two Imposed superp a p l l $trips
I
A,
8, Figure 1
444
ANALYTICAL CHEMISTRY
Elutor
(7) Kornel, L., J . Clin. Endocrinol. & Jfetab. 20, 1445 (1960). (8) Kornel, L., Ibid., in press. (9) Kornel, L., Kleber, J. W.,Conine, J. W.,“Program of the 45th hIeeting of the Endocrine Societ’y,”p. 20 (1963). (10) Kornel, L., Eik-Xes, I
