Quantitative Application of Sample Dispersion in Potassium Bromide for Infrared Analysis of Steroids HARRIS ROSENKRANTZ, PAUL POTVIN, and PAUL SKOGSTROM Worcesfer Foundation for Experimental Biology, Shrewsbury, Mass. ,The limitations of infrared analysis of steroid mixtures dispersed in potassium bromide were studied using a rectangular die. Plots of density vs. concentration for androgenic, estrogenic, and corticosteroid hormones yielded approximately linear relationships. Certain characteristic frequencies gave better results than others, as did concentrations below 300 y. Plots of the change in ratio of characteristic bands vs. per cent concentration of pure, two-component mixtures also gave good straight-line relationships. Known mixtures were estimated to within 8% of the actual concentration. Sample size, solvent transfer, and the particular steroid affected the acceptability of the spectra.
S
INCE THE ANNOUNCEMEKT of
the potassium bromide pellet technique (14, 16) for infrared spectroscopic analysis, this preparative procedure has received considerable application. Successful refinements in the size and shape of the pressed material have permitted significant diminution in sample quantity (1, 2, 9, I S ) . A series of standard infrared spectra have been recorded for corticosteroids using this technique (8). Few examples of quantitative application have been published. Browning, Wiberley, and Nachod (6) have discussed many of the variants of this procedure in a quantitative analysis of atropine and scopolamine. Kirkland ( 1 1 ) has examined the influence of dispersion techniques on the analysis, and quantitation in an inorganic problem has been announced by Corbridge and Lowe (6). Schwarz and coworkers (15') have utilized three characteristic bands to determine desoxyribonucleic acid quantitatively. The present paper evaluates the possibilities of quantitative analysis of steroid hormones in purified mixtures dispersed in potassium bromide. Reports from other investigators and experience in this laboratory have revealed certain difficulties in this method. Jones ( I O ) has adequately pointed out that inhomogeneously dispersed systems cannot obey the law of Bouguer-Beer as closely as solutions can. In the present work the true molecular extinction coefficient was not sought. It was hoped that repetition of identical manipula-
tory procedures would prevent variation in the deviations from the true extinction coefficients due to any density gradients in the pressed forms. Three other possible problems had to be considered. These were the possibilities of bromide influence on linkages of the compounds, formation of water of hydration, and trapping of solvents during crystallization. Farmer (7) has stated that heating and prolonged grinding result in hydroxyl-halide influences on the spectra of acids, phenols, and solid alcohols. Barker and coworkers (3) observed variations in the spectra of glucosides but demonstrated that this is due to the formation of hydrates in the potassium bromide film (4). The present investigation showed similar spectral shifts in infrared curves of some steroids dispersed via methanol, so, in addition to taking precautions against moisture, no solvent was used for transferring the substances. METHODS AND EXPERIMENTAL
Samples were accurately weighed on a microbalance and mixed with 18.4 mg. of potassium bromide which had been prepared as a fine powder by hand grinding for 20 to 30 minutes in a glass mortar. Because long intervals of manipulation might result in compound alteration, the steroid-potassium bromide mixture was stirred for only about 3 minutes with a microspatula in a conical tube. The mixture was transferred and compactly pressed into the die orifice with a microspatula; transfer loss was approximately 7%. The die accommodated 18.4 mg. of potassium bromide. A preliminary description of the die has been published (13). A picture of the unit, both dismantled and assembled, is shown in Figure 1. All components are made of machine steel hardened to Rockwell C-59-60 and the die steel contains 5% chromium. The narrow plunger (22 X 0.6 mm.) is given added strength by sandwiching it between two blocks of steel a t the end in contact with the hydraulic press. The die is mounted in a holder similar to that sold by Baird Associates, Inc. The surfaces which form the rectangular steroid-potassium bromide prism are lapped to a high degree; approximately 8% of material is extruded. This unit may be conveniently evacuated in a metal chamber
similar t o that manufactured by Baird Associates for their dies. Experiments in which atmospheric pressure, press pressure, and time were variables disclosed that evacuation for 5 minutes followed by pressing (Carver hydraulic press) at 2000 pounds per square inch for 3 minutes gave clear prisms. The vacuum was maintained during prism formation, the pressure being about 0.1 mm. of mercury. The thickness of the pressed form was approximately 0.406 & 0.012 mm. The rectangular prism was recovered from the die plunger and was shortened approximately 8% to fit the microholder shown in Figure 1. (A more recent model of the die eliminates the necessity of shortening the pressed prism, as Its dimensions are similar to the microholder.) This holder was machined to fit the microadapter for Perkin-Elmer infrared spectrometers of the Model 12C or 21 types. The rectangular prism was weighed to determine the actual quantity of material to be irradiated. The design of the holdfrrmade it possible to assume that approxlmately 75% of the material was in the beam pathway. The microholder eliminated 9% of the radiation reaching the detector; while an additional 8% loss in radiation occurred on the insertion of a potassium bromide pressed prism. It appeared convenient to operate the Perkin-Elmer Model 12C instrument a t a slightly increased gain. Two characteristic bands were selected! for each compound for the construction! of calibration curves. The ordinate IO
represented log and the abseissrr, denoted concentration in micrograms. 10 (100% transmittance) Fas calculated from transparent regions as closely as. possible and on both sides of the bands. under study. I was obtained from subtracting the band intensity from IO a t the point where the base line intercepted the band. The estimations were made from the lined divisions on the recording paper. A similar preparative procedure was employed in the experiments on mixtures of pure steroids. Calibration curves were derived from plots of the change in the intensities of bands char-
concentration of each component. The total wei ht of both components did not exceef 300 'y. The applicability of this procedure to "01.30, NO. 5, MAY 1958
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biological extracts was evaluated by adding cortisone and cortisol, separately and together, to cow adrenal homoge nates (5 mg. per gram) and citratec whole blood (5 mg. per 100 ml.). Tht homogenate proteins were precipitatec in acetone, and the aqueous acetont was concentrated and submitted to L 70% methanol-ligroin partition. Thf residue of a methylene chloride extracl of the aqueous methanol phase wat chromatographed on silica gel; tht eluate containing the polar steroids was examined in the infrared. Furthei chromatography in the tolnene-pro pylene glycol system for partition or paper was also applied. The blood aliquot8 were extracted with ethyl acetate and the residue from the solvent was chromatographed as above. RESULTS
The 845- and 882cm.-' bands of dehydroepiandrosterone were used for estimating the concentration of the steroid. The lo base line intercepts were a t 860 and 830 for the former, and a t 895 and 860 for the latter band. The 872cm.-' band of testosterone was similarly used; the 1115-cm.-' band was much less dependable. This variation could not be assigned to increased opacity of the rectangular prism, because measurements a t both freqnencies were made on the same pressed prism. This indicates that Io was not sufficiently accurate for the calculations involving the 111&cm.-' band. The Io base line intercepts were a t 885 and 850 for the 872cm.-' hand, while those for the 1115cm.-' band were near 1150 and 1100 em.-' This situation also existed for cortisone for bands near 1052 and 1135 cm.3, which gave higher densities than expected. The base line intercepts were 1080 and 1000 and 1150 cm.-', respectively. The opposite result of decreased densities occurred for the 8 9 2 and 899-cm.-' bands of cortisol. lo determinations were made at 910 and 875 cm.-' for both bands. Estradiol-178 was selected as a representative of the steroids containing a benzenoid ring. Two characteristic bands, 918 and 930 em.-', yielded a good relationship between density and concentration. The base line intercepts were identical for both bands, 940 and 900 em. -1 Twwomponent systems were also analyzed with pressed rectanplw prisms. For mixtures of cortisol and cortisone a plot was made of the change in the ratio of intensities of bands characteristic of cortisol (866 ern.-'; ZO intercepts, 900 and 850 cm.-*) and cortisone (878 ern.-'; Zo intercepts, 900 and 850 om.-l) us. the per cent concentration of the compounds in the mixture. A straight line was obtained, n.ith values in the upper half of the ahscissa reflecting increasing cortisol concentration and those in the lower half
976
ANALYTICAL CHEMISTRY
Figure A. B. C.
1.
Die for forming potassium bromide rectangular prisms
Exploded vlew Holder for redangular prlmr Assembled die re.iting on steel block against which p r i m Is formed
representing increasing cortisone concentration. Known mixtures containing 32 and 46% cortisol were analyzed by the curve; the results showed 30 and 43'% cortisol, respectively. A value a t 84% cortisol which markedly deviated from the straight line shows the effect of increased opacity of the pressed prism a t elevated total concentrations of both components. This is also seen in calculation CUNW of single components a t levels above 300 y . A good straight-line relationship was obtained for a similar plot for mixtures of estradiol-178 (888 cm.-l) and estriol (875 em.?); la intercepts were 900 and 860 ern.-' Synthetic mixtures containing 28 and 58% estradiol-178 analyzed 32 and 65%, respectively, for this component. Analysis for these two components in the presence of each other could be useful, as they are often present in the same urinary fractions. Another difficult pair of steroids to resolve in mixtures is dehydroepiandrosterone and epiandrosterone. A curve was plotted representing intensity changes in a band near 1220 cm.-' for dehydroepiandrosterone and one near 1235 an.-' for epiandrosterone (lo intercepts, 1240 and 1220 cm.-l). Synthetic mixtures containing 34 and 72% debydroepiandrosterone analyzed 34 and 72%, respectively, for this component. Bands between 900 and 800 em.-' gave smooth curves but not straight lines. A similar situation occurred in mixtures of dehydroepiandrosterone and testosterone. Obviously, suitable bands must be selected for best results. Results on the steroid fractions from adrenal homogenates were not useful, either becanse the fractions were too impure or separation had occurred between cortisone and cortisol. Analysis of the steroid residue from blood indicated the recovery of approximately 78% cortisone and 65% hydrocortisone in the eluate containing both steroids. Colorimetric estimation of the com-
pounds in resolved zones on paper chromatograms was in good agreement with the infrared analysis: 707, cortisone and 60% hydrocortisone. DISCUSSION
Experiences in this laboratory have afforded knowledge about the advantages and limitations of sample dispersal in potassium bromide for infrared analysis. The die constructed for this purpose has been in use for nearly 2'12 years without adjustment or repair. Because its design approximates the dimensions of the slits, microgram samples may be inspected. Orientation of the pressed prisms immediately in front of the entrance slits of the spectrometer obviates the requirement of a beam condensing unit (8). The rectangular shape of the formed prism favors complete illumination of the sample. The clarity of the pressed forms was reproducible but dependent on the type and concentration of the compound. Certain substances caused opacity a t concentrations near 150 y while others gave transparent prisms at levels near 300 y. Sudden variation in opacity could be detected by a decreased 100% transmittance line. In addition, marked deviation of the straight line toward increased densities in a plot of density us. concentration revealed unusual opacity of the p r e p aration. Presumably such opacity involves the crystal size. Some compounds gave useful infrared spectra a t approximately 30-y levelse.g., estradiol-170, dehydroepiandrosterone, and cortisone-while others had to be examined a t concentrations of 150 r--e.g., cortisol, progesterone, and testosterone. Where the limitation of sample quantity is exacting, the 25% loss of material due to transferring, trimming of extruded material, and shortening of the pressed prism to fit the holder was a disadvantage.
However, the reproducibility of these manipulations offset some of this disadvantage. I n studies employing solvents for the more quantitative transfer of samples, several worth-while observations were made. Methanol, methylene chloride, and aqueous solutions were used. The volumes transferred to the powdered potassium bromide were between 0.01 and 0.1 ml. The routine evacuation time was sufficient to remove the solvents but longer periods were applied in the case of water. No solvent could be detected by its absorption in the infrared curve. The crucial test for the presence of solvent not detectable by its own characteristic absorption was the appearance of alterations in the spectrum of the compound under investigation. Barker and coworkers (4) have demonstrated the formation of the monchydrate of glucose in potassium bromide disks. Farmer (7) has evidence for the involvement of the bromide or chloride in pressed pellets containing organic acids. In the present study opportunity was available for recording spectra of the same steroid prepared in a potassium bromide prism as a dry and a solution admixture. Some steroid spectra, such as cortisone, dehydroepiandrosterone, and etio acid of cortisol, were altered by exposure to methanol; others, such as cortisol and etio acid of ll-dehydrocorticosterone, were not. Caution should be exercised when sample transfer via a solvent is involved. This does not seem to be necessary for all substances, but no
set rule is available for segregating different molecules according to their ability to bind solvents. As the spectral alterations observed could be ascribed to the use of a solvent, it was felt that binding of the steroid with the bromide probably did not occur in these compounds. Another advantage of this procedure was that the total time required for the quantitative preparation of a rectangular prism was about 15 minutes, and the pressed forms could be stored or the sample recovered easily. Application of the method to chromatography eluates indicated that fractions which give poor infrared curves because of contamination do not readily lend themselves to analysis for the steroid components. Eluates which yielded crystalline mixtures of steroids permitted estimations of each compound to within 20% of the true concentration. Apparently, relatively clean residues must be obtained before quantitative estimations can be made. After this work was completed Kirkland (12) reported the use of a similar rectangular die which afforded good quantitative results and similar handling advantages. ACKNOWLEDGMENT
The authors wish to thank W. A. Patterson of Baird Associates, Inc., for the loan of a rectangular die which was used as a model for improvement for the particular needs discussed here. Thanks are also due to Dwight Priest of the Parker Manufacturing Co. for use of the facilities of his plant.
LITERATURE CITED
(1) Anderson, D. H., Smith, R. G., ANAL. CHEM.26, 1674 (1954). (2) Anderson, D. H., Woodall, N. B., Ibid., 25, 1906 (1953). (3) Barker, S. A,, Bourne, E. J., Neely, W. B., Whiffen, D. H., Chem. & Znd. (London) 1954, 1418. (4) Barker, S. A,, Bourne, E. J., Weigel, H., WhifFen, D. H., Zbid., 1956, 318. (5) Browning, R. S., Wiberley, S. E., Nschod. F. C.. ANAL.CHEM.27. 7 (i955j. Corbridge, D. E. C., Lowe, E. J., Ibid., 27, 1383 (1955). Farmer, V. C., Chem. & I n d . (London) 1955, 586. Hayden, A. L., ANAL. CHEM.27, 1486 (1955). Ingebrigtson, D. N., Smith, A. L.? Zbid., 26, 1765 (1954). Jones, R. N., J . Am. Chem. SOC.74, 2681 (1952). Kirkland, J. J., ANAL. CHEM.27, 1537 (1955). Ibid., 29, 1127 (1957). Rosenkrantz, H. in Glick’s “Methods of Biochemical Analysis,” Vol. 11, p. 21, Interscience, Kew York, 1955. Schiedt, U., Reinwein, H., 2.Naturforsch. 7B, 270 1952). Schwarz, H. P., hilds, R., Dreisbach, L., Mastrangelo, S. V., Science 123, 328 (1956). Stimson, M. M., O’Donnell, M. J., J . Am. Chem. SOC. 74, 1805 (1952).
6
RECEIVED for review June 21, 1956. Accepted January 8, 1958. Supported in part by a grant from the Medical Research and Development Board, Office of the Surgeon General, Department of Army under Contract No. DA-49-007-MD-184, and by contract AT(30-1)-918, U. S. Atomic Energy Commission.
Extraction and Colorimetric Determination of Chromium with 1,5-Diphenylcarbohydrazide JOHN A. DEAN and MARY LEE BEVERLY Departmenf o f Chemistry, Universify o f Tennessee, Knoxville, Tenn.
b A rapid, selective, and accurate colorimetric determination of chromium is based upon the solvent extraction of chromium(V1) from aqueous 1 N hydrochloric acid with 4-methyl-2-pentanone and development of the magenta color of the chromium-l,5-diphenylcarbohydrazide complex in the extract. The absorbance of the color body is measured 15 minutes after mixing a t 540 mp. Beer’s law is followed. The optimum concentration range extends from 1.0 to 10.0 y of chromium in 6 ml. of solution. Very large amounts of iron and moderate amounts of
vanadium offer no interference. The method is particularly applicable to cast iron and steel samples whose chromium content is low.
T
REATXENT Of a weakly acidic SOlUtion of chromium(V1) with 1,5diphenylcarbohydrazide (6) has found wider application than other colorimetric methods for chromium. Several elements interfere, including vanadium(V) and iron(II1) which give brown colors with the reagent. Bernhardt (3) extracted the chromium-1,5-
diphenylcarbohydrazide complex with 1-hexanol or cyclohexanol, but all the metals that interfere with the usual aqueous color development remained troublesome. The solvent extraction of chromium(VI) from aqueous 1N hydrochloric acid with 4-methyl-2pentanone is a convenient and rapid method for isolating chromium from many elements (4, 11). Following the extraction step, the magenta color of the chromium(VI)-1,5-diphenylcarbohydrazide reaction is developed in situ and measured 15 minutes after mixing. A similar treatment after exVOL 30, NO. 5, MAY 1958
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