V O L U M E 2 5 , NO. 7, J U L Y 1 9 5 3 Add paper pulp and filter through a rapid filter into a 100-ml. volumetric flask. Wash with warm water until the volume of the solution is near the mark of the flask; cool and dilute to the mark. LIMESTONE.Transfer 1 gram of sample to a platinum crucible or dish and ignite to remove organic matter if this is necessary. Then transfer to a suitable evaporating dish, cover, add 10 ml. of 1 to 1 hydrochloric acid, heat to boiling, and boil until the sample is decomposed. Rinse cover and sides of dish and evaporate until the odor of acid has disappeared. Add 1 ml. of 1 t o 3 hydrochloric acid and 4 ml. of hot water and digest for a few minutes. Add 45 ml. of hot water and continue the sample treatment as for portland cement. CEMENTM O R T ~ R STreat . the ignited sample as for limestone, but add 20 ml. of 1 to 1 hydrochloric acid and boil for 5 minutes hefore evaporation. Take up the evaporated residue with 10 nil. of 1 to 9 hydrochloric acid, heat for 5 minutes near boiling, dilute with 40 ml. of hot water, and continue the sample trentmmt as for limestone. RESULTS
Table I11 she)! s a comparison between the chemical and flame analyses of %vera1 cements, stones, and mortars. The chemical analyses with one exception were obtained following the usual dehvdration of silica with hydrochloric acid, precipitation of iron and alumina with ammonia, double precipitation of calcium witli owlate, and double precipitation of magnesium with am-
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monium phosphate, and final ignition and weighing as Mg21’,0i. The chemical values for magnesium oxide of the portland cements were also corrected for manganous oside found in the pyrophosphate. The data show an excellent agreement between the chemical and flame analyses. Only the higher magnesium oxide contents (over 6%) show deviations much in excess of 0.1%. However. even these results are acceptable for most purposes. The determination of magnesium in these materials can bc made in about 1 hour; a single operator can carry out twelvca determinations in the usual working day. ACKNOWLEDGMEh-T
The authors wish to thank W. C. Taylor, chief, Chemical Section, General Repearch Division, Owens-Illinois Glass Co., foi encouragement and helpful criticism received in this work. LITERATURE CITED
(1)
Beckman Instruments, Inc., South Pasadena, Calif., “Instructions for the Hecknisn Spectrophotometer,”Bull. 259 (Septernber 1951).
RFCEIVED for review h-ovember 4,1952.
Accepted April 22, 1953
Comparison of Infrared Absorption Spectra of Steroids Obtained on Solid Films and Mulls and in Solutions HiRRIS ROSEXKRANTZ AND LEONARD ZABLOW ?’he Worcester Foundation for Experimental Biology, Shrewsbury, Mass., and the National Institute of Mental Health Cooperative Research Station at the Worcester Foundation, Public H e u l t h Serrire, Federal Security .4gency, Worcester, Mass. The variability of infrared spectra obtained on the same steroid in different states has not previously been evaluated. Cross comparisons could be made among spectra obtained on steroids prepared as solid films, as mulls, or in carbon disulfide solutions when tendency for hydrogen bonding was not profound. Where bonding occurred, significant absorption changes were observed between 9 and 10 microns. Despite these changes, the major portion of the fingerprint region (8 to 9, and 10 to 13 microns) suggested identity among the spectra. Hydrogen bonding occurred in solid films and mulls
I
N R E C E S T J ears the infrared absorption spectra of many steroids and structurally related compounds have appeared in the literature (I, 8, 6-8). Infrared analysis has been a fundamental technique in the elucidation and identification of the structures of steroidal hormones and their metabolites ( 2 , 6). I t has become increasingly desirable that infrared curves obtained on these biologically important compounds be easily comparable despite the variation in instrumentation and film preparation The early 1% ork of Furchgott, Rosenkrantx, and Shorr (1 ) contained absorption curvee which were recorded on a manually operated spectrophotometer, the compounds being studied as melted or deposited films. The published spectra of Jones and Dobriner (6) primarily are of the fingerprint region of compounds observed in carbon disulfide solutions. Recently, a group of isosteroids was studied by Josien, Fuson, and Cary ( 7 ) and the infrared spectra that were obtained m-we represented as lines of dif-
and no structural alteration was seen in nielted films of steroids that had relatively low melting points and contained less than four oxygen atoms. Spectra may be compared to a significant extent, irrespective of the preparative method employed. This could avoid unnecessary duplication of curves, especially for workers who are not in a position to compile an extensive catalog of spectra. Use of published curves may lead to identification of an unknown compound where no more pure steroid is available for study in a manner identical to that used in studying the unknown.
ferent lengths appearing at particular frequencies. The samples were prepared as Kujol mulls or in solutions of carbon disulfide and hexachlorobutadiene. More recently, Rosenkrantz, Milhorat, and Farber (8) have obtained the infrared absorption curvec: of compounds in the ergostane and cholestane series, the materials being examined in the solid state. In none of the above investigations, where the same compound was studied by different workers. are the infrared spectra superimposable in all respects. -4lthough there is good agreement between the early curves of Furchgott, Rosenkrantz, and Shorr arid those of Jones and Dobriner, several discrepancies exist, some of which may be assigned to the different instruments used. Hoa-ever, no evaluation has been made of the variations in the absorption spectra obtained when a steroid was studied in the solid or solution state. The present study has attempted to clarify this issue. I t is hoped that the data will demonstrate the sperific ad-
ANALYTICAL CHEMISTRY
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WAVE NUMBER, Chi.-’
vantages of the different methods of preparing a steroid for infrared analysis and the extent of comparison of the infrared curves.
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The infrared absorption curves were recorded on a Perkin and Elmer Model 12-C spectrometer with automatic slit drive. Sjx steroids were selected on the basis of melting point, solubility in carbon disulfide, and structural complexity. Androstane, Asandrostene-3(fl)-ol, dehydroepiandrosterone, desoxycorticosterone, A4-androstene-3,17-dione, and corticosterone were studied as mulls and solid films and in carbon disulfide solution where possible. All spectra were recorded between 2.5 and 13 microns, the curves being aligned a t 3.4 microns, the carbon-hydrogen linear vibration. WAVE NUMBER, CM.-1 1300
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Infrared Absorption Spectra of A5-Androstenol-3p
Figure 2.
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b. Mineral oil mull e. Deposited film from chloroform d. Carbon disulfide solution
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Figure 1. Infrared Absorption Spectra of Androstane a. Melted film b . Mineral oil mull e. Carbon disulfide solution
The absorption curves shown in Figures 1 to 6 are tracings of part of the automatic recordings. Intensity variations are due partially to differences in sample quantities used. Jones ( 4 ) has pointed out that reference to “absorption bands or peaks” in most of the infrared spectra in the literature is not exact, as the bands are actually transmittance minima. In the present study the curves were prepared as in the past to facilitate comparison between them and previous published spectra. They are thought of in terms of absorption. RESULTS
Because the purpose of this study is to compare the infrared absorption curves obtained on each compound prepared for analysis by various techniques, each of the six steroids is discussed individually. Androstane. This substance has a low melting point, and attempts to deposit it from solvents resulted in the formation of a melted film. Therefore, Figure 1 does not contain an individual curve for a deposited film. The absorption curves of androstane were nearly identical. Slight differences were explained by variation in the amount of material analyzed. The entire fingerprint region for all three spectra would have permitted identification of this molecule in a cross-comparison. Apparently in a molecule as simple as androstane, which contains no substituents other than hydrogen on the steroid nucleus, there are no significant variations in the infrared spectra, the technique used being of no consequence. Ab-Androstene-3fl-01. The four spectra of this substance shown in Figure 2 appeared to have some dissimilarities, a major discrepancy being observed between 9 and 10 microns. This region of the infrared is concerned with carbon-to-oxygen linkages in which the carbon only has single bond linkages (1). As hydrogen bonding could transmit some effect to the carbon-oxygen linkage, it seemed reasonable that some changes would occur in the 9- to 10-micron region. Hydrogen bonding has been observed in steroids in the solid state ( 1 ) and was observed by the
shift of the absorption band arising from the hydroxyl group. This phenomenon also was seen in the present study, the hydroxyl vibrations occurring near longer wave lengths in all the spectra except that of the solution. Possible bonding effects could explain the fact that the solution and mull spectra contained only one intense band near 9.5 microns, vhile the other two spectra had a doublet near this wave length. Josien et al. ( 7 ) have reported hydrogen bonding in steroids in Sujol mulls. This is not unexpected, as such mulls consist of microcrystalline particles which may behave like crystals of a deposited film. I t would appear that bonding in mulls does not occur to the same extent and intensity as in solid film preparations. I t is obvious in Figure 2 that bands between 9 and 10 microns have become more intensified in the spectrum of the melted film. To eliminate the possibilities of a change in the molecule, the material from the melt was recovered in carbon disulfide and was shown to give an identical spectrum with that of untreated material in carbon disulfide. I t was concluded that the variations in this region were characteristic of the melted film. Doublets near 9.8 and 11.3 microns also differentiated the other spectra from that of the me!t. .4 comparison of spectra of the mull and 1300
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IDEHYDROEPIAN DROSTERONE
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Infrared Absorption Spectra of Deh ydroepiandrosterone Melted film Mineral oil mull Deposited film from ehloroform Carbon disulfide solution
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V O L U M E 2 5 , NO. 7, J U L Y 1 9 5 3 deposited film could have estahlished identity, hut the spectra of the solution and melted film \I-ould have raised considerable doubt. Dehydroepiandrosterone. As some discrepancies had occurred in the spectra of Ab-androst~ne-38-01,it was believed that similar findings might he observed in. the spectra of dehydroepiandrosterone, especially since this compound contained an additional oxygen group. As in the example of androstane, the curves of dehydroepiandrosterone would have permitted identification on cross-comparison of all preparations. Only one significant change occurred. The hand near 9.50 in the spectrum of the solution appeared near 9.42 in the melt and deposited film, while this same hand was near 9.32 microns in t,he mineral oil mull. Recovered material from the melt \\-as subsequently analyzed in carbon ditulfide and disclosed the restoration of the 9.42. band to approximately 9.50 microns. This demonstrated, as in the case of 1:-androstene-38-01, that the variation that occurred between 9 and 10 microns did not result from structural alteration. A~Androstene-3,17-dione. As it was not clear why A5-androtene-38-01 should have easier hydrogen bonding opportunities than dehydroepiandrosterone, A4-androstene-3,17-dione was studied. If hydrogen bonding did account for changes in the 9 t'o 10 micron region, then the spectra of all preparations of Al-androstene-3,lT-dione should he identical. since little opportunity for bonding is present in this molecule. The spectra in Figure 4 reveal the close similarity of the curves. any of which could be used for identification of A4-androstene-3,17-dione. This result would suggest that apparently A5-androstene-3p-oldoes hydrogen-bond more easily than dehydroepiandrosterone. 1300
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revealed important differences (Figure 6 ) . Similarly to A5-androstene-3fi-ol, the variations occurred primarily between 9 and 10 microns in the spectrum of the melt. There was good agreement h e h e e n the curves of the deposited film and the mineral oil mull, hut the spectrum of the melted film had an intense hand near 9.18 which occurred near 9.38 microns in the other absorption curves. In addition, the latter contained absorption bands of medium intensity near 9.8 and 10.04 microns, which appeared only as side inflections in the former spectrum. Significant differences occurred near 11.1 and 11.6 microns where a triplet and a doublet, respectively, in the spectra of the deposited film and mull seemed t o be merged into one band in the curve of the melt. I100
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Infrared Absorption Spectra of Desoxycorticosterone
(I. Melted film b. Mineral oil mull
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Infrared Absorption Spectra of A*-Andmstenedione-3,17
Melted film b . Mineral oil mull e. Deposited film from chloroform d . Carbon disulfide solution a.
Desoxycorticosterone. This steroid was selected for study because it is soluble in carbon disulfide, has an intermediate melting point, and contains three oxygen groups. The results that mere obtained can be seen in Figure 5. The spectra of the melt, deposited film, and carbon disulfide solution were identical in all respects. The similarity of the mull spectrum to the others was striking but certain discrepancies were apparent. Absorption bands of very weak intensities near 8.35, 9.65, 9.T5, and 10.29 microns and the absence of the broad band near 10.5 microns differentiated the spectrum of the mineral oil mull. In addition, the single absorption band near 11.55 microns in the other spectra appeared as a doublet in the spectrum of the mull. This observation was checked using other samples of desoxycorticosterone and mineral oil, but the same results were obtained. At present no explanation is available for this discrepancy. Corticosterone. This steroid was not studied in carbon disulfide solution because of poor solubility. Unfortunately, comparison of the spectra of all the corticosterone preparations
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Infrared Absorption Spectra of Corticosterone Melted film Mineral oil mull Deposited film from chloroform '
The material used for the melted film was recovered and prepared for another analysis as a deposited film. This new spectrum was identical to that of the melt and not to the deposited film or mull, demonstrating that a definite structural change had occurred in the corticosterone molecule. This was not too surprising, as this compound has a high melting point with many oxygen-containing groups which might tend to make it unstable to the melting process. Despite the alteration that had occurred on melting, the spectra of corticosterone obtained from different sources were identical when prepared as melted films. HOKever, it is not recommended that compounds with high melting points be prepared as melting films irrespective of oxygen content. DISCUSS103
The data that have heen presented have afforded some evaluation of the preparative techniques used in infrared analysis. I t
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A N A L Y T I C A L CHEMISTRY
,
hap been slioivii that comparison of spectra can be made between qolid films and solutions for some steroids and not for others. There are some shifts in going from solution to solid states, and although this did not interfere with identification of a number of steroids, variations among other hydrocarbon curves may introduce serious doubt as to identity. Jones (6) has suggested that variations in the spectra of closely related structures-e.g., replacement of a methyl by an ethyl group-at times are of the same magnitude as variations observed in changes of state. It would be entirely misleading to compare spectra obtained on different states of such similar compounds. The steroids of low or moderately high melting point have been shown to be structurally unaltered during preparation of melted films. The spectra of the melted films discussed here were identical with solution spectra, except in molecules which had profound tendency for hydrogen bonding. Such bonding was the major cause of spectral differences and occurred between 9 and 10 microns. Since absorption between 8 and 13 microns characterizes a molecule specifically, an extensive similarity of curves between 8 and 9 and 10 and 13 microns, irrespective of discrepancies between 9 and 10 microns, would be strongly suggestive of identity. Examination of Figure 2 reveals that the 10- to 13-micron regions of the different preparations of A5-androstene-3p-ol were comparable. Despite the dissimilarities between 9 and 10 microns, it is suggested that the curves are interchangeable on the basis of the 10- to 13-micron region and could be used for identification. The curve of corticosterone (Figure 6) obtained from the melt preparation was shown to arise from a change in structure and this was reflected not only in the 9- to 10-micron region but also between 10 and 13 microns. Intensification and merging of absorption bands clearly indicated the spectra Lvere not comparable. Therefore, if band positions and relative intensities are apparently the same in the fingerprint region, then despite variations between 9 and 10 microns, where bonding plays a significant role, it would appear that curves obtained from different preparative methods could be compared successfully in many cases. Preparation of unknoivn compounds for comparison with a group of known steroids via their infrared spectra should be performed in an identical manner. The melt technique has the advantage of permitting the recording of a complete spectrum on a small sample ( I to 3 mg.) of material with a minimum of handling. Infrared studies of solutions permit analysis of microquantities of steroidal material only nhen a microcell is available ( 3 ) .
Transfer of a sample from one solvent to another in order to obtain a complete spectrum is inconvenient and not' always is n suitable solvent available. Carbon disulfide has proved esceedingly useful for Cis-steroids (6). Josien, Fuson, and Cary ( 7 ) have applied Xujol mulls for their observations on normal and isosteroids. ;Ibsorption bands of good int,ensities are obtained on mineral oil mulls containing 3 mg. of substance. Preparation of a mull is simple, but recovery of the steroid from the mineral oil may be troublesome. The most general technique for preparing a steroid for spectroscopic analysis is deposition of a film. Empirical trials with different solvents usually yield crystalline or glassy films which are suitable for analysis and a complete spectrum can be obtained 0110.5 to 2 mg. of substance. ACKNOWLEDGMENT
The aut,hors wish to express their gratit,ude to the following for samples of crystalline steroids: The Ciba Pharmaceutical Co., Summit, S.J., for androstane, Aj-andaostene-3/3-ol, and A'-androstene-3, li-dione, and Oscar Hecht'er, Robert Jacobseii, and Frank Ungar, Worcester Foundation for Esperimental Biology, dhrewsbury, Mass., for desosycorticosteroiie, dehydroepiandrosterone, and corticosterone. LITER4TURE CITED
(1) Furchgott, R. F., Rosenkranta, H., and Shorr, E., J . B i d . C'bem., 163.375 (1946). (2) Ibid., 171, 523 (1947). (3) Hardy, ,J. D., Wilson, H., and Dobriner, K., Federation Proc., 8, 204 (1949). (4) Jones, R. N., A p.. p l . Spectroscopu, 6, No. 1 (1951). . (5) Jones, R. N., personal communication. (6) Jones, R. S . , and Dobriner, K., Vitnmins and H O F , ~ L O / 7, ~CS. 293 (1949).
(7) Josien, 31. L., Fuson, S . ,and Cary, A. S., .J. Aiu. C ' h P m . Soc., 73, 4445 (1951). (8) Rosenkrantz, H., Milhorat, A. T., and Farber, AI.. J . B i d . Chem.. 195,509 (1952). RECEIVED for review Kovexnber 8, 1952. Accepted April 27, 19.53. Investigations aided by a grant from the U.S. Public Health (C-321) Siprvice and supported in part b y contract KO.DA-49-007-MD-184, Medical Research and Development Board, Office of the Surgeon General, Drpartment of the Army, and in part by the Permanent Science Fund of t h e .itnerican Academy of Arts and Sciences.
Spectrographic Determination of Residual Impurities J. E(. IIURWITZ Division of Physical Metallurgy, Department of Mines and Technical Surceys, Ottawa, Ontario, Canada
S
PECTROGRAPHIC analyses are often requested for materials for which no standards are available because of the lack of other accurate chemical methods. The general procedure is to prepare standard solutions or powders which resemble the smiples in chemical composltion. However, sometimes the materials used in preparing synthetic standards contain unknown residual amounts of the elements to be determined. The presence of these elements will introduce errors in the quantitative determinations if they are not accounted for. Several methods of background and blank corrections have I)een proposed (1, 3-6). This paper stresses that background wri ections are important when residual impurities are determined. Furthermore, these determinations may be made even though the residual concentrations are below the visual limits of tion of which the spectrographic technique is capable. tl(5tc.c
B4CKGROUND CORRECTIONS
The apparent intensity of any spectral line is composed of two parts-the background intensity and the real intensity of the spectral line. The real intensity may be emitted by two sources of atoms of the same element: atoms that are present as residual impurities and atoms deliberatelk added. Only the background and apparent intensities in the form of ratios can be derived inimediately from niicrophotonieter readings and the emulsion calibration curve without further calculation. Because background intensity may cause a serious nonlinearity of the working curve, coirections for this effect must be made. The methods of background corrections described by Honerjager-Sohm and Kaiser (4)and Pierce and Sachtrieb ( 5 )are both mathematically and experimentally sound. Since the usual procedure at the hlineq Branch i- to use the logarithm o f the in-
