Fluorescence and Absorption Spectra of Some Corticosteroids in Sulfuric and Phosphoric Acids
*
JOSEPH W. GOLDZIEHER and PAIGE K. BESCH Department of Endocrinology, Southwest Foundation for Research and Education, Sun Antonio, Tex.
b The fluorescence properties of some corticosteroids were studied in various acids in an attempt to develop fluorometric quantitative procedures for these substances. Both the absorption and fluorescence phenomena are influenced by acid concentration, time, and temperature of reaction. Sufficient flexibility is provided by these variables to permit altering the desired experimental conditions. The corticosteroids studied were corticosterone, hydrocortisone, cortisone, tetrahydrocortisone, and tetrahydrohydrocortisone.
F
techniques are often more sensitive by one or two orders of magnitude than corresponding colorimetric methods. Thus they are finding increasing application, as in the measurement of microgram and ultramicrogram quantities of steroids. Strong acids, such as sulfuric and phosphoric acids, react with steroids t o produce complex molecules which have characteristic absorption bands. The absorption spectra of these steroid-acid complexes have been studied in great detail (2, S, 5-7, 9-13, 15-20), These solutions sometimes fluoresce with great intensity; characteristics of the estrogen-acid fluorescence have been examined by numerous workers ( 2 , 3, 7, 9-12, 14). I n 1954, when the fluorescence characteristics of a variety of other steroids in sulfuric acid, 85% phosphoric acid, or 98% formic acid n-ere examined (If?), it appeared that certain steroids-such as corticosterone and hydrocortisone, might lend themselves well to fluorimetric analysis. I n the same year Sweat (18) described R highly sensitive technique for measuring hydrocortisone by means of sulfuric acid fluorescence. The intensity of fluorescence emission is a function of the amount of energy trapped by the fluorescent molecule. It depends, therefore, not only on the intensity of the source of exciting radiation, but also on the efficiency with which this radiation is absorbed. Thus both the characteristics of the light source and the absorption spectrum of the irradiated solution are important factors in achieving maximum excitation efficiency. Studies of the absorption spectra of steroids in strong LUOROJIETRIC
962
ANALYTICAL CHEMISTRY
acids have usually been carried out in an effort to devise methods for steroid identification or to correlate the observed absorption phenomena with molecular structure, but not with the intent of locating bands of maximum energy absorption in particular spectral regions. The absorption spectra of the five selected corticosteroids in various acids have been studied from a different point of view and in somewhat greater detail than had been done previously. Formic acid, 98%, proved to be a poor reagent for the production of fluorescence (12); 85% phosphoric acid was far superior and in some instances surpassed the fluorescence prov.) sulfuric acid. duced by 90% (v. I n 1955 Nowaczynski and Steyermark (16) independently published several papers describing a large series of absorption spectra obtained by reacting steroids with 85% phosphoric acid to which had been added double the quantity of phosphorus pentoxide required to make 1 0 0 ~ ophosphoric acid. Following their suggestion, further studies were pursued with this polyphosphoric acid.
ing steroid, possibly cortisone. Corticosterone also contained an impurity (presumably 110-hydroxyprogesterone) which was detected in the 8-hour toluene-propylene glycol system. It was removed by several recrystallizations from acetone. The ethyl alcohol used for dissolving the steroids was allowed to stand in the dark with a large excess of m-phenylenediamine for a week or more, after which it was distilled in an all-glass system. Procedure. A hundred micrograms of steroid in 0.2 ml. of alcohol was placed in a long borosilicate test tube, t o which 4.0 ml. of polyphosphoric acid was added. The solution was agitated vigorously. One group of tubes was allowed to stand a t room temperature and absorption spectra were determined a t 2 and 24 hours; another group was heated in a water bath a t 100" C. (with care to avoid entrance of moisture) for 20 minutes, cooled in ice water, allowed to come to room temperature, and read immediately and after 24 hours; the third group was kept a t 100" C. for 1 hour, cooled as before, and read a t once and a t 24 hours. All steroid solutions were read against the appropriate reagent blank.
Methods and Materials. Absorption spectra in the range from 210 to 650 mp were determined in a Beckman DU spectrophotometer using standard 1-em. silica cells. Polyphosphoric acid was prepared by adding 79 grams of phosphorus pentoxide to 100 grams of reagent grade (85 to 87%) phosphoric acid. The mixture was shaken gently for a half hour and filtered through glass wool. Five corticosteroids were studied: corticosterone, hydrocortisone, Cortisone, tetrahydrocortisone, and tetrahydrohydrocortisone. The purity of these steroids was investigated a t a concentration of 200 y per sq. em. by paper chromatography in four systems (ligroin-propylene glycol for 24 hours; toluene-propylene glycol for 8 hours; toluene-propylene glycol for 72 hours; and chloroform-formamide for 8 hours). The rate of migration was followed by ultraviolet light, 2,4-dinitrophenylhydrazine, blue tetrazolium, Zimmerman reagent, and antimony trichloride reagent. The samples of cortisone, tetrahydrocortisone, and tetrahydrohydrocortisone were homogeneous in all chromatograms. I n one chromatographic system the sample of hydrocortisone contained a small quantity (less than 0.591,) of a second reduc-
Results. Major changes of the absorption spectra are produced by varying development conditions, as illustrated in Figures 1 to 5. The dotted cuives indicate incubation a t room temperature for 2 hours; broken lines indicate incubation a t 100' C. for 20 minutes; solid lines indicate incubation a t 100' C. for 60 minutes. The absorption spectra of the Corticosteroids in polyphosphoric acid are a t a concentration of 25 y per ml. At room temperature corticosterone, hydrocortisone, and cortisone show a strong absorption band a t 290 to 295 mp; this apparently corresponds to the 275- to 290-mp sulfuric acid and 275- to 290-mp phosphoric acid band which has been related to the A4-3-keto configuration. Present data indicate that the intensity of the absorption band diminishes progressively upon heating. If this thermolability holds true in other compounds with the A4-3-keto configuration, it may be of some value for identification purposes. I n general, reaction a t higher temperatures tends to diminish absorption below 300 mp (except with the tetrahydro compounds) and to increase the
+
300 WAVE L E N G T H (m))
Figure 1 . Absorption spectra of corticosterone in polyphosphoric acid
degree and coniplexity of absorption above this point. This is of practical significance, for intense sources of exciting radiation are far more readily available in the 350- to 450-mp range than in the shorter ultraviolet. In particular, corticosterone and hydrocortisone develop absorption bands in the 380- to 390- and 460- to 480-mp regions upon heating in polyphosphoric acid a t 100' C. With hydrocortisone the 460- to 480-mp absorption develops more strongly, while with corticosterone the 380- to 390-mp band predominates and in addition an over-all increase in absorption in the region below 255 to 265 nip appears. Cortisone develops two minor absorption bands a t 340 and 370 mp after 20 minutes a t 100" C., and these merge into a single prominent band at 370 nip on heating for 60 minutes. Whether this band has the same structural significance as the 380- to 390-mp band observed with corticosterone and hydrocortisone, but with the superimposed hypsochromic effect of the CII ketone group of cortisone cannot, of course, be determined in such a small series of compounds. The tetrahydro derivatives of cortisone and hydrocortisone show no absorption in the 290- to 295mp region a t room temperature. Both display a broad band of low absorbance with a maximum a t 390 to 400 mp. Tetrahydrohydrocortisone also exhibits a a-eak absorption band in the 500-mp region, Heating intensifies the absorption phenomena in the visible range, but produces a far greater augmentation of absorption in the ultraviolet range below 350 mp with the appearance of several absorption bands and inflection points. These studies were not intended t o clarify structural interpretations of absorbance phenomena, but certain structural aspects deserve brief mention. Linford (16) has suggested that an 11hydroxy group on an A4-3 keto corticosteroid shows an absorption band in the 370- to 392-mp region. Results indicate that a band of this type is not present when corticosterone and hydrocortisone react a t room temperature,
400 WAVE L E N G T H
500 (my1
600
Figure 2. Absorption spectra of hydrocortisone in polyphosphoric acid
but appears after 20 minutes a t 100' C. However, tetrahydrocortisone, which lacks both the 11-hydroxy and the A4-3 keto configuration, also shows absorption in this region after 60 minutes of heating. This merely emphasizes the difficulties of interpreting absorption in terms of molecular structure. Taking into account slight differences in reaction procedure, the present results confirm the findings of Nowaczynski and Steyermark. I n most instances the curves obtained by these authors are intermediate between curves for 20- and 60-minute heating. Because their experiments were carried out by heating in an oven a t 107' C. for 20 minutes, compared to the w t e r bath a t 100' C., this difference may be expected. Redetermination of the absorption spectra after 24 hours revealed no change in any of the solutions heated to 100" C. This is in contrast to the solutions reacting a t room temperature which underwent appreciable changes (1, 19). FLUORESCENCE SPECTRA
Materials and Methods. An automatic recording spectrofluorometer of new design consisted of a 1000watt Hanovia xenon-mercury arc and a Bausch 8: Lomb monochromator (Type 33-86-45-01) incorporating a 1200 lines per mm. grating of 250-mm. focal length as a source of monochromatic exciting radiation. The monochromatic beam \vas collimated by a long-focus Bausch & Lonib 33-86-53 quartz lens and directed a t the side of a square silica cuvette polished on all sides and similar in dimensions to the standard Beckman cuvette. The sample cell was held in front of the aperture of the analyzing monochromator in a housing which provided great flexibility for experimental arrangement of its optical components. The analyzing monochromator, a Perkin-Elmer Model 99 doublepass type, employed a 60 X 72 mm. crystal quartz prism. A shielded 1P21 (or a 1P28 for certain applications) photomultiplier, selected for high sensitivity, was used as a detector. Its chopped output, amplified by a Perkin-
WAVE LENGTH (mpl
Figure 3. Absorption spectra of cortisone in polyphosphoric acid
Elmer bIodel 107 13-cycle amplifie?, wne registered on a Brown Electronik recorder modified for the purpose. The instrument was provided with an automatic wave length drive and wave length marker; the latter was calibrated against suitable mercury, sodium, and potassium emission lines over the 200- to 759-mp range. No satisfactory permanent standard of fluorescence intensity was available; a piece of Corning fluorescent uranium glass cut to the size of the standard cuvette was tried but fluoresced far too intensely to serve t h e purpose. It was impossible to obtain a more dilute sample of this material. As a consequence, all intensity measurements are relative, and no quantitative comparisons between experimentcarried out a t different times are permissible. Spectra have not been corrected for the spectral response of the phototube. The corticosteroids, dissolved in 0 2 ml. of ethyl alcohol, were pipetted into long borosilicate glass test tubes; 5 ml. of the appropriate acid was added. After shaking, the tube vias inimersed in a boiling water bath for 20 minutes, cooled in ice water, and allowed t o stand a t room temperature for an hour. The acids used were 85% phosphoric acid. polyphosphoric acid (as described above), and sulfuric acid (Du Pont. reagent grade) diluted to 90% v.) 80, 70, 65, and 60%. Whenever possible, and particularly when the viscous polyphosphoric acid was used, the test solution was poured into a silica cuvette and allowed to stand for a half hour. I n this way a great deal of scattering was pliminated. as shown in Table I. (17.
VOL. 30, NO. 5, MAY 1958
+
963
'.OI
I'O3
w
m
-I""I""I""I""l""l""1
500
400
300
200
WAVE
LENGTH
600
(mpl
cence spectrum of a n excited molecular species is dependent on the energy absorbed and independent of the wave length at which the energy is supplied. This must be qualified with respect to the Stokes effect: Under most experimental conditions the fluo-
Table 1. Effect of Standing on Intensity of Scattering in Polyphosphoric Acid Intensity" of Scattered
Time, Min.
Exciting Radiation
0 15 30 45 60 80 1020 5
Arbitrary units:
115 7'2 27 25 21 20 22 436-rn~irradiation.
Table II. Fluorogenic Efficiency of Exciting W a v e Lengths on Corticosteroids in Polyphosphoric Acid
Compound Corticosterone
Fluorescence Exciting Spectrum Peak Wave JT'ave Length, length, InMfi ml tensity
436 390 315 290 240 Hydrocortisone 436 390 325 280 Cortisone 436 370 330 290 Tetrahydro436 hvdro375
520 520 526 520
86= 12 13 2
524 524 524-536 ... 524 524
765 13 11 2 89= 76
...
524 526
...
65a 52
Tetrahydro436 528 72a cortisone 375 528 55 a Arbitrary intensity, constant for each steroid. 964
ANALYTICAL CHEMISTRY
300 WAVE
Figure 4. Absorption spectra of tetrahydrohydrocortisone in polyphosphoric acid
Selection of Excitation Wave Length. Theoretically, the fluores-
200
400 LENGTH (m))
500
600
Figure 5. Absorption spectra of tetrahydrocortisone in polyphosphoric acid
rescence spectrum mill be of longer wave length than the exciting radiation. If the excitation wave length chosen falls Iyithin the span of the fluorescence spectrum of a particular substance, only that part of the fluorescence spectrum will appear which is of longer wave length-i.e., lower energy-than the exciting radiation. This is often the explanation for different colors of fluorescence seen at different excitation wave lengths. As a consequence, studies of the fluorescence spectrum should include excitation at a wave length sufficiently low not to interfere with display of the total fluorescence spectrum. Because under most conditions the spectrum has the shape of one or more Gaussian distribution curves, a suitably low excitation wave length is not difficult to select. -4 further consequence of Stokes' law is the possibility that irradiation with a relatively long wave length-e.g., 436 mp-may conceal the existence of fluorescence phenomena in the ultraviolet region. S o useful fluorescence spectra have been observed in the ultraviolet in the considerable number of steroid-acid mixtures that have been investigated. The validity of these theoretical considerations was tested by preparing solutions of each of the corticosteroids in polyphosphoric acid at a concentration of 10 y per ml. and irradiating each with light of wave lengths corresponding to the wave lengths of the major absorption bands shown in Figures 1 to 5. The data summarized in Table I1 show that the wave length a t peak fluorescence intensity is independent of that of the exciting radiation. I n the few instances where the fluorescence intensity of a compound was appreciable at several exciting Kave lengths, the peak intensities were equalized by adjusting amplification. The fluorescence spectra were identical in their entirety-Le., the curves could be superimposed. I n every instance the fluorescence intensity was highest when the 436-mfi mercury line was used for irradiation. This indicates that the characteristics
of the particular source used (specifically, the extremely high intensity of the mercury lines as compared to the xenon continuum) are of greater practical consequence than the selection of spectral regions of maximum absorbance. For most applications of spectrofluorometry a simple, readily available, high-intensity mercury source with appropriate isolating filters will be adequate if not actually superior to expensive and far more complicated sources providing a continuum and requiring a quartz monochromator. In some commercially available instruments, where resolution has been sacrificed for extremely high sensitivity, the intensity of the exciting radiation is not a n important consideration, and the source used is a matter of choice. It is also possible that nonspecific fluorescence may respond in a manner different from that of the important compound, thus permitting the calculation of correction equations; in instances of this kind, n-ide latitude in the choice of excitation wave length may be an advantage. Efficiency of Various Acids in Fluorescence Reaction. After establishing that t h e 436-mp mercury line is the most effective means under the present experimental conditions of exciting fluorescence with steroids dissolved in strong acids, the efficiency of various acids as fluorogenic systems was studied. Formic arid is a poor fluorescence medium for a nide variety of steroids (12). Phosphoric acid (85%), polyphosphoric acid, and the Bates and Cohen sulfuric acid procedure ( 2 ) were compared. The latter consists of heating the steroid tvith 1 ml. of 90% (I-. sulfuric acid, cooling, and diluting R itli 4 nil. of 65y0 + v.) sulfuric acid. With each steroid, the peak intensity of the sulfuric acid fluorescence spectrum was arbitrarily set to 100 scale units, and the spectra of the 85% phosphoric acid and polyphosphoric acid reactions were compared to it. The fluorescence intensities a t peak wave length are shown in Table 111. With the exception of corticosterone in polyphos-
+
(17.
17.)
phoric acid, sulfuric acid appeared to be the superior agent. Because sulfuric acid performed so favorably, can be redistilled for e\treme purity with great ease ( I S ) , and is less viscous and therefore easier to handle than polyphosphoric acid, more detailed studies of its fluorogenic properties mere performed. Concurrent n ith these studies of the corticosteroids, investigations of the rstrogen-sulfuric acid readion (4) indicated that the tno-stage procedure of Rates and Cohen did not offer any advantage over single-stage reactions n ith a suitable concentration of sulfuric acid. Consequently further experiments were limited to the reaction of corticosteroids (at the same concentration of 10 y per ml.) in 90, 80, 70, and 607, (v. v.) sulfuric acid a t various temperatures and for various time intervals. Using hydrocortisone in 90% sulfuric acid developed for 20 minutes a t 100" C. as the standard, fluorescence spectra were determined for the five corticosteroids reacted in four concentrations of sulfuric acid a t 23" C. for 2 hours and at 100" C. for 20 minutes, The peak fluorescence intensities are recorded in Table IV. The individual behavior of each corticosteroid under thcse varying conditions is clearly evident. For corticosterone and hydrocortisone, room temperature reaction is more advantageous than high temperature developmmt ; in 90% acid the t m develop about equal fluorescence, while in 70% acid corticosterone it is nearly five times as fluorescent as hydrocortisone. After heating a t 100" C. for 20 minutes, corticosterone and hydrocortisone lose their pre-eminent fluorescence, and all the corticosteroids with the exception of cortisone have about the same order of fluorescence intensity. The superior performance of corticosterone and hydrocortisone after room temperature reaction is not in accord with predictions made on the basis of absorption spectra, which show greater absorbance in the 436-mk region after high-temperature reaction in either sulfuric or polyphosphoric acid, S o explanation for this di crepancy i immediately apparent. Table IV shows that the optimum system for corticosterone fluorimetry is room temperature reaction in i O % (v. v.) sulfuric acid for 2 hours, irradiation with the 436-mk mercury line, and measurement a t 525 m p . The concentration of acid is fairly critical. For hydrocortisone, 80% sulfuric acid is apparently superior, although the difference over the range of 90 to 70% acid is relatively small. Because corticosterone and hydrocortisone are the major secretory products of the adrenal gland, experimental situations might arise n here it would
+
+
be sufficient to measure the sum of these compounds (on a weight basis) without actual separation. The two compounds behave differently under changing development conditions, so it should be possible, with some sacrifice of sensitivity, to find a point a t which both substances fluoresce with equal intensity. An experimental series described in Table V indicates that a temperature of 45" C. for 20 minutes will fulfill these requirements. On the other hand, the problem may demand that contamination of one steroid by the other be minimized during fluorimetry. I n the nieasurement of corticosterone, room temperature reaction and io% acid will minimize the effect of contamination by hydrocortisone, while in measurement of the latter, 100" C. reaction in 90% acid will minimize contamination by corticosterone. It may conceivably be desirable t o measure the sum of all the corticosteroids 1%-ithoutseparation. The data of Table Is' do not indicate the presence of a sulfuric acid system in m-hich the fluorescence intensity of cortisone can be raised to the level of the other four corticosteroids. If cortisone is omitted from consideration, a sulfuric acid system using acid somewhere bet-a-een 70 and 807, and high-tempera-
Table IV.
ture reaction might fulfill these requirements. It is evident, therefore, that the conditions for the development of fluorescence may be tailored to meet the requirements of the particular experimental situation. Quantitative Aspects of Fluorescence Spectra. Prior t o the developnient of a spectrofluoiometer which incorporated iiieans for electronic amplification, certain problems of quantitation could not be attacked successfully. Amplification of the signal by
Table 111. Fluorogenic Efficiency of Various Acids Reacted with Corticosteroid
Sulfuric Acid
+
+ v.)
85'7, Phoi-
phoric Acid 80 69 12
Cortisone 100a 42 Tetrahyd '0hydro100a 61 62 cortisol e Tetrahj-d '0cortisol e 10 o a 21 8 .Irbiti ary intensity setting for each steroid.
Fluorogenic Efficiency of Sulfuric Aciti 90% (v.
Polv-
(rj phds(90-56 phoric Compound 6 3 7 ~ ) Acid Corticostrirone 100e 135 Hydrocortisone 10W 88
a t Various Concentrations
-4cid Concentrr tion 807 0
__
70%
60 70
Intensity Wave Kave Wave Wave arbitrary length, length, length, length, Compound units mp Intensity mp Inteisity mp Intensity m p A. Development in sulfuric acid at 23" (I!. for 120 minutes Corti258 525 535 525 9.5 525 60 325 costerone Hydro248 530 2 0 526 34 ... cortisone "0 532 Cortisone 8 ... 3 ... 2 ... 2 ... Tetrahydrohydro74 525 58 525 , ;8 525 19 cortisone Tetrahrdro2 .?I 525 6 ... 2 ... 1 cortisone B. Development in sulfuric acid at 100" 12.for 20 minutes Corti41 540 40 537 530 121 530 costerone Hydrocortisone 100a 537 76 530 ::8 525 69 ,526 10 ,545 5 ... 3 ... 2 ... Cortisone Tetrahvdrohydro28 525 cortisone 46 525 44 525 :i2 525 Tetrahydrocortisone 47 595 33 325 :!0 325 14 525 a -1rbitrar)- intensity standard, identical for A and R. Fluorogenic Efficiency of 80% Sulfuric Acid under Various Conditions o f Incubation 23" C. for 40" C. for 60" C. for 80" C. for 100" C. for 120 Min. 20 A h . 20 3Iin. 20 l l i n . 20 llin. Compound I,,, M p I M p I M p I M p I M p Corticosterone loo5 525 77 525 21 525 12 525 12 530 Hydrocortisone 58 528 55 530 32 530 29 525 24 537 Table V.
5
Arbitrary intensity standard.
VOL. 30, NO. 5, MAY 1958
965
i
I
E NGTH
Figure 6. acid
Fluorescence spectra of estrone in sulfuric
increasing the width of the optical slits introduces a certain amount of distortion due in part to the change in resolution of the instrument. The use of an interrupted photomultiplier signal and subsequent alternating current amplifier has removed these difficulties. It is recognized, however, that the 13cycle Perkin-Elmer system (originally designed for the slow response of thermocouples) involves complicated electronics as a result of the lorr-frequency alternating current; the cost and complexity of these components could be greatly reduced by the use of a fast chopper and commercially available sialile linrw alternating current amplifiers. (In a current modification of a new spectrofluorometer design a direct current thermocouple is being incorporated into the instrument so that measurements can be made of input energy.) The fluorescence spectrum varies in shape depending on the concentration of the fluorescent molecular species, just as the absorption spectrum does under analogous circumstances. This is illustrated by various concentrations of estrone in sulfuric acid in Figure 6, A . (Similar families of curves 11ere determined for the corticosteroids, but because the problem of self-absorption has not yet been considered this point was illustrated with a steroid showing intense fluorescence a t low concentrations.) The fact that these curves are identical except for amplitude may bc verified by increasing the electronic g:iin of the instrument as shown in Figure 6, B , ahere it has been adjusted for each sample so that the peak intensity of all the samples is approximately the same. The identity of the spectra at various concentrations is evident. In Figure 6, -4 and B , the concentrations of estrone are 1.25, 0.625, 0.313, 0.125, and 0.025 y per nil. of 85% sulfuric acid. llruch information may be obtained 966
(rn,u)
ANALYTICAL CHEMISTRY
WAVE L E N G T H
(m))
Figure 7. Fluorescence spectra of corticosterone in sulfuric acid
by equalization of peak fluorescence intensities through amplification--e.g., when a fluorescence spectrum with two peaks is observed, increasing dilutions of the fluorescent compound, compensated by increasing amplification, is the siniplest way of deterniining whether there are two fluorescent molecular species present or whether there is simply a strong band of self-absorption simulating two fluorescence peaks. This may occur with estradiol-17p or estriol under certain conditions. I n studying the quantitative aspects of a fluorescence reaction a i t h a filtertype fluorometer, deviations from linearity a t increasing concentration may occur, and they are customarily ascribed to self-absorption. This may be an incorrect assumption (8) and must be proved by absorption measurements carried out over a spectral distribution comparable to that of the analyzing filter, a tedious and cumbersome task. d simpler way is to record the fluorescence spectrum a t several concentrations, keeping the peak fluorescence intensity constant. This is illustrated for 33.3 and 1.67 y of corticosterone in 90 65% sulfuric acid, amplified approximately to the same peak intensity (Figure 7). d diminution of fluorescence occurs with greater concentration in the region between 455 and 480 mp, rrhich corresponds to the absorption band of corticosterone in sulfuric acid. A filter fluorometer using an analyzing filter with a band pass a t 520 to 560 mG should not experience any distortion of linearity in the calibration curve as a result of self-absorption. The precise determination of peak fluorescence wave length may be difficult in the case of substances of low
+
fluorescence intensity unless high sensitivity or amplification is available, as illustrated by the lonest and highest curves of Figure 6, A . SUMMARY AND CONCLUSIONS
Both the absorption and fluorescenet) phenomena TT hicli occur when steroids react with strong acids are influenced by acid concentration, time, and temperature of incubation. Sufficient flexibility is provided by these variables to permit the tailoring of the reaction to the desired experimental conditionse.g., the fluorescence reaction with sulfuric acid can be adjusted to minimize errors in the measurement of one steroid due to contamination with another. The fluorescence intensities can be adjusted so that the major corticosteroids (except cortisone) ha1 e nearly the same fluorescence intensity. niaking feasible an approximate measurement of the total corticosteroid content of a nii.;ture on a n-eight-forweight basis. The fluorescence spectrum has bren studied in relation to the exciting radiation and its independence from the wave length used for excitation verified. The implications of this fact with respect to the design of fluorometers arc clear: For most purposes an inexpensive mercury arc with appropriate interference filters should prove as useful as a rontinuum source yielding a selection of less intense exciting wave lengths by means of a quartz monochromator. The utility of signal amplification by electronic means has been illustrated in studies of the fluorescence spectrum as it is affected by concentration change and self-absorption effects.
Ibid., p. 182. Bauld, W., Engel, L. L., Goldzieher, J. W., Can. J . Biochem. and Physiol. to be published. Bernstein, S., Lenhard, R. H., J . Org. Chem. 18, 1146 (1953). Ibid., 19, 1269 (1954). Braunsberg, H., J . Endocrinol. 8, 11 (1952). Brsunsberg, H., Osborn, S. B., Anal. Chim. Acta 6. 84 11952). Diczfalusy, E., Act‘a Endoc;inol. Suppl. 12, 1 (1953). Finkelstein, M., Ibid., 10, 149 (1952). Goldzieher, J. T.,Endocrinology 53, 520 (1953). Goldzieher. J. W., Bodenchuk. J. M.. Kolan, P., A N ~ L CHEX. . 26, 853
ACKNOWLEDGMENT
The authors are greatly indebted to Kenneth Savard, University of Miami for the analyses of purity of the corticosteroids, to Merck, Sharp & Dohme, and Schering Corp. for their generous supplies of steroids, and to Sabri el Farra and Taylor Chandler, Jr., for technical assistance.
LITERATURE CITED
(1) Axelrod, L. R., J . A m . Chem. Soc. 75, 6301 (1953). (2) Bates, R. W.,Cohen, H., Endocrinology 47, 166 (1952).
(lCI54). \ - - - - I -
(13) Goldzieher, J. W.,Bodenchuk, J. M., Nolan, P., J . Biol. Chern. 199, 621 (1952).
(14) Kalant, H., Biochem J . 63, 101’ (1956). (15) Linford, J. H., Can. J . Biochem. and Physiol. 35, 299 (1957). (16) Sowaczynski, W. J., Steyermark, P. R., Arch. Biochern. BLophys. 58, 453 (1955). ( l i ) Kowaczynski, W. J., Steyermark, P. R., Can. J . Bzochem. a n d Physzol., 34, 592 (1956). (18) Sweat, M. L., ASAL. CHEX.26, 7i3 (1954): (19) Zaffaroni, A , J . Am. Chern. Soc. 72, 3828 (1950). ( 2 0 ) Zaffaroni, A., Recent Progr. Hormone Research 8 , 51 (1953).
RECEIVED for review September 13, 1957. Accepted December 30, 1957. Study supported by funds supplied by the School of Aviation Medicine, U. S. Air Force, under contract AF 18(600)-921.
Volumetric Determination of Aluminum in Presence of Iron, Titanium, Calcium, Silicon, and Other impurities H. L. WATTS Alcoa Research laboratories, Aluminum
Co. o f America, Easf Si. louis, 111.
b In highly basic solution, aluminum forms soluble sodium aluminate, and iron and titanium precipitate as hydroxides. When aluminum reacts with fluoride, the hydroxide which combines with the aluminum cam be released for titration with standard acid. The titration can b e performed in the presence of the precipitated hydroxides. Interference from calcium can b e eliminated b y precipitating i t as the oxalate. By limiting the sample size, aluminum can be determined in the presence o f silicon dioxide. The method i s fast, precise, and accurate.
T
CLASSICAL procedure for the determination of aluminum oxide (or aluminum) in bauxite involves the determination of R203 (the ignited residue obtained in the ammonium hydroside precipitation method), ferric oside, titanium dioxide, and phosphorus pentoside. The amount of aluminum oxide is calculated by subtracting the sum of the other oxides from the Rz03 value. This method is slow and accuracy of the aluminum value depends upon the accuracy of the R203and other determinations. Also, the accuracy is adversely affected by the presence in the R 2 0 n precipitate of small amounts of impurities that are not determined. Many authors have advocated a preliminary digestion with sodium hydroxide, follon-ed by filtration of the insoluble iron and titanium hydroxides. However, the highly basic solution necessary to convert all of the aluminum hydroside to soluble sodium alumiHE
nate is often difficult to filter, and the accuracy and precision are seldom acceptable. Killard and Diehl(6) briefly described a private communication from M. L. n’indle for the routine volumetric determination of aluminum in the presence of iron, cobalt, and nickel. The solution was adjusted to pH 7 , potassium fluoride was added, and the released hydroxide was titrated back t o p H 7 with standard acid. A volumetric method for the determination of aluminum in the presence of fluoride, zirconium, and uranium was developed by Paige, Elliott, and Rein (4). Paulson and Murphy (6) used an indicator method for the determination of aluminum after its separation from iron and chromium. I n both methods, the reaction, though not quite stoichiometric, is represented by the equation : A102-
+ 2 H20 + 6 F-
+
AlFs---
40H-
+
(1)
The highest recommended aluminum concentration in either method is 11 mg., and the largest amount of potassium fluoride is 20 ml. of a 7% solution. A fast, accurate method for the determination of aluminum in bauxite and other aluminous materials was desired. Because bauxite often contains as much as 55% aluminum oxide, good precision would require a method with which amounts of aluminum larger than 11 mg. could be titrated. Also, it was desired to avoid the separation of iron and other impurities.
Busliey ( 1 ) showed a series of tunc's for the titration of sodium aluminate solutions with hydrochloric acid. I n Figure 1, a curve for free sodium hydroxide shows that the equivalence point, a t vhich aluminum hydroxide starts to precipitate, occurs a t pH 9.9 for 0.11 gram of aluminum oside. Bushey found that the pH of the equivalence point decreases with lower concentrations of aluminum oside. Therefore, a t pH 10 and above, aluminum is present as soluble sodium aluminate. When the pH of a sample is adjusted to above 10, iron and titanium precipitate as the hydroxides and aluminum dissolves completely as sodium aluminate. This reaction is the basis of several methods, including those involving filtration of the hydroside precipitates (3). HoFT-ever, a separation is unnecessary for the iron and titanium concentrations present in bauxite. Aluminum can be determined volumetrically by titration of the hydroside released by the addition of fluoride (Equation 1). The pH values of 10.0, 10.5, and 11.1 were investigated as the start and finish points of the titration. As shown in Figure 1, the pH relationship permitted sharper end points a t pH 10.0 than a t any higher pH. Impurities-e.g., iron, fluoride, and phosphorus-have considerably less effect a t pH 11.1 than a t pH 10.0. However, on actual samples of bauxite, no significant difference in accuracy was obtained whether the start and finish points were a t p H 10.0, 10.5, or 11.1. Because the sharper VOL. 30, NO. 5, MAY 1958
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