Phosphoryl Chloride Enhancement of the Fluorescence of Steroids in Sulfuric Acid SIR: Numerous compounds of biological interest fluoresce under various conditions. Goldzieher, Bodenchuk, and Nolan (3) have published an extensive list of the emission maxima of steroids in sulfuric acid activated by the 436-mp mercury line. Goldzieher and Besch (2) have recently published a critical study of the conditions for fluorometry of hydrocortisone, cortisone, and their “tetrahydro” derivatives. Sweat (5) reported t h e quantitation of hydrocortisone and corticosterone by measurement of their fluorescence in sulfuric acid. The fluorescence of the estrogens under various conditions is a well known phenomenon. However, many steroids in sulfuric acid have a fluorescence of such low intensity as to be valueless for quantitative purposes. The addition of phosphoryl chloride to the sulfuric acid used for fluorometry causes increased intensity of Auorescence of certain steroids. This was particularly so for the estrogens when compared t o the dilute sulfuric acid reagents of Jailer (4) or Diczfalusy (1). Aldosterone, digitoxin, and estrone showed a greatly increased intensity of fluorescence with the modified sulfuric acid reagent. The enhancement of fluorescenee by phosphoryl chloride was evident for substance S at room temperature. Some of t.he cortical steroids such as cortisol and cortisone did not show an increase on treatment a t room temperature. Corticosterone showed a slight increase at room temperature, but there was a loss on heating. The treatment with phosphoryl chloride caused little or no change in the wave length of the maxima of the activating or emitted light, when compared to the spectrum of the steroid in sulfuric acid alone. Data obtained with varying concentrations of steroids with phosphoryl chlo-
Table I.
Steroid Substance S
S-Glycol’ D-L-Aldosteronec Corticosterone Cortisone Cortisol
Phosphoryl Chloride-Enhanced Fluorescence of Steroids Fluorescence Intensity, Arbitrary Units/r Wave Length,a M p 30% POCla Activation Emission H,SO, only in H2SOI Not heated 460 510 0 88 7 2 450 500 1 6 8 0 450 510 8 0 11 2 460 510 16 0 21 G 410 480 4 0 1 5 460 510 48 0 32 0 I
Heated l l O o , 30 min. Estrone 440 480 so 0 Androsterone 450 490 2 2 Digitoxin 460 510 6 4 Progesterone 460 520 20 Testosterone 460 515 2 4 4 All determinations made on Farrand Model 104224 spectrofluorometer.
150 0 8 0
12 0 3 0 2 7
* 4Pregnene-17a,20&2l-triol-3-one.
Synthetic aldosterone supplied by Ciba Pharmaceutical Products, Inc , through Endocrine Study Section, Sational Institutes of Health. ride in sulfuric acid indicate that the intensity of fluorescence is proportional to the concentration of the steroid, subject t o limitations such as self-absorption because of too concentrated solutions. Table I lists preliminary results obtained with representative steroids using 30% phosphoryl chloride in concentrated sulfuric acid under stated conditions which are not necessarily optimal for the steroids listed. The values represent actual fluorescence intensity above that shown by the blank. Blanks have not been higher than that shown by sulfuric acid alone. Table I presents the maximal wave lengths of the activating and fluorescence spectra obtained by scanning the solutions from 220 t o 650 mp, on the Farrand Model 104244. Activation a t 436 mp does not appear t o give optimal fluorescence for all the steroids under the conditions used in these experiments.
LITERATURE CITED
(1) Dicxfalusy, E., A d a EndocrinoZ. CopenhagenSuppl. 12,85 (1953). (2) Goldzieher, J. W., Besch, P. K., ANAL.CHEX 30,962 (1958). (3) Goldzieher, J. K., Bodenchuk, J. N., Solan, P., Ibzd., 26, 853 (1954). (4)’ Jailer, J. W.,J . Clin. Endocrind. 8 ,
564 (1947). (5) Sweat, &I. L., A X I L C H E v 26, 773 (1954). JOSEPH C TOKCHSTOXE ROBERTA KEISMAN ARNOLDF MARCANTONIO JOHN W. GREESE,JR. De artment of Obstetrics and 8ynecology and Robinette Foundation, Department of Medicine School of Medicine University of Pennsylvania, Philadelphia, Pa. RECEIVED for review Julv 7 , 1958. Accepted J u I ~29, 1958. fVork supported in part by USPH Grant A-1509, the Heart Association of Southeastern Pennsylvania and the John G. Clark Memorial Fund.
Near-Infrared Data on Oximes at 2.78 Microns SIR: Several recent contributions from this laboratory have reported the application of near-infrared spectrophotometry t o the determination of terminal unsaturation (d), terminal epoxides (4), and phenolic hydroxyl (3). During our research on further applications of the near-infrared region (1.0 to 3.1 microns), we have run a variety of oximes in t h e fundamental hydroxyl
region and determined the wave length of their absorption maxima and their molar absorptivities (Table I). This information may be of value t o others engaged in research involving these compounde. T h e data in Table I indicate that oximes have an extremely intense fundamental hydroxyl band at 2.78 microns. The molar absorptivities are three to
four times those of most alcohols and hydroperoxides, and are roughly equivalent t o those of phenols ( 3 ) . From the limited data, it appears t h a t dilution affects the absorptivities of the oximes of ketones more than the oximes of aldehydes. With the oximes of ketones, it will thus be desirable to work from a calibration curve. Because of the high molar absorpVOL 30, NO. 10, OCTOBER 1958
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