(9) Haines, W. J., liecent Progr. in Hormone Research 7, 255 (1952). (10) Hollander, T’. P., Vinecour, J., AXAL.CHEM.30,1429 (1955). (11) Huang-hlinlon, Wilson, E., Wendler, N.L., Tishler, M., J . Am. Chem. SOC. 74,5394 (1952). (12) Kliman, B., Peterscn, R. E., J . Bzo2. Chem. 235, 1639 (1960). (13) Peterson, R. E., Ibzd., 225, 25 (1957).
(14) Peterson, R. E., Proc. Symposium o n Advances in Tracer Applications ~j T r i t i u m New England Suclear Corp.,
Boston, Mass., p. 16 (1958). (15) Simpson, S. A., et al., Helv. Chim. d c t a 37, 1163 (1954). (16) West, C. D., Damast, B., Pearson, 0. H., J . Clin. Endocrinol. and Metab. 18, 15 (1958). (17) West, W. G., J . B i ~ l Chem. . 238, 94 (1963).
(18) Zaffaroni, A., Recent Progr. in Hormone Research 8 , 51 (1953). RECEIVEDfor review August 17, 1962.
Accepted May 27, 1963. This investigation was supported by grants from the American Cancer Society and Xational Cancer Institute, C.S.P.H.S. Portions of this work are included in a Thesis by J. R. Seely submitted in partial fulfillment of the requirements for the Ph.D. degree in the Department of Biological Chemistry, University of Utah School of Medicine.
Chromatographic Separation of Mixtures of Vitamin D2, Ergosterol, and Tachysterol2 ANTHONY W. NORMAN and HECTOR F. DeLUCA Department o f Biochemistry, University of Wisconsin, Madison 6, Wis. b Two new methods for the chromatography of vitamin D and the other compounds produced by the ultraviolet irradiation of ergosterol or 7dehydrocholesterol c re described. One involves thin-layer chromatography and i s able to separate at least six compounds from such an irradiation mixture. The other, a silicic acid column chromatcgraphic procedure, adequately separates mixtures of at least 25 mg. of vitamin D and its parent irradiation mixture into at least four compcments.
D
a n attempt to prepare and purify radioactivcb vitamin D, the lack of suitable chromatographic systems for the isolation, identification] and demonstration 01 radiochemical purity became apparent. This was particularly evident when the isolation of vitamin D from a mixture resulting from the ultraviolet irradiation of ergosterol or i-dehydro :holesterol was desired. The fact that these compounds differ primarily in conf guration rather than in functional group or molecular weight makes their chromatographic separation very difficult. -1 number of paper chromatographic techniques for vitamin D have been delised ( l b ) employing either quinoline-imprcgnated papw ( 1 , 14) or rwersed-phase paper 12, 18). Unsatisfactory resolutions, long development times, and limited capacity were found to be the primary drawbacks of t h e v techniques. Column chromatography has been used lvith varying degrees of succes. Alumina (4, 171, Floridin ( 6 ) ,and Celite (2, 3, 6, 11) have been the primary adscrbants. These methods were devised for the separation of classes of compounds rather than individual componenti. I n the case of the alumina columns 14, 17‘) difficulty has been experienced in reproducibly URING
“weakening” the alumina. Gas chromatography of vitamin D has also been carried out (21). However, it is not satisfactory for a study of vitamin D metabolism] since interconversions of the compounds occur during sample vaporization prior to adsorption on the column. It was therefore evident that to prepare, identify, and study the metabolism of vitamin D, more satisfactory chromatographic procedures were necessary. This paper describes two methods] one employing a silicic acid column and one utilizing thin-layer chromatography, both of which are capable of separating milligram quantities of vitamin D from its parent irradiation mixtures. METHODS
Thin-Layer Chromatography. Thinlayer chromatography apparatus and Cab-0-Si1 silica gel (Research Specialties Co., Richmond] Calif.) were employed in all t h e experiments described. T h e silica gel was applied t o t h e plates in t h e manner designated by t h e manufacturer. This gives a uniform layer approximately 250microns thick. The silicic acid on the plates was activated at 140’ C. for a t least 16 hours before use. All compounds employed for the R , determinations mere crystalline. Vitamins Da and Da, ergosterol, 7-dehydrocholesterol, and dihydrotachyqterol were commercial preparations. Ergosterol acetate, cholesterol acetate, and vitamin Dz-3,5,-dinitrobenzoate were prepared in this laboratory and recrystallized three times to a constant melting point ( 1 7 9 O , 11601 and 149” C., reipectivelg) which agreed satisfactorily n-ith literature values (8, 9,20). The irradiation mixture was prepared in the following manner. One hundred milliliters of a n ergosterol .elution in peroxide-free diethyl etliet (50 lug. per 100 nil.) was placed in a 250-ml. round-bottomed quartz f l a k connected
to a reflus condenser. This was exposed to ultraviolet rays from a Model S-2303 Hanovia Alpine sun lamp (Hanovia Lamp Division, 100 Chestnut St., Kewark, N. J.) a t a distance of 15 cm. for 8 minutes. The solution was then stored a t -25’ C. under nitrogen until used for chromatographic analysis. Approximately 10 to 20 fig. of the compound in 2 p1. of organic solvent was applied to t h e silicic acid on the plates, 2.8 em. from t h e bottom. The plates were placed in a glass chromatographic chamber so t h a t they were immersed to a depth of 1.0 em. After approximately 35 minutes, when the solvent front was 10.0 em. above the origin, the plates were removed from the chamber, allowed to air-dry 5 to 10 minutes, then sprayed with either 0.2% K h f n 0 4in 1.0% KazC03or 0.2M H2S04. The plates sprayed with the N2SO4 mere heated n i t h two 250-watt heat lamps (General Electric Co.) for 15 minutes to develop the spotq. The &So4 spray gives a permanent record, but greater quantities of compound are needed for detection relative to the Kbln04 spray. The compounds separated by the thinlayer technique were idenbified by comparison of the R f values with standards and by the folloTving technique. Initially 1 to 2 mg. of the ergosterol irradiation mixture was streaked in a very narrow band across the complete width of the plate a t the origin. The separation was carried out as described previously. After drying, the plate was covered with a sheet of paper t h a t left 1-cm. strips exposed on both edges. These were sprayed to locate the bands of separated compounds. Then, wing the bands on the edges of the plate as markers, strips of unsprayed silicic avid were scraped off with a microscope slide. The compounds were eluted with 95 to 100% recovery from the silicic acid scrapings with acetone, the silicic acid was removed by centrifugation, and the acetone evaporated with it stream of nitrogen and replaced n ith 95% ethanol. tTltrariolet absorption of all fractions VOL. 35, NO. 9, AUGUST 1963
1247
was finally determined. The curves observed for vitamin Dz, ergosterol, and tachysterol agreed satisfactorily with those reported in the literature (8,IS). Adsorption Values. BioRad silicic acid, - 325-mesh (California Corp. for Biochemical Research, Los Angeles 63, Calif.), was used for t h e determination of adsorption values and as t h e adsorbant for column chromatography. The most feasible solvent system for t h e separation of vitamin D and ergosterol was systematically studied by determining the adsorption coefficients, utilizing a modification of Trappe's method (19). Five milligrams of vitamin Dz or ergosterol was shaken n i t h 1.50 grams of silicic acid in 10.0 ml. of the solvent under examination. Various concentrations of diethyl ether (analytical reagent grade), in redistilled Skellysolve B (Phillips Petroleum Co. preparation, primarily n-hesane), b.p. 65-70' C., were used for the determinations. After being shaken at 5-minute intervals for 1 hour a t 24' C., the silicic acid was allowed to settle and 1.0-mi. aliquots of the separated fluid were withdrawn for analysis. The solvent was evaporated with a stream of nitrogen and the residue was dissolved in 95% ethanol. The absorbance of vitamin Dz at 264 mb or ergosterol a t 282 mp was measured in a Cary 11 spectrophotometer and the degree of adsorption of the two compounds calculated for the various solvent combinations. Column Chromatography. Columns 58 x 1.5 cm. were prepared by slurrying 24.0 grams of BioRad silicic acid, -325 mesh, in redistilled Skellysolve B. The column was compressed gently under a pressure of 10 p s i . Columns prepared in this manner had a holdup volume of 20 ml. and a flow
Table I. RI Values of Vitamin D and Related Compounds Following ThinLayer Chromatography Solvent" Compound A B CVitamin D, 0 . 3 3 0.44 0.15 Vitamin D; 0.32 0.44 0.15 Ergosterol 0.27 0.35 0.12 7-Dehydrocholesterol 0 . 2 7 0.35 0.12 Dihvdrotachvsterol 0.49 0 . 7 5 0.24 Chdesterol 0.30 0.41 . . . Cholesterol acetate 0.98 0.99 0.86 Ergosterol acetate 0.97 0.99 0.79 Vitamin Dz-3,5-dinitrobenzoate 0.96 ... 0.56 Irradiation mixture Spot I (tachysterol) 0.46 0.64 ... Spot I1 (vitamin D) 0.33 0.44 Spot I11 (ergosterol) spot IV spot v
spot VI
0
0.27 0.12 0.09 0.00
0.35 0.20 0.15 0.00
...
... ... ...
A = 10% acetone in Skellysolve B, B = lOOv/, chloruform; C =
Solvent:
10% A c o t o n c i n n - H e x a n e
0
z $ De
ERG.
IRRADIATION
M I X T URE
ERG.
1248
ANALYTICAL CHEMISTRY
VIT.
D2
ERG.
Figure 1. Thin-layer chromatography of ergosterol solution subjected to ultraviolet irradiation Solvent.
10% acetone in Skellysolve 8 (v./v.)
Left to right. Columns 1, 2. 3, 4 .
Vitamin D2 and ergosterol Ergosterol 5, 6, 7, 8. lrrodotion mixture 9 , 10. Vitamin Dz 1 1 , 12. Vitamin D2 and ergosterol
rate of 1.0 ml. per minute under a pressure of 150 cm. of water. Samples of up t o 25 mg. were dissolved in B volume of Skellysolve B never exceeding 10 ml. and allowed t o flow on the column by gravity. Elution was carried out with 10% diethyl ether in Skellysolve B (v./v.). Ten-milliliter fractions were collected. Although vitamin D absorbs maximally at 264 mp and ergosterol absorbs maximally at 282 mp, both compounds absorb significantly at 270 mp. Therefore, in order to avoid two absorbancy readings, the absorbance at 270 mp was determined on all fractions after the solvent had been removed with nitrogen and replaced with 9501, ethanol. The peaks found by the monitoring at 270 r n p were precisely identified by determining the ultraviolet absorption curve from 200 to 300 mp on all the fractions in question. The curves observed for vitamin D2, ergosterol, and tachysterol agreed satisfactorily with standards and those reported in the literature (8, 1s).
Tritium radioactivity, where measured, was determined on a n aliquot of the ether-Skellysolve B fractions by standard techniques in a Packard Model 314EX liquid scintillation counter. A counting solvent containing 100 mg. of dimethyl POPOP [dimethyl-2,2-p-phenylenebis-(5-phenyloxazole)] and 3.0 grams of PPO (2,5-diphenyloxazole) per liter of toluene (analytical reagent grade) was employed.
v./v.;
byo acetone in Skellysolve €3, v./v. Spots of irradiation mixture labeled I-VI refer to chromatogram shown in Figure 1.
0 O C I
RESULTS AND DISCUSSION
Thin-Layer Chromatography. The R I values for t h e compounds ex-
amined are reported in Table I. T h e three most effective solvent systems were found to be 5 % acetone in Skellysolve B (v./v.), 10% acetone i n Skellysolve B (v./v.), and 100% chloroform. The RI values reported are averages of a t least nine determinations. Some variation in the R f values from plate to plate was noticed; but this variation was not great enough to warrant the inclusion of standards on every plate. This difference was felt to be due to irregularities in the thickness of the silica gel and to temperature fluctuations. Another variable affecting the R/ values was the length of time the plates were left at room temperature after removal from the oven. If the plates were allowed to stand more than approximately 45 minutes, the RI values markedly increased. This was probably due to absorption of moisture from the air by the silicic acid, thus weakening the acid's adsorption capability. Figure 1 demonstrates a typical chromatogram of an ultraviolet irradiation mixture of ergosterol. Six compounds can be found in the crude irradiation mixture. Only spots I, 11, and 111 (Table I) could be identified as tachysterol, vitamin D, and ergosterol, respectively. The other spots (IV, V, and VI) were present in small quantities relative to spots I, 11, and 111, and as a consequence were not identified. I t ih conceivable that one of the unidentified minor components is lumisterol, since it is definitely known to be present
I
/
I
/
I
,
HABSORBANCE 0-0RADIOACTIVITY
/
I
,
I
,
/
l
1
1
200,000CPM A
55 50
0
5
2 40 5 45,
35 I 30 f 25 20 15 5
5 ; IO c
IO 20 -30 40 50 PERCENT ETHI-R IN n-HEXANE
5
Figure 2. Adsorption of vitamin DZ and ergosterol on silicic acid 0 Ergosterol
0 Vitamin
D 5 m g . o f viiamin DB or ercosterol equilibrated with 1.50 grams of silicic acid in 10.0 ml. of solvent under examinaticm
0
FRACTION NUMBER (IOrnl)
Figure 3. Silicic acid column chromatography of H3-ergosterol subjected to ultraviolet irradiation 0 Absorbance a t 270 rnM Radioactivity Fractions 1 to 80 eluted with solvent of 10% ether in Skellysolve B (v./v.] Fractions 81 to 100 eluted with acetone
0
in the irradiation mixtures of ergostcrol (’7). The other n-inor components may be previtamin D) and 5,6-transvitamin Dz, as they are also believed to be formed from irradiated ergosterol (13). The same type of chromatogram was observed when the 7-dehydrocholesterol irradiation mixture was subjected to thin-layer chromatography. The chief advantage of the thin-layer chromatographic system is its remarkable ability to separitte compounds of the vitamin D family. The best procedure formerly a v d a b l e (11) gave only three spots with our ergosterol irradiation mixture ar, compared to the six and sometimes seten spots observed with the present system. Other advantages unique to this method are the rapidity with which separation may be carried out and the wide range in amount of compound which may be separated. -4mounts of material as low as 1 pg. have been detected with the KMnOl spray; yet up to 2 mg. have also been spread across the plate and chromatographed. Column Chromatography. The d a t a in Figure 2 demonstrate why it has been difficult h i devise column chromatographic methods for the successful separation of vitamin D2 from ergosterol or other similar compounds. T h e differcnce in degree of adsorption between vitamin D) and ergosterol on silicic acid is exceedingly small. A t first glance i t would appear that no particular solvent stands out as more favorable for the separation of vitamin Dz and ergosterol. However, if the data in Figure 2 are utilized in the manner suggested by Johnson (IO), it can be calculated readily that to obtain a good sep,tration, a solvent having the properties of 10 to 20% diethyl ether in Skellysolve U must be used. If solvents with much more than 20% of diethyl ether in Skellysoh-e I3 are employed, inordinately long columns would be required. These long columns would have prohibi1,ively slow flow
rates for any practical use. On the other hand, if less than 10% diethyl ether in Skellysolve B is used, extremely large volumes of solvent would be needed to elute vitamin D2 and ergosterol from the column. The data of Figure 2 are aIso useful in predicting the order of elution of vitamin D and ergosterol from a column. Since vitamin D is not adsorbed as strongly as ergosterol on silicic acid, i t would be expected that vitamin D would appear in the effluent before ergosterol. As a consequence of these considerations, the solvent combination of 10% diethyl ether in Skellysolve B (v./v.) was feit to be the most useful for the separation of compounds of the vitamin D family on columns of silicic acid. Figure 3 shows a typical chromatogram of a mixture resulting from the ultraviolet irradiation of ergosterol labeled with tritium (16). This same type of chromatogram was found when irradiated 7-dehydrocholesterol was chromatographed. Three major identifiable peaks-vitamin D, tachysterol, and ergosterol-were observed. The last peak was obtained when the column was “stripped” with acetone. It gave no identifiable ultraviolet absorption curve and is probably a mixture of several compounds, as is indicated by the very large amount of radioactivity present in these fractions. The difference in specific activities among vitamin D2, tachysterol, and ergosterol probably results from the loss of H8 hydrogens that occurs in the rearrangement of the double bonds under the activation with ultraviolet light. A small unknown peak routinely appeared in the region of fraction 10. A slight variation in the exact position of compounds in the eluent has been observed when different batches of UioRad silicic acid are used. However, these dif-
ferences have not been great enough to entail altering the concentration of diethyl ether in the Skellysolve B eluent. It is not known why the order of compounds eluted from silicic acid columns differs from the order of compounds separated on silica gel thinlayer plates. The difference may be due to the different types of silicic acid employed in the two procedures. Furthermore, the thin-layer chromatography silica gel has a binding agent mixed with it, so that the silica gel will adhere to the glass plates. The two chromatographic procedures described in this paper provide a very sensitive and reproducible means of separating members of the vitamin D family. It is hoped that these techniques will prove useful in the elucidation of possible metabolic conversion products of vitamin D. Moreover, both methods provide new techniques for critically evaluating the chemical purity of vitamin D. ACKNOWLEDGMENT
We are indebted to Henry Scott and Lloyd Hein, Wisconsin Alumni Research Foundation, Madison, Wis., for the use of the Hanovia ultraviolet light and to Harry Steenbock for his continued interest. One of us (A. N.) was the recipient of a Kational Institutes of Health predoctoral fellowship, No. 13,654. LITERATURE CITED
(1) Davis, R. B., McMahon, J. M., Kalnitsky, G., J. Am. Chem. SOC.74, 4483 11952).
(2)%e%itt, ‘ J. B., Sullivan, ICI. X., ANA? CHEM.18, 117 (1946). (3) Ewing, D. T., Schlabach, T. D., Powell, M. J., Vaitkus, J. W., Bird, 0.D.. Zbid., 26. 1406 (1954). (4) Festenstein, G. N.,’ Morton, R. A., Biochem. J. 60, 22 (1955). VOL. 35, NO. 9, AUGUST 1963
1249
(5) Fujita, A,, Numata, K., J . Vitamino[. 4, 299 (1958). (6) Green, J., Biochem. J . 49, 45 (1951). (7) Havinga, E., Verloop, A., Koevoet, A. I,., Rec. Trav. Chim. 75,371 (1956). ( 8 ) Huber, W., Ewing, G. W., Kriger, J., J . A m . Chem. SOC.67,609 (1945). (9) Inhoffen, H., Ann. 508,81 (1934). (10) Johnson, M. J., "Manometric Techniaues." R. H. Burris. W. 1%'. Umbreit. J. 'F. itauffer, eds., Burgess Publishing Co., RIinnea olis, Minn., 1959. (11) Jones, S. Morris, W. W., Wilkie, J. B., J. Assoc. Ofic. Agr. Chetnzsts 42, 180 (1959).
h.,
(12) Kodicek, E., Ashby, D. R., Biochem. J . 57, XI1 (1954). (13) Koevoet, 9.L., Verloop, A., Hwvinga, E., Rec. Trav. Chim. 74, 788 (1955). (14) 'Kritchevsky, D., Kirk, M:R.,J.Am. Chem. Soc. 74, 4484 (1952). (15) Mcllahon, J. M.,Davis, R. B., Kalnitsky, G., Proc. SOC.Exptl. Biol. M e d . 75, 799 (1950). (16) Xorman, A. W.,DeLuca, H. F., in preparation. (17) Shaw, R. H., Jefferies, J. P., Analyst 82,8 (1957). (18) Terepka, A. It., Chen, P. S.,
Jorgensen, B., Endocrinology 68, 996 (1961). (19) Trappe, W., Biochem. 2. 305, 150 (1940). (20) Iyindaus, A . , Ber. 36, 3752 (1903). (21) Ziffer, H , VandenHeuvel, W. J , Haahti, E. O., Homing, E. C., J . Am. Chem. SOC.82, 6411 (1960). RECEIVED for review February 11, 1963. Accepted May 23, 1963. Published with the approval of the Director, Wisconsin Agricultural Experiment Station. Research supported by PHS grant ilM 05800.
Use of Phosgene in the Determination of Hydroxyl Groups in Polymers DAVID G. BUSH, LEO J. KUNZELSAUER, and STEWART H. MERRILL Research laboratories, Eastman Kodak Co., Rochester 4, N. Y.
b
A new analytical procedure is described for the determination of organic hydroxyl groups. Because the method i s capable of high precision and accuracy, it has been applied particularly to the determination of terminal hydroxyl groups in polymers. The hydroxyl-containing material reacts with phosgene to form the chloroformate. After removal of the excess reagent by volatilization, the chloroformate is hydrolyzed with aqueous alkali. The chloride content of the hydrolyzate, which is equivalent to the original hydroxyl content, is titrated with silver using potentiometric techniques. Water does not interfere. The relative error of the method is less than
2%.
f
M
are available for the determination of the hydroxyl content of organic compounds (10). However, if a method is desired for the analysis of terminal hydroxyl groups in a polymer, many of these general methods are no longer satisfactory. This arises from the fact that as the molecular weight increases, the terminal hydroxyl content decrease5 and a highly precise micromethod is necessary for terminal hydroxyl analysis. Some of the general hydroxyl methods have been applied to hydroxyl endgroup analysis in polymers. Several worker5 (5,7 , 8 ) have measured the hydroxyl conce~itratioii i i i poly(propylerie glycols) by infrared spectrophotometry. Lithium aluminum hydride has been used to measure the hydroxyl content of epoxy resins (14). The infrared method and the hydride method both have the dislzdvantage that water interferes in the determina-
1250
ANY METHODB
ANALYTICAL CHEMISTRY
tion. Therefore, for precise results the sample must be anhydrous or the water content must be measured by a separate technique, usually the Karl Fischer procedure, and the final results corrected. Variations of the acetylation method of Ogg, Porter, and Willets (12) have been used by a number of workers. A variety of acylation reagents (2) were evaluated for use in determining the hydroxyl content of epoxy resins. Acidcatalyzed acetylation has been used to determine the hydroxyl number of polyoxyalkylene ethers (16). These methods are all indirect, the amount of hydroxyl is calculated from the difference between the blank and the sample titrations. As a result, high precision for samples of low hydroxyl content is difficult to achieve. Other methods involve the synthesis of a new compound by the reaction of the hydroxyl group with some reagent which contains an easily characterized group. This product is then isolated and analyzed. Some workers (8, 16) have exchanged the hydrogen in hydroxyl groups of poly(ethy1ene terephthalate) for deuterium. The hydroxyl in this same polyester was also measured from the bromine content after acylation with bromoacetyl bromide (6). Another method is based on the conversion of the hydroxyl end groups of polyesters to acid groups by reaction with succinic anhydride (4). These methods have the disadvantage t h a t the polymer may be fractionated with regard to molecular weight during isolation from the excess reagent. The present method is not limited by any of these disadvantages. The chloroformate of the polymer is formed by reaction of the hydroxyl group with
phosgene. The excess reagent is removed by volatilization and the chloroformate is hydrolyzed. The hydrolyzate is analyzed for its chloride content. Water does not interfere. No separation is required which might fractionate the polymer. The final argentometric chloride analysis is direct, accurate, and highly precise. EXPERIMENTAL
Reagents. 1,18-Octadecanediol, m.p. 96' to 98' C., was obtained by reduction of the corresponding diester (1). I-Octadecanol, Eastman Grade, was recrystallized from ethanol and from benzene at 10' C., m.p. 58' to 59" C. Triethylene glycol, Eastman Practical Grade, was fractionated, taking the product boiling a t 167" C. at 15 mm., n D 2 j = 1.4542. All organic solvents used were Eastman Grade. Unless otherwise noted, all chemicals used were reagent grade. Apparatus. The potentiometric titrations were made with a Beckman Model G pH meter. The indicator electrode was a Beckman silver billet type, k39261. The reference electrode was either a Beckman glass electrode, 40498, or a Beckman saturated calomel electrode, $39170, n ith the saturated solution of potas-ium chloride replaced by a saturated solution of potassium nitrate. Procedure. Weigh accurately a sample containing 0.3 t o 0.8 mmole of hydroxyl into a 100-ml. two-necked. round-bottomed flask. Add 50 ml. of a n aprotic solvent (such as benzene, dichloroethane, trichloroethane, or 1,2 dimethoxyethane) to the flask. .Idd a stirring bar, place the flask in a hood over a magnetic stirrer, and start the stirrer. Fill a dry-ice condenqer and place it in one neck of the flask Connect the other neck of the flask to a phosgene tank. Condense
