Analysis of the Photochemical Isomers of Ergosterol by Column Chromatography Re& Mermet-Bouvier Groupe d’Etudes des Effets des Rayonnements sur les Structures M o l h l a i r e s , Dkpartement de Biologie, Centre d’Etudes NuclPaires de Saclay, B.P. N 0 2 , Bztiment 142, 91190-Gif-sur-Ycette. France SEPARATING THE PHOTOCHEMICAL ISOMERS of ergosterol is a tricky experimental problem, since the compounds have the same functional groups and molecular weight. The work carried out by Windaus ( I ) , Velluz ( 2 ) ,and Havinga (3) showed that the ultraviolet irradiation of ergosterol made at a temperature below 10 “C forms five isomers : previtamin D1, lumisterol?, tachysterol*, and toxisterols I and 11. If the irradiation temperature exceeds 10 “C, three additional isomers are formed, which are vitamin Ds and suprasterols I and 11. Several chromatographic methods were used for separating the ergosterol isomers [thin-layer (4-8), paper (9, IO), gaseous phase (11-14), and column chromatography (6, 15-18)]. This last method is described in this paper. Norman and D e Luca ( 6 ) separate vitamin Dl, tachysterolz, and ergosterol on a silicic acid column; the elution is effected with a 10% diethylether mixture in Skellysolve B (v/v). Shaw and Jefferies (15) separate the isomers into three groups on a column of activated alumina, using a solution of 6x acetone in petroleum ether (B.P., grade 40 to 60) as eluant. Kobayashi (16) uses a column containing alumina and separates the lumisterol,, tachysterol*, and ergosterol; the elution is made by a 2 : l mixture of n-hexane and diethylether. Our method for separating the photochemical isomers of ergosterol is described below.
Relative ahswbance
(1) A. Windaus. “Les vitamines et les hormones,” GauthierVillars, Paris, France, 1938, p 91. (2) L. Velluz, Confirence at Leyden University, April 24, 1959. (3) E. Havinga, Chiniio, 16 ( 5 ) , 145 (1962). (4) N. A. Bogolovski, I,. 0. Shnaidman, and E. N . Kutznetsova, Med. Prom. SSSR.19,41(1965). (5) T.Kobayashi, Vitaniiiis (Kyotu),34, 482 (1966). 35, 1247 (6) A. W. Norman and H. F . De Luca, ANAL. CHEM., (1963). (7) G. Sanders, Thesis, Leyden University, 1967. (8) R. Mermet-Bouvier, J . C/ironiotogr., 59, 226 (1971). (9) E. Kodicek and D. R . Ashby, Bioclzem. J., 57, XI1 and XI11 (1954). ( I O ) J. W. Copius Peerebom. J. B. Roos, and H. W. Beekes, J . Chromutogr., 5 , 500 (1961). ( 1 1 ) H. Ziffer, W. J. Vandenheuvel. E. 0. Haahti, and E. C. Homing. J . Amer. Cheni. Soc., 82, 641 1 (1960). (12) T. Kobayashi, J . Vitumiiiol. (Kyoto), 13, 255 (1967). (13) R. Mermet-Bouvier, J . Chromutogr. Sci., in press. (14) P. P. Nair, C. Bucana, S. De Leon, and D. A. Turner, ANAL. CHEM.. 37,631 (1965). (15) W. H. C. Shaw and J. P. Jefferies, A d y s t (Loiidon), 82, 8 (1957). (16) T. Kobayashi, J . VitumOiol. (Kyoru), 13, 258 (1967). (17) A. W. Norman, J. Lund, and H. F. DE Luca, Arch. Bioc/iem. Biophys., 108, 12 (1964). (18) J. Green, Biochem. .I., 49,232 (1951). 584
ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
F’revitamrn Lumisterol
T
Tachysterol
E
Ergosterol
E
EXPERIMENTAL
Standards. The ergosterol was supplied from the Sigma Corporation of St. Louis, Mo. Its purity was checked by gas and thin layer chromatography.
P L
0
Elution volume n ml
,
1
80
160
210
+ 323
Figure 1. Alumina column chromatography of the photochemical isomers of ergosterol Apparatus. An apparatus made by the Seive Company of Rambouillet, 78--France, is used. It comprises a quartz circulation vessel located between a grating monochromatorwhich can be adjusted electronicaJly on two different wavelengths between 2000 and 7000 A-and a photomultiplier. The fraction collector includes 185 test tubes which are changed according to either a time presetting, o r a drop counting or else keeping a volume constant. A cooled container houses the fraction collector, the circulation vessel, and the photomultiplier. The maximum length of the column is one meter. Irradiation Method. The various isomers are obtained by irradiating, at 5 “C, a n ergosterol solution in diethylether (10 g per 1.) with two ultraviolet sources; a Mazda TG 16 type germicide lamp which, by its 2537 A emission, furthers the production of tachysterol,; and a Bausch and Lomb high pressure mercury vapor lamp fitted with two filters. One of these filters consists of a quartz tank full of distilled water to reduce the intensity of infrared radiation, and the other one is a MTO-J280a type filter which absorbs the short wavelength ultraviolet radiation and thereby causes lumisterolz to form. Column Chromatography. By using columns filled with alumina of 1 cm in diameter and 56 cm in length, and columns
T
P : Previtamin L : Lumisterol T : Tachysterol E : Ergosteral
Figure 2. Silica gel-alumina column chromatography of the photochemical isomers of ergosterol
Relotive abswbance
E
T a r 2 A : laxistera12 A P : Prwitamin
L :Lumisteral T : Tachystcml E : Ergasteral
Figure 3. Silica gel-alumina column chromatography of the.photochemica1isomers of ergosterol of decreasing diameter such as those described by Fischer (19). previtamin DP and lumisterol, could not be Separated (Figure 1): when the eluate corresponding to the previtamin D2 peak was analyzed by gas chromatography, a mixture of previtamin Dz and lumisterolz was found-on the other hand, the two other isomers separated (tachysterolz and ergosterol) were found to be chromatographically pure. Many tests were made with various column products (Florisil, silicic acid, Bio-si1 CW, Sil-R, A1208, etc . . . ) of different origins, particle size, and Brockman numbers, with no better results. The tests which proved to be conclusive were then carried out in the following way: the elution solvent was kept the same as the one used in the irradiation medium--i.e., diethylether-in order to avoid changing solvent and even to prevent the composition of the irradiated medium, which is variable with time and oxidation, from changing during this operation. A column of 1 cm in diameter and 60 cm in length, with a Teflon (Du Pont) tap was used and was filled with a mixture of 29 grams of silica gel (Sigma) containing about 1 2 z alumina (Sigma, ref. S. 7875), with a particle size of 325 mesh or smaller, in diethylether, with n o previous treatment. In each chromatographic assay, 1.5 to 2 ml of __
(19) G. A . Fischer and J. J. Kabara, A m l . Biochem., 9, 303 (1964).
the irradiation medium were used and were eluted only with diethylether. After a few of these successive assays made with the same column, the quantities of each of the isomers obtained are sufficient to give infrared spectra and t o make a gas chromatography analysis (20). The fractions collected with this column were 95 of the total. RESULTS AND DISCUSSION
Figure 2 shows a typical chromatogram, resulting from the analysis of a n ergosterol mixture irradiated for a few hours. For longer periods of irradiation, a n additional isomer is seen (Figure 3) whose eluted fraction, when tested in ultraviolet spectrophotometry, provides a s p y t r u m with a maximum absorption in the vicinity of 2520 A, as in the spectrum of toxistero12 A (21); its analysis by gas chromatography with different temperatures of the injector gives two distinct peaks and this proves that this isomer undergoes a thermal reaction in the injector and then changes into two isomers similar to previtamin and vitamin DP. (20) R. Mermet-Bouvier, Thesis, Rennes University, 1972. (21) P. Westerhof and J. A. Keverling Buisman, Rec. Trau. Chim. Pu~s-Bus, 75, 1243 (1956). ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
585
-
--
-
Ielativc abrcrbance
Toxisterol2 A
Relative absorbance
Prcvitamin Vitamin
Viamin 02
02
Lumisterol
....... . ...
L
-----
Tochptmi Ergosterol
10
BO
120
*
Elution volume In ml
Figure 5. Silica gel-alumina column chromatography of the previtamin Dz-Vitamin Dz
Wave length
I nm I
Figure 4. Ultraviolet absorption spectra of the photochemical isomers of ergosterol The ultraviolet spectra of each of these five eluted isomers is given in Figure 4. With highly irradiated ergosterol solutions, another isomer-difficult to isolate from previtamin D2-was evidenced but no toxisterolz B (21) was found. With this method it might be possible to confirm the thermal equilibrium constants between the previtamin and vitamin Dz as determined by Legrand (22), since these two isomers are clearly separated by our method (Figure 5). They are obtained by heat-boiling and recirculating a solution of vitamin Dz,in ethyl alcohol ( 5 grams per liter) for 90 minutes. As t.he molecular extinction coefficient values of these two bodies are known ( 2 3 , it is possible to make a quantitative analysis of these two eluted fractions by ultraviolet spectrophotometry. CONCLUSION
The method described above enabled five isomers to be separated for the f i s t time (in elution sequence: toxisterol? (22) M. Legrand and J. Matthieu, C. R. Acad. Sci. 245, 2502 (1957). ( 2 3 ) W. H. C. Shaw, J. P. Jefferies, and T. E. Holt, Aiialyst (Loizdon), 82,2 (1957).
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 3 , MARCH 1973
A, previtamin D?, lumisterol?, tachysterol?, and ergosterol) and a new isomer to be evidenced. This method, which has also been used successfully in the photochemical analysis of vitamin D.. (24), has several advantages: no action o n the irradiation medium, the column may be used several times in succession, good reproducibility and low solvent consumption, and the solvent used affords direct ultraviolet spectrophotometric analysis in the spectrum absorption range of the various isomers (2400 to 3100 A), The present method, compared with the other ones we tried (8, 13), also shows several advantages: no oxydation of the products, since the solutions were quickly deposited on top of the column; separation of isomers better than the one obtained with gas chromatography and thin-layer chromatography; very sharp separation of previtamin from Dz vitamin, which would have been impossible with gas chromatography; greater amount of each of the isomers obtained; quick quantitative dosage of the eluated fractions through ultraviolet spectrophotometry; separation of previtamin from lumisterol, which would have been difficult to obtain, even by conventional chemical methods such as esterification with 3,5-dinitrobenzoate. By comparison with conventional chemical methods requiring several operations o n the irradiated medium (such as evaporation, esterification, saponification), the present method needs no action on the irradiated mixture. RECEIVED for review March 13, 1972. Accepted October 19, 1972. Part of the thesis submitted to Rennes University, June 1972. (24) R. Mermet-Bouvier, C . R. Acad. Sci., in press.
