THE CHROMATOGRAPHIC SEPARATQQN OF {RRADiATED ERGOSTEROL T116335 éar fhe Degree of M. S. MiCfi’zfifiaN STAW CQLLESE Liiiiarz Eiroc‘fi Kimbafi 1950 0-169 Date This is to certifg that the 4 ‘ A“- ~ A t‘.‘ I thesis entitled _ _-- "Chromatographic Separation of Irradiated Ergosterol". presented l‘HJ Lillian Kimball has been accepted towards fulfillment of the requirements for .A- _M_'_S_-_— dcgree in PhflSiCal Chemistry /‘ I / t “ .‘e \ 1/. Major prolCSsuI’ . .~. ‘______.“—o‘-u -_.—--.L)e__-.‘ . —-—-———_... ———-¢—-—_—_ _ ._._.__ THE CHROMATOGRAPHIC SEPARATION OF IRRADIATED ERGOSTEROL By Lillian Broch Kimball A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of NMSTER OF SCIENCE Department of Chemistry 1950 WY m. 7544 K m A CKI\IO’«"."LE D G TEN T The writer wishes to express her sincere appreciation to Doctor Dwight T. Ewing for the able guidance and helpful suggestions offered in the course of this work. ********** ******** *****# **** ** a: t". (EvHITital TABLE OF CONTENTS PART I THE CHRONATOGRAPHIC SEPARATION OF CALCIFEROL BY MEANS OF VARIOITS ADSOIBEIIL .OOOOOOOOOOOOOOOOOOOOOOOOOOCOOCOOOOO TEATERIAL MID EQTTIPTJMTToooooooooooooooo000000.000.ooooo Ergoster01..............0.0...OOOICOOOOOOOOOOOOOO GaloiferOIOOOOIOOOOOOOOOOOOCOCOOOOOOOOOOOOOIOOOOC mherQQOQOIOOOOOOOOOOO0.0.0....OOOOCOOOOOOOOOOOOC AlCOhOIOOOOOOOOCCOOOOOOOOOOOOOOIIOOOOOOOOOOOOOOCC HexaneCOOCCOOCOOCOOOCOOOOOOOOOOICOOOOOOO000...... Silica gel-OOOOOOOOOOOOOOOOOOOOOOOOOCOOOOOOI...IO. Chromatograph tubes.............................. Irradiation source............................... Collection of fractions.......................... Determination of absorption spectra.............. P‘lrpose...OOOOOOOOOIOOOOOIOOOOOIOOOOOOCOOOOOOOOOO ProcedurBOCOCCOOOOOOOOO.0...00.000000000000000... :RAESIILTSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. DISCIJSSIOI‘ICOIOOOOOCOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOCOC SlegimIQYOOOOOI0.0000000000000000.000IOOOOOOOOOOOO0.0... LITETRARTIE CITED...OOOOOOOOOOOOOOOOOOOOOOOOOOIOOOOO... PART II THE CHROIATOGRAPHIC SEPARATION OF IRRADIATED ERGOSTEROL.... PlerOSSOCOQOOOCO...0....0.0.0.0...OOIOCOOOOOOOOOC Procedureooooo000.000.00.00.coo.0.000.000.0000... Calculation of Irradiation Time Per Mblecule..... R-E‘SULTSCCOOOO00.0.0000...00.0.0.0...OOOOOOOOOOOOOOOOOC DISCITSSIONOO0....OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO SITI‘aql'll-ARY...OOOOOCOOOOOOOOOOOOO0.0.0.0000...00......O... LITEi-{ATITE CITEDOOOOOOOOOOOOOO0.0000000000000000000000 PAGE uqqmmmmtbrhmmm 10 ll 13 18 19 26 27 29 31 32 58 47 TABLE I. II. III. IV. I. II. III. IV. V. VI. VII. VIII. LIST OF TABLES PART I DATA FOR DIFFEILEB‘TT ADSOILBLITIA‘S ITSEDO 0 o a o o o o o o o o o o o o o o o o EXTINCTION VALUES AT 265 NU FOR FRACTIONS OF ELUANT RECOVERED FROM DIFFERENT ADSORBENTS................. VOLUNE OF ELUATE NECESSARY TO RECOVER CALCIFEROL FROM TLAGNFSOL AS RATIO OF ETHER TO HEILANE IN ELUANT IS ‘J’ARIEDOOOOOIOOOOOOOOO‘OOOOOOOOOOOOOOOIOOOOOOIOOIOOOOO EXTINCTION AT 265 NU FOR DIFFER {T FRACTIONS OF ELUATE RECOVERED FROM MAGNESOL COLUMN AS RATIO OF ETHER TO IIECAI‘IE IN ELTIAI‘TT IS VARIEDOOOOOOOOOOOOOOOOOOOOOOOOO. PART II DATA FOR RUNS WITH DIFFERENT IRRADIATION TIT-33.0.0.0... ABSORPTION SPECTRA FOR TYPICAL FRACTIONS IN RUN NO. 1. ABSORPTION SPECTRA FOR TYPICAL FRACTIONS IN RUN NO. 2. ABSORPTION SPECTRA FOR TYPICAL FRACTIONS IN RUN NO. 3. ABSORPTION SPECTRA FOR TYPICAL FRACTIONS IN RUN No. 4. ABSORPTION SPECTRA FOR TYPICAL FRACTIONS IN RUN NO. 5. ABSORPTION SPECTRA FOR TYPICAL FRACTIONS IN RUN NO. 6. CALCULATION OF ABSORPTION SPECTRA FOR SUBSTANCE ELUTED I‘Jlm CALCIFEQOL..C..OCOCOOOOOOO0.0.0.000....OCOOCOO. PA 14 15 16 17 39 4O 41 42 43 44 45 46 PART I PART I THE CHROMATOGRAPHIC SEPARATION OF CALCIFEROL BY TIME OF VARIOUS ADSORBENTS Chromatographic processes are especially effective for the separation of certain mixtures and the isolation of certain compon- ents for purposes of identification. The chromatographic method was first used by Day in 1897 in work with Pennsylvania earth oil. He later noted that if crude oil were forCed upward through a column of fuller's earth the sequence from.top to bottom would be: saturated aliphatic hydrocarbons, then aromatics and unsaturated substances, and, finally, nitrogen and sulfur compounds (1). The method was further developed and systematized by Tswett so he is usually considered to be the inventor of chromatography (2). many modifications of the method have since been tried, some with astonishing success. The success of the chromatographic method is based on some difference in the affinity of the adsorbent toward the different components present in the mixture to be separated. The affinity of an adsorbent for the adsorbate can be altered by varying the solvent and the preparation of the column. Components which cannot be separated using a particular adsorbent may oftentimes be separated by some other adsorbent which is more preferential in character. The theory, limitations, and applications of the chromatographic process in general are fully'explained in the works of Zechmeister (3) Strain (4) and others and so were not discussed in this paper. -1- The chromatographic technique has been applied in this labora- tory to the separation of various vitamins from.the impurities associated with their production. Bullard (5) showed that ergosterol could be separated from calci— ferol using superfiltrol as an adsorbent and an eluant composed of a 50:10:1, ratio by volume of hexane, ether and ethyl alcohol. Baker (6) investigated various adsorbents with regard to their use in the separation of vitamin Difrom'the other components of fish oils and found that superfiltrol and possibly magnesia were suitable when a 50:lO:1 ratio by volume of hexane ether and alcohol was used as an eluant. Powell (7) used chromatographic separations on superfiltrol for removing vitamin D from ergosterol in the determination of the 2 vitamin D content of various fish oils. He made use of the same eluant as Baker. Chen (8) by varying the eluant used and changing the amount and type of solution used to prewash the column showed that the adsorption of vitamins A and D and ergosterol by alumina and superfiltrol could be altered. MATERIAL AND EQUIPIENT Since the materials and equipment used in both parts of this in- vestigation were identical they will be described fully in Part I of this paper and will not be repeated in Part II. Ergosterol: The ergosterol was a good commercial grade received from Parke Davis and Company, lot F-103-48, and was prepared by the Mbntrose Chemical Company. Calciferol: The calciferol used was pure synthetic vitamin D in crystalline form, each gram containing a minimum of 40,000,000 U.S.P. units of crystalline D and was obtained from the Winthrop Chemical Company, 2 Inc. From this calciferol a stock solution was made up containing 0.0166 grams of calciferol in each 200 ml. of ethyl alcohol. Another stock solution was made up containing 0.0174 g. of calciferol in each 200 ml. of solution. Three milliliters of one of the stock solutions were used for each run in Part I. Ether: A c.p. grade of anhydrous ethyl ether was distilled immediately prior to use over sodium hydroxide and sodium sulfite. Approximately 30 g. of sodium hydroxide and 30 g. of sodium sulfite were used for 500 ml. of ether. The distillate was collected and the absorption curve determined using the Beckman spectrophotometer. In order that the ether be suitable for use it was necessary that it transmit down to 215 mu. Alcohol: A c.p. grade of ethyl alcohol was purified in the following manner: twenty g. of potassium.hydroxide and 10 g. of silver nitrate were added to one liter of the alcohol and was allowed to stand for one week with occasional shaking. The alcohol was decanted from the flask and distilled. The distillate was collected in 100 ml. fractions and the absorption curves were determined on the Beckman spectrophotom- eter. Only these fractions which transmitted down to 220 mu were used. If upon addition to the ether, hexane mixture the alcohol showed that water was present (water gives a milky color to the mixture) the alcohol was further treated with amalgamated aluminum and allowed to stand for 24 hours before being redistilled. Skellysolve B was purified by passing it through a column 24 inches in height and 1.5 inches in diameter containing activated silica gel. Fractions of about 100 ml. each were collected and the absorp- tion curves determined on the Beckman spectrophotometer. Only those fractions which transmitted down to 215 mu, and which showed no absorp- tion due to benzene were used. All other fractions were set aside and passed through the silica gel a second time. Approximately 300 ml. of hexane could be obtained from one column using 500 ml. of skellysolve. The hexane was stored in a dark bottle and remained stable for several weeks. -4- In Part I of the investigation different ratios of hexane and ether were used as eluants. In Part II the only eluant used with the exception of run No. B was a mixture containing a ratio by volume of 50 parts hexane, 10 parts ether and 1 part alcohol. This mixture will hereafter be referred to as 50210310 Silica gel: The silica gel used for purification of the skellysolve was P.A. 100 Refrigerant Grade purchased from the Davison Chemical Company and was suitable for use as purchased. Once used, the silica gel was re- activated by washing it in a Buchner funnel with distilled water until no odor of hexane remained. It was then air dried and then placed in an oven maintained at 2500 for 24 hours. After removal from the oven the silica gel was kept in an air tight container until used. The following adsorbents were used in the chromatographic process and were used without further treatment. 1. Alcoa Activated Alumina Grade E80 Mfd. by Aluminum Ore Co. 2. Granular Adsorptive magnesia=# 2652 California Chemical Co. 5. magnesium.Silicate 34 Lot No. 5. 4. Pacific Silicate Co. Ltd. Chemical Dept. 5. Silica Gel - finely divided. 6. Superfilterol - The superfiltrol used was a finely divided bentonite clay obtained from.the Filtrol Corp. -7. Magnesol A Chromatograph tubes: The chromatographic tubes used in Part I had the following dimensions inside diameter upper portion 8.0 mm. inside diameter lower portion 4.0 mm., length, upper portion 17.0 cm., lower portion 8.5 cm. The upper portion of the chromatographic tube used in Part II was 21.5 cm. in length, the lower portion 11.0 cm. The inside diameter of the upper part of the tube was 17.7 mm. and the inside diameter of the lower portion.was 4.4 mm. Irradiation source: For Part II of this investigation a 500 watt Hanovia low pressure mercury vapor lamp was used. This lamp operated on a current of 9 amps. The radiation was unfiltered. A quartz ribbon cell with a thickness 0.21 mm. and volume 0.29 ml. was used as an irradiation chamber. The cell was arranged as shown in photograph (I) so that the cell was parallel to the mercury vapor lamp. The solution to be irradiated was poured into the reser- voir, upper right in the photograph, and the irradiated mixture was collected in the flask, lower left. Collection of fractions: 1. Throughout the first part of this investigation the fractions eluted from.the chromatographic column were collected using the appara- tus shown in photograph (II). The eluate from the column was allowed to drop into the vials contained in the suction flask. The vials could be m0ved by means of the handle at the top of the suction flask. -6- H u 21.n— PLATE ll Suction.wws applied by means of a water aspirator connected with an open end manometer. 2. In the second part of this investigation fractions were col- lected using a Technicon Automatic Fractionator. The upper portion of this instrument is shown in photograph (III). The tubing from the top of the chromatographic column.and the top of the separatory funnel leads to the carbon dioxide tank which supplied the necessary pressure and at the same time excluded air from the system.during the chromatographic process. Determination of absorption spectra: All absorption curves were determined using a Beckman spectro- photometer equipped with quartz cells. Purpose: It was the purpose of this investigation to study the adsorption of calciferol by various adsorbents and to determine the possibility of effecting a chromatographic separation of calciferol from.the other irradiation products of ergosterol. The absorption spectra of the fractions eluted was used as a means of determining the effective- ness of separation. Procedure: 1. The chromatographic column was prepared in the usual manner using enough adsorbent so that the column.when packed was 8 cm. in height. The apparatus was set up as shown in photograph (II). -7- PLATE III 2. Then 3 ml. of alcoholic stock solution of calciferol was pipetted into an erlenmeyer flask. The solution was evaporated to ‘dryness over a water bath (temp. 60° C.) with the aid of suction. The residue was taken up in 3 ml. of eluant. 3. The chromatographic column was washed with 10 ml. of ether. At this time the water aspirator was adjusted so that a drop rate of one drop every three seconds was attained. The pressure differential was marked on.the mercury manometer and was thereafter kept constant by adjustment of the water aspirator. 4. When the level of the ether in the chromatographic column dropped to a mm. of the top of the absorbent the calciferol solution was added and a clean vial was placed under the column by rotating the vial holder. The eluate from the prewash was discarded. All other vials were calibrated to hold 3 m1. 5.1 The flask was rinsed with 3 m1. of eluant. The resultant solution was added to the column when the level of the original calci- ferol solution dropped to a mm. of the top of the adsorbent. 6. ‘When the level of the solution used for rinsing the flask reached a mm. of the top of the adsorbent the developer was added and the chromatogram.developed until enough 3 m1. fractions had been col- lected to insure the complete elution of the calciferol from the column. The number of fractions necessary was ascertained for new adsorbents by making a survey run or by comparison with previous runs. 7. The vials containing the desired fractions‘were placed in a suction flask and evaporated to dryness over a water bath and the residue was taken.up in 4 ml. of the eluant. -8- 8. The absorption curves of the resulting solutions were deter- mined on the Beckman spectrophotometer using 4 ml. of the eluant as 8. blank. 9. The extinctions of the various fractions were plotted as functions of the wave length. -9- RESULTS The extinction at 265 mu is directly proportional to the amount of calciferol present in the fraction. Table I shows the different adsorbents, the ammunt of adsorbent used, the pressure differential maintained, and the volume of eluant collected in the comparison of the adsorbents. The data in Table II show' the extinction values at 265 mu for theC>FJCDCD e #401!me H‘DU‘IDJCD C30) rec» 0.024 0.143 0.357 0.499 0.437 0.431 0.377 0.229 0.174 0.075 0.034 0.283 0.820 1.00 0.621 0.136 0.036 0.075 0.135 0.207 0.308 0.415 0.435 0.475 0.364 0.219 0.095 0.049 -17- 3. 4. 5. 6. 8. L. H. D. 1! F. LI TERATURE CI TED W. Miner, Ed.; Annals of the New York Academy of Sciences 49, 141-326 (1948) W. Miner, Ibid. Zschmeister, Principles and Practice of Chromatography, J. Wiley and Sons, N. Y. (1941) H. Strain, Chromatographic Adsorption Analysis, Interscience Publishers, Inc. New York, N. Y. (1942) J. Bullard, if. S. Thesis, Michigan State College (1945) H. Baker, M. S. Thesis, Michigan State College (1944) . J. Powell, 1. S. Thesis, Michigan State College (1946) Chen, 321.8. Thesis, l-iichigan State College (1950) -18... PART I I PART II THE CHROMATOGRAPHIC SEPARATION OF IRRADIATED ERGOSTEROL The study of ergosterol and its irradiation products has been the subject of a voluminous number of research papers, since Pohl (1) and Windaus (2) first reported, in 1927 that the impurity contained in sterols, which after irradiation possessed antirachitic properties, was ergosterol or a similar sterol. Ergosterol had been isolated in 1811 by Braconnot and was rediscovered by Tanret in 1889 (3) but the structure of the molecule was unknown. After forming and irradiating various esters of ergosterol 'Windaus (4) found that the irradiated esters possessed no antirachitic properties. He found, that if the irradiated esters were reconverted to sterols that the sterols produced did possess antirachitic proper- ties and he therefore concluded that the hydroxyl group present in the sterol was responsible for these properties and that irradiation affected ergosterol and its esters in the same manner. Windaus also concluded from.measurements of solutions of irra- diated ergosterol that solvents pervious to the radiation had little effefit upon the irradiation process (5). He found that the specific rotation of a solution of irradiated ergosterol varied directly with the time of irradiation, finally reaching a maximum.positive value at which time the antirachitic properties of the solution also reached a maximum. “With further irradiation the specific rotation of the solu- tion decreased in magnitude finally becoming negative with the gradual -19- disappearance of antirachitic properties. If oxygen were excluded two‘ isomers of ergosterol. were formed, both containing three double bonds and one hydroxyl group, one with a positive and one with a nega- tive specific rotation, the oneletter isomer being the active one.(6) In 1930 Windaus (7) reported that suprasterol 1. and 2., the products formed when ergosterol was irradiated with a mercury vapor lamp for 50 hours at 75° C. were stable to air. These two compounds were shown to have no absorption over 260 mu. They seemed to be formed simultaneously as neither one could be converted into the other by means of irradiation. A toxic compound was reported by'Windaus (8) to be present in irradiated ergosterol and was characterized by showing a maximum.ab- sorption in the region of 247 mu. At the same time a compound (later known as tachysterol) showing main absorption from.280 mu to 290 mu was postulated as originating from vitamin D. The presence of the toxic compound was substantiated by Morton and others (9) who showed that after 150 mdnutes irradiation of an alcoholic solution of ergosterol the absorption spectra of the irra- diated material showed a substance exhibiting a strong maximum.at 247 mu. Up to this time all efforts to separate the irradiation products of ergosterol had been more or less unsuccessful. 'Windaus (10) had made futile attempts to separate the irradiation products of ergosterol 12y extraction from methyl alcohol and benzine solutions. The separa- 13ions based on chemical reactions did not yield products sufficiently ENure for quantitative work. -20- Windaus (ll) believed that in the conversion of ergosterol into vitamin D by the photochemical process the molecular formula, the hydroxyl group, and the 3 double bonds remained unchanged and that the formation of vitamin D was due solely to some steric or structural rearrangement of the molecule which increased the spatial size and gave it a characteristic absorption maximum between 270 and 265 mu. Vitamin D1 was eventually separated (12) from irradiated ergosterol (405% conversion of the ergosterol us ed) by treating the irradiated mix- ture in Etzo with citraconic anhydride for ten days and crystallizing the D1 from acetone. Vitamin D1, was finally shown to be a molecular addition compound of lumisterol and vitamin D2 (13). It was found that upon heating D1 a short time with acetic anhydride and subsequent cooling lumisteryl- acetate crystallized out of the solution and the lumisterol could be recovered by saponification of the acetate, while the acetate of vitamin D2 remained in solution. The lumisterol, an isomer of ergos- terol, showed absorption maxima at 265 and 280 m1. Upon further irra- diation with a magnesium spark lumisterol could be converted into vitamin D2 but the reverse reaction could not be made to occur. Thus lumisterol was shown to be an intermediate between ergosterol and vitamin D2. Vitamin D2 was then added to lumisterol in varying amounts and the melting points of the various mixtures was determined. The final melting point diagram shown below showed that when lumisterol and vitamin D2 were added together in equal amounts a molecular addition -21- Iproduct was formed. The melting point of this compound and its absorp- ‘bion.spectra coincided with those for vitamin D1. 125° 120° ///\ 1150‘: l/ ' P + \ 110° ‘ " , 0 20 40 60 BO 100% Lumisterol 'Vitamin D2 Lumisterol, tachysterol, and calciferol (l4)'were agreed to be 'the intermediates between ergosterol and the suprasterols but the order of conversion for some time remained doubtful. Setz (15) as a result of his investigations concerning the influence of different wave lengths of radiation on the conversion, stated that it appeared that light of longer wave lengths seemed to lead chiefly to lumisterol which could be converted onLy slowly to tachysterol and vitamin D2. He reported that shorter wave lengths made it possible to skip the lumisterol stage with the immediate formation of tachysterol. Tachysterol is ten times more sensitive than ergosterol to radiation between 300 and 360 mu and the conversion to calciferol is rapid. 'With radiation from.a mercury vapor lamp filtered through xylene which absorbed the greater portion of the radiation of wave lengths less than 280 mu he found that lumisterol and vitamin D2 were formed in constant proportions. With radiation from.magnesium.light (chiefly between 278 and 280 mu) -22- jpractically no lumisterol was formed and the resulting products were :mainly tachysterol and vitamin D2. Today it is generally agreed that the conversion takes place in the following manner (16). Ergosterol——) Lumisterol—> Protachysterol—e Tachysterol ——> Ca1ciferol-————9-Toxisterol Suprasterol I. Suprasterol II. This is the sequence proposed by Dimroth (17) except that protachy- sterol has been added between lumisterol and tachysterol. Protachy- sterol has not been isolated in the pure form but spectroscopic studies of irradiated solutions shoW'that an intermediate is formed in the irradiation process which on standing is converted into tachysterol. This sequence was further substantiated by the investigations of Heilbron and Spring (18) which showed lumisterol to be tetracyclic in structure and tachysterol and calciferol tricyclic in nature. The chemical structure of ergosterol and its irradiation products was for a long time a subject of much investigation. One of the first structures for ergosterol contained two six membered and two five membered rings with a single side chain of the formula CllHZS’ three double bonds, and one hydroxyl group (19). It was finally shown.that ergosterol was a sterol because it yields Y-methyl-cyclo-penteno- phenanthrene {upon dehydrogenation with selenium) (20). The reaction of ergosterol with ozone (21) yielding methyliso- propyl acetaldehyde shows the double bond on the side chain to be -23- between carbon atoms 22 and 23. The two double bonds in the ring are between carbons 5 and 6 and carbons 7 and 8. The attachment of the hydroxyl group to carbon atom.three was shown by the oxidation of acetylated ergosterol with chromic acid (22). This position had been favored by Danielli and Adams (23) after measurements of the surface potential of films of ergosterol during irradiation. The changes in surface potential were surmised to be due to a change in the tilt of the hydroxyl group. It is now believed that with the addition of radiant energy to the ergosterol molecule an activated molecule results which is lumis- terol. With further addition of radiant energy the bond between carbon atoms 9 and 10 breaks giving rise to double bonds between carbon atoms 5 and 10, 6 and 7, 8 and 9 and tachysterol is formed. These bonds rapidly rearrange giving a more stable state which is cal- ciferol. In this state the double bonds are located between carbons 5 and 6, 7 and 8, and 18 and 10. (24) Thus the conversion is the direct result of absorption of radiant energy by the molecule. Various methods of irradiation have been in- vestigated with ergosterol in the solid state in solution and in the vapor state (25,26,27). Jendrassek used a method whereby solid ergos- .terol in contact with a solution containing vitamin D or ergosterol was irradiated and the solution containing the ergosterol was led away by dialysis fresh solution being continuously added.(28) Trufanov (29) believed that a 2% solution of ergosterol in benzine irradiated for four hours, at which time 50% of the ergosterol was -24- converted, gave the best result with regard to the production of D 2. Bills, Honeywell, and Cox (30) after irradiating solutions of ergosterol in ether, cyclohexane, and alcohol with a mercury are found that the same general absorption curves resulted. However, the potency of the ether solutions was about twice that of the other solutions. It now seems that the activation takes place more rapidly in ether solution. Irradiation of solid ergosterol (31) has not proved successful 'because the vitamin D is formed only on the surface of the crystals. 2 With further irradiation the D is decomposed before the provitamin 2 in the middle of the crystal can be affected. The investigation of irradiation in the vapor state has not been sufficiently investigated to allow any definite conclusions as to the value of the process. The most efficient technical method new in use is the method whereby an ether solution flows into special quartz irradiation chamber buih: concentrically around a mercury vapor lamp (32). The effect of temperature of the solution being irradiated upon the conversion.was investigated by'Webster and Bourdillon (33). They irradiated solutions of ergosterol at 77.a°, 30.60, 1°, -1e°, and approximately - 1830 and - 1950 C. They concluded there was very little change in the activity of products produced in any of the first four cases. The wave lengths of light used for irradiation have been investi- gated at some length and it has been more or less agreed that wave -25.. lengths between 275 and 300 mu produce the best yields of vitamin D with the smallest amount of by products. Kon, Daniels and Steenbock (34) reported that for the 256, 265, 280 and 293 mu lines the quantity of radiant energy necessary to form an amount of vitamin D to cause demonstrable results in rats was 700 - 1000 ergs. The data obtained under the most careful conditions indi- cate that 7.5 x 1013 quanta will produce one U.S. Pharmacopoeia unit of Vitamin D. However in the active region the energy required depends upon the wave length. In 1933 Bacharoch and others (35) proposed that the following E 1%, 1 cm. values for ergosterol and calciferol be adopted. E 1%, 1 cmn for ergosterol at 281 mm not less than 320 and for calciferol at 265 and E %, 1 cm. not less than 470. At the time of this investigation there were available no E 1%, 1 cm. values for any of the other products of the irradiation process. Though curves for all of the products (attributed to Brockmann) are published by Rosenberg (36) the original data was unavailable so that the curves could be used only in a qualitative manner. Purpose: In this part of the investigation an attempt was made to separate the irradiation products of ergosterol by chromatographing the crude irradiation mixture using superfiltrol as an adsorbent and a solution of 50:10:1 for developing the chromatogram. A special quartz ribbon cell was used as an irradiation chamber and irradiation was carried out on ether solutions of ergosterol with a mercury vapor lamp. -2 6- The ether solutions were irradiated for different lengths of time and the resultant mixtures were chromatographed on superfiltrol. One milliliter fractions of eluate were collected and the absorption curves of the various fractions determined. Procedure: 1. 0.25 g. of ergosterol was dissolved in 100 ml. of freshly distilled ether and cooled in an ice and salt mixture. 2. The other solution was irradiated in the following manner: a. The mercury lamp was turned on until maximum light intensity was attained. b. The air jets focused on the cell were turned on to keep the air around the cell in circulation and thus cool the irradiation cell. c. The cell and reservoir were rinsed with 50 m1. of freshly distilled ether. d. The ether solution.was poured into the reservoir and allowed to pass through the cell. The rate of flow was maintained a constant by regulation of the head of the solution in the reservoir. 3. The total irradiation time was measured with a stop watch. 4. For irradiation times longer than £3 minutes the solution was passed through the cell more than once. 5. The mercury vapor lamp was turned off. 6. One ml. of the irradiated solution was removed for absorption analysis. -27- 7. The remaining solution was evaporated to dryness over a water bath (temp. 60° c.) with the aid of suction. 8. The residue was immediately taken up in 6 m1. of 50:10:l. 9. One tenth of a milliliter of this solution was reserved for analysis. 10. The remaining solution was chromatographed in the following manner on a column containing 12 grens of superfiltrol which had been prewashed with 50 ml. of 50:10:l. 8.. b. 'Jhen the level of prewash solution was about 1 mm. above the top of the column the solution to be chromatographed was added slowly to the column. (Note: If the adsorbent is disturbed the resulting bands will not be even.) The flask was then rinsed with. 3 ma. of 50:10:l and this solution.was added to the column. 0. When the level of the solution reached within a mm. above the top of the column the eluant was added to the column and elution.was continued until the de- sired number of fractions were collected. (Note: in the early part of this investigation it was assumed that 30 fractions would be sufficient. It was later decided to increase the number of fractions collected until it was certain that the extinction of the last fractions were neglegible. This number turned out to be about 70.) 11. Each fraction consisted of 1 m1. of eluate or 43 drops. 12. The fractions were collected using a Technicon Automatic Fractionator. 13. The fractions were diluted to 5 ml. with 50:10:1 and the absorption curves for each fraction determined by means of a Beckman spectrophotometer. When necessary, fractions were further diluted so that the extinction values in the range from.400 mu to 220 mu did not exceed 0.900 extinction units. 14. The extinction values for the separate fractions were plotted as functions of the wave lengths. Calculation of irradiation time per molecule: The time necessary for the total other solution to flow through the cell into the receiver was considered to be the total irradiation time. When it was necessary to pass the solution through the cell more than once (explained in the procedure) the total irradiation time was the’sum,of the times necessary for the solution to pass through the cell. Example:in Run No. l the solution was passed through the cell once. The stop watflh was started when the ether solution was poured into the reservoir and was stopped when the last of the solution.had run into the receiver. This took 12 minutes 22.8 seconds and this is the total irradiation time. In Run No. 2 the solution.was passed through the cell twice. The first time the solution was passed through 11 minutes and 50 seconds elapsed. The solution passed through the cell the second time in 11 minutes and 10 seconds. The sum of these two periods of time is 23 minutes. This is the total irradiation time. In each run the volume of solution irradiated was 100 ml. The volume of the quartz irradiation cell was measured by Kirn (37) to be 0.29 ml. It was assumed that the rate of flow of solution through the cell was constant. In order for 100 ml. to flow through the cell it was necessary for the cell to empty ICC/0.29 times. It was evident that each molecule would be in the cell for the length of time neces- sary for the cell to empty. Thus the irradiation per molecule is obtained by dividing the total irradiation time by loo/0.29. Example: Run No. 2 Total Irradiation time = 23 minutes Irradiation per molecule 23-+—l29 = 0.062 minutes or .2. 3.71 seconds -30- RESULTS The pertinent data pertaining to each run has been tabulated and is shown in Table I. The procedure for each run was the same except that for zero irradiation time the ergosterol was recrystallized from benzene and ethyl alcohol according to the method prescribed by Huber, Ewing, and Kriger (38). The irradiation time for Run No. l was 12 minutes 22.8 seconds or 2.15 seconds per molecule. Table II shows the extinction values of fractions 8, 10, 14, 16, 19, 22, 25, 28. The absorption curves for these fractions are graphed in Figure 1. Run No. 2 was irradiated for a total of 23 minutes or 2.71 sec- onds per molecule. Table 111 gives the extinction values for some of the typical fractions obtained after chromatographing. The absorp- tion curves for these fractions are graphed in.Figure 2. Table IV gives the extinction data for Run No. 3. The ether solution was irradiated for a total of 30 minutes and 35 seconds. This irradiation is the equivalent of 5.32 seconds per molecule. The ab- sorption curves are graphed in Figure 3. Tables V and VI give the extinction data for various fractions for Runs Nos. 4 and 5 which are irradiated 8.88 and 9.52 seconds per molecule respectively. The graphs for these runs are represent ed in Figures 4 and 5. These graphs are only representative for'the different runs for size does not permit reproducing them completely without foregoing legibility. -31- Extinction M /a\ \ (22) 60 (D O \fl 0 a O (25)\ 30 m 20 / (28) 10 F.“‘e.._: _j:__ _ _ (8) (10) 2:20 240 260 280 300 326 lave Lev Ch in nu 0 Figure 1. Irradiati - ave 7.15 sec. per molecule. Extinction 180 160 140 120 100 60 40 20 [QB (16) I 7 \\\ \/ [a a 27) fl III-IIIII-I 2»o 2 O 310 340 370 400 lave Length in mu Figure 2. Irradiation Time 3.71 sec. per molecule. Extinction 14 120 100 80 60 40 2O 22 19 (16) 4 ( 7 4 6 (39 ( 5) 220 250 280 310 340 370 400 Wave Length in mu Figure 3. Irradiation Time 5.32 sec. per molecule. l6) 275 / 250 { 221 / AM I r Extinction H m c> 125 100 H \\ i 75 J) ‘ 9 50 J < 3 25 \ (12) O o 257) 2 o 310 340 370 400 Wave Length in nu Figure 4. Irradiation Time 8.88 sec. per molecule. Extinction 27 24 210 180 / 150 ‘l ( :2) 120 24 9o 39) so, 7 30 ‘3 raefi‘”"‘l 3o) ézo 250 280 Bio 340 370 leve Length in nu Figure 5. Irradiation Time 9.53 see. per molecule. 12~ 100 (42 80 S 3 g 6. ‘ A 23‘ g (38) V\ l 4 ( a) . " . (31 ‘ / (28 \\~ _. _ ‘_ 0 ‘~ h‘__ 0 250 2 O 310 340 370 400 Figure 6. Irradiation Time Zero. lave Length in nu 60 \n O Extinction a O (A) 3O 2O 10 0 A ¢ Figure 7. C) -E5 250 260 310 340 370 400 Wave Length in mu Calculated absorption curve of substance eluted uith calciferol” (A) Absorption curve of fraction 16 Figo 2 a (8) Curve if only calciferol were present, (C) A minus B. DISCUSSION The graphs shown in Figures 1-6 would seem to indicate that the amount of calciferol produced by the irradiation of ergosterol under the conditions herein described reaches a maximum when the irradiation time per molecule is approximately 8.88 seconds. The conditions of the experiment did not allOW'for closer adjustment of the time of irradiation. It is difficult to state the above positively because as the irra- diation time was increased the absorption spectra of the fractions eluted showed that the calciferol band was overlapping more and more with some other substance. Since the first sixteen or seventeen fractions eluted in each run show little or no distortion of the cal- ciferol curve, between 230 and 300 mu it seems probable that calciferol could be separated from the interfering products by chromatographing fractions 17-25 a second time. In this way the curve of the second substance eluted from the column might be determined. It seems evident that the increase in absorption of the various fractions in the range from.about 232 mu (see Figures 1-5) down to 222 mu is caused by some product produced in the course of the irradiation because this increase is not characteristic of the curves obtained from.the ergosterol which was not irradiated. The only irradiation products of ergosterol which show this char- acteristic increase in absorption beloW'23O mu are the suprasterols. The extinction value for the suprasterols at 265 mu is zero. If it -32- is assumed that the extinction at 265 for fraction 16 in Run No. 2 is due only to the calciferol present in the fraction (see Figure 7) then the extinction values at other points between 265 and 222 mu can be calculated for the pure calciferol. This calculation.has been shown in Table VIII. If the pure calciferol curve is subtracted from the experimental one curve C results. This curve compares favor- ably with the curve reported for suprasterol. The same calculations have been carried out for other fractions and the resulting curve has the same characteristics. By comparison of the emtinction.values Obtained for fraction six- teen in the different runs it can.be noted that as the irradiation time is increased the distortion of the calciferol curve between 234 and 222 mu is increased. The increased distortion is due to an in- crease in concentration of the substance having an absorption spectra like that illustrated by curve C in Figure 7. The fact that the concentration of this substance increased with irradiation time is also to be noted from.the number of curves in each run.showing this characteristic distortion. In.Run E0. 2 only the absorption spectra for fractions 9 to 26 show this distortion.while in Run No. 4 the absorption spectra for all fractions from.12 through 66 ShOW’the same effect. This increase in concentration with irradia- tion time flurther supports the supposition that the increase in absorp- tion from 234 to 222 mu is due to the presence of suprasterol for it is logical that as the time of irradiation is increased some of the calciferol would be decomposed to form suprasterols. The rate of elution of the substance also supports the theory that it is suprasterol. According to theory, if all other variables are held constant those substances which contain the most rings should be eluted last but those having the greatest degree of unsaturation should be eluted last. Assuming the structure of suprasterol to con- tain two double bonds in a spiro-oyclo-pentane formula and one double bond in the side chain (59), and assuming of calciferol to have a ruptured cyclo-pentenophenanthrene structure containing three double bonds and a double bond in the side chain, it is evident that though calciferol is the most unsaturated, the suprasterol molecule contains more rings, thus the two molecules should migrate down.the column at approximately same rate. This indeed proves to be the case. Thus the presence of suprasterol along with calciferol in the first fractions eluted from the column seems fairly evident. Definite proof, however, has not been established since a complete separation of the two components has not been effected. With an. irradiation time of 51 minutes (see Figure 4, fraction 22) the calciferol band is overlapped by a band containing one or more other substances. The result is that the curves in this region show a maximum.absorption at 286 mu. If a hypothetical calciferol curve (assuming high or low concentration of calciferol) is subtracted mu. Such a curve could not result from.the addition of a lumisterol curve to a tachysterol curve or a toxisterol curve or any combination of these for both of the first two exhibit principal absorption at 280 from.such a curve a smooth curve is obtained having a maximum.at 286 mu while the absorption peak for toxisterol is at 248 mu. 1 i Since similar fractions for the other runs show a similar maximum at 286 mu it is improbable that this absorption.maximum is due to experimental error. Therefore, it seems evident that this absorption maximum is due to some intermediate formed in the conversion of ergos- terol to calciferol which has not yet been isolated or to some error inherent in the method used. In the runs where the number of fractions collected exceeded thirty it can be noted that fractions 30 through 40 shOW'an absorption peak at about 250 mu. It was at first assumed that this peak was due to the presence of toxisterol. However, the run for zero irradiation time shows a similar series of curves. The presence of calciferol is not evident from the absorption spectra of the unirradiated sample. Thus the maximum.at 252 mu could not be due to toxisterol which presupposes the presence of calciferol. Thus the substance causing this maximum.could not be a product of the irradiation process but rather a result of a reaction of the eluant with the adsorbent, a reaction of the eluant with the ergosterol, an impurity present in the ergosterol, or some decomposition reactiai of the ergosterol on the superfiltrol column itself. All of the possibilities were investigated separately. First a run.was made to determine whether the Solvent was causing the elution from the absorbent of some substance showing maximum absorption at 252 mu. The column was packed in the usual manner and was washed with 50 ml. of 50:10:1. Then a solution of 50:10:1 was passed through the column and 66 one m1. fractions were collected. The absorption spectra -35- of each fraction was determined. The extinction values obtained were all so low, less than one extinction.unit, that it was evident that the absorption maximum.at 252 mu was not the result of an affect of the solvent on the adsorbent. Next a run was made to determine whether or not the peak at 252 mu was caused by some affect of the alcohol on the ergosterol or the adsorbent. The column was perpared in the regular manner and 0.13 g. of ergosterol in 100 ml. of ether, evaporated to dryness, was taken up in 6 ml. of a solution containing 50 parts hexane and 10 parts ether and added to the column. Only half of the usual amount of er- gosterol was used assuming 50% conversion would take place with irradiation under ideal conditions. It was noted that in the chromatographic process when 50:10:1 was used as an eluant a blue band appeared about one third of the way down the column. As elution continued the band migrated down the column and was eluted when.between 30 and 40 ml. of eluate had been col- lected. These fractions, 30-40 showed a yellow pigmentation. A 50:10 hexane ether solution.was used for elution. Seventy-six one ml. fractions were collected. During the chromatographic process it was noted that the blue band did not appear and the eluate showed no yellow color. The absorption spectra of each fraction.was determined. The curves obtained still showed a maximum at 252 mu. The possibility that the ergosterol was impure was next investi- gated. The ergosterol to be used was recrystallized from.benzene and ethanol. Then 0.13 g. of ergosterol was dissolved in 100 ml. of ether, -35- evaporated to dryness, taken up in 6 ml. of 50:10:1, chromatographed in the regular manner and 70 fractions were collected. After the absorption spectra had been determined it was evident that the maxi- mum at 252 mu was still present and had in no way been affected by the recrystallization process. To determine whether decomposition of the ergosterol was occur— ring on the column a second run was made like the one above. ‘When 138 ml. of eluate had been collected in 13 different portions the absorption spectra of the several portions were determined. The curves for the last three fractions were typical of pure ergosterol. These last three fractions, corresponding to the last 26 ml. of eluate were evaporated to dryness taken up six ml. of 50:10:1 and chromato- graphed in the regular manner. The absorption curves from the eluted fractions showed the same maximum.at 252 mu as before. Therefore, it can only be concluded that some reaction as yet undetermined occurs between the ergosterol and the adsorbents which is responsible for this maximum.in.the absorption curve. The unconverted ergosterol is the last substance to be eluted from the column but it is not possible to recover the ergosterol in the pure state by this method due to the interference of the sub- stances eluted with ergosterol in fractions 33 to 40. The ergosterol absorption curves substantiate the presence of a maximum occurring in the region of 330 mu as reported by Hogness but this was not in- vestigated further. -37- 2. 3. 4. 5. SUMMARY There is substantial evidence that suprasterol is eluted from the column along with calciferol in the first portion of the eluate. It seems possible to separate calciferol from the ergosterol and its irradiation products other than the suprasterol by chromatographing part of the eluate a second time - approxi- mately fractions 17 - 26. The absorption.xaximnm.at 252 mu occurring in fractions 33 - 40 is not due to toxisterol as at first assumed but to some reaction between the ergosterol and the superfiltrol. The substance causing the absorption maxinum.at 286 mu might be isolated by a second chromatographic process. The maximum amount of calciferol seems to be produced with an irradiation time of 8.88 sec/molecule. -38- TABLE I DATA FOR RUNS WITH DIFFERENT IRRADIATION TIME Run Total Total Vol. Irradiation per No. Irradiation Eluate molecule Time ml. calculated 1 12. min. 22.8 sec. 28 2.15 sec. 2 23 min. 27 3.71 sec. 3 30 min. 35 sec. 76 5.32 sec. 4 51 min. 78 8.88 sec. 5 54 min. 38.2 sec. 39 9.52 sec. 6 0 72 t 0.00 Concentration of ergosterol was 0.25 grams per 100 ml. of ether. Volume of irradiation cell was 0.29 ml. One ml. fractions were collected. In Run No. 6 the concentration of ergosterol was 0.13 grams per 100 ml. of ether. TABLE II ABSORPTION SPECTRA FOR TYPICAL FRACTIONS IN RUN NO. 1 Irradiation Time 2.15 sec./molecule wave Fraction Length in mu 8 10 14 16 19 22 25 28 226 0.054 0.612 18.4 63.8 64.4 45.9 23.1 20.8 230 0.054 0.640 17.2 58.6 59.1 42.3 21.4 18.7 234 0.050 0.630 17.5 57.2 58.0 41.7 20.0 17.8 238 0.049 0.576 17.2 57.7 59.1 43.0 20.8 16.9 242 0.045 0.518 18.8 60.9 63.2 45.4 21.9 17.4 246 0.042 0.468 20.2 63.4 68.1 47.4 22.8 18.4 250 0.037 0.418 21.8 68.8 73.5 51.6 24.2 18.8 254 0.037 0.369 23.1 71.8 78.0 55.5 26.0 18.8 258 0.034 0.347 25.5 75.6 82.5 59.1 28.7 19.8 262 0.034 0.330 26.4 76.6 84.8 62.4 30.7 20.3 266 0.033 0.335 28.0 76.8 85.6 65.8 32.2 20.0 270 0.034 0.339 29.3 74.9 83.4 66.1 33.5 19.8 274 0.033 0.340 28.4 70.4 80.9 64.8 34.7 19.8 278 0.034 0.356 27.9 66.2 74.3 60.9 34.7 20.8 282 0.035 0.379 27.4 59.6 68.0 59.1 34.0 20.3 286 0.035 0.366 24.6 52.2 60.9 53.0 32.2 19.2 290 0.032 0.336 20.7 44.6 51.6 48.4 29.1 17.8 294 0.032 0.321 18.8 36.9 42.9 39.1 24.5 16.8 298 0.031 0.303 15.5 31.2 36.1 34.0 22.9 14.7 302 0.027 0.248 10.9 24.4 27.9 27.9 18.9 12.9 306 0.024 0.199 8.59 18.0 19.1 18.5 13.3 9.87 310 0.021 0.160 6.99 13.3 13.5 13.0 9.87 7.91 The extinction values in these tables were obtained in the follow- ing manner. For these fractions showing a maximum.extinction of less than 1.00 in the range between 220 and 400 mu the extinction values were read directly using the Beckman spectrophotometer. All other fractions were diluted so that all the extinction values were between 0.400 and 0.900 and the extinction values were calculated by multiplying the values read on the instrument dial by the dilution factor. -40.. TABLE III ABSORPTION SPECTRA FOR TYPICAI.FRACTICNS IN RUN NO. 2 Irradiation Time 3.71 sec./molecu1e Wave Fraction Length in mu 14 16 19 22 25 27 222 9.93 65.4 124. 56.8 19.5 14.4 226 0.1 59.5 111. 51.7 19.1 14.9 230 9.45 52.2 101. 48.8 18.3 13.8 234 9.93 54.7 99.9 48.7 18.4 14.1 238 9.30 56.4 105. 51.0 19.2 14.6 242 9.93 61.0 114. 55.0 21.1 15.5 246 1.0 67.1 126 60.2 22.8 16.3 250 11.9 73.3 138. 66.8 25.6 17.8 254 12.5 78.6 147. 72.2 27.9 19.2 258 14.2 83.7 157. 78.2 31.3 21.7 262 15.3 86.5 163. 84.5 36.3 24.7 266 16.7 88.0 163. 88.1 39.8 27.3 270 17.5 86.7 160. 90.0 43.1 30.1 274 18.0 84.5 157. 92.7 47.8 32.6 278 18.0 79.5 145. 89.6 48.8 34.1 282 17.3 72.5 133. 85.2 48.1 34.6 286 16.0 65.8 124. 84.9 50.4 35.2 290 14.2 55.7 104. 75.3 46.5 32.9 294 13.0 46.7 83.4 59.3 38.8 28.8 298 11.3 40.4 72.9 53.3 35.7 26.3 302 9.15 32.4 57.9 45.2 31.2 22.8 306 7.45 22.9 35.7 27.3 20.6 16.3 310 5.67 16.4 22.5 14.6 12.1 11.8 320 1.55 4.18 6.51 4.49 5.58 6.51 340 0.620 1.24 1.71 1.08 1.86 2.64 360 0.620 0.930 1.24 0.465 0.930 1.39 380 0.465 0.620 1.08 0.155 0.775 1.08 400 0.465 0.465 .775 --- 0.465 0.775 -41- TABLE IV ABSORPTION SPECTRA FOR TYPICAL FRACTIONS IN RUN NO. 3 Irradiation Time 5.32 sec,/molecu1e wave Fraction Length mu 12 14 16 19 22 27 33 39 46 60 75 220 56.7 65.1 78.4 89.6 94.0 43.5 28.7 39.0 42.9 22.3 14.3 222 55.3 ---- ---- --—- ---- 41.7 27.6 ---- ---- ---- ---- 226 53.3 58.0 68.5 77.2 80.8 39.5 26.3 36.7 40.2 20.1 12.7 230 51.0 55.3 64.4 71.1 75.8 38.3 26.3 36.4 38.6 19.7 12.8 234 48.5 51.9 62.6 69.7 74.2 36.4 25.9 36.7 39.7 20.3 13.0 238 44.3 51.7 64.7 72.5 76.2 37.5 27.8 37.6 41.7 21.7 13.0 242 42.6 53.6 68.5 77.6 83.3 42.9 31.6 40.9 46.1 24.8 15.2 246 43.4 56.3 71.9 86.1 90.6 43.4 31.0 44.7 51.3 27.2 15.6 250 46.2 62.8 80.1 94.2 99.6 49.0 34.2 49.4 58.6 32.0 19.2 254 49.0 67.1 84.1 103. 108. 53.8 36.3 51.4 65.6 36.8 21.5 258 48.7 69.4 88.9 113. 116. 58.2 39.2 54.5 72.9 43.6 25.6 262 48.1 72.5 95.1 115. 123. 62.6 41.6 57.3 80.4 49.3 29.2 266 46.7 73.5 98.0 120. 127. 67.4 43.6 55.6 81.5 51.6 30.8 270 44.8 72.5 97.3 121. 129. 70.5 46.7 62.0 83.7 63.2 38.9 274 42.9 71.0 98.5 120. 128. 76.3 47.0 56.4 88.1 57.5 35.2 278 40.2 67.5 95.6 116. 126. 77.5 48.8 57.5 89.5 59.1 36.2 282 36.4 61.0 88.0 111. 121. 79.7 53.5 64.4 100. 66.7 40.9 286 32.9 56.9 84.3 102. 106. 84.0 52.2 47.7 70.1 47.1 27.8 290 27.7 48.2 72.0 90.0 101. 70.0 41.1 41.7 61.2 39.6 25.0 294 23.4 40.8 60.6 74.7 83.5 60.9 39.2 42.6 63.0 41.4 25.7 298 20.1 34.7 51.7 65.0 72.5 53.9 29.8 20.4 33.3 22.0 13.3 302 16.4 27.4 41.6 51.6 59.0 43.7 23.3 16.3 15.8 11.6 5.73 306 11.9 19.2 28.8 34.9 37.5 28.5 16.4 12.1 12.1 7.90 3.72 310 8.53 13.0 19.2 23.4 25.0 18.8 13.3 9.91 9.30 6.66 3.40 320 3.10 5.11 7.60 8.83 10.1 9.76 8.99 7.28 6.98 5.26 1.86 330 2.01 ---- ---- —--- ---- 2.01 2.17 6.04 ~--- 4.80 ---- 340 ---- 1.70 1.55 2.17 2.64 ---- ---- ---- 5.27 ---- 2.94 350 0.775 ---- ---- ---- ---- 2.33 3.57 3.10 ---- 3.56 ---- 360 ---- 1.24 0.775 1.24 1.39 ---- ---- ~--- 2.64 ---- 1.24 370 0.465 ---- ---- -—-- ---- 3.88 4.65 1.39 ---- 2.32 ---- 380 ---- 1.08 0.775 0.930 0.620 ---- ---- ---— 2.64 ---- 0.620 390 0.465 ---- ---- ---- ---- 1.08 1.55 .930 ---- 1.39 ---- 400 ---- 0.930 0.465 0.155 .620 —---' -;-- ---- 1.55 ---- 0.620 -42- TABLE V ABSORPTION SPECTRA FOR TYPICAL FRACTIONS IN RUN N0. 4 Irradiation Time 8.88 sec./mo1ecule ___ wave Fraction Length ___mu 12 14 16 19 22 28 55 38 41 49 55 220 18.5 106. 210. 146. 59.5 26.4 58.4 98.5 61.8 11.9 10.8 226 16.5 96.1 190. ---- 57.2 27.0 ' 57.1 100. 59.6 11.5 9.50 250 16.1 90.0 176. 122. 56.9 25.7 56.7 98.2 59.6 11.0 8.99 254 16.5 89.7 176. 122. 57.8 21.7 57.6 104. 62.5 11.5 9.76 258 16.1 95.0 184. 128. 40.0 20.8. 59.5 111. 65.5 12.9 9.76 242 16.1 101. 201. 140. 45.6 21.6 66.7 121. 70.1 15.8 12.4 246 16.5 110. 220. 155. 47.8 25.1 68.6 150. 76.7 19.9 15.0 250 16.9 119. 259. 160. 55.5 25.7 71.1 141. 85.7 27.0 19.2 254 17.2 127. 259. 180. 59.0 24.0 71.6 148. 95.2 52.6 22.0 258 17.7 124. 275. 196. 66.4 25.1 66.3 150. 102. 45.2 29.0 262 18.0 140. 287. 216. 77.8 25.7 62.4 155. 110. 51.8 33.8 266 18.4 140. 294. 198. 85.0 25.6 55.9 151. 111. 55.5 56.7 270 18.9 158. 294. 206. 92.4 25.7 48.0 161. 127. 71.6 46.0 274 19.2 155. 294. 224. 106. 24.5 42.9 152. 120. 65.6 42.1 278 19.5 127. 275. 220. 110. 25.4 45.7 154. 122. 67.5 45.2 282 19.2 117. 255. 219. 111. 28.1 47.1 166. 155. 77.4 49.9 286 17.7 109. 242. 229. 121. 28.5 40.5 157. 106. 51.9 33.6 290 16.6 92.6 204. 208. 114. 27.9 57.5 124. 94.0 45.1 28.2 294 14.7 76.5 182. 177. 94.9 27.1 57.7 122. 94.0 45.7 50.1 298 15.2 67.3 147. 169. 90.2 27.8 50.4 89.4 65.7 22.2 15.2 502 11.0 54.6 122. 146. 81.9 25.9 24.5 68.0 43.8 6.66 6.04 506 8.99 57.5 75.5 89.2 52.7 21.6 20.9 55.2 56.0 4.65 5.25 510 6.82 25.8 45.6 46.0 31.6 20.6 18.8 45.6 50.1 5.88 5.41 520 1.86 ---- ---- ---- ---- 15.5 11.9 50.7 ---- 5.26 2.94 550 ---- 5.41 7.75 12.7 15.5 15.2 ---- 27.9 21.2 2.79 2.65 540 0.775 ---- ----‘ ——-- ---— -—-- 5.58 ---- ---- ---- -——— 550 1.59 3.26 2.48 2.17 5.10 —--- 17.1 15.2 2.63 1.55 560 0.465 -—-- -—-- ---- ---- —--- 5.10 ---- --_- .---— ---- 570 1.24 1.42 1.86 1.55 1.59 ---- 8.07 6.84 2.02 1.86 380 1.08 ---— --—- ---- ---- ---- 1.59 ---— _——- ---- ---_ 590 0.465 1.42 1.59 0.620 0.950 -——- 5.72 2.69 1.47 1.59 400 0.155 0.465 1.65 1.59 ---- -—-- 1.28 ---- 1.79 1.21 --—- -43- TABLE VI ABSORPTION SPECTRA FOR TYPICAL FRACTIONS IN RUN NO. 5 Irradiation Time 9.52 sec./molecule Wave Fraction Length mu 14 16 17 19 22 24 27 30 33 36 39 222 45.4 186. 204. 146. 69.6 37.1 21.4 23.0 38.2 45.1 55.2 226 41.6 166. 183. 132. 64.2 34.5 20.0 21.7 37.7 45.3 55.2 230 38.5 154. 169. 123. 60.3 32.9 18.6 19.6 35.4 45.3 56.3 234 38.3 152. 166. 122. 61.2 33.2 18.1 17.5 33.2 46.4 60.0 238 39.2 155. 173. 127. 63.8 34.5 17.7 16.0 33.0 48.9 64.2 242 42.8 166. 187. 138. 69.2 36.9 18.5 16.7 36.3 54.8 71.8 246 46.5 183. 204. 151. 75.3 39.8 19.5 17.4 38.2 57.8 78.8 250 50.9 199. 222. 164. 84.0 44.4 21.3 18.1 40.5 62.7 86.6 254 55.2 213. 239. 178. 93.5 50.1 22.9 18.9 40.5 62.0 90.3 258 58.7 225. 252. 193. 104. 56.1 25.7 18.6 37.4 57.9 90.7 262 61.7 231. 262. 206. 116. 64.7 28.8 19.6 35.8 55.3 91.6 266 64.2 232. 264. 212. 126. 72.3 32.1 19.6 30.7 47.7 86.4 270 65.3 227. 261.. 216. 136. 79.7 35.8 20.1 27.4 43.6 88.8 274 65.3 222. 241. 221. 149. 89.2 39.4 20.9 25.4 38.6 80.2 278 64.2 205. 226. 208. 149. 92.8 42.1 22.3 26.2 39.4 82.5 282 60.3 187. 209. 197. 147. 93.3 43.4 23.4 28.1 43.2 90.8 286 56.3 173. 196. 193. 154. 99.6 45.7 23.7 25.6 35.8 70.7 290 50.3 147. 166. 166. 140. 92.2 44.1 23.1 24.2 32.6 63.8 294 44.5 119. 135. 134. 115. 79.0 41.1 23.0 24.2 32.9 64.6 298 39.4 103. 118. 120. 107. 73.7 40.9 23.6 22.6 25.7 42.9 302 33.2 81.7 94.3 99.5 93.6 65.9 37.5 21.9 20.0 20.6 32.4 306 25.8 52.5 58.9 59.9 60.2 45.7 30.4 18.9 17.1 17.4 27.2 310 19.2 34.7 35.5 33.3 35.5 31.6 27.6 18.4 16.4 15.8 23.1 320 4.97 9.60 10.7 11.3 15.3 17.7 12.0 14.6 11.8 10.7 14.6 340 1.71 2.15 12.79 .2.79 [3.72 i4.97 '6.50 “5.27 4.65 5.12 8.84 360 1.08 1.08 1.55 1.39 1.39 2.17 2.64 2.33 1.75 2.64 4.50 380 1.08 0.930 1.24 1.08 1.08 1.71 1.86 1.71 1.39 1.55 2.32 400 0.775 0.775 0.930 0.775 0.930 1.39 1.55 1.39 1.08 1.08 1.24 -44.. TABLE VII ABSORPTION SPECTRA FOR TYPICAL FRACTIONS IN RUN No. 6 Irradiation Time Zero wave Length mu 28 31 34 38 42 58 222 5.89 14.6 18.0 22.6 18.0 6.20 226 5.73 15.0 18.6 22.6 16.3 6.05 230 5.73 16.1 20.6 23.4 15.6 5.43 234 6.51 18.7 23.9 26.2 15.3 5.27 238 7.91 22.0 28.2 29.4 16.3 5.74 242 9.62 26.2 34.7 35.2 20.1 7.45 246 10.4 28.7 38.5 38.9 25.0 9.92 250 11.3 31.2 43.4 46.4 35.2 15.3 254 10.8 30.2 43.9 49.4 43.1 20.0 258 9.76 26.5 40.9 53.6 57.5 28.2 262 9.15 23.9 38.9 57.0 69.1 34.6 266 7.29 17.8 31.6 55.0 75.4 38.5 270 5.74 12.4 27.2 61.1 96.0 49.7 274 4.81 9.45 21.4 53.3 87.2 44.7 278 5.42 9.15 21.5 54.4 89.6 46.2 282 5.74 10.1 24.3 62.3 103. 52.7 286 5.27 8.22 17.0 42.5 69.4 34.8 290 4.65 7.13 14.4 35.6 57.2 28.7 294 4.65 7.28 15.0 37.8 60.6 30.4 298 4.19 5.42 8.37 19.2 29.6 13.4 302 2.95 3.72 3.72 7.60 10.8 3.57 306 1.86 2.64 2.33 4.80 5.73 1.39 310 1.86 2.33 2.02 4.34 4.96 0.930 320 1.39 1.55 1.24 4.03 4.81 0.930 340 0.930 1.08 0.620 3.88 5.27 0.775 360 ---- 0.775 0.310 ---- 3.41 0.310 380 02775 0.465 ---- 1.71 2.17 0.310 400 0.775 0.465 ---- 1.39 1.24 0.310 -45- TABLE VIII CALCULATION OF ABSORPTION SPECTRA FOR.SUBSTANCE ELUTED WITH CALCIFEROL wave Length A B C D an 222 ---- 65.4 30.6 34.8 230 0.158 55. 41.9 13.1 240 0.217 58.6 56.2 2.40 250 0.280 73.0 72.5 0.50 255 0.307 79.9 79.4 0.30 260 0.329 85.2 85.2 0.00 265 0.340 88.0 88.0 0.00 A B Extinction values obtained from a pure calciferol curve. 8 = Extinction.values for fraction 16, Figure 2. 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