hl H I'll E ‘ WW 4 l — -— — CHROMATOGRAPHIC SEPARATION OF VITAMINS D Thesis for the Degree of M. S. MICHRGAN STATE COLLEGE Fu-ho Chen 1950 0-169 This is to certify that the thesis entitled "Chromatographic Separation of Vitamins D" presented by Fu-ho Chen has been accepted towards fulfillment of the requirements for M— degree tum]. Chemi stry Mmc Major profegor Date W959.— CHROMATOGRAPHIC SEPARATION OF VITAMINS D by Fu-he Chen A Thesi- Submitted to the School of Graduate Studies of Michigan State College of Agriculture and. Applied Science in partial fulfillment of the requirements fer the degree of MASTER OF SCIENCE Department of Chemistry 1950 CHEMISTRY DEPT. T 54%} c 5 vs ACKNOWLEDGMENT The writer wishes te express her appreciatien to Dr. Dwight T. Bring fer his guidance and valuable suggestions. M a! '1 g *3 ‘ u *4- N I. II. III. IV. V. TABLE OF CONTENTS INTRODUCTION EXPERIMENTAL 1. Treatment of Solvents and Materials 2. Procedures RESULTS TEBLES DISCUSSIONS SUMMARY FIGURES BIBIJOGRAPHY Page 11; 21 22 I. INTRODUCTION The chromatographic adsorption method for the separation of certain substances was originated.by the Russian botanist M. Tswett in 1906. He discovered that the pigments in the extracts of green leaves form a series of green and yellow'bands in.a certain sequence or order when a solution of the mixture was filtered through a glass tube filled with precipitated chalk. Moreover he found that complete separation of the pigments from one another could be effected only by eluting the adsorbed substances with fresh solvent or with mixtures of solvents. The process by which the different bands can be washed down the column at different rates has been called deweleping. Further study has been made by many scientists. Zechmeister and Cholnoky (21) and Strain (16) wrote treatises on chromatography. Theories have been developed.by Wilson (20) and by Martin and Synge (11). And the measurement of a quantitative nature has been made by Cassidy (h, 5). Upon these basic discoveries and theories, it has been con- cluded that the efficiency of a chromatographic separation was related very closely to the nature of the adsorbent, adsorbed material, the solvent and the physical conditions in the column. Many attempts have been made to separate mixtures of similar or dissimilar substances from each other by the chromatographic method. Vitamin A was removed from Sterol and Vitamin D by Walff (19), Ritsert (in), Marcussen (10), and Ewing it a}; (9). The separation of Vitamin D from the irradiated solutions of previtamins and natural oil was completed by Miller (12), De-litt and Sullivan (7), and Powell (13). The separation of pure Vitamin D from pure ergosterol had been also studied.by Baker (1) and.Ballerd (2). The purpose of this investigation was to study the factors which may increase the efficiency of separation of Vitamin D2 from Vitamin D3: also of separation of these from the mixture containing Vitamin A and ergosterol. II. EXPERIMENTAL 1. Treatment of Solvents and Materials Diethyl other: .A commercially good grade of anhydrous diethyl ether was distilled in the presence of sodium hydroxide and sodium sulfito to remove any water and peroxide which might have been present. The transmittance of the distillate should have been greater than 60% at 230 mu. néfiexane: The commercial product of Skelly-solve was purified for n-hexano. The purification was accomplished by chromato- graphing the solvent through an antivated silica gel column packed to a height about 2/} m. with an effective inner diameter of h cm. The transmittance of the eluted hexane should be greater than 92% at 230 mu. Ethyl alcohol: To each liter of anhydrous ethyl alcohol 20 grams of potassium hydroxide (solution effected by first die- eolving into a minimum amount of wateri and 10 grams of finely powdered silver nitrate were added. The mixture was shaken in an amber bottle and allowed to stand one woek'befere distilling. The transmittance of the distillate should have been greater than 99% at 265 m. Silica gel: It must have been activated at approximately 250'C for at least four hours. After it had been used once, it could be reactivated by washing in a.Buchner with a small amount of distilled water until the coder of hexane was no longer noticeable. n It was dried at room temperature and reactivated according to the ab eve procedure. Stock solutions .o_f_ the testing materials: A very pure grade of Vitamin D2 (calciferol), Vitamin D3, Vitamin A ester of acetate and also a very pure commercial grade of ergosterol were undo up in aléholic solution with definite concentrations. Alumina: The activated alumina used is “Grade A and mesh minus 80". It was the product of the Aluminum Ore Company. Superfiltrol: It was finely divided activated bentonite clay, obtained from the Filtrol Corporation. It was very important that all the solvents and solutions used should be free from any trace of benzene and other impurities which had high absorption value in the ultra-violet range and might have interfered with the absorption curves of the substances that were determined in this experiment. 2. Procedures The rate _o_f_ migration of Vitamin 132 and Vitamin 2-5 _a_5 determined by the concentration (extinction) o_f_ the fractions from a chromatographic column: The chromatographic column was made of pyrex with an inner diameter about 7 mm. in which ’4 grams of alumina was packed to a height of 8 cm. The method of prepara- tion was similar to that used by Ewing _e_ti a; (8). The column was fixed to the fraction collector which was connected to an 5 aspirator for 1 cm. mercury pressure. Then it was prewashed with 10 ml. of diethyl ether. Three ml. of the alcoholic stock solution that contained 0.00026 5 of Vitamin 132 and 0.000253 g of Vitamin D3 (about 10,000 units of each) was evaporated to dryness on a hot water bath at about 70.0 under a reduced pressure. The residue was taken up in 3 ml. of the chromatographic developing solution which was made up of various proportion of hexane and other, or hexane and alcohol. The solution was added.and followed.by another 3 ml. portion of the solvent for rinsing the flask on to the column. The column was not allowed to become dry at any time which might have destroyed the equilibrium between the phases of adsorbent, solute and solvent and then broken the band. Further additions of the same solvent was made to elute the adsorbed substance. Collected the eluent into 3 ml. fractions and hav-i—ng diluted 14-. with 1i ml. for determination .r the ab- sorption curves on.a.Beckmann spectrophotometer Model D U. Separation of Vitamin D2 and Vitamin 22: Mixture of equal amounts of Vitamin D2 and Vitamin D3 (0.000261 g and 0.000253 g, respectively) was taken up in 3 ml. of developing solvent and chromatographed through the alumina column. The experiment was carried out following the procedure given in the above material. For a long- or column, more diethyl ether was used for prewashing and the quantity increased with the same proportion as the increase of the length of the column. 6 Chromatographic separation of Vitamins D2, 1-33., A, and ergosterol: By alumina column: -- The column must have been prepared as described in the above. It must have been mounted on the auto- matic fraction collector as shown in the picture on next page: then prewashed with 10 ml. of diethyl other. A carbon dioxide tank was attached to furnish 1 cm. mercury pressure. The mixture of the alcoholic stock solution of 0.000171; g. of Vitamin D2, 0.000169 g. of Vitamin D3, 0.000199 g. of Vitamin A, and 0.000108 g. of ergosterol was evaporated to dryness and residue was taken up in 3 ml. pure hexane, then followed by 3 ml. hexane for rinsing the flask. The column was developed with 20 ml. of hexane and eluted with the mixed solvent of hexane and other. The oluent was collected in 1 n1. fractions and diluted to 1t ml. for determination of the absorption curves on a Bockmann spec trephetmeter. By superfiltrol: -- The chromatographic column was packed with superfiltrel to a height of 8 cn., then prewashed with a certain amount of the mixture solvent which was used for developing. The residue from the stock solutions was taken up in the mixed solvent and the column was developed with the same solvent. The procedures employed were the same as used in the above. Since through the superfiltrol column, the solution passed rather slowly, a 10 cm. mercury pressure was required. be at. L41 ~I !' l -—i—e——~g TE J muffle} .7” “I i i III. RESULTS The rates of migration of Vitamin D2 and Vitamin D3 on an alumina column with various mixed solvents of hexane and other, and hexane and alcohol were compared in Tables I and II. Separation of Vitamins D2 and D} had been tried on various lengths of alumina column with various solvents. Results were tabulated in Table III. Results of separation of Vitamins D2, D3, A, and ergosterol on an alumina column were listed in Table IV. The effect of quantity of prewashing solvent on the separation of Vitamins D2, D3, A and ergosterol on a superfiltrol column were shown in Table V. The percentage of recovery of the adsorbed substance ceuld.be calculated from the additivity (2) of the extinction of each fraction of the eluont. Use was made of the equation E(1%, lcm.) . 1- Io I Is where E(1%, lcm.) was the extinction of 1 g. solute in 100 ml. solution measured through a cell of 1 cm. thickness, 1 was the thickness of the cell in centimeter, and c was the concentration in grams per 100 ml. solution. Having rearranged and modified the equation lo Io I c_i( E l , 1cm. 1 where £(1eg Io/I) was the summation of the extinctions of the fractions 8 at a certain wave length and E(l%, lcm.) was a constant for a given substance in a given solvent at the given wave length. mm0.0 mm mm Hm0.0 mm0.0 00 0m mm0.0 0~0.0 an ma mw0.0 ~HH.0 0m0.0 0m0.0 so an m0H.0 mNH.0 ~m0.0 mm0.0 am an me.0 m0m.0 mm0.0 Hm0.0 w: an 0am.0 mam.0 ~w0.0 m~0.0 mm0.0 n: ma mmm.0 00m.0 aaa.0 NHH.0 Hm0.0 m~0.0 m H on .0 0mm.0 maa.0 m0H.0 Hm0.0 mm0.0 mm0.0 m H zam.0 maa.0 mmm.0 0:0.0 m0a.0 Hm0.0 wm0.0 210.0 mm NH mam.0 0mN.0 mmm.0 0mm.0 mam.0 mma.0 mm0.0 0~0.0 mao.0 mm an no .0 mw0.0 0w:.0 mma.0 Hma.0 amm.0 maa.0 wm0.0 ama.0 a:0.0 0m 0H ama.0 .0 mmm.0 amm.0 mam.0 0mm.0 mmm.0 0ma.0 H0N.0 mw0.0 am m 0m0.0 : 0.0 mma.0 0~:.0 mom.0 mam.0 no.0 m0m.0 mmm.0 mma.0 :m w ~s0.0 mn0.0 m0m.0 HmN.0 m:m.0 0am.0 m0m.0 mm~.0 mam.0 ~m0.0 mom.0 mn0.0 am a «No.0 m00.0 mw0.0 mw0.0 mma.0 wmm.0 mam.0 00m.0 mm~.0 ~w0.0 m:m.0 Hm0.0 ~w0.0 an m mm0.0 mm0.0 w~0.0 maH.0 HNH.0 m0:.0 Ham.0 maa.0 mmw.0 “00.0 nam.0 ma m H20.0 100.0 mw0.0 0ma.0 maa.0 man.“ 0am.0 amm.0 mmm.0 00H.0 NH m mm0.0 ma0.0 H00.0 am:.0 ww0.0 mom.” ma:.a mm0.a m mHH.0 mmm.0 a0w.0 ~m~.a m m m 0.0 n a no m0 mg mm mm mg mg m0 m0 an mg mm mm mm .wuwmml n.aeeeen I3... 0 onion .a i m 0::00 .n n m onion .a u m 00-0: .a a m 0~-0m .a u m oniom .a u m 0ma0H .a i m a. .H. a . .95 .3an and “my cannon Mo 92505: no numebaom A»: Mn can we anaemflb 93m moanmmhMopmaohno Seam enemas mo omeapomuh He is mmm um dozen—wanna «H Handy 10 .nmofipaom .Ha N a“ unoHpomahn Omoco ON mm $0.0 2.5.0 am an NHH.00 HHH.0 mm mm HmH.0 :mH.0 am mm me.0 w~m.0 mm Hm 0~H.0 mmm.0 0m on 0wH.0 1:3.0 mm mm mmH.0 m~0.0 mm mm anH.0 gm am 00H.0 mm mm on mm m: mm m~0.0 m: m ~m0.0 z: mm Hon.0 m: Hm me.0 mm .0 0m 0m H0N.0 mm .0 w mH mam.0 Hnm.0 mm wH mmm.0 an NH ~H~.0 mm 0H 0m mH mm mH mm H mm0.0 :00.0 am NH NMH.0 00H.0 mm HH mHm.0 mmH.0 mm0.0 0m 0H 0H:.0 amH.0 mm0.0 moH.0 wH m mHm.0 wmm.0 NH:.0 mmH.0 0H m mom.0 mmm.0 Nmm.0 Hmm.0 :H a HmH.0 ma~.0 m-.0 0mH.0 mH0.0 NH 0 aHo.0 m:m.0 0MM.H HMH.0 0H m :H0.0 m .0 .0 m mmm.H m m a m m H .mm mg m0 m0 m0 mm .mm. mg no we A.Hav.e.ecn acHaeeea .u . .u i. o on common mu common ma enemas no :\ amm 0 E an 0 H a H4 «H H.n.cHM|mm Hcacefimr .H. H.» ..z N .Honeoq dad ogmm M0 Gavan“: he nudobHem gawk a 0mm ma umHsmpr oasmnwndmmmAMopmsonmo Bonn amenaa mo ooHvomuh mo.ma mmm as moHaoanHfi ”HH mummy TABLE III: Results of Separation of Pure Vitamin D and Vitamin D 11 with Various Solvents and Column. 2 3 Length of Adsorbent column (cm.) Solvent Result Alumina "A" 8 hexane-other no separation (30-70) Alumina ”A” no hexane-ether no separation (30-70) Alumina ”A" 8 hexane-other no separation (20-80) Alumina 'A" 24 hexane-ether no separation (20-80) Alundna "AP 8 5% alcohol no separation in hexane Magnisol 8 hexane-ether no separation (90-10) Alumina 2“ hexane-ether no separation (it _._. 20) (20-80) TABLE IV: Using.Alumina Column. Length of column (cm.) Solvent 2.5% alcohol in hexane 1.0% alcohol in hexane hexane—ether (6040) hexane-ether (50-50) hexane-ether (1-2) hexane-ether (10-90) 12 Separation of Vitamins D from Vitamin A and Ergosterol % of VitaminsD recovered 25 28 hl 13 TABLE V: Separation of Vitamins D from Vitamin.A and Ergosterol Using Superfiltrel Column of 8 cm. Length with Different Quantity of Prewashing Solvent. Solvent Vol. of prewashing % of VitaminsD solvent (ml.) recovered hexane-other-alcohol 30 0 (50-10-1) hexane-ether-alcehol 15 67 (50-10-1) hexane-ether-alcehol 6 90 (50-10-1) hexane-ether-alcehol 3 87 (50-10-1) ' hexane-ether 30 9M (50-10) hexane-ether 15 95 ( 50-10) IV. DISCUSSIONS Effect of polarity of solvent on the rate of migration of Vitamin D2 and From the results obtained with Tables I and II, the extinction at 265 mu.of each 3 ml. fraction was found. In Figures I and II the extinction at 265 mu.of each fraction was plotted against the volume of eluent recovered, and the points on the curves were located at the end of each fraction. The curves showed the relationship that with the solvents containing higher concentration of other and alcohol, samples would pass through the column with less quantity of eluont; also the extinction of the fractions around the maxima was greater than those with the solvents containing less other and alcohol. Further- more, comparing the curves of Vitamin D2 and Vitamin D3 in the same solvent, it could be noted that Vitamin D2 will pass through the column earlier than Vitamin D3 if solvent used was relatively rich in other and in alcohol. The difference between the rates decreased as the con- contration of ether and alcohol in hexane decreased; until at a cer- tain concentration, the above order reversed. This phenomenon might be explained by the following theories. It was observed that the rate of migration of a solute through an adsorption column.was a function of the adsorption isotherm. The adsorption sequence was also a function of the isotherms of the solutes (h, 5, 5 ). According to the tfihries of Strain (16), the more polar the substance, the stronger it was adsorbed and the higher the band was formed on the column. But the sequence or adsorption order of the bands was not the same under all circumstances. It varied with the 15 solvent and the adsorbent, with the kinds of substances adsorbed, and with the conditions in the column. It could also change with the alteration of one or more factors such as the adsorbent, the solvent, the temperature, the concentration of solutes, the presence of an impurity, and the hydrogen ion concentration (17). In examining the structural formula of Vitamin D2 and Vitamin D3, Ho Vitamin D2 Vitamin D3 they were found to differ only by a double bond and a methyl group on the side chain. The presence of the double bond in a compound increased the polarity. Thus Vitamin D2 should.bo more polar than Vitamin D3 and would form.a band above the latter. Since both of the bands formed by Vitamins D2 and D3 were colorless, it was not possible to follow the order or sequence visually. Nevertheless Vitamin D2 did appear in the eluent earlier than Vitamin D3 in solvents containing higher percent- ages ef ether or alcohol in hexane. So it seemed that in the presence of other or alcohol it might have influenced either the relative positions or the rates of migration of Vitamin DE and.Vitamin D3, or both. Compared with hexane, other and alcohol were more polar in nature. Then polarity of the solvent could be responsible for the fact that the more polar the substance to be absorbed, the greater would be the rate of migration in a more polar solvent. The desorp— tion is carried on by the displacement of the adsorbed substance on 16 the adsorbent by the solvent which was more polar than the adsorbed substance (16) and the band formation was due to the equilibrium be- tween the phases (18) as follows: Stationary phase -—————A nubile phase T______ s lutes solutes 1/ ‘ " 1 selven e—A adso bent solvent The rate of migration of the adsorbed substance on the column possibly could have been increased by increasing the affinity between the sol- vent and the adsorbed substance. Effect 2: the solvent used for introducing_the sample: Hydrocarbons such as n-hexane and isoctane were considered as non polar compounds which possess little or no eluting power on.a chromatographic column. If the sample was introduced by a non polar solvent, the effect of elutien during adsorption might have been eliminat- ed and the band formed would have been sharper. 'In other words it needed loss quantity of solvent to elute it.) This effect was noted.by comparing the curves in Figures III and IV. Vitamins D2 or D3 intre- duced.by pure hexane orisoctance were more concentrated in the eluent than those introduced by the developing solvent. This effect might have facilitated the separation .r the mixture. In separating Vitamins D2 and D} from ergosterol on an alumina column, better results were ob- tained by introducing the mixture in pure hexane instead of using the developing solvent, as shown in Figures V and VI. Further study was necessary'before this proposal ceuld.be adopted. 17 Separation :3 Vitamins 2: Based on the previous results obtained in this experiment, Vitamin.D2 might have been completely or partially separated from Vitamin D}, by choosing a solvent which caused a greater difference between the rates of migration of the two and a column which could increase the distance between the bands as they reached the bottom of the column. So a more polar solvent and a longer column were chosen for this purpose. Unfortunately, the curves obtained by plotting extinction at 265 mu of each fraction against volume of eluent showed only one maximum which suggested that the two substances might form only one band eluted together or the two bands formed could be very close and overlapping. lbreever, the adsorption curves of Vitamins D and D were too much alike to determine whether the eluont was 2 subs nee. era. a pureAmixture. The results did not indicate separation. Vitamins D2 and D3 could be separated quantitatively from Vitamin A.and ergosterol by a superfiltrol column, (Table V). Vitamin .A was adsorbed on the column and ergosterol could be eluted afterward. By an alumina column, Vitamin.A could also be removed from the rest while ergosterol followed.Vitamins D too closely and.caused overlapping. Figures V, VI and VII showed that Vitamins D came out in the earlier fractions of the eluent and ergosterol in the latter portions. The mixture of the three appeared in the middle portions and the percentage of ergosterol contained in the mixtures increased as the number of fractions increased. 18 Effect :5 prewashing :£_guperfiltrol column: Prewashing affected the affinity between adsorbent and the 8d! sorbed substances. It was probably connected with the water contact’; of the adsorbent, (15). Alcohol, because of its structural similarity, could replace water in the adsorbent and deactivatdd the latter. Ether, because it could dissolve water less readily than alcohol, gave no appreciable effect on prewashing. When a superfiltrol column was pre- washed by 30 ml. of hexane-ether—alcehol (50-10-1), it lost its ad- sorption power. Then Vitamins D2 and D3 and ergosterol eluted together from flhe column in the first portion (Figure VIII). Nevertheless, Vitamin.A still remained in the column. When it was prewashed with 30 ml. of hexane-ether (50-10), this effect was not noted (Figure XI). The purpose of prewashing was to remove the residue contained in the adsorbent (13). From an 8 cm. superfiltrol column, the residue washed off showed the absorption curve as given in Figure XIII. During washing, a yellow band was formed on the column which migrated down with further prewashing. If 30 ml. of the solvent, hexane-ether- alcohel (50-10-1), was added, it was enough to wash the band from the column and destroy the adsorbility of the column. The yellow band had been collected and its absorption curve made as in Figure XIII. If alcohol was not present in the solvent, the yellow band did not appear. In the solvent of hexane-ether-alcohel (50-10-1), the yellow band did change into blue color when the samples were introduced. Also the absorption curve showed the difference (Figure IX, curve No. 8). Whether the blue band was the combination of the substance in the yellow band and the adsorbing material added or the decomposed substance 19 formed on the column, could not be concluded from this experiment. From the results listed in Table V, the trial of prewashing tho superfiltrol column with 6 m1. of the developing solvent hexane-ether- alcohol (50-10-1) gave the higher percentage of recovery of Vitamins D2 and D3 from the mixture. When 3 ml. of the solvent was used which was about sufficient to wet the column, the earlier fractions of the eluent were contaminated with the residue washed from the column. When 15 ml. of the solvent was used, the latter fractions of the eluent contained the blue band (Figure IX). In both cases the percentage of recovery of pure Vitamins D2 and D3 was decreased. However, the de- veloping solvent hexane-ether (50-10) which did not contain alcohol, gave better results than that containing alcohol (Figures VIII, IX, X, XI AND XII). In Figure XII the solid lines represented those fractions containing pure Vitamins D while the broken lines represented those contaminated with impurities. Effect of different grade ‘o_{ alumina on the rate 2: migation: Alumina of Grade (E n 20) had also been used for chromatographing pure Vitamin D2, with the procedures as described in (III, 1). When hexane-ether (30-70) was used as the developing solvent, Vitamin D2, in this case, would not appear until 50 ml. of eluent had passed. It also required more solvent to elute the adsorbed substance that alumina grade "A" did. Furthermore, the extinctions in each corresponding fraction were less. In other words, it could be considered that the band formed was wider. Nevertheless, it still possessed the same property that the increase of the other content in hexane increased the rate of migra- 20 tion and also narrowed the band. Evaporation of stock solution _o_f Vitamins 2: In evaporating the alcoholic stock solution of Vitamin D2 and D3 on a hot water bath, care was necessary. Preferably, the solutions should have been evaporated to dryness within as short a time interval as possible. If the time for evaporation was too long, a yellow oily residue would be the result instead of white crystals in the flask. This substance would pass through the chromatographing alumina column without being adsorbed and would show an absorption curve as in Figure XIV. Whether this was a thermally decomposed.product or a product formed in some other manner had not been further investigated. 1. 2. 5. V. SUMMARY The rates of migration of Vitamin D2 and Vitamin D3 on an alumina column were influenced by the polarity of the developing solvent. They were decreased as the polarity of the solvent decreased. Comparing the rates of migration of Vitamin D2 and Vitamin D3 in the same solvent, that of Vitamin D2 was greater than Vitamin D3 in the more polar solvent. The order was reversed as the polarity of solvent decreased. .A sharper band formed if the substance was introduced by a non- polsr solvent than by a more polar one on an alumina column. Separation of Vitamin D2 from.Vitamin D3 did not seem possible with solvent hexane-ether and hexane-alcohol using an alumina column. Vitamins D could be separated from Vitamin.A and ergosterol quantitatively by means of a superfiltrol column and partially by an alumina column. Solvent of hexane and ether (50-10) gave better recovery of pure Vitamins D than solvent of hexane, other and alcohol (50-10-1) from a chromatographic superfiltrol column. The adsorbability of superfiltrol was affected by the prewashing solvent containing alcohol, thus having influenced the percentage of recovery of pure Vitamins D. ‘Using a larger quantity of this. prewashing solvent it deactivated superfiltrol and caused no separation of Vitamins D and ergosterol. ”‘- . . .ouu .en.oa oamoamav .uum oouow .Amaco Has .u-n on-a“ .AoHMeAm~ ”nun .oouo. .wovoaa. .n-= . .o . ov «any mu-m oauom . oceans "nun .oa-oa . «so He ”acceptance .aoanos ca ensued: Auw nonuo use 23 ensue: Saigon .uvnnn ma case»: . ma adso»: nod—Ion .H on: an .HI 5 voaenooom agenda Ho oasao> we co on no as on on on o «.o v.0 s s. O O 13 ‘3 H 592 an “0110011 «Am o.H o.H ¢.~ .n on 4 Rm .0 .Aonvdaav an an 4 mm.o .Amvufiuv mm c« d nu . ovdamv a: nu d um .32. 3 a: 5 4 we .3 Amy ensue: upco>aom on 00 mdaav .uco«un can onoune> ca 4% Honooae can .uovuu a cuaeudb . No cuaoua> ”0H lam .HH endunh .HI cu venenooom acosnu uo olddo> o o O é; um g9; no uotqoutixi o H N a Induction Extinction c: I§“:fifjr 0.2 I 4 15 / - ‘qL::. E O s . b 12 18 24 30 35 42 Volunsvof lluont Recovered in mi. Figure III. lolvcnt used,hoxano-ether (40-60); Sample introduced in mixed solvent , and in pdro hexane -----. (1)o(2). Vitamin D2; (3)&(4). Vitamin D 3. 0.8 I\ t \ \ 0.6 t \ \ \ 0.4 41 1 x \ \ 0.2 \ O 5“ 8 ' 1e 15 20 24 28 30 Volume of lluont Recovered in mi. figure IV. Solvent used, 1.5} alcohol in isoctsno; Vitamin 02 introduced in mixed solvent , and in pure iooctono n-—-- . { Ixtinotion \ ' o“ \ n 230 2&0 250 260 270 280 290 300 Isvo Length inlni. Figure V. Absorption curves of fractions of eluent from chrometogrephin o mixture Vitamins D2, D3, L, and ergosterol on s Inins column with solvent hexnne-othe (50-50). Sample introduced in mixed solvent. ' Ixtinction 1.2 0.8 ...,,/, // gaff? \ (12) 230 zoo 250 260 270 280 290 no Iavo Length in .1- I'iguro VI. Absorption curves of fractions of eluent from chromatographing a mixture Vitamins 9:, n}, A. and ergosterol on alumina column with solvent nomano- other (50-50). sample introduced in pure hexane. Extinction 1.6 1.‘ 003 // (5) 0.6 / / A A / / “4/ / ° Xi /( (in) mag/j it w// Rik) M 230 240 250 260 270 280 290 300 Iowa Lomtn in m. Figure VII. Absorption curves of fractions of eluent from chromatographing a mixture Vitamins D , oz; 4, and orgosterol on alumina column with solvgnt nano- other (1-2). Samplo introduced in pure hexane. Extinction 1, ' / /*"K\ \ \ \ \58) \\\\\X IN \\ ‘W~a 230 2100 250 260 270 280 290 300 Iavo Length in 3:. figure IX. Absorption curves of fractions of eluent from chromatographing a mixture Vitamins Dz, , A, and ergosterol on superfiltrol column with sol ont hexane-ether-alcohol (50-10-1) . Used 15 ml. of sol- vent for prewashing. .03 a-.. :7 3 .3. ”Sufilfi Tune :1 48 5-265 I; to. 3 . av "acobaoo .3159... :1 on A3 :2 .3 A -363 I; .3 o sameness oceans) mama cleaoo Houuaauuoann co oununas ucunasnuouseouno souu vouobooou n onus-pub .HHK one-«h .Hm ca couobooem aconan no slouch ma 3 r «a 2 3 ) “ All. ’1’ \1 \o\\.\\. Jill/1 1.1.! [”7 m‘.g\m / / ,, s m / n w to... m. . u E 3 a a. I 7. any a 0.0” \ a < 3 A v . ANV God ‘a 1-6~ 1.4 P O O \ \ (1) lxtinction 9 on 9 cu 0.4 0.2 \\ m \‘HN \ g. 3L 300 230 2‘0 250 260 270 200 290 '1" hut-h in no Figure XIII. (I) Absorption curve of aching solvent from superfiltrol column. (2 Absorption curve of tho yellcw'band on superfiltrol coluan. H O 0‘ Extinction H 10 O on \\ is 2_o 240 90 260 270 280 2’0 Wave Length in mm. Figure XIV. Absorption curve of the yellow residue obtained from evaporating alcohollic Vitamins D solution. 1. 5. 6. 7. 9. 10. 11. 12. 13. 11+. 15. 16. 17. 18. 19. 20. 21. BIBLIOGRAPHY Baker, D. 3., M. S. 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(Interscienco Publishers, Inc., New York, 19145). Strain, H. 151., Ind. Eng. mien... Anal. Ed., lg, 605 (1916). Strain, fi. 3.. Anal. Chem., 3;, Ill (1950). Walff, L. 2.. Vitamin Forsch., I, 277 (1938). Wilson, J. 11., J. Am. Shem. S.c.,‘ §2_, 1583 (19110). Zechmeister, L. and L. Gholnoky, Principles and Practice of Chromato- graphy. (Chapman and Hall, Ltd., London, 19111). /‘ OCT 1 . 51 in! 14 fl . .. . v40... .~.el s.- .O‘AI‘ podfi~_I/. o.. u. . . v . n 'c 'l h .....c. .. a c .3. ..£..f.. .... . A,‘ ' lll‘llll'lllllllllll l lll lllllll l ' 31293 0244