THE USE OF Efl‘iER {N TEE CHROMATQQELMEH‘C SEPARA??QN OF VITAMJN DE AND ERGOSTEROL The-sis for the Degree 6f M. S. MICHEGAN STATE COLLEGE Richard Coieman Pinkerton 1943 Thixhtoeerufgthatthe thesis entitled The Use of Ether 1n the Chromatographic Separation of Vitamin D2 and Ergosterol presented by Richard Coleman Pinkerton has been accepted towards fulfillment of the requirements for M.S . degree LChemistry A 7% Major prom?!“ Date May 27. 1948 '2 . r .‘, y. (I f' ‘I. I if, .7"; b. Zn L .3; ‘ .‘ r... , figs”:- W - v.45: -.— .._-,_..a.p 1,..- ha- WEE ' |u1.\l\|ll|. lt\|\.llll|l.l.l I): ‘I.‘.\.\ 'v‘ll‘l‘l‘l I'.‘ ll“|¢lu']l\ »'.|II.I!I‘. It" '41:! I {“ll‘ [[‘(l‘ll‘nr‘ll [4[llllll!l I ,II mm (/F XTITJ‘ZLILILl D2 A1] \ g; _\1 h») i 4135‘— L by Richard Ccleman Pinkerton Submitted to the Graduate School of Eichigan State College of Agriculture and Applied Science in partial ful?iinent cf the requirgments for the degree of ,— I‘EIXSTLK ("F S 013 HOE DeDartment of Chemistry ma . .-.~.r\ 'an-y TC. I A . O’e’ 6/ (>8 \ I (7/? 8' ACKUCPLBDGHLNT The writer wishes to express his appreciation to Professor DWight T. Ewing for his guidance and valuable suggestions. EM 0’ 0W8 Introduction 1. Observation- on Ohroutopephic Separation Iron Binary Solvente Ehe Action of Binary Solvents on “Super riltrol' Column Ihe Chronetogrephic Behavior of calciferol And hgoeterol in Binary Solutione Diecuuion Summary 11. A Method for the Seperetion of Oelciferol Iron Synthetic Mixtures of Calciferol end. lrgoeterol Ueing the Binery Solvent Horne and Ether Procedure Diecueeion Emery Li tereture Cited Page 1. 3. 5. 8. 11. 14. 14. 25. THE USE (F ETHSR IN THEP ‘ipunT,erHLIC S JP.ARA1ICN CF VITAKIN D2 AND ERGCSTERCL The development of methods for the chromatographic separation of calciferol frcm sterols was the result of a search for a physical—chemi- method for the estimation of the vitamin D content of both fish oils and erradiated ergosterol. Ta kins (l) employed a hexane-ether solution to separate vitamin.A from the vitamin D in fish oils, using a column of "Super Filtrol," which is an activated bentonite clay. The sterols had been removed from the sans! nified oil previcusly by precioitation with digitonin. Analysi is for vitamin D i: as macle by the antimony triohloride method. {ingsl ey (2) found that the addition of a small amount of ethanol to the hexane—ether mixture improved the separation from vita- min A. The rate of movement of hands down the column was also increased. He found that a mixture of 50 volumes of n—hexane or Skellysolve, 10 volumes of ethyl ether and 1 volume of ethyl alcohol gave the best re- sults. He followed the chromatographic separation fram vitamin A with a separation from the sterols on short column using a hexane-benzene so- lution. Both of these workers used the antimony trichloride reaction which is not specific for Vitamin.D. Baker (3) used the ultraviolet aosorption spectrum of calciferol in determining the potency. This permitted a more detailed study of the chromatographic process. It had the disadvantage that the solvents used dissolved same material out of the "Super Viltrol" v also had an absorption spectrum in the ultraviolet. Meanwhile, Carlson (h) studied the adsorption characteristics of calciferol, ergosterol and cholesterol on "Super Filtrol" in various binary solvent mi: {tures. Results showed that when the proportion of l. alcohol in a hexane—alcohol solution was increased, the adsorption of both calciferol and ergosterol fell off rasidly. lith increasing proportions of ether in a hexane-ether solution, the adsorption decreased less rapidly, but ergosterol was adsorbed more than calciferol at any fiven per cent of ether. Bullard (5) made a survey of the "SO-lC—l" method to determine its efficiency in separating ynthetic mixtures of calciferol and ergosterol. She recovered amounts of calciferol ranging from 93-99%. She also investi- gated several binary solvent mixtures and found that pure ether or an ether—alcohol mixture also rave partial chromatograqhic separations. She concluded that only the "SO-lO-l" combination gave satisfactory results. in her work, corrections were made either by running a blank column as e did Baker or by using a sufficiently large amount of oaloiferol and erCosterol so that the correction could be minimized by dilution. The presence of the interfering substance from "Super Filtrol" made accurate detection of small amounts of caloiferol difficult. The purpose of the work reported in this thesis is twofold. First, the effects of the various Solvents and binary mixtures are examined with the intention of providing an explanation of the banding phenomena. Separations from binary mixtures are studied in order to find a more satisfactory solvent combination. Second, having found a successful mixture, 3 more quantitative method is developed for the chromatoiraphic separation of calciferol from synthetic mixtures of ergosterol and oalciferol. Particular attention is directed toward finding a better method of correction for the interfering substance from "Super Filtrol" and toward more successful separations of small amounts of the two ood- pounds. ('I- LBSBPVATICNS LN CHR-KAT-UKAHHIC Sgfinhai ;13 FROM BINARY sonvanrs The Action of Binary Solvents on "Super Filtrol" Columns Before an investigation of the chromatographic behavior of ergoster- ol and calciferol'was made, the characteristic action of the solvents on "Super Filtrol" was COnSidered. The solvents used, singly and in binary mixtures, were n-hexane, benzene, ethyl ether, ethyl alcohol and iso- propyl alcohol. The hexane used was either Eastman "Hexane" (from Petroleum) (Practical) or was obtained from Skellysolve B. It was purified by taking the 65—690 fraction and removing the benzene present by chromato- graohing on a column of silica gel activated at a temperature of 2250 C. It was hen redistilled, again taking the 65-690 fraction (6). The test its purity was its ability to pass light at 230 mu. on the Beckman Fr) 0 Spectroototelometer and the absence of extinction maxima at 2h8 and 25h mu. \ The benzene used was C.P. anhydrous, thiophene free. The ether used has dried over sodium to remove both water and alco- hol and was distilled from ferrous sulfate just before each operation. The ethyl alcohol used was refluxed over silver oxide, distilled, dried over aluminum amaljam and redistilled (7). The iso~propyl alcohol used was dried over aluminum amalgam and distilled. The solvents may be considered in two classes, polar and non-polar. The non-polar liguids hexane and benzene behave almost identically on columns of "Super Filt'ol." Both are easily desorbed by polar liquids 3. and both impart a dark gray shade to the column. Then either is passed through a column, a darker gray area or band is formed extenaing from the top. The width of this band is proportional to the Volume of solvent used, and its presence is correlated vith the diss>lvinz out of some factor in the "Super Filtrcl" which has an absorption spectrum in the ultraviolet. then this area extends to the bottom of the column, the hexane (or benzene) is practically free of this material when it leaves the tube. The adsorption characteristics in this band are different from those of the area below it, as will be shown later. The polar solvents, ether, ethyl alcohol and iso—proply alcohol, all dissolve a large amount of material from the "Super Filtrol." They easily displace the non-polar liquids and the alcohols will desorb ether. Ethyl ether gives a gray shade to the column be ween the slate-grai imparted by benzene or hexane and the nearly white shade of the clay when it is net with the alcohols or water. The shade serves as a quali- tative index of the polarity of a Solvent. All the polar liquids observed clog up the chromatograph column to such an extent that they are unfit for practical use unless they are mixed with a n n—polar liquid. then binary mixtures of ethyl or iSo—prooyl alcohol and benzene or hexane in low alcohol Concentrations are placed on a column, an addition- al band is develnoed which is characteristic of the presence of the alco- hol. This band as ears as a fine line and is usually tan or bratn in Color. Its distance from the top of the Column is proportional to the Volume of solvent used and to the Concentration of alcohol. If the con- centration is less than two volumes per hundred of hzxane or benzene this narrow band occurs within the ride gray area characteristic of the non- polar solvents and is obscured. If the concentration is areater the alcohol line occurs in the area below. Its position in the tube for the first 3 on. can be accurately predicted, given the volume of solvent which has passed and the cancentration of the alcohol. This band plays an inoortant oart in the "SO—lO-l" method of separation. The volume of solvent necessary to aush this line to the bottom of the column is equal to the total volume of the uashin: and elutinq solutions used in that method. Binary mixtures of ether and hexane or benzene show no such banding nheiunenon. The chr matcgraoh tube as ears the same as when benzene or h-xane alone are used, exceot that the shade is lighter. It was found by uptical analysis that the quality as well as quantity of material dissolved from "Super Filtrol" varied according to tie solvent used. In the case of binary mixtures, the tyne of material coding from the tube varied through the individual run. The material in the first portion of eluant of a hexane—ether system was found to he more of the type which is found dissolved from hexane aLone. In the later portions the quantity rapidly decreased but the quality was that of the substance which dissolved out of ether. The Chromatosraphic Behavior of Calciferol and Lrgosterol in Rinary Solutions Preliminary surveys of the chrgmatwyradhic behavior of calcifercl and ergosterol in binary solvents were made by Bullard (5). All solvent combinations seemed u.sUitable except for the already successful ternary combination of 50 voluies of hexane, 10 volunes of ether and 1 volume of alcohol. She reported that a separation could be made from an ether- alcohol system, but that it was not cumnlete. This choice of solvents 5. has been found impractical because it close the Colu n as Well. She al- so noted that a system of hexane and ether retained both compounds on the nrosencs of alcohol was column. ho quantitative Work was reported. The 1 th ught essential. Tnmkins had previously used a hexane—ether mixture to separate vitamin A from vitamin D (l). Kingsley (2) used aloohol in addition mainly because it gave a sharp band which could be used as a ref rence line. He developed his columns unti' this line reached the bottom of the tube. This practice became standard for chromatogravhing erradiated ergosterol solutions. It was decided to reinvestiyate these systems, and also systems cintaining benzene, to determine what qualities of the solvent were necessary for separation. Attention was devoted to the mixtures hexane-ether and hexane-alouhal to find out'why negative results were obtained. From pure hexane or benzene both calcife cl and ergosterol are strongly adsorbed (a). hence, when they were chromatogravhed, both were removed and banded in the top of the tube. Here they r mained, resisting any further elution. Combinations of benzene and hexane were found to give the same result, although they had been successfully used previous- ly in the separation of sterols in fisn oils (2). During the runs, oalcifercl changed from orange to purple, while ergosterol changed from pink to purple, indicating some change in the chronophoric groups during development. If a column was saturated with ergosterol, that portion which was adsorbed in the da k gray area mentioned before was purple in color while that excess which came in contact with the lower area was pink. This excess could be washed through a short column, but proved to give an entirely different abscrotion saectrum that that of ergosterol. The material ads»rbed in the uoner area could never be completely eluted. — n A - - 3 ~ ~ ‘ a — o a s GalClichl or ergosternl in etnyl alcohol iorned no VlSlble bands I O o and passed quickly thr u h the column, considering the extreme clogging effect of these polar Solvents open the clay. This was expected since neither are afsorbed from pure ether or pure alcohol (h). then calciferol or er osterol were chrgmatihraohed using a hexane solution oontainin: small amounts of ethyl alcohol (l-B volumes/loo), both banded in the fine alcohol line discussed before. ISO-propyl alcohol contains a secondary alcohol group, as oo ergosterol and calciferol. It was tried next in the same Concentrations, hoping that it would provide a more suitable oolar couocnent in the solvent n'xture. Results mere the same. Ihen benzene tas substituted for hexane, the colors of the bands developed in he aloohol line were slifhtly different. Yet both com- pounds concentrated id'in that petition. In every case, the develooing solution which passed through the column before the line reached the bottom of the ube has deVoid of any material except the substance dissolved from the "Suaer Filtrcl." The portion of eluant containing this band Contained larte amounts of material possessing a strong ultra— violet ahsorption spectrum. That portion of eluant collected after the band had completely passed thrtugh the column contained insignificant amounts of material. Sudan III, which was used previously as a marker, also banded in the alcohol line, althou h it is of an entirely different molecular species. hot only‘was a chrzmatograohic separation impossible, but the material which came thrcu;h the tube when either calciferol or ergosterol was placed on the column had apoarently decomposed. Its Ultraviolet absorption spectrum bore little reseutlance to either cononund. However, in the case of ergosterol, slight extinction maxima u”re retained at 270 and 2ol mu. The bands formed iere all strongly colored and the eluant 'v was yellow rather than clear. In hexane—ether syscehs, contrary to previous TCSUltS, a separation Has pcssible. ho bands were visible, but if very lar e -ucunts (greater than 0.5 mg.) cf ergostsrcl Kare used, a di?£“se blue area develcped in the too cf the tube. Calcifercl came thrcuyh the column in an undecom— posed form, but the ergcstercl could never bezreCcvered. Since no reference band auqeared, it was necessary to develop the tube by noting when a certain c;lume had passed. The choice of the arnner vvlume will he discussed in Part II. A benzene—ether system'was also found successful. The Column behsvior was poerer than then hexane vas used. The flow of eluant turcu h the tube was prnhihitively slow and the column developed fractures and air pockets. The greatest difficulty in using benzene was encountered in ontica analysis. The eluant had to he evaucrated to dryness and taken up in alcohcl several times in order to remove the last traces of benzene. If weights of calcifercl less than 0.5 m8. were used, the interference in abscrption was still too great to make a quantitative extimate cf the reCovered material optically. Larger amtunts of calciferol permitted greater dilution and the effects of benzene could be eliminated. Discussion It has been stated that the type cf material which comes thrcugh the chromato: ash cclunn, dissolved from the "Super Filtrol," varies with the solvent used. In the case of a binary solvent such as hexane-ether, the type of material varies Curing the same run. Carlscn has shown (h) that ethyl alcohol is selectively adsorbed on "Suner Fi"trol" from hexane-alcohol mi tures when the c ncentration of alcohol is less than r—,. o 1 .‘ 1 c n v, - 1 v. - - . c 259 by'weignt. Ltaer is poSitively aCSUTbeC iron hexane—ether mixtures when the eth r content is less than L2? by teibht. These chanyes in the quality of ma erial leavine the tn 3 might therefore be expected. In making any Correction for the optical a s ration of this substance, the volume of wash soluticn used in a chromatoeram must be kept constant. This not only keeps the amount of dissolved material within certain limits, but keeps the quality of the absorption swectru; iniform. KnOLin; that ethyl alCchcl is quite strgnply adsorbed from heXane on "Super Filtrol," an explanation of the presence of the alcohol line might be offered here. During the initial part of a chromatograoh using a hexane—alcohol mixture, more material Would be dissolved from the too of the Column than if hexane alone were used. The alcohol Content should then decrease, as it is selectively adsirbed. Some of the diSSolved material would then be rode asited, since its solubility‘vould be in reased. Also, any impurity oresent which is soluble in small alCthl conchltraticns but which is relatively insoluble in hexane alone will be handed in the rexion'whrre the aloohol content begins to decrease. As has been shown by observations on both hexane-alCchol and benzene—aloohol solutions of erposterol, calciferol and Sudan III, the banding depends upon the position of the alcchol line. This means that the position of the hand depends noon the volume of wash Solution used be- fore the particular compound is placed on the c luan and upon the alco— hol concentration of the binary m xture. This type of banding is pecvliar to systems Containing more than one liquid in the solvent mixture. Then a single s,lvent is used, the material to be chromato— graphed.will initially band at the too of the column if it is adserhed at all. In the solvent hexane—aleshol, the most innortant factor in banding seems to be the LCSthiufl characteristics of the molecule 9. regarded as a function of changing alcohol concentration. Calcifercl and ergosterol behave similarly in adsorption under varying concentrations of r"! alcohol in hexane (h). lhis explains why no separation was detected. '7' 1 it tas observed'that ergoster;l chro ma-sg‘aphed from hexane or ben— zene solutions was retained more strongly when it was adsorbed in the uoper portion of the cvlumn. Its Color was blue in the under area, pink in the lower region. A difference in oh on the "Suoer Filtrol" surface ndght aCCount for the variation in color. ‘Then Sudan III is adsorbed on "Super Filtrol" frum hexane it is deep blue in color. If sodium carbon— ate and a small amount of water are added, the Sudan III is desorbed and returns to its normal red Color. If the miXLure is then acidified.with HCl solut'on, the Sudan III is rearscrbed and changes back to blue. Yet this oil soluble dye is not normally an indicator. The process is apparently quite reversible. This would indicate that "Super Filtrol" normally presents an acid surface. It is knctn that erfosterol is sensitive to the hydroren ion, although there is only a slight change in color in the visible range. Acid causes rearrangements of the double bonds in the molecule (8). A COMQaTlSeH may now be made between the "SO-IO—l" method and the separation frum a hexane ether solution. In the "ED—lU-l" method, ergosterol bands in the alcohol line. Calciferol passes throush the Column ahead of the eryusterol because of the presence of ether. This means that the effective column length is decreased, since the aloohol line reaches the hi dle of the column before the compounds are added. The advantare is that the line fo ms a convenient reference pvint for stopping the development. In a hexane-other chr nat:graoh, er.osterol is adsorbed at the top of the column and does not travel damn the tune very fast. The full 10. —-\ length of the column is emolg ed in thes s Q)arat i n. Again, calciferol passes through because it is deserted more effectively by the ether. Rate :3 flov of the el ant is sL T:er, but this disadvantage is removed by using a shorter c lLuLn. The :vera lL rnflulinf time is less. Smaller amounts of material may he eJllat , as will be sh tn in Part II. A binary systern suitable.for nuantitative separations must possess at least twu characteristics. First, the separation must be reasonable ctnplete on a column of practical length. Secsnd, if analysis is to be macie optically, the material appearing in the eluant must have an ab— sorption spectrum unchanqed by its Contact vith the Column. It is pointed out that in all successful separations, ethyl ether was th essential in redient. Systems in ahieh ether was a sent did not satisfy either of the eb m>flm mo mvoooopm Immuwrm Qde ca wmam mmfimmflm Hohnpmompn OHpnM esp Cd HopoMfloaso Mszpm thpo HJHvMfloaxo man.mfl>zH @cwfi 06H; HohvmwoAnr mafia mcmumm Umflmup Coopo Hosooqm op oSU masao> Immvflc: op mdam vamn mcflm CH Hohopmcxhfl he Hoamoaw 0:0; ammOHmlomH UQHMHQ cushm Hogcoad 09 mdd mo Amzpm mm Izmdfizm op mzam Camp mcflm CH HohmMHoamo msam onwmmm nfizfioo mo mop msam Gfi Umflpcmww man: op xnfla ma Haw II mso: Hohmpmomkm 0Com QESHJQ mo gap mamhzg :fl UngQmwm anon op mgcmpo mfl Haw II acoz HohOMflono mammmm mafia pqwdam qH cwhm>oomm GOwahmawm amflpopmu poaoo Coamepom Uswm vasomfioo pmo>aom =qyzequ magma: ad aifimqfi RE acuiwmqqfi E 8 .m.v._H...MJH_S.a m6 ofieEflufléfi33.: a? 5 mfiuHBdbwfimflu H mqmde 12. 'possible. h. 'Zhen the combinatien hexane-ether-alcehcl is used, calcifercl passes thr ugh the cciumn due to the or sence cf ether, while erycctcrcl bands in the line due to alcchel. 5. Unless ether is present, the a scrjti n aBSerticn Spectra of calciferel and ergcsterol are altered by their centact tith the Coluw and cannot be identified. If other is used, calciferel passes thrsugh the column unaffected. l3. II " l‘r!n"” v: (v? f. ‘ 11' ‘W V” .1 MP“. “‘..‘ z‘rT‘ ‘1, ' inf-"PH." rlvf p1" , '1' 1r I. .‘.J. 3...»; v.5. van-L1,“. *4le .1“); .JIJG VL...L._.1L4.VLI Jul“... .‘..L .4 HLAOI..LL.}. “.7. 0 ~. ~r ._1".;-v.~'==*-‘ f‘,"\ ‘rwv‘ Q£.IL‘1‘.J¢L.'. rEmQLnflJ ALL}! LJkLiaJI-i Having decided upnn a hexane-ether mixture as the mast suitable binary solvent fer use in the chrsmetoyraphic sedaratien of Vitamin D2 and ergosterLl, a series cf separations were run on fsur synthetic mixtures of the tum; c-npeunds. Five trials were hide with each mixture, varying: the height cf the sawle in each 0 see to Oete r Line the lini- taticns of the c lunn. The ansunts sf calcifercl recevered frtm the ch10 :1atLgraus were dettrm ned ovtically en the Decknan Swectrephctelo- meter and are reperted in Ta bles II— Procedure The ChrcmateLragh Tube.—- The body cf the Chrsnatc;raph tube was made frax‘. ass tuninr havin: an outside dia Ieter of 10 mm. A slight cons trictien'\.as ulaced betreen the bcdy cf the tube and the outlet in crder to suopurt a Cutten plug. .In additiun, an adaptor'was cmnstructed to fit the tube to a 25 ml. Erlenmeyer flask and to permit partial evac- uation (Fig. 1). Packing. -- A Cctt_n elm; was placed in the tube and "Super Filtrel" was packed in it t; a desth of h cm. While the tube and receiving flask were being evacuated. A dif.ere :tial pressure of - 10 mm. was used threuqhout. The finely divided clay'was settled by gently tapping on the tube with a pencil. The Solvent. —— A mixture of five vtlumes of hexane and one volume he sclvents P3 of ether was made up just befsre eachsseries pf segarutiens. lb. w CHROMA’I'O GRHPH TUBE “ SUCTION :— JUPEB F/L TROL” TUBING- 4cm. COTTON PLUG- K co NJTRICT‘ION Fig. l.-- Deteil of Chromatograph Tube and the Adaptor used to Permit Evacuation of the Receiving Flask. were purified as described in Part I. The other had been distilled the same day. This rir‘ure was used in vashin; the colunn, developin. the elm onat Jam and makinf all dilutions of the sample. The Sample. —— A kn mn mivture of or~cstercl and calcif ml was made fl up frem SL;Ck Solutitns 61 the c noounfis and va3o ated to cur nes s, using the aspire or. The re sic:ue uas disso lv tin a 1 known volume of the hexane— ether mi ture. Five dilu t aietltns of a “liture Erfosterol l. 2. ‘-.'."eight of Calcificr .l in my. placed on O.h292 column Veight of Ergoste*ol in mg. placed on column (Both are dissolved 'in 1 ml. Of hexane— ether mixture) El. of alcohol in which zecovery from AD column‘was dissolved Uncorrected extinction of recovery at 26h mu. 0.506 0.5 Uncorrected extinction of rectvery at 230 mu. 0.3335 0.3 Corrected extinction O) of recovery at 260 mu. 0.5l0 0.5(5 lei nt of Calciferll recov red, in my. 0.036 0.0863 Per cent of Calciferol reCLVered 101.6 100.5 Average Per Cent QeCUVery fur Five Triels l7. 0.0858h 0.0878 Trial 3. 1L. 0.0h292 0.02861 0.0039 0.02927 h h 0.h12 0.202 0.082 0.3h3 0.0012 0.0296 95.9 103.0 100.50 of 09.0% Celciferol and 50.63 5. 0.02lu6 0.02295 TABLE Iii Chromatographic Separations of a Lixture of 59.03 Calciferol and 00.6% Brgcsterol l. 2. teight of Calcifercl 0.0292 0.03580 in mg. placed on column ‘ieight 0f grggster 1 in mg. placed on 0.2927 0.05320 colmm1 (Both are dissolved in 1 . of hexane- ether mixture) E1. of alcohol in which recovery from 00 8 column was dissolved Uncorrected extinction of rCCCVery at 200 mu. 0.530 0.5125 Uncorrected xtinction of recovery at 230 mu.0.315 0.330 00 rected extinction of recovery at 200 mu. 0.505 0.073 Lcifiut of Calciferol TGCLVOTed in ms. 0.032 0.0303 Per cent of Calciferol recovered 100.6 93.6 Average Per cent Reecvery for Five Seaples Trial .3. 0. 0.00292 0.02106 0.02927 0.1000 0.539 0.2955 0.3515 0-2155 0.091 0.2533 0.0020 0.02166 97.8 100.9 99.00 5. 0.01073 0.00732 0.1725 0.150 0.1307 0.0112 100.1 Cl‘xromato;'raphic Semaratiwns of a .ki‘ture ff 75.13 ’Iigrlcijerol and 20.9,; Sr; sterol Trial 1. 2o 3. LL. 5. Keight of Calciferol in my. placed on colum1 Height of Ergcs ercl in mg. placed on Column (30th are dissolved in 1 m1. of Lexane- ether mivture) Kl. of alcohol in thich recovery fram column was dissolved Uncorrected extinction of recovery at 200 mu. Uncorrected extinction of rccavery at 230 mu. Corrected e tincti n of recovery at 250 mu. Height of Calciferal recovered in mg. Per cent of C'lcifercl recovered Average Per cent Recovery for Five 0 I C 19. angles 0.00292 0.02106 0.0102 0.00710 0 0. 51 0.207 \J‘I 0.336 0.201 0.033 0.252 0.013 0.0215 96.2 100.3 99.02 0.01073 0.00355 0.177 0.158 0.131 0.0112 100.0 ‘ T111 3 1.12; V ‘ *- - ‘— . C_‘- .-‘~\r”‘ -. I.‘A .0 --‘.'1r4- ~--“ CkerIlatQ. ,I‘8.h).'.lC 1.152.104 (3.0.1.0118 Us. 8. . 1210‘er (4.1. rzasterol l. 2. height rf 0 in mg. . 01a co lunn Height of Arrest in mg. 913090 on 0.00530 r0 1 0.00833 0.00997 column (30th are diss 1V<3d in 1 ml. of hexane— ether mixture) Ml. of a1 >hcl in which rcccv:rv from 00 8 C“lm31inas dimmlaed UnC2rrected e"t.nctimn of recovery at 200 nu. Uncorrected extinction of rec vely at 230:0. 0.270 0.302 Corrected e::t:ncti n of recavery at 200 mu. Leiwht cf Calciferol recavered in mg. Per cent of Cale ifer;l TOCLVUTCd 0.500 0.003 0.0325 1 Average Per cent ReC0Ver for Dive Satples 20. i) o 3 3 CC.” I .1. U. 0123“? 0.00003 iferol 0. 0.02106 0.00202 0.2055 and 9.73 0.1915 0.200 100.16 2 5. 0.01073 0.00121 Extinction at 206 nu. Extincti on at 23 O 1.111 a 3200/3230 AV ra e E26h/i230 xSarfle on column dissolved in 0 n1. 0‘ -1 :101- -‘., .. 1 ' . : .2 \ r- .- LXLlheblOn Characteristics cl Lu stance .0 -‘_‘ 1‘ p -. f‘.,,,- ‘ .‘ .- J-“ - LlssolVed iron "eager 81101.1" 0. Q; residue% 0.0315 0.0705 of residue* 0.001 0.135 I \(7—1 0 LVLU ts of residue fram 12 ml. of eluant from a blank ethyl alcohol. mah ‘ TYT LJLiJJ-JA’J u .L. "‘ \ ~ ' . \ f‘ - ‘ ’ n W A f‘ '. n-'~ ' in ~ "'5" 1 1'“ . -’ ‘VJ‘ « ' n 1“ A;SUT}tl n VuiJJS ilr ehlci-er1l “eonveiee -r.m a Llthre o: h9.hfl / "I f A ‘I “r “ZINE c'\-.~'-. ‘f 1‘ . _~‘~'- .A cx', 7 “ 4-,- ‘ p - . ~ Cal-J—C‘l-.L \ijJ. b.1— C»¢'L(A SJ 0 VI.) .Jl‘fd -l:" UVI‘ ‘.'l CLAIC- \J “ \J‘JQ Vkliwies Have Length ?rt?ncti n of Currecti n Cipreeted. E‘x/26h ij/Zéh A- i in mu. Total Residue* Extinctien fu‘fire Calciferol —;.055 —0.0Sl —Q.Oh7 0.1éO _o.oh5 -0.0hh “'0 o 0‘43 0.5u5 0.573 2&0 0.230 ~0.0u2 O. 99 0.730 0 019 250 0.2h8 -0.0hl 0.207 0.01) 0.852 252 0.255 —0.030 0.2l7 0.355 0.0‘5 25h 0.263 -0.033 0.225 0.qu 0.9 0.275 0.280 —0.037 _3.o3é .1), 035 —o.03h 0.969 0.905 200 0.207 -0.033 0.250 1.000 l.J00 *The residue frum an uri'inal 0.02lh5 mg. uf Chicifurfil V2: dis 5 null/’80 O 111 E1 t Lnl ‘f L ml. Cf ethyl ale L_L lave length AXLifiCfil n cf C rrecti0n Cerreeted 0 A/2éh u A/2éh in mu. T00;— Zecidue% Extinctian fM'RHe alcif“ 268 0.230 —0.032 0.203 0.976 0.975 270 3.275 -0.031 0.260 0.961 0.900 272 0.265 —0.030 0.235 0.925 0.906 276 0.233 -0.029 0.220 0.875 0.855 276 0.200 -0.029 0.211 0.631 0.799 273 0.225 —0.023 0.197 0.776 0.733 260 0.203 -0.027 0.161 0.713 0.675 232 0.19” —0.027 0.167 0.658 0.603 230 0.175 -3 026 0.109 0.537 0.537 286 0.156 —).OZb 0.132 0 520 0.672 23; 0.100 —0.023 0.117 0.061 0.007 290 0.120 —0.022 0.102 0.602 0.306 292 0.109 —0.021 0.003 0.306 0.293 290 0.095 -0.020 0.075 0.295 0.202 296 0.082 —0.019 0.063 0.208 0.196 298 0.070 -0.017 0.053 0.200 0.158 300 0.067 -0.016 0.060 0.173 0.126 m - TV" ,‘l/"1.'1 y“ T 1.-.:PLL‘J ‘J.L..L (UkliAILuL‘LLl) The residue frcm an cririnal 0.02l06 mg. cf calciferel was dissolved in a total cf 0 ml. of ethyl ale hel. 23. exf/Ncr/ON OF MATERIAL //v VML. 0F EtOH ‘8 I ‘ M L. o o O m 2&0 240 260 360 270 .230 290 300 WAVE LENGTH IN ”7,44. Fig. 2.-- Extinction Curves of (1) Totel Residue in 12 ml. of Eluant from a Chromatographic Separation of a Mixture of 49.4% Celciferol - 50.6% Ergosterol and Containing 0.02146 mg. of Calciferol, (2) Substance Dissolved from "Super Filtrol" end (3) Corrected Celciferol Recovery Curve. Extinction values are for the total solids dissolved in 4 m1. of ethyl alcohol. fl 1 ‘- 3 _. + 1 , . + a .. O .2 _. -‘ .1 _ l 1 i I 1 1 230 270 ’20’0 , 260 270 280 290 300 VflhdethhSTH AN n00. Fig. 3.~- Comparison of a corrected celciferol recovery curve (1) with tie adsorption curve of pure celciferol (2). The calciferol was recovered from a 49.4% calciferol - 50.6% ergosterol mixture. TABLE VIII A Con-arisen 0f the Litincticn Values fur the Cuhetdnce .‘ LisselveC fr,n "Buyer biltrcl" as Calculated fr-m the Currecticn Ftrnule ‘ ‘ 0‘ \ 1 . .(< ~‘ ‘ - .- . ’3“. ,K ‘ r1 \" 4-" r‘ -- n ‘ u a dLC the ValuGS expectec 1r m 00:.0110t10n Dilution Factor 00 8 0 0 0 0 fix. of Galciferol 0.0292 0.0053 0.00292 0.02861 0.02106 0.01072 Placed on Column *xtincticn at 260 cu. 0f the Sutstence Per cent 0 Leifercl biss:1ved frcm "Su7er Filtrcl" frtm Ferdula in Iixture 09.0 0.036 0.005 0.061 0.001 0.033 59.0 0.025 0.0025 0.003 0.0022 0.002 75.1 0.013 0.023 0.030 0.035 0.006 90.3 0.012 0.025 0.030 0.023 0.0305 Extinction at 260 mu. of the Substance Calculated frum the Averaged 3100K Determinaticns and the Liluticn Blank Determinaticns 0.000 0.013 0.030 0.036 0.036 0.036 Containing No Calciferclv Dilution Factor 00 03 :7 pr pr 5% Discussion The Column -- The aiaunt 0f "Suier Filtrel" used mas measured by the le1gth 0f the c luin rather than by uei ht. A check Sh died that the M01 nts 0f Super Filtr l in c.luans packed t1 tre same length did net vary by more than 53 and that the variations in the results 0ktained here in nu may used as a c;wprenise. separation, while longer lea ths Volumes (0 running time. slower at the The Lash which 1as suff Filtr 01" which ‘assed the constant. the advantages 0 0.1. The Elutinf initial m1. had of the calciferal had large That the cheice was average caleifer 1 rev purity, --3 ., from a mixture cf car elated with the mxsh and (cvelahla“ amount salutien and V , 7- amounts Cl 311 CO'. 1L vs riatiens in wci ht. A length of 0cm. was Shorter c luan lengths did not five canalete required not only an increase in the salutiuns used but unduly increased the The rate of fl:: thraugh the celunn was 0 1.1ireraelv end of a run as clog in: increased. Vx’r Solution. -- The volume of wash solution “as fixed at 8 ml., icient to dis sclve eut most of the substa ince from "E.uper interferes tith optical analysis. fter this volume had of this material dr peed sharaly and 3.ained almo st Althnu5h more mashing might have redeved an additiunal aaeunt, c be Cained vauld he effset by a greatly increased veluae an imi in running time. Valune. It was faund that after the saaple in the been develfiped 11th 11 ml. ng1e, all but a small fra ctien flask. After this volume > ’ passed inte the receiving er 'ctei 1 (or a m eificatien) started t0 c “ —.( . ‘ _‘ " H h9.ip ceiC1feILl. .itn miltnreo C,Jualulflu Sfluiidl WUI Cents hf ergostervl, the slight distertirn in the rcLien begund 26h mu. eininishes. S unwary A method has been tested for the Chromatcgraahic separation of calciferul and ergccter l en "Super Filtrcl" using a hexane-ether sol- uti n. The evera 9 yield sf calciferol for the'ttenty separations tried was 99.87%. The four synthetic mi tures used C ntained h9.h, 59.h, 75.1 and 90.35 calcifercl. Examination of the a erage recoveries fgr each cf the fcur mi tnres sh:«s that the column {ave the same quanti— tative results regardless of the ancunt of erggstersl present. The quality of the reCuvered pr.cuct was hette when the per cent ergtsterel in the mixture was smell. in unts of calcifer;l separated by the column ranged fr,m 0.4 to 0.01 mg. The smaller column used enabled the separation cf such small quantities and also decreased the amount of material used. 1. 2. 3. 4. 5. 5. 8. LIME CITED Ionkins. I. 8.. Ph D. Thesis. mehigan State College. 1942. Kingsley. G. V.. H: D. Thesis. Michigan State College. 1942. Saber. D. 3.. KS. Thesis. Michigan State College. 1944. Carlson. C. Wu Ph D Thesis. Michigan State College. 1946. Bollard. L. J.. 11.8. Thesis. Michigan State College. 1945. Ewing. Kingsley. Brown and lust. Ind. and Eng. Chem. Anal. 1... g. 301 (19.3). Weissberger and Freshener. Cram Solvents. p. 123. Oxford University Press. 1935. Rosenberg. H. B... Chemistrl and. W 31 1h: Vitamins. p. 359. Interscienoe Publishers. Inc.. It. 1.. 1945. Oser. Melnick and Peder. Ind. and Eng. Chem. Anal. 10... 29. CHFZMBTRY DEVI. T612.015 203321 \ P655 Pinkerton 321 93 02446 7874