MI | 5 t 146 215 THS A CRITECAL STUDY OF THE SPECTROPHOTOMETRIC METHODS F'OR THE EVALUATiON OF THE POTBNCY OF IRRADlATED ERGOSTEROL Thesis for the Degree of M. S. MICHIGAN STATE COLLEGE Donald Hart Baker 1944 A CRITICAL STUDY OF THE SPECTROPHOTOMETRIC METHODS FOR THE EVALUATION OF THE POTENCY OF IRRADIATED ERGOSTEROL by Donald Hart Baker A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE Department of Chemistry 19% The author wishes to express his gratitude to Dr. D. T. Ering, under whose direction this investigap tion was carried out, and to Parke, Davis and Company, whose fellowship made this work possible. TABLE OF CONTENTS Introduction Equipment and Reagents Description of Procedures Study of the Length of Time Required for Saponification Distribution of Vitamins D in Ether Portions During Extraction Study of Adsorbents Natural Oils Superfiltrol Correction for Ultra-violet Measurements Study of Calciferol and Ergosterol Summary of Oil No. 3772 Irradiated Ergosterol in Corn Oil Irradiated Ergosterol in Alcohol Irradiated Ergosterol in Fish Oil: A Itudy of Corn 011 Study of Sudan 111 Summary Literature Cited FM”?! ' QWJUI’ZL 10 14 15 15 21 27 27. 29 30 46 INTRODUCTION Numerous attempts have been made to quantitatively esti- mate vitamins D by chemical means, usually colorimetric. One of the most reliable colorimetric reactions is that between vitamins D and antimony trichloride in chloroform solution. This reaction yields at 500 mu.an absorption maximum.whose extinction (1%, 1 cm0 is directly prOportional to the amount of vitamins D present. Unfortunately, a number of other comp pounds react with antimony trichloride, and their absorption at 500 mu is sufficient to prevent an accurate estimation of vitamins D potency. Vitamin A, sterols, carotenoids, and pigments are among these compounds which interfere and which also usually occur with vitamdns D in natural oils. Therefore, after the selection of the antimony trichlor- ide colorimetric reaction for the determination of the vita- mins D potency, the most important problem was that of es- tablishing a method of purification whereby the vitamins D might be separated from the interfering’compounds mentioned above. Tomkins (1) used the following procedure: saponifica- tion of the oil, cooling and treating with digitonin to re- move sterols, chromatographing to separate the vitamin A from vitamins D. The vitamins D were then determined colori- metrically as mentioned earlier. Saponification with.N/2 a1- coholic potassium hydroxide serves as an important means of separation since the vitamins D are contained in the .2- non-saponifiable fraction. In the chromatographing vitamin A, carotenoids, and pigments are adsorbed onto activated ben- tonite clay from a hexane-ether solution. Kingsley C2 & 3) modified Tomkin's method until the following procedure resulted: saponification; extraction of the non-saponifiable fraction with ether; removal of vita- min A, etc., by chromatographic adsorption, The vitamins D and sterols were then determined colorimetrically with anti- mony trichloride. The vitamins D were removed from an ali- quot of the sample by chromatographic adsorption, and the sterols determined colorimetrically. The difference in the two Log Io/I measurements was then used to calculate the vitamins D potency. Kingsley calculated that a factor of 19,300, when multiplied by the E (1%, 1 cm) of the antimony trichloride reaction at 500 mu, would give the number of vitamins D units per gram. YOung (4) verified Kingsley's method for natural fish oils containing vitamins D, but he found wide deviations be- tween the physical chemical assay and the bioassay when Kingsley‘s method was tried on samples containing irradiated ergosterol. Young suggested that some modifications of Kingsley's method might be used on samples of irradiated ergosterols. Rage (5) modified Kingsley's method for natural oils by substituting a swirling of the sample with the activated bentonite clay for the second chromatograph, or sterol cor- rection. This modification not only shortened the procedure .3- but also simplified it since it had been difficult to separate out all of the vitamins D by ordinary chromatographing. Naturally incomplete removal of vitamins D would cause a low value for the calculated potency. Kingsley's method with Hage's modification still was not successful when applied to samples of irradiated ergosterol. EQUIPMENT AND REAGENTS The adsorption columns must be carefully and uniformly .prepared to obtain reproducible results. The tubes for the chromatographic separations are made by sealing a 6 cm. length of 7 mm. Pyrex tubing to the bottom of a 1.6 x 15 cm. (0.625 x 6 inch) Pyrex test tube. These tubes are cleaned before use by soaking in sulfuric acid-dichromic acid solue tion, rinsing with distilled wgter and with alcohol, and finally drying in an oven. The suction apparatus is designed so that a bank of 8 columns can be develOped simultaneously, controlling the pressure with the aid of an Open-tube mercury manometer attached to a suction flask. unless otherwise specified, the adsorbent used is a finely divided grade of activated bentonite clay (Superfiltrol, obtained from The Filtrol Corp., 315 West 5th St., Los Angeles, California). The adsorption columns for the first chromatographic separation are prepared by placing a small wad of cotton in the bottom of one of the adsorption tubes, pressing this down firmly, and adding enough of the adsorbent so that, when very firmly pressed down with a piston (a glass rod with a cork on one end) under 6 cm. of suction, the height of the packed .4- column will be 3 cm. A second and equal portion of the ad- sorbent is then added and pressed down as before to give a hard, level surface. It is important that there should be no air pockets, as they cause irregularly shaped adsorption bands. To aid in overcoming this, the piston-head cork used in packing the columns is slightly smaller than the inside diameter of the Pyrex tube. This also helps avoid loosening of the adsorbent by suction when the piston is raised. Unless otherwise stated, all extinction measurements of the antimony trichloride reactions were made with 1 cm. cells on a Bausch and Lomb visual spectrOphotometer. Absorp- tion curves in the ultra-violet region were measured with 1 cm. quartz cells on a Beckman quartz spectrOphotometer. Too great emphasis cannot be placed on the purification of the solvents used in this work. For example, peroxides in ether, a fairly common occurrence, will cause a large error in measurements since they give a definite color reaction with.antimony trichloride. Alcoholic potassium hydroxide is prepared by dissolving 28 grams of C. P. potassium hydroxide pellets in 95 percent ethyl alcohol to give 1 liter. USually, however, this so- lution is made Up in 100 or 200 ml. lots as needed. C. P. ethyl ether is used without further purification for extracting the saponified oils and for elution of ad- sorption columns. The anhydrous ethyl ether for the chromatograph is puri- fied by washing C. P. ethyl ether with 1 percent ferrous -5- sulfate solution to remove peroxides, then 10 times with dis- tilled water to remove alcohol, drying with phosphorus pent- oxide, decanting, and storing over sodium. This ether is distilled as needed and kept over ferrous sulfate. The Skellysolve is purified by washing twice with con- centrated sulfuric acid, allowing it to stand for 24 hours each time over the acid, then washing twice with 10 percent sodium carbonate solution, and once with a mixture of 10 per- cent sodium carbonate and 5 percent potassium permanganate solution. .The Skellysolve is allowed to stand over this mix- ture for 24 hours. Then it is washed 15 times with.distilled water, the reagent decanted into a dry flask, and dried over sodium for at least.24 hours. The dried solvent is then dis- tilled (68 to 70 deg. C.), the first 5 percent and the last 10 percent of the distillate being discarded. The absolute ethyl alcohol is a high-grade commercial product. C. P. chloroform is washed thoroughly with 7 approxi- mately equal portions of distilled water, dried over anhydrous potassium carbonate, decanted, and distilled, discarding the first and last 10 percent of the distillate. The purified chloroform is kept with activated charcoal and filtered as needed. C. P. thiophene-free benzene is dried over sodium, dis- tilled, and shaken with Superfiltrol before use. The antimony trichloride reagent is prepared by dis- solving 18 grams of C. P. antimony trichloride in 100 m1. of ~6- the purified chloroform and then adding 2 ml. of redistilled acetyl chloride. The ether is tested for peroxides by adding a few milli- liters to a mixture of potassium iodide and starch solution. The presence of peroxides will be indicated by the appearance of a blue color. The chloroform is tested for chloride ions by adding a few milliliters to a silver nitrate solution made slightly acidic. A white percipitate indicates the presence of chlor- ide ions. The chloroform is tested for phosgene by adding a small quantity to a saturated barium chloride solution. An Opaque film between the chloroform and water layers indicates the presence of phosgene. Due to the instability of purified chloroform, it is not advisable to prepare more than a week's supply of chloro- form and antimony trichloride reagent at one time. DESCRIPTION OF PROCEDURES The following procedures were used in this investiga- tion: Procedure A (Kingsley's method with Hage's modification) Step 1. Weigh out sample containing 4,000 to 100,000 U. S. P. Units. Step 2. Add 10 ml. of alcoholic potassium hydroxide if the sample weighs 1 g. or less, or 10 ml. per gram if more. Step Step Step Step Step Step Step Step Step Step 3. 5. 6. 7. 8. 9. 10. -7- Place a short stem funnel in neck of flask to serve as condenser. Saponify in water bath for one hour or more at 70 to 75 deg. C. Swirl frequently. Cool and add 20 ml. of water for each.10 ml. of potassium hydroxide. Extract with ethyl ether in separatory fun- nel using one 40 ml. portion of ether and three 20 ml. portions. Combine ether extracts and wash with six 50 ml. portions of water. Do not agitate during the first three washings. Filter washed extract through anhydrous sodium sulfate to remove water. Rinse separatory funnel with ether. Evaporate to dryness under reduced pressure, using hot water bath. Dissolve residue in 5 ml. of the following mixture (50-10-1): 50 parts Skellysolve, 10 parts anhydrous ether, and 1 part abso~ lute ethyl alcohol (by volume). 11. Prepare 6 cm. adsorption column and wet with 12. 10 ml. of 50-10-1. Add sample, 5 ml. of 50-10-1 for rinsing flask, and 55 ml. for developing the column. Each addition of solvent is made just before the t0p of the column becomes dry. Step 13. Step 14. Step 15. Step 16. Step 17. Step 18. Step 19. Step 20 0 St ep 21. Step 22. -3- USe differential pressure of 6 cm. of mercury for chromatographing. Dry by drawing air through column for 5 to 10 minutes. Remove adsorbent down to 2 mm. below the vitamin A ring, which is yellow or orange. Elute the remainder of the column with 10 to 15 ml. of ether. Evaporate combined filtrate and eluate to dryness (see step 9). Dissolve the residue in 10 ml. of chloroform. Add 10 m1. of antimony trichloride reagent to 1 m1. of the chloroform solution, swirl 30 seconds, fill absorption cell, and deter- mine the extinction at 500 mu.on the Bausch and Lomb visual spectrophotometer exactly 3 minutes after starting to add the reagent. This reading represents vitamins D plus sterols (D plus S). Evaporate 1 ml. of the chloroform solution (see step 9). Dissolve the residue in 25 ml. of B281 (2 parts Benzene, 1 part Skellysolve by volume). Add Superfiltrol (the amount which will fill a 5/8 inch test tube to the depth of 1 inch. -9- Step 23. Allow to stand for 50 minutes with very fre- quent swirling. Step 24. Filter and rinse with two 10 ml. portions of B381. Step 25. Evaporate filtrate to dryness (see step 9). Step 26. Dissolve residue in 1 ml. of chloroform. Step 27. Repeat step 19. This reading represents the sterols (e). To calculate the potency in D units per gram: 1. Subtract sterols from vitamins D plus sterols, (D plus S)-(S). 2. Determine the E(1%, 1 cm.) of the difference. 3. Multiply the E(l%, 1 cm.) by 19,300. The result is the potency in D units/gram. Procedure B (Direct)- Steps 18 and 19 of Procedure A. Procedure C (Saponified) Steps 1 to 9 inclusive, 18, and 19 of Procedure A. Procedure D (Saponified and First Chromatograph) Steps 1 to 19 inclusive of Procedure A. Procedure E (First Chromatograph) Steps 10 to 19 inclusive of Procedure A. Procedure F (Second Chromatograph) Steps 21 to 27 inclusive of Procedure A. In case, during the abovementioned procedures, it was de- sired to measure the absorption of the sample in the ultra-violet -10- region the procedures were modified in the following manner: instead of dissolving the sample in chloroform prior to adding the antimony trichloride reagent the sample was dissolved in absolute ethyl alcohol, and the resulting solution was divided into two or more portions. For the ultra-violet measurements one portion was diluted with more absolute ethyl alcohol until the proper concentration for the most accurate measurements was reached. For the antimony trichloride reaction measure- ments another portion was evaporated to dryness, taken up in chloroform, and the extinction measured as described previ- ously. When the samples consisted of irradiated ergosterol, the length of time for saponification was shortened to 30 minutes. Hereafter, it may be assumed that the saponification time for samples of irradiated ergosterol was 30 minutes unless stated otherwise. STUDY OF THE LENGTH OF TIME REQUIRED FOR SAPONIFICATION When it was noticed that samples of irradiated ergosterol in corn oil with. alcoholic potassium hydroxide during saponi- fication became homogeneous sooner than had been observed for samples of natural oils, it was decided to make a brief in- vestigation to determine the length of time required for com- plete saponification. The oils selected for this study were #3772, a solution of pure calciferol in corn oil the bioassay of which is 200,000 D units/gram and Be 103, irradiated ergosterol in corn oil, -11- which had a bioassay value of 700,000 to 750,000 D units/gram. Table I shows the measurements made on samples of these oils run by Procedure C with varying lengths of time for saponifi- cation. As a result of this investigation, it was decided that 30 minutes would allow sufficient time for complete saponi- fication and that 30 minutes would be adOpted as the standard saponification time for samples of irradiated ergosterol. DISTRIBUTION OF VITAMINS D IN ETHER PORTIONS DURING EXTRACTION To make sure that all the vitamins D were being extracted after saponification, a brief investigation was made as to the amounts of vitamins D extracted by each of the four portions of ethyl ether used in this part of the procedure. The same two oils were used in this study as were used in the preceding study of saponification times. Procedure C was followed except that the portions of ether used in ex- traction were kept separate after extraction and were treated as separate samples thereafter. Table II shows the measure- ments made on the portions of ether after extraction. Since the Log Io/I measurements of the third and fourth ether portions of both oils were so small that they would be attributed to the insensitivity of the eye in reading the instrument at low or zero extinctions, it was decided that extraction with four portions of ether as described in Pro- cedure A was sufficient to extract all of the vitamins D con- tained in the amounts of sample as were ordinarily used in this investigation. -1 2... TABLE I Sample Time of Calculated Oil Weight Saponification Log 1 e/ I “1%, 1 on.) D u/ g 3772 0.060g. 3 minutes 0.48 8.80 170,000 e a 6 I O.u9 8.98 173,000 . . 10 ' 0.ns 8.80 170,000 ., N " 15 " 0.1V] 8. 62 166,000 a I 30 fl o.ne 8.80 170,000 a I #5 " 0.“? 8.62 166,000 Be 103 0.0203. 1 minute 0.47 25.8 n98.000 ' ” 6 minutes 0.53 29.2 563,000 n u 11 a 0.53 29.2 563,000 " " 15 " O. "-8 26.“- 510.000 " " 3O " O. 48 26. N- 510. 000 u a as u 0.53 29.2 563,000 " " 60 " 0.52 28.6 552,000 -13- TABLE II Sample Ether 011 Weight Portion Log Io/I 3(1%, 1 cm.) D u/g 3772 0.060g. 1 0.u2 7.80 150,500 2 0.0% 0.73u 14,150 3 0.025 0.u5s 8,850 n 0.025 0.u58 8,8;0 Be 103 0.0203. 1 0.53 29.2 562,000 2 0.075 n.12 79.500 3 0.025 1.37 26.n00 t 0.030 1.65 ' 31,900 599.300 -14- STUDY OF ADSCRBENTS A study was made in which the effectiveness of various ad- sorbents was compared to that of the adsorbent, Superfiltrol, normally used. The 011 B 568%, a standard high D fish oil having a bioassay value of 17,000 D units/gram, was used in this investigation. All samples contained 0.500 g. of this oil. The procedure used was Procedure A, Kingsley's method with Hage's modification. Table III shows the measurements which resulted from the treatment of this oil using the adsorbents listed. It can be seen from Table III that all the samples of Super- filtrol behaved in a similar manner and that, therefore, any of the types tested might be used for the analysis of a sample of unknown potency without modification of Procedure A. when Magnesia or Magnasol was used as the adsorbent, both the D plus S and S measurements were high as compared to those obtained when Superfiltrol was used. This would seem to indi- cate that the first chromatograph did not succeed in removing as much of the interfering substances as did Superfiltrol. Howb ever, in the cases of both samples of powdered Magnesia, the difference between the D plus S and 8 measurements was only slightly less than that difference obtained when Superfiltrol was used as the adsorbent. In the Cases of Alumina and Magnesium Silicate, there seemed to be little or no difference between the action of the two chro- mstogrsphs. Obviously, these adsorbents could not be UBO‘Biithv out a drastic modification of the present procedure. -15- It may be concluded from this study that different adsorb- ents vary considerably in their effectiveness in removing in- terfering substances and that a different type of adsorbent might be used only after a modification of the present procedure. NATURAL 011.8 The natural oils listed in Table IV were run by Procedure A, Kingsley's method with Hage's modification. As can be seen from the table there is, for the most part, quite close agreement be- twsen the experimentally determined D units/gram and the bioassay values. SUPERFILTROL CORRECTION FOR ULTRA-VIOLET MEASUREMENTS Late in the course of this investigation, it was observed by C. W. Carlson, who was carrying on related studies in this laboratory, that when certain solvents were passed through an. adsorption column containing Superfiltrol the filtrate contained a substance, apparently eluted from the Superfiltrol, which had an appreciable absorption in the ultrapviolet region. When this observation waslmdds known, it was decided to ascertain whether or not any substance was eluted from the Su- perfiltrol when the specific mixtures of solvents used in this investigation came in contact with the Superfiltrol. Therefore, a "blank" was run on each of the two types of chromatographic adsorption. Fifty-five milliliters of the 50-10-1 mixture (the total volume used normally) was passed through a 6 cm. adsorption column, approximately the bottom two cm. of the column eluted with ethyl ether, the filtrates ~16- combined and evaporated to dryness, and the residue taken up in absolute ethyl alcohol to 25 ml. The absorption in the ultra-violet region was measured for this solution on the Beckman quartz spectr0photometer. Then the solution was di- luted 1:1 with alcohol and the absorption measured. Finally, a 1:3 dilution of the original alcohol solution was made and the absorption measured. The results of these measurements are shown in Figure I. The same procedure was carried out with the second chromatograph except that 25 m1. of the 8281 mixture were swirled-frequently for one hour with the amount of Superfiltrol normally used in this operation, filtered, rinsed with two 10 ml. portions of the same solvent, evaporated to dryness, and the residue taken up in absolute ethyl alcohol to 25 ml. The same dilutions and.measurements were made as in the case of the first chromatograph. The results of those measurements are shown in Figure II. As a result of this study, it was decided that a correc- tion must be made in ultrapviolet measurements of a sample whenever that sample has been treated by any procedure involv- ing the use of Superfiltrol. It is believed that this may best be done by running a "blank" simultaneously with the sample through all the operations involving Superfiltrol. As may be seen from Figures I and II, the absorption of the substance eluted from the Superfiltrol varies inversely with the final volume of the solution, and after absorption has been measured for any one concentration, it may be calculated for any other -17- TABLE III ‘dOOIbent L08 Io/I E(1¢, lcm.) D u/g Superfiltrol plus 8) 0.5 . . 0.858 16,600 Superfiltrol Lot 63 0.54 “0.1 5.5; 0.902 17,400 Superfiltrol Lot 63 0.5 ”0.1 ‘UTET 0.902 17,u00 Superfiltrol Lot A 0.50 -0011 5.3? 0.858 16,600 Superfiltrol Lot B 0.u8 -0008 ‘UTEU 0.880 17.000 Superfiltrol Lat O Coll-8 -000 . 0.958 16,600 Superfiltrol Lot 1-202 0.51 -0 11 -Uf¢0' 0.880 17,000 Adsorptive Powdered Magnesia, Lot 26u1 1.10 “Os ‘07§;' 0.81u 15,700 Adsorptive Powdered lagnesia, Lot 2642 1.08 “Os 0 . 0.836 16,100 Adsorptive Granular Magnesia, Lot 2652 0.90 ‘002 . 1.u7u 28,u00 -18... TABLE III (cont'd.) Adsorbent Log Io/I E(1%, Ian.) D u/g M 1 “A" 1.0 w“ .2 . 1.386 26,750 algginz, 80 mesh (0(p1us B) 0.15 r e ‘ - 8 -0 1 75.75% 0 o H nesium Silicate 5% 0.37 “Os g o 0. 308 5,950 I esium Silicate #3 , Lot 3 g. 8 11;: 0. 286 5,520 -19- ' Chloroform probably contaminated TABLE IV Oil No. Type of Oil 3:?giz E(l%, 1 cm.) D u/g Bioassay ‘ 20923 §§2§d3§§ High 0 1.000 3. 0.152 8,900- 15.000 1.000 3. 0.195 9,500- 1.000 3. 0.59h 11,500- 1.000 3. 0.616 11,800- 1.000 3. 0.7n8 11.100 1.000 g. 0.737 1H,200 1.000 3. 0.771 1h,900 1.000 3. 0.81% 15,700 0.50003. 0.771 1u,900 0.50003. 0.70} 13,600 0.50003. 0.792 15,300 0.50003. 0.771 14,900 0.50003. 0.81h 15,700 0.50003. 0.771 1n,900 0.50003. 0.771 1n,900 0.50003. 0.792 15.300 0.50003. 1.012 19,500 P 68u6 Standard High D 0.800 3. 0.990 19,100 20,000 Fish 011 0.800 3. 0.990 19,100 0.50003. 1.056 20,u00 0.500 3. 1.078 20,800 3 568a Standard High D 0.50003. 0.880 17,000 17,000 Fish Oil 0.50003. 0.902 17,u00 0.50003. 0.921 17,800 -20- TABLE IV (cont'd.) 011 No. Type of 011 3:?§%§ E(l%, 1 cm.) D u/g Bioassay 21273 High 0 Fish 011 1.000 g. 0.583 11.250 12,000 28283 Haliverol D 1.000 g. 0.h73 9,100 11,500 29263 High 0 Fish 011 1.000 g. 0.561 10,800 13,500 50750 Albacore 0.70003. 0.628 12,100 12,650 0.70003. 0.613 11,800 507st Skipjack 0.70003. 1.385 26,700 2h,750 0.50003. 1.385 26,700 50731 Yellow Fin 0.70003. 0.566 10,900 10,810 0.70003. 0.597 11,500 50810 Blue Fin 0.70003. 0.707 13,600 11,100 0.70003. 0.738 lu,200 5083A Yellow Tail 0.70003. 0.660 12.700 13,800 0.70003. 0.692 13,300 50854 Bonita 0.7000g. 0.755 lh,600 17,150 0. 70003. 0. 738 1’4, 200 -1b-‘t ’1 .1 Wavelength (mu) ' Fig. I- Extinction (Log Io/ I) of the substance eluted fron.8uper- filtrol, in absolute ethyl alcohol, plotted against wavelength. . 1- Procedure 8, final volume 25 ml. 11- Procedure 3, final volume 50 ml. 111- Procedure E, final volume 100 mla 0.8 v l ‘ 'l 0.71\ i 0.6 l I: I i \ | 13095 i. g) \ 110'“ 4\. \u/r’fi‘e. I I: ‘ \K S} 0'5 > \\ \ ' 2 I II N“ :§33. Extinction (1%, 1 cm. I 1 . r Fig. V- hxtinction (13, 1 cm.) or a calciztrol-eroosterol mixture in absolute etnyl alcohol plotted agfllnst wavelength. The original solution contained O.;;Q§ ng. calciferol/ml. and 0.0510 ng. ergoaterol/nl. . .- tr . V? q” 1 ' 1’ . I- Ire-r. ‘ch d, «Oaavbll’dra lon. \J'VJV—.—-\.Q./ JU AA . l\vo 1“) I \ ‘ .7:- u 0‘ .“' 1307/0/14». .11.. -01 n)‘ I? ’e 11 " ‘ «a ‘ s ‘O \s -“"r‘- [1-1" 1 "‘- *- ir0-€‘Atlre J, “Jaabe-Lbr. 41—):‘0 U.U~-----.-\JQ./ JV 0“ . \.,Ull~al “7 3- ‘ . . _"_. ‘.'.‘/_J.IIA‘JI\) [:10 \..~’l H) T ‘ w-- n ‘ . . 1r ’ ‘ .v‘e ‘" IE.‘ 3,- ‘ r‘ n A“‘ -ane'l‘Ar-e u’ g)...?...ur Lav... 5".h'ulfau'LIU0/ LK'J .u“. \.,'J.“.a/ ‘- "v [a -‘ r‘ ‘lr lu-logo/..U .l- \.31 ., 1 ‘ '- .‘a ~ \ .: . c‘D‘O .. acr.cc.eu -hr 001.1,--.n o. -uper.1.-1ol -A-8- “'31”: g: 5“ C‘-(“L k4. v: ' 32C) 4111' -Il‘t.)l. 1.- l w cl (F.1ltlul a - A a l:- .ro-e hr, 5 \-.crel ,3 r:c.1un, N. ,. ‘ - ‘ ,JII'-..‘..’ . )2 9-11.1.--“ -1 a. c:.1-t:-. \ ..- A T ‘. .‘: - a )A.("~ u'ztlalz J.-'\ (1"7;./1J‘ J1». \..~“d/ / I ‘ _ 1 4‘ .Afi. 'u.’ 1U- I.. \-~- / Ivr- '- A V .L 1321' :‘u A .-r “a a: .v‘ -n -z‘ .-‘..";-L ,1. ‘ , . \ all I - J I: {5‘ ‘JI‘L ~- 1".‘ e-A t‘ k-a . 1‘ A A .. -. I _ ' ‘, r, ‘ ‘ I \_ \ccl ./ ' .41 1 - 1‘11 1.3:. 1. {l -J-‘ .10 {:3 U r x C. C: A) N 0 ID (,1 O 3; Q‘IU L) f‘) Havelcnstn (an) VI- Extinction (13, 1 cm.) of Oil No. 3772 in absolute ethyl alcohol plotted against wavelength. I- Procedure 3, concentration 0.5005./100 ml. II- Urocedure D, Joncentration 0.2ocg./100 ml. Corrected for absorption of :uperfiltrol lIa- same as 11 except uncorrected for absorption of Superfiltrol III- Procedure A (Sterol Sorrection) Corrected for absorption of superiiltrol .Concentration O.4525-/100 ml. 1118- same as Iii except uncorrected for absorption or superflltrol - “A..- “fix“ 0.. .‘ .' .-. :- ~ -«T— a'...n..'o “2' ~ g —- --W_ s s ...... Wavelength (mu) fig. 111- Extinction (13, 1 cm.) of a #5772-ergoeterol mixture in absolute etnyl alconol plotted against wavelength. Sue original mixture contained 0.3330g. of #3772 and 25 ml. of ergorterol (3.2T5mgo/nl.) I- .TDCCJJTB 3, Jon: ”tration: 0.lOZlgo/100 ml. (doliu)‘ 5.657go/103 ml. (sol'n)‘ ll- rroCe.ure D, lousentration: 0.22083./103 ml. (solid) 14.jdéo/130 Al. (;ol'n) ’ Jorxectel ior acaorption oz superfl trol lie- -818 as 11 except unzorrcctel {or absorption of superfiltrol 1-1- 1r33~14re A \cterol Jcrrection,, Joncentration: 0.30245./100 ml. (oolid) 24e926'e/100 ml. (Sol'n) Sorreztei :.r Lbslrption of superfiltrol ..18- -ane as i.i except uncorrecte. Lor absorption of superriltrol 0 \.Jli., - craze of #3772 an. pare ergosterol/103 ml. \ :l'n} - Jrs-a or or-oinal aol'n/lou ml. -24.. removed as expected by the first chromatOgraph. As may be observed in the graphs of Figures III to VII inclusive, the true nature of the changes which take place during any of the procedures involving adsorption with Super- filtrol cannot be determined until a correction has been made for the absorption due to Superfiltrol. It is regrettable that the necessity for making this correction was discovered so late in the investigation that other oils could not be re- examined before the investigation was discontinued. However, it is the opinion of the author that the application of this correction to samples of irradiated ergosterol will greatly decrease the difficulties hitherto encountered in experimen- tally determining the vitamins D potency of such oils. It may be concluded from this study that the first chro~ matograph plays an important part in the separation of cal- ciferol from interfering substances. No correlation between the bioassay value of an oil and its ultrapviolet absorption may be made unless the oil has been chromatographed. 8111011111 or 011. N0. 3772 011 No. 3772 is an 160x dilution of calciferol in corn oil, having a potency of 200,000 D units/gram. When samples of irradiated ergosterol were treated, this oil was used as a check in order to verify the purity of the solvents and reagent and the effectiveness of the operations undertaken. Table VI shows the results of measurements of the antimony trichloride extinction for various procedures. Figure VI shows typeical absorption curves in the ultrapviolet region for the same pro- cedures. .25- TABLE VI Sample Ptocedure Weight “1%, 1 cm.) D units/gram H 0.060 3. 11.55 223,000 0.050 3. 9.23 178,000 0.050 3. 9.90 191,000 0.050 3. 9.55 183,000 0.06003. 8.80 170,000 0.06003. 9.73 188,000 0.06003. 9.90 191,000 0.06003. 9.73 188,000 0.06003. 9.90 191,000 0.06003. 8.62 167,000 0.05003. 9.45 183,000 0.06003. 8.62 167,000 0.06003. 7.90 152,500 0.05003. 8.36 161,000 0.05003. 9.90 191,000 0 0.0u0 g. 9.62 186,000 0.060 g. 9.51 18h,000 0.060 3. 7.61 lu7,000 0.060 3. 8.80 170,000 0.060 g. 8.62 167,000 0.06003. 6.97 135,000 0.06003. 8.80 170,000 0.06003. 8.80 170,000 0.06003. 8.25 159,000 182,000 av. TABLE VI (cont'd.) Sample -26- Procedure Weight r(1%, 1 cm.) 0 units/gram 0 0.06003. 6.60 127,000 0.05003. 8.36 161,000 0.05003. 8.14 157,000 0.02003. 8.80 170,000 0.05003. 8.80 170,000 0.05003. 9.24 178,000 0.0500g. 8.58 166,000 164,000 av. D 0.060 g. 6.42 124,000 0.060 3. 7.89 152.000 0.060 3. 7.89 152,000 0.05003. 7.26 140,000 0.05003. 7.26 140,000 142,000 av. -27- IRRADIATED ERGOSTEROL IN CORN OIL Perhaps the most extensive phase of this investigation was the study of samples of irradiated ergosterol in corn oil. A large number of samples of this type of oil were run by various procedures, and measurements were made both of the antimony trichloride extinction at 500 mu and of the absorp- tion of the alcoholic solution in the ultrapviolet region. Almost all of the samples had been purified to some extent before they reached this laboratory; many were in their final, commercial form. All with'but one exception, Frtron, had been irradiated while in solution. Ertron was irradiated while in crystalline form. As may be observed from Table VII, there is little or no correlation between the bioassay values and experimentally de- termined D units/gram for Procedure B and only slightly more for Procedure 0. In agreement with the conclusions drawn from the study of calciferol and ergosterol, considerable coree1a~ tion appears to exist between the bioassay values and D units/gram for Procedure D. Figures VIII to x inclusive show typical ultra-violet ab- sorption curves for samples of irradiated ergosterol in corn oil. IRRADIATED ERGOSTEROL IN ALCOHOL A considerable length of time was also devoted to the study of irradiated ergosterol in alcohol. However, in the case of these samples, few, if any, had been purified to any -03- extent before they reached this laboratory. Particular attention was paid to oils No. 43573, 43583, 43593, 43603, 45013, 45023, 45033, and 45043. These had been irradiated for different lengths of time; the irradiation times being 60, 45, 33, 15, 15, 33, 45, and 60 minutes re- spectively. Table VIII shows the results of measurements of thetantimony trichloride extinction for the abovementioned oils and those others which fall into the same category. Figures 11 and XVIII show the various ultra-violet absorption curves for these oils when run by different procedures. All oils in this group were diluted with absolute ethyl alcohol before treatment. Most were diluted 1 ml. to 100 m1.; 011 No. 64174 was diluted 1 ml. to 25 m1.; various dilutions were made of oils No. 96872 and 97182. In Table VIII, the E(l¢, 1 cm.) values were calculated for the dilutions, but the D units/gram values are for the original solutions. IRRADIATED ERGOSTEROL IN FISH OILS A number of oils of the type irradiated ergosterol in fish oils were treated by various procedures, mainly Procedure A. Table II shows the results of the measurements made of the an- timony trichloride extinctions. As may be seen from the table, Procedure A gives results which are lower than the bioassay values. The Procedure D values are also lower than the bio- assay values, but these values divided by the bioassay values and multiplied by 100 fall in very closely with similarly calculated values for samples of irradiated ergosterol in corn -29- oil (see Table VII). Thus, it would appear that Procedure D, perhaps with some modification, would be the best of those procedures tried so far with which to treat samples of ir- radiated ergosterol. Obviously, some other factor besides 19,300 would be required; however, once constant results were gained by using this procedure, a different factor would be merely a.matter of arithmetic. A STUDY OF CORN OIL A brief study was made of corn oil in order to ascertain if it interfered in the antimony trichloride reaction or if it had a significant absorption curve in the ultrapviolet re- gion. Table I shows the effect of corn oil and another simi- lar oil on the antimony trichloride extinction; the corn oil used is commercial “Masola Oil". Figure III shows the ultra-violet absorption curves for the corn oil when treated by various procedures. As Table 1 indicates, corn oil has a definite effect on the antimony trichloride reaction; however, the effect is quite small when compared to the weight of sample. In agree- ment with Young (4), it was found.by this author that the I(l%, 1 cm.) of corn oil decreased after saponification. This was also true of the other oil tested. In any event, it is not believed that corn oil has an appreciable effect on samp— les of vitamins D of the potency usually met. Figure III shows that corn oil has a very definite ultrapviolet absorption curve. When treated by Procedure B, -30- measurements cannot usually be taken below 250 mu due to the high absorption of corn oil below that wave length. The ab- sorption of corn oil in the ultra-violet region also decreases after saponification. After the discovery of the necessity for making a correction for Superfiltrol, it was noticed that the ultrapviolet absorption curve for corn oil when treated by Procedure D was very similar to that for the correction. Consequently, it may be assumed that corn oil is largely, if not completely, removed by treatment with Procedure D. STUDY OF SUDAN III When attention was first focused on ultrapviolet ab- sorption measurements, it was decided to determine if the dye Sudan III, used as a.marker for the first chromatograph in Kingsley's method, had an appreciable absorption of its own. As can be seen from Figure XX, the dye has an absorption sig- nificant enough to prohibit its use in the adsorption column. Consequently, the orange-colored corn oil layer in the ad- sorption column was used to determine where the column should be cut for elution with ethyl ether. Previous investigation had demonstrated that the corn oil and Sudan III layers occur in the same place in the adsorption column. TABLE v11 Oil No. Procedure ggglli: “1%, lcm. ) D u/g_ Bioassay D“§i§.§.§3° Hg 2 B 0.040 3. 23.9 462,000 450,000 102.6 Hg 3 B 0.040 g. 23.9 462,000 450,000 102.6 Sample F B 0.060 g. 27.9 538,000 440,000 122.5 45120 B 0.050 3. 8.24 159,000 250,000 63.6 B 0.060 3. 8.71 168,000 67.2 61691 H 0.060 3. 6.05 117,000 200,000 58.5 65751 B 0.050 3. 10.32 199,000 250,000 to ’ 275,000 B 0.060 g. 10.73 207,000 13192 B 0.050 g. 9.89 191,000 250,000 76.4 H 0.060 3. 9.63 185,000 74.0 78272 B 0.060 g. 8.06 157,000 225,000 69.8 B 0.060 3. 8.53 164,000 73.0 84742 B 0.060 3. 11.57 224,000 275,000 81.5 B 0.060 3. 11.65 225,000 81.8 15153 B 0.060 g. 10.10 195,000 179,000 to 196,000 34983 B 0.07003. 8.33 161,000 275,000 58.7 B 0.06003. 8.80 170,000 61.8 35563 B 0.06003. 10.10 195,000 250,000 78.0 B 0.06003. 10.88 210,000 84.0 36323 B 0.06003. 8.62 166,000 275,000 60.4 B 0.06003. 8.43 163,000 59.3 36962 B 0.07003. 8.48 164,000 200,000 82.0 B 0.07003. 8.17 158,000 79.0 38133 B 0.07003. 7.40 143,000 125,000 111.3 TABLE VII (cont'd.) Oil No. Procedure 3:31.123 E(l%,lcm.) D u/g Bioassay %a:s:30 B 0.06003. 8.25 159,000 127.2 42943 B 0.07003. 8.96 173,000 160,000 108.0 B 0.06003. 8.80 170,000 106.3 42953 B 0.06003. ,9.17 177,000 175,000 101.0 B 0.06003. 8.80 170,000 97.1 42963 B 0.07003. 6.60 128,000 150,000 84.6 B 0.07003. 6.77 131,000 87.3 Ba 103 ' B 0.015 3. 48.1 929,000 920,000 101.0 B 0.030 g. 46.9 905,000 98.4 B 0.010 g. 45.5 880,000 95.7 Bb 103 B 0.040 3. 28.7 554,000 500,000 110.8 B 0.030 3. 28.9 558,000 111.6 H 0.020 3. 28.9 558,000 111.6 So 103 B 0.035 3. 24.4 471,000 480,000 96.0 B 0.025 g. 1 25.3 . 488,000 101.5 Bd 108 B 0.010 3. 75.8 1,460,000 1,000,000 146.0 B 0.010 3. 80.2 1,550,000 155.0 B 0.01003. 74.8 1,440,000 144.0 Be 103 B 0.030 g. 41.9 810,000 775,000 104.5 B 0.015 3. 42.8 827,000 106.7 B 0.015 g. 45.8 884,000 114.0 Bf 103 B 0.020 3. 47.5 917,000 920,000 99.5 B 0.010 3. 47.3 915,000 99.3 Bg 103 B 0.020 3. 35.5 685,000 625,000 109.5 B 0.020 3. 36.6 706,000 113.0 TABLE VII (cont'd.) 011 No. Procedure gglgh: E(l%,l cm.) D u/g Bioassay D uigagsigo 9031 B 0.02003. 34.7 670,000 B 0.02003. 33.0 637,000 B 0.02003. 30.3 585,000 B 6727 B 0.02003. 35.2 678,000 B 0.02003. 34.7 670,000 B 0.02003. 30.8 595,000 B 6851 B 0.02003. 28.6 552,000 B 0.02003. 28.6 552,000 B 0.02003. 25.9 500,000 B 6975 B 0.02003. 28.1 542,000 B 0.02003. 27.5 530,000 B 0.02003. 25.3 488,000 A 14290 B 0.01003. 79.2 1,530,000 Ertron B 0.0128g. 43.2 834,000 B 0.0106g. 44.4 857,000 50254 B 0.05003. 10.56 204,000 B 0.05003. 11.12 217,000 59824 B 0.02503. 29.1 562,000 B 0.02003. 31.3 605,000 Hg 2 c 0.040 3. 19.8 381,000 450,000 84.6 Hg 3 c 0.040 g. 18.4 356,000 450,000 79.1 Sample r c 0.060 g. 23.3 450,000 440,000 102.3 45120 0 0.050 g. 7.47 144,000 250,000 57.6 c 0.060 3. 6.05 117,000 46.8 61691 C 0.060 so 5.04 97.000 200,000 48.5 -34... TABLE VII (cont'd.) Sample D u/g x 100 Oil No. Procedure Weight E(l%,l on.) D u/g Bioassay Bioassay 65751 c 0.050 3. 10.65 205,000 250,000 to 275,000 0 0.060 3. 8.98 174,000 13192 c 0.050 3. 9.24 178,000 250,000 71.2 c 0.060 g. 7.88 152,000 60.8 78272 c 0.060 g. 7.96 154,000 225,000 68.5 c 0.060 3. 7.06 136,000 60.5 84742 c 0.060 g. 11.57 224,000 275,000 81.5 c 0.060 g. 9.52 184,000 67.0 15153 0 0.060 g. 8.45 163,000 179,000 to 196,000 C 0.060 g. 8.65 167,000 34983 0 0.06003. 5.87 113,000 275,000 41.2 0 0.06003. 6.42 124,000 45.1 35563 0 0.06003. 7.80 151,000 250,000 60.4 0 0.06003. 8.06 155,000 62.0 36323 0 0.06003. 6.23 120,000 275,000 43.7 0 0.06003. 7.15 138,000 50.2 36963 0 0.06003. 5.50 106,000 200,000 53.0 0 0.06003. 6.28 121,000 79.0 38133 0 0.06003. 5.50 106,000 125,000 84.8 0 0.06003. 6.05 117,000 93.6 42943 0 0.06003. 6.43 124,000 160,000 77.5 0 0.06003. 6.97 134,000 83.8 0 0.05003. 7.92 153,000 95.6 0 0.05003. 7.26 140,000 87.5 -35- TABLE VII (cont'd.) Sample D'ugggx 100 Oil No. Procedure Weight E(1%,l cm.) D 915 Bioassay oassay 42953 0 0.06003. 6.60 128,000 175,000 73.2 0 0.06003. 6.97 134,000 76.5 0 0.05003. 7.04 135,000 77.1 0 0.05003. 7.04 135,000 77.1 42963 0 0.05003. 5.06 98,000 150,000 65.3 0 0.07003. 5.82 112,000 74.7 0 0.05003. 6.60 127,000 85.3 Ba 103 c 0.020 3. 34.6 687,000 920,000 74.7 c 0.020 3. 38.5 743,000 80.8 c 0.020 g. 36.9 712,000 77.3 Bb 103 0 0.030 3. 21.3 410,000 500,000 82.0 c 0.030 3. 26.1 504,000 100.8 Be 103 c 0.035 g. 18.9 364,000 480,000 75.8 c 0.040 3. 20.1 388,000 80.8 c 0.040 g. 20.1 388,000 80.8 Bd.103 c 0.010 g. 51.7 997.000 1,000,000 99.7 c 0.010 3. 52.8 1,020,000 102.0 0 0.010 3. 52.8 1,020,000 102.0 Be 103 c 0.020 3. 30.2 584,000 775.000 75.4 0 0.020 3. 33.0 637,000 82.2 0 0.020 g. 38.0 733,000 94.6 Bf 103 c 0.020 3. 31.3 605,000 920,000 65.7 c 0.020 3. 38.5 743.000 79.7 B3 103 c 0.020 3. 26.9 520,000 625,000 83.2 c 0.020 3. 31.4 607,000 97.0 -30- TABLE VII (cont'd.) 011 Do Procedure figmple 2% x 100 , ght E(l%,l cm.) D dig» Bioassay cassay c 0.020 3. 28.6 552,000 88.3 9031 0 0.02003. 19.3 373.000 B 6727 0 0.02003. 21.4 413,000 B 6851 0 0.03003. 22.0 424,000 B 6975 0 0.03003. 17.6 339,000 1 14290 0 0.01003. 62.7 1,210,000 Ertron 0 0.02133. 35.2 680,000 0 0.02133. 35.7 688,000 52054 0 0.06003. 10.10 195,000 0 0.06003. 9.90 191,000 59824 0 0.02003. 28.0 542,000 0 0.02003. 28.0 542,000 63824 0 0.05003. 10.78 208,000 0 0.05003. 11.22 214,000 63834 0 0.05003. 3.30 63,500 0 0.10003. 2.42 46,500 Hg 2 0 0.040 3. 13.75 265,000 450,000 58.9 Hg 3 0 0.040 g. 11.55 223,000 450,000 49.5 Sample I c 0.060 3. 17.8 344,000 440,000 78.2 Ba 103 0 0.020 3. 29.2 563,000 920,000 61.2 D 0.020 g. 29.7 573,000 62.3 Bb 103 o 0.030 3. 16.5 318,000 500,000 63.6 B0 103 D 0.040 3. 13.6 262,000 480,000 54.5 0 0.040 g. 14.3 276.000 57.5 Bd.103 o 0.010 3. 42.9 830,000 1,000,000 83.0 -37- TABLE VII (cont'd.) Sample D‘u x 100 Oil No. Procedure Weight E(l%,l cm.) D u/g Bioassay B oaseay Be 103 D 0.020 g. 23.1 446,000 775.000 57.5 D 0.020 g. 27.5 530,000 68.4 Bf 103 D 0.020 g. 31.4 606,000 920,000 65.8 Bg 103 D 0.020 g. 18.7 366,000 625,000 57.7 0 0.020 3. 20.9 403,000 64.6 Ertron D 0.0213g. 29.5 570,900 D 0.0213g. 30.0 578,000 vi. ‘1 v'.| A A 7'~ _-4v .5.» \l .1 M J l I") .- O L\ v 1 2:: j; :\ 2’3} 3'. D 277 A’ 23) 2‘30 5‘». - .H. --_;fi. .avelengtn (an, (ll, 1 c1.) of -11 go. A 14290 in ELBOlJLO .14. I”- 3xtlnzt -n ‘L 1.1 l ... , .--,_ . ., .. n. , .,. a C -v {ALD-sd k’A—uvtd v.11..‘ ”Etiefitllgbal. .rkwue-fl Ujfiroce‘ldee U. \ . -v - I' ‘~ ‘ .23..‘,'-10I'-01)I. v0») 3:40'ag'd .25. ' 1 O ‘ ~ a _.._' , \- we-aa. ’ \ 0 1' law I) r\ I \ s—v / w H r" ’ O. .J / {1 1.‘ ./ IJ - I ed +1 r’ (J r, \: ‘24 ' . H ‘ ‘y/I ‘ ‘_) 7'3 WI 0 1" _ ‘ lJ L z o l A I ‘ fl - N AW 1! -. ~ 2,4 5'30 .4; “2.1.. ._.'\._,' ,. ., ...I.l ~\ I'I' ‘7"?1‘3.1&‘J!1 /‘ LEA Fig. A- axtlnction (1L, 1 on., or all be. ad lui in absol.te ethyl alcau01 leLLed awalnet WnVelvnptn. free; a by .rlce;ar~ J. Janeentrktlun Jot/7’7. /l._wJ 131- ,s ///-.» -08- TABLE VIII Sample 011 No. Procedure Amount E(1%, 1 cm.) D unitségram Bioassay 96872 B 1 m1. 0.977 1,430,000 1,000,000 to B . 1 m1. 0.985 1,440,000 1'250'000 B 1 m1. 0.861 1,230,000 S 1 m1. 1.065 1,560,000 0 1 ml. 0. 574 840,000 0 1 ml. 0.910 1,340,000 0 1 ml. 0.836 1,226,000, 0 1 m1. 0.863 1,265,000 0 1 m1. 0.917 1,340,000 0 1 m1. 0.714 1,280,000 0 0.4 ml. 2.17 1,300,000 0 0.4 m1. 2.105 1,260,000 r 1 m1. 0.985 1,440,000 r 1 m1. 0.920 1,350,000 r 1 m1. 0.310 371,000 r 1 m1. 0.310 371,000 97182 B 1 ml. 0.59} 860,000 1,000,000 S 1 ml. 0.566 830,000 B 1 m1. 0.675 810,000 0 1 ml. 0.546 801,000 0 1 m1. 0.492 722,000 0 1 m1. 0.468 690,000 0 1 ml. 0.478 701,000 r 2 ml. 0.1347 161,000 r 2 ml. 0.1550 185,000 -39- TABLE VIII (cont'd.) Sample Oil No. Procedure Amount E(l%, 1 cm.) D units/gram Bioassay 64174 B—F 0.03143. 23.5 455,000 43573 B 1 m1. 0.392 757,000 B 2 m1. 0.364 703,000 B 2 m1. 0.378 730,000 0 3 m1. 0.126 243,000 0 3 m1. 0.160 308,000 0 3 ml. 0.160 308,000 r 2 m1. 0.245 473,000 r 2 m1. 0.189 365,000 43583 B 1 m1. 0.770 1,485,000 B 1 ml. 0.770 1,485,000 0 1 m1. 0.588 1,135,000 0 1 m1. 0.588 1,135,000 0 1 ml. 0.588 1,135,000 r 1 m1. 0.532 1,025,000 r 1 ml. 0.448 865,000 43593 B 1 m1. 0.518 1,000,000 B 1 m1. 0.742 1,435,000 B 1 m1. 0.728 1,405,000 0 1 m1. 0.588 1,135,000 0 1 m1. 0.560 1,080,000 0 1 m1. 0.546 1,055,000 r 1 m1. 0.532 1,025,000 a 1 ml. 0.407 785,000 TABLE VIII (cont'd.) -40- Sample Oil No. Procedure Amount “1%, 1 cm.) D units/gram Bioassay 43603 B 1 m1. 0.504 973,000 B 1 m1. 0.532 1,025,000 43603 c 2 m1. 0.392 757,000 0 2 m1. 0.364 703,000 0 2 m1. 0.371 716,000 r 2 ml. 0.280 540,000 r 2 m1. 0.273 527,000 45013 B 2 m1. 0.385 743,000 B 1 m1. 0.392 756,000 0 2 m1. 0.301 581,000 0 2 m1. 0.287 553,000 r 2 m1. 0.308 595,000 E 2 ml. 0.315 608,000 45023 B 1 m1. 0.407 785,000 B 1 m1. 0.434 838,000 0 2 ml. 0.294 567,000 0 2 m1. 0.294 567,000 r 2 m1. 0.336 648,000 r 2 m1. 0.301 582,000 45033 B 1 m1. 0.770 1,485,000 S 1 m1. 0.743 1,435,000 0 1 ml. 0.602 1,162,000 0 1 ml. 0.588 1,134,000 r 1 m1. 0.532 1,025,000 r 1 ml. 0.490 945.000 41- TABLE VIII (cont'd.) Sample Oil No. Procedure Amount E(l%, 1 cm.) D units/gram Bioassay 45043 B 1 m1. 0.925 1,785,000 B 1 m1. 0.910 1,755,000 0 1 m1. 0.770 1,485,000 0 1 m1. 0.742 1,430,000 r 1 m1. 0.617 1,190,000 E 1 m1. 0.562 1,085,000 - .rhifi -_-_..- - - .__.____.M—.~.-————-—-—— 1!? -.‘-—--.~‘ 1 ‘ W 1:! ,/" fl -h‘."00e-o . ./ \‘.. ‘r/ w 0 ‘ Q 17 4 “*’——o\ k r w ’ . ‘ .. ' ‘LI \‘\‘\ . ,n. 10. ,/ ‘ \ ‘- ‘ I’ s e \ ” ‘ K. 0"... ‘ ‘ ll \ .' I. . o“ O 9" ..-‘. -‘ ‘ a ,I ‘ ,- _._ ...... ,- ' III \ \ . ‘> “. ‘ I v k 5‘ \ . \ \\ ‘ \ rt 1‘) .. 0" \ ‘ r0 ‘\ .. k, 7" J ‘\ \ \J ‘. \ L3 1. “‘ 1') H 14 ,r (a B 7 .Q 0’ Q ‘ >1 “ -1 , \ O \5 \ 5 5 \ ‘\ a .. - 1+ “aw-u. 5. .9“ ‘ L] k‘ M... O .. uh..—.-h- s ‘\ 5 *-. " I 2 b———————o-——-—-—0“-*"’ ’ w“ “I. ‘ M's ,-. .- .; ,0 “U c.- L : .3 2, 3 2CD 27v 7700 I Fig. XI- Axtinction (ll, 1 cm.) of £11 50. 43575 plotted against wavelenbtn. I- :roceuure 3, Jonoeutretion C.OZQég./IOO ml. in absolute q ethyl alconol * II- Procedure 0, Sensentratiun 0.045755./100 ml. in absolute ethyl alconol III- Procedure E, Concentration 0.0540g./100 ml. in absolute ethyl alconol IV- rrocedure F, Concentration 0.1625./l00 ml. in absolute ethyl alcohol O A a o L‘ as W 280 270 280 290 300 wavelength (mu) _-_—_ L‘ .- . - ,- '1‘. XII- Extinction (1%, 1 cm.) of Oil No. 45553 plotted against wavelength. I- Procedure 8, Concentration O.07355./IOO ml. in 953 ethyl alcohol II- Procedure 0, Concentration 0.0277g./100 ml. in absolute ethyl alcohol III- Procedure E, Concentration 0.04l66./130 ml. in absolute ethyl alcohol IV- Procedure F, Concentration 0.l664g./100 ml. in absolute ethyl alcohol I. I 1 0:11.). 9! xtlnctlon (2' 3" :4 \"a‘ \ 24 » afrv’" \\ 22 lr" //‘ \ 2'3 ’/ I" ’ L 1 12, 10 L its 1 I! 0 ~ . .. . . s---. I . ' . O . ‘ .e-“‘“‘ .‘ 4 —‘ . .yd‘“ . "4 s ‘ e ‘1 '3‘ “IV ”A.“ A \ AVA A r A - w o. —,):) LHO we ;)V -'..4’ S—e—v unis! IV» Fig. XIII- thinction (1:, l 33.) 31 Lil “o. 4?;;’ plotte; against wavele-Jtn. 1- Procedure Q, Concentration 0.3354,. ice ml. in 58;-ilze etnyl alconol II- Prose-are J, :O-TSLLFHLIOH 3e9/; I e 13o Li. in “V:JL.Z” etnyl alcohol 111- ;roce1urc i, Zonce;tration C.Cwle€./l;3 3-. in a.eo13:e ethyl alcohol .. 1:. I. 3 IV- Erocedire F, Sonoentration 0.07;,9./ILJ 11. in ruSJlaLt ethyl alcohol 230 240 250 260 270 280 290 300 ‘w'are length (mm) h...“ ‘__.-_ — _-..-- —— ..._.._- -. --_~___..-_...... 4 ‘ ————_ ._._—-_ .a.._—._.—__ --_--—. Fig. XIV— Extinction (l%, 1 cm.) of Oil No. 45603 plotted against wavelength. 1- Procedure 8, Concentration 0.0201g./100 ml. in 95% ethyl alcohol II- Procedure C, Concentration 0.04l255./100 ml. in absolute ethyl alcohol III- Procedure E, Concentration 0.0550g./lOO ml. in absolute ethyl alcohol IV- Procedure P, Concentration 0.1650g./100 ml. in absolute ethyl alcohol O :3 l (f ...\ '4’. ‘4. _.a u - .b a a A A; . A a -- ' fl \ ‘ J e "’ a] ’ " ,4 A ,o ,4- ,; -‘{A\ A A" A .fl‘ A A . --- -74 can a;a . “ ‘ \ h t‘-;‘ .--.. \IM’ 1 '- .e . -_ ‘1 Q . e , 3... o- -11 no. ~;.. r.otoed ageinst I I ‘ ., . ‘x ’1“'\ lx‘ -‘ ' b l . sew -. ‘4‘ Is. k.-. &¥Jg. V... --¢.. 1“ b 80 Lane ‘ . . ‘ _ l W '. -‘ 9 _.“e vr‘ ‘VAV k.» “3;. ‘vv “l. ;a; art‘vaISe \, . . ~ ‘. ‘ .,‘ . \‘ . -»‘. . .3 , :e y. - . V.-.- -sA. ls {la'CVAufibe ‘v‘~ ‘ I ‘ I“I ‘w .‘ - 9..-; . \..§‘\.‘ .1 fix» A. ‘5‘ |‘~DV&‘ie ; s‘ ‘7 7. .2 "$-. .gv—O'h'fi. -L .i"-. ”"1 ‘ .»‘-¢—~~‘O-'. -..__.._... 1 l “7' *1 I . - ' 250 240 250 260 270 2.1:) 250 303 Wavelen'*n (mu) as” Fig. XVI- Extinction (1?, 1 cm.) 0: til 50. 45025 plotted against wavelength. I- Precesure 8, Joncentration c.07zyg./l;o ml. in 95S etnyl alconol I II- .roceiure 3, Concentration 0.OCJBSU./IJO ml. in absolute ethyl alconol III- :roce.ure E, Concentration C.OC/3°./lCC -l. in absolute ethyl alconol IV- Proceiure F, Concentration O.lo545./100 ml. in absolute etnyl alcOnol 1 E 3 II 1.--. ~ . . pm); b-a 0\ re (D 111 I p... .1:- .Extinctioh mini rd .4 O N \ a) I O\ . __..—. _....’ £— 1... ! L 1 f 250 240 2:0 260 270 250 290 E )00"' Harelength (mu) ._-- _--.—.-._—._-_—_—-_--- —' .. c.-- Fig. XVII- Extinction (12, 1 cm.) of Oil No. 45055 plotted against wavelength. I- Procedure 8, Concentration 0.0247g./100 ml. in 95% ethyl alcohol II- Procedure C, Concentration 0.027lgo/100 ml. in absolute ethyl alcohol III- Erocedure 8, Concentration 0.04065g./100 ml. in absolute ethyl alcohol IV- Procedure F, Concentration 0.1626go/100 ml. in ablolute etnyl alcohol ...... ...... 2250 23b 7?‘ 28o .. , 4...- .-_. -7“ -_-_+ _.. mum-19*: » , ,._ we . O .- “ --- 0.- Fig. XVIII- Extinction (L%, l on.) of Oil No. 45045 plotted againat wavelength. ' I- Procedure 8, Concentration C.O§)Og./lOO II. in abaolute ethyl alcohol 11- Procedure 0, Concentration 0.0275g./100 al. in abaolute ethyl alcohol IlI- Brocedure E, Concentration 0.0§)Og./loo ml. in ahaolute ethyl alcohol IV- Procedure F, Concentration 0.066lg-IIOO ml. in absolute ethyl alcohol 32,753 4071 60c wu,_’ ~42- TABLE 11 1% Oil No. Type of Fish Oil Srugge— 3:111:21]; Elem D u/g Bioassay $435330 56391 Halibut Liver 011 B 0.0603. 23.5 453,000 225,000 201.5 0 0.0603. 21.1 u07,000 181.0 D 0.0603. 7.88 152,000 67.5 31523 Cod Liver 011 A 0.50003. 5.32 102,500 250,000 M1.0 0 0.50003. 6.60 127,500 51.0 35783 Halibut Liver 011 A 0.10003. 3.96 76,h00 A 0.10003. n.62 89,000 A 0.10003. 1.52 89,000 39215 Fish Liver 011 1 0.10003. 3.96 76,u00 125,000 61.2' 1 0.10003. 2.53 “8,000 38.& A 0.10003. 3.96 76,#00 61.2 #0713 Fine Liver 011 A 0.10003. 5.72 110,000 150,000 73.4 1 0.10003. n.62 89,000 59.1 A 0.10003. 5.72 110,000 73.1 13023 Cod Liver 011 A 0.05003. 5.68 109,500 250,000 43.8 0 0.05003. 7.15 138,000 55.3 13125 Halibut Liver 011 A 0.10003. 5.17 100,000 160,000 66.2 A 0.10003. n.07 78,500 19.1 A 0.10003. 8.95 95.500 59.7 6007u rieh Liver 011 1 0.1003. 3.7h 72,200 A 0.2003. 3.7% 72,200 I T .. ...... u . . , . . . , » I . . . . e—o—.————.— — { bro-“.0 -n-g a , 1 , - .fl ‘ - - o . r . . . h“ ._o~ 4 .... p—g-._4,- ~ . . , ..— 230 240 95o ::o :73 :30 2,0 6 wavelengcn ;:u, 0.20 {Extinction (1%, 1 cm.) 9 H U 230 240 253 230 27: 2;; 2;o aavelenuth (nu) Fig. XIX- Extinction (1?, l on.) of Corn oil in absolute ethyl alcohol plotted against wavelength. I- Procedure B, Concentration 0.18753./1oo ml. 11- Procedure C, Concentration 5.5555./100 ml. III- Procedure D, Concentration 5.0003o/lOO ml. ~F\-‘ ’ ,uu xtinction (H3, 1 cm.). 3“ A-l 20 ,o/ ,1 ’ t . [/I” [/0 \ 13 4/ "I? 16 ,’ 111 fi-, 14 - 5." l2, —*_. A Fig-“fl t I 10 5i o _ . IV ,, “e . -—- .- '. ..« ‘“ 0% r-e . r..—o—~—-o O-~4 " -¢ . .. t -o 0 qt ‘7 233 240 3:3 233 T7? 3.: :1; Fig. XII - thinction (1;, l 31., 3f til V0. 4?;;j glotte; 8_3lu5t Haveleudtn. i— ITOCCque ;, Concentration O.cjelb.ll-o ml. 1; ape--nzs etnyl alc0nol II- rroseaare J, :OJTQLLrHLlQH 3.09] i o/lyu .1. ix reeJL,:e ethyl alcohol 111- :roceiure 5, Jonce;tratisn QOQJIng0/lyq al- in neeelate ethyl alcohol a /“'\ IV- Eroceiire 3, Concentration 0.07;5g.;iog :1. in ;.eolu;e etnyl al:0nol _ _. _-_-_...._—_____- LL'L_V_ b... e—e.- L O-n‘. H e -e—e 1._,.’+—-— . . o L e c Q . a . . * .- u . - o H a“... H— - . . . — F- H— qw- — -— - e . . . ._T_,~.._. 250 240 250 260 270 280 290 Wavelength (mu) Fig. XIV- Extinction (1%, 1 cm.) of Oil No. 45603 plotted againat wavelength. 1- Eroceoure B, Concentration 0.020lg./lOO ml. in 95% ethyl alcohol II- Procedure C, Concentration 0.041255./lOO ml. in abaolute ethyl alcohol 111- Procedure E, Concentration 0.0550g./lOO ml. in absolute ethyl alcohol IV- Procedure P, Concentration 0.16505./lOO ml. in absolute ethyl alcohol -u. . .. ._-L ....) n'~ V on il', 1 11.331. ix: H H H O L; . ‘LCJ. et.._. (. U l... 2 I) J {a 0 :5: :70 ac 2,20 1?- Litinst;:n \l., 1 c1.) of eil 50- «5.15 glottad against ion C-OflO;-,IOC :1. in absolute y l (J ( 91 'T‘ ( V (A t (a ’2 ‘9 '1 " \ ~ ffi '\ -‘ - , - I'VCC A ‘re a" .“1.Cef..vr‘i:l~:l‘. \J'wy -IJ°.' -vv ml. 13 Busolute ~I '. a 7* ‘ ‘ "ll".r “ ‘ v ‘ ‘ 111‘.\ v-fi‘ 'II “‘N‘ t o-‘- ..Ov'.-A.e a, JJeer~ ~.‘\--)x. v.\‘ s . v' “L. 1.0 abeLS-A e V ' \ . ' ‘ 9'. . “ r l ' l . , r \' ‘ axe ~ ‘1. ’.J..v.r"~.a~).'. \.LL\.\ ./ ‘\~ .a . ll; 808V. «‘Be 0‘ e \ Wavelength (no, V Fig. XVI- Extinction (11, 1 ca.) 02 Lil Lo. #5025 plotted aoainet wavelength. I- PIOJBJure 8, Concentration 0.07393./15o ml. in 953 etnyl alconol Il- .roceiure C, Concentration O.CCQZSU./lOO ml. in absolute ethyl alconol III- :roce.ure E, Concentration 0.0ijjg./lcC al. in absolute ethyl alconol IV- rroceiure F, Concentration O.ld«4¢./lOO ml. in abeolute etnyl alcOnol .—- Extinction (11._l one). be ya be no Jr ca r4 0 L) . . e - 0 _‘ ' ~ ~ _ _ _ -._..-..—._.- L -- n“\‘ . I! k" ‘ . *‘4‘3 v AV ~‘V—04‘._. “w ; 'v : _ ‘_ ____,_-- -" "fi -“+‘~ .90 240 220 260 270 250 290 ' 50°... Wavelethn (mu) Fig. AYII- Extinction (13, 1 c2.) of oil Jo. 95035 plotted against wavelenéth. I- rroceiure 8, Concentration 0.0247éo/100 ml. in i3? ethyl alconol II- Procedure C, Concentration 0.027lgo/lOO ml. in absolute ethyl alcohol Iil- :rocedure E, Concentration 0.040655./lOO ml. in absolute ethyl alcohol IV- Procedure F, Concentration 0.16263./IOO ml. in absolute etnyl alcohol - . . . , _ , . . _. - . I. . - -..T - -.. ..— . . 1 . . l . . t 1 . . . 7 . . . _ ' , , , . . . f . - . t - .._ ‘ - e . s - 4 - l - . e a 9 ‘- ‘ - 0 - ‘3‘ ‘ 1 . . . . . . . . l . . . . 3 . "‘f . - a... - . . 1 ._- 11.1.. .-1._--..._.._.-._---..__,-...--._‘......-..-- 1 e - s ! . '. . ' 22 1.11111, 1 cm.) H I“ 43' U\ ‘I till I" N Extin ' ' L ' : ' l"'"° ' -.-...4...” ' F'_;__ 230 2110 250 : 260. _ ; :270‘ ' "t It: :t‘:'t'f':‘:2?¢-_-+_._. -...-- g 3 - .,,,. ...... ......-. --.. _ .. -..-.. .. ...... . . . _ 1-. '“""j“‘"”TTE*“T‘T““?.L-¢-1 'i' 1-1- ‘2-”u'. Ianlangth(m) : 1ft. :f;;;;“1‘ ‘1- 'I' . , ' ___________ 0:. .. - I. _~L__:_-‘ Fig. XVIII- Extinction (1%, 1 cm.) of Oil No. 4504) plotted against wavelength. ‘ 1- Procedure 3, Concentration 0.0)§Og./lOO ml. in absolute ethyl alcohol 11- Procedure C, Concentration 0.0275g./lOO ml. in absolute ethyl alcohol 111- rrocedure 2, Concentration 0.0§§Og./lOO ml. in absolute ethyl alcohol IV- Procedure F, Concentration 0.066lgo/100 ml. in absOlute ethyl alcohol :11 lo. W713 3302; ‘4312 600', ~42~ TABLE IX 1% 011 No. Type of Fish Oil 23:26.. 33211;: Elem D u/g Bioassay 36133:? 56391 Halibut Liver 011 8 0.0603. 23.5 153,000 225,000 201.5 0 0.0603. 21.1 uo7,000 181.0 D 0.0603. 7.88 152,000 67.5 3&523 00d Liver 011 A 0.50003. 5.32 102,500 250,000 ui.o D 0.50003. 6.60 127,500 51.0 35783 Halibut Liver 011 1 0.10003. 3.96 76,u00 A 0.10003. n.62 89,000 A 0.10003. n.52 89,000 39215 Fish Liver 011 1 0.10003. 3.96 76.noo 125,000 61.2' A 0.10003. 2.53 n8,000 38.u A 0.10003. 3.96 76.n00 61.2 #0713 Fish Liver 011 1 0.10003. 5.72 110,000 150,000 73.1 A 0.10003. n.62 89,000 59.1 1 0.10003. 5.72 110,000 73.1 #3023 Cod Liver 011 A 0.05003. 5.68 109,500 250,000 u}.8 n 0.05003. 7.15 138,000 55.3 13125 Halibut Liver 011 1 0.10003. 5.17 100,000 160,000 66.2 1 0.10003. n.07 78,500 #9.1 1 0.10003. n.95 95.500 59.7 6007u riah Liver 011 1 0.1003. 3.7u 72,200 A 0.2003. 3.71 72,200 —-——_~.-——-— _.—-———_— fiv— . o—a-p—TLMQ-o- o». I. naveleugtn (:u) *0 0 ie 23 .4 fartinctioh (1- ' ‘\\ .._.m£ii 0.12 ~ ' ‘~vee“* 1‘7- - . - .-.—-....-1 . - 1..-- ...4 23o 2A0 250 243 27: 2Q; 230 200 eavelenuth (nu) Fig. XIX— Extinction (13, l on.) of Corn Oil in absolute ethyl alconol plotted against wavelength. 1- Procedure 8, Concentration 0.18753./1oo ml. II- Procedure C, Concentration 5.3555./lOO ml. III- Procedure D, Cancentration 5.0006./lOO ml. 43- TABLE x Sample Oil Procedure Weight E(1%, 1 cm.) D units/gram Corn Oil 8 3.0003. 0.382 7,370 B 2,0003. 0.u23 8,180 0 3.0003. 0.117 2,830 0 3.0003. 0.117 2,830 Special #1 B 2.0003. 0.190 9.150 0 3.0003. 0.323 6,230 0 2.0003. 0.330 6,370 e . . I. a i . 1 v . 9 - . : -”';" -1- ~' ‘ ,7- -—--<- ”.-...- e .- -2- ‘ ~ >>>>> o - -—--.1 T I 1 i. ‘ ' . a . _ 1 1 , , -1 _ , -31 - I - I-e t, . C . A ...... é. _._Lu_Jimi , . H r 5 __-R - H \J _ 3 q d 6’ O 4 -31 51., I ' 3‘3 \ _ i h.) 2 Vw‘-‘. 1 . . , .‘fi ' r \ I 0 2:0 :13 250 2:0 :7: :33 2,0 300 {s flJVSICLQtS (:1) f- 0.32 1\ )-< .e—‘l \. e z: -. \1 e 0-39 \\ H 1 \ "n \ ...? 0'- H V A r» ‘0‘ \ 11 u ya...“ 1 ‘ O ‘\\ \‘ -.- I; \ \. £3* 0 I) ’I\\\.‘__—-—". ......“‘ O. l._ \ \ g x w. p ‘.\ \‘_ ‘ Ill 0003 , L. ""‘.---4 0‘ o-v—O h“; _- ’ .5-“ . ‘ ‘re OeO‘? 2:3 240 :50 :‘o :7: 2;; 290 =et eeve-enwtn (nu) ”...- - .-——— Fig. XIX- Extinction (1C, 1 ca.) of Corn til in alconol plotted aJainet wavelength. ‘ w I- Procedure 8, 30ncentration 0.10750./ioC 31. II- Proceiure C, Concentration 5.5j59./lCO ml. III- Procedure D, COncentration 5.OOCO./lOO ml. inction (Lon Io/I) .) 1.3 7 1 1 1 1.: \ . 1 1 is“! ..l 1.3 \ ‘ I / \‘ l k ' f ... “‘.. ' L. U } ‘*‘ ' "i .‘.\ ' \ I “ v ._ 'a 9 .' i ' I - ! LOW ' ‘——f "f' A \':‘ P"~ r‘w‘ n‘n ’ ..,J .."C e .e ._ J 2;,L; a-v 293 503 f .;;e e.utn (au‘ 7 ‘,, , ‘ ‘ ‘ 1 . ‘I ‘ . ~Q ' ’. _ ,‘ _ ‘- 9‘“. C...“ XL‘ ‘2 .4.a \“i‘u lo/I/ \JI ~JJ.‘ :1 -‘I 111 ayb\J“4t,e et..Jl ei_;.:l 1 ott . wine: Levelenytn. frert~d by rrocecure S- ~44- SUMMARY Thirty minutes was shown to be ample time for complete saponification of samples of irradiated ergosterol. Four portions of ethyl ether, for the extraction of saponified samples as described in Procedure A, were shown to be sufficient for the extraction of samples of irradiated ergosterol. In the study of adsorbents, considerable difference was shown to exist in the behavior of various adsorbents. Dif- ferent samples of Superfiltrol acted alike, but, aside from magnesia, none of the other adsorbents examined could be used in this investigation without some modification of the pres- ent procedures. ‘ Twelve natural oils were run by Kingsley's method with Hage's modification. For the most part, there was close agreement between the experimentally determined potency and the bioassay value. It is not felt that further modification of the procedure is necessary for natural oils. After investigating the ultraeviolet absorption of a sub- stance eluted from Superfiltrol during chromatographing, it was shown that a correction must be made for the absorption of this substance in the measurements of the ultrapviolet ab- sorption of oils run by certain procedures. This substance has a definite ultrapviolet absorption curve the intensity of which varies inversely with the concentration of the solution. In the study of calciferol and ergosterol, it was demon- strated that calciferol and ergosterol are unchanged by -45- saponification. However, ergosterol is held back during chromatOgraphing, while calciferol goes through the chroma, tograph column. Corn oil was also shown to be removed by saponification and chromatographing. It is believed that any method for determining the potency of irradiated er- gosterol must have saponification and chromatographing in- corporated in it. Numerous samples of irradiated ergosterol in corn oil were treated by various procedures. Practically no corre- lation was found to exist between the experimentally de- termined potency and the bioassay values unless the samples were saponified and chromatographed (Procedure D). This re- sult is in agreement with the conclusion reached after the study of calciferol and ergosterol. Eight samples of irradiated ergosterol in fish oils were treated by various procedures. Most values obtained were lower than the bioassay values. However, results ob- tained by using Procedure D compared favorably with those obtained by using the same procedure on samples of irradiated ergosterol in corn oil. Corn oil was found to have considerable effect in the measurement of the ultrapviolet absorption of irradiated er— gosterol and a slight effect on antimony trichloride extinc- tion measurements. The effect in both cases was less after saponification. It was shown that the dye Sudan III could not be used as a marker for the adsorbent column since enough of the dye goes through the column to interfere with ultrapviolet measurements. (1) (2) (3) (it) (5) -46- LITERATURE CITED Tomkins, Ph. D Thesis, Michigan State College, 1942 Kingsley, Ph. D. Thesis, Michigan State College, 19H2 Ewing, D. T., Kingsley, G. V., Brown, R. A., and Emmett, A. D., "Physical-Chemical Method for Determination of Vitamins D in Fish Liver Oils", Ind. & Eng. Chem., ii, 301 (19n3) Young, Ph. D. Thesis, Michigan State College, 19%} Bags, M. s. Thesis, Michigan State 0611e3e, 19113 Jul2'45 r“ bx) 0 MN We? DIN 21349 11'." 2 3 '50 on t. "30 JUN 13 0 '51' 01020 _ as- 0.0‘ F‘}(L$l' ul-I'I’.‘ 160041 Baker Bi,7 lllllllllllHillllUNIIIHN‘IIIIIHIIHHIHIIHilllllllllllll 31293 02446 7254