Determining Vitamins D2 by Two Physical Chemical Methods

used successfully by Ewing, Kingsley, Brown, and. Emmett {6) for determining the vitamins D content of fish liver oils, has been found by several inve...
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Determining Vitamins D2 by Two Physical Chemical Methods D. T. Em I\G 4 \ D \I. J. €'OB ELL, *Michigan S t a t e College, East Lansing, Mich.,

K. A. BHORY

AVD

i. D. E\I\IETT, Parke, Daeis a n d Co., D e t r o i t , Mich. the sample is treated directly with the antimony trichloride reagent as prepared by Yield, Russell, and Zimmerli and the potency of the original sample is calculated from the extinction at 300 mp of the treated sample. Potency values for 49 irradiated ergosterols in corn oil and five irradiated ergosterols in fish liver oil, as determined by the chromatographic method, are given with the corresponding bioassay values. Results obtained for 51 irradiated ergosterols in corn oil, when using the colorimetric method, are compared with their bioassay values.

Two methods for determining the ,itamin Dz content of oil solutions of irradiated ergosterol are described. In a spectrophotometric method the nonsaponifiable fraction of the oil is chromatographed, using a column of Superfiltrol and a solvent composed of a mixture of 30 parts of hexane, 10 parts of ether, and 1 part of alcohol. Interfering substances are adsorbed on the Superfiltrol while the vitamin D1 portion passes through the column. The vitamin Di content is evaluated by measuring its extinction at 263 mp. .icolorimetric method is proposed in which the nonsaponifiable fraction of

must be removed or a suitable reagent must be used which, although it may not be specific for vitamins D?, will not introduce an appreciable error due t o its reaction with the interfering substances. With this in mind tITo methods are proposed by the authors: I n the first method the interfering materials are removed from the nonsaponifiable fraction of oil solutions of irradiated ergosterol by a chromatographic procedure which is a modification of the method used by Ewing et al. ( 5 ) . The vitamin D2content of the purified solution is then evaluated from its extinction a t 265 nip. The second method is a colorimetric procedure using the modified antimony trichloride reagent of Nield, Russell, and Zimmerli ( 7 ) . The nonsaponifisble fraction of the sample is treated directly with this reagcnt and the potency of the original sample is calculated from the measured extinction a t 500 mp of the treated sample. The effcct of interfering substances is considered negligible.

.kRIOUS methods have been proposed for determining the vitamin D: content of irradiated provitamins D solutions and of fish liver oils. ahexten-ive review of these methods has been given ( 5 ) . Yield, Russell, and Zimmerli (7, 12) found that by adding acetyl chloride to the antimony trichloride reagent of Brockmann and Chen ( 3 ) ,its sensitivity could be increased and the presence of zinc, tin, or antimony had a tendency t o stabilize the color reaction. They also observed that sterols having double bonds in the side chain did not affect the reaction and those having double bonds in their ring structures gave the following absorption coefficients: El% 1 om. El% 1 om.

2 . 2 a t 500 mp

2 double bonds

Dz and Ds

El% 1 om.

1800 at 500 inlr

1 double bond

4YD

7 a t 515

inM

Thus, the effect of aterols upon the reaction with the modified reagent appears to be small. Shantz (10) found that to obtain reproducible results by the antimony trichloride reaction, temperature, light, and concentration must be rigidly controlled. In these laboratories, the two-step chromatographic procedure, which was used successfully by Ewing, Kingsley, BroTvn, and Emmett (6) for determining the vitamins D content of fish liver oils, has been found by several investigators (2, 6, 11) t o be inapplicable to irradiated solutions of ergosterol. Hage ( 6 )modified the second chromatographic step of this procedure for assaying fish liver oils by swirling the benzene-Skellysolve solution of vitamin D, and sterols with Superfiltrol instead of using a column o,f the adsorbent t o separate the sterols from the vitamins D fraction. For assaying irradiated erogsterols in volatile solvents, Powell (9) further modified this method by completely eliminating the second chromstographic step. He found that by using a longer column of the adsorbent and prewashing with the solvent, vitamin D, could be separated, quantitatively, from crude irradiated ergosterols in volatile solvents and the chromatographed material gave an ultraviolet absorption curve similar to that of a standard calciferol. DeWitt and Sullivan (4)also separated vitamins D from both fish liver oils and irradiated ergostkrols by chromatographic procedure. 4 column made up of a 1 t o 1 mixture of magnesia and diatomaceous earth ITas used as the adsorbent and petroleum ether as the solvent. Separation was achieved by eluting, separately, the fluorescent zones observed on the column when exposed t o ultraviolet light and determining the vitamins D content of the eluates colorimetrically, using as the reagent antimony trichloride dissolved in ethylene chloride. Fiom this review it is evident that in an accurate physical chemical method for determining the vitamin D2 content of oil solutions of irradiated ergosterol, either all interfering materials

EQUIPMENT AND REAGEh-TS

,

Adsorption Columns. A 9-cm. column of Superfiltrol is used except for the fish liver oils, when the length is 11 cm. I t is prepared by the method of Ewing et al. ( 5 ) . The packed column must be thoroughly JTashed with the chromatogra.phic developing solution before the sample is added. Various lots of Superfiltrol, with which the authors have n-orked, give different results in the chromatographic separation. This effect, probably due to a difference in activity, seems to be associated with the color of the material; the whiter gradesof Superfiltrol give 9 better chromatographic separation than the gray colored grades. Spectrophotometers. For ultraviolet measurements a Beckman spectrophotometer (quartz) equipped with a hydrogen discharge tube is used. Either a Bausch & Lomb visual spectrophotometer equipped lvith a Martin's polarizing unit and 1-cm. glass cells or a Beckman spectrophotometer is employed for making measurements in the visual range. Ethyl Alcohol (absolute). h C.P. grade of absolute alcohol is purified by distillation, after being decanted from a silver oxide precipitate, prepared by adding 3 grams of potassium hydroxide and 1.5 grams of silver nitrate per liter of alcohol. Further purification is obtained by adding activated aluminum amslgam, decanting, and redistilling. The purified solvent should transmit t o 215 mp. Alcoholic Potassium Hydroxide. Fourteen grams of highethyl grade potassium hydroxide are dissolved in 500 ml. of Q5yG alcohol which has been purified in the manner described for absolute ethyl alcohol. Ethyl Ether. dnhydrous ether (c.P.)is used as obtained. It should transmit to 219.5 mp and be free of peroxides. Hexane. h commercial grade of hexane is redistilled to remove any residue. The distilled product should transmit to 210 mp.

317

ANALYTICAL CHEMISTRY

318 Chromatographic Developing Solution. This solution is made up from the purified reagents described above by taking 50 parts of hexane, 10 parts of anhydrous ethyl ether, and 1 part of absolute ethyl alcohol. Chloroform. Small amounts of alcohol are removed from C.P. chloroform by washing seven times with an equal volume of distilled water. It is then dried over anhydrous sodium sulfate, decanted, and distilled; the f m t and last lOy0 of the distillate are discarded. Antimony Trichloride Reagent. This reagent is prepared fresh for each day's run. Eighteen grams of C.P. antimqny trichloride are dissolved in 100 ml. of purified chloroform. After this solution is filtered, 2 ml. of redistilled acetyl chloride are added. PROCEDURE

Preparation of Sample. SallrPLEs I N OIL. Oil samples are weighed into small glass capsules and then placed in 125-ml. Erlenmeyer flasks containing 10 ml. of alcoholic potassium hydroxide. (When the chromatographic-ultraviolet absorption curve method was used 1-gram samples containing 100,000 to 1,500,000 units per gram were successfully run. For the antimony trichloride method a large enough sample to contain about 20,000 units is most convenient, but samples containing only 500 units can be determined accurately, as shown by Table IV.) A short-stemmed funnel is then placed in the neck of the flask and the sample is saponified in a water bath a t 70" C. from 0.5 to 1 hour or until saponification is complete. Then 20 ml. of water are added to the saponified solution and the nonsaponifiable fraction is extracted in a separatory funnel, using five 20-ml. portions of ether. The combined ether extracts are washed with a t least six 50-ml. portions of water or until the ether interface is clear and the water layer is not alkaline to phenolphthalein. The first three washings are made without shaking, to prevent the formation of an emulsion. The washed ether extract is then filtered through an anhydrous sodium sulfate filter pad into a 125-m1. Erlenmeyer flask and, after the separatory funnel is rinsed and the filter pad washed with 25 ml. of ether, the combined ether portions are evaporated to dryness, using gentle suction and a water bath a t about 50" C. SAMPLES IS VOLATILE SOLVEXTS.If the sample is an irradiated ergosterol in a volatile solvent, all the above procedure can be eliminated and only the solvent needs to be removed by evaporating to dryness, using suction and a hot water bath. The dry residue from the above procedure can then be treated by either of the two following methods: Chromatographic Ultraviolet Absorption Curve Method. The residue is taken up in 10 ml. of the chromatographic developing solution. This is allowed to percolate through the prepared 9- or 11-em. column of Superfiltrol which has been previously washed with 40 to 60 ml. of the developer and has not been allowed to become dry. 9 10-em. differential in pressure is maintained throughout the whole procedure. The flask is rinsed with 3 to 5 ml. of the developing solution, which is added a t once to the column. By means of a shortstemmed separatory funnel which is fitted t o the tube, the developing solution is added to the column drop by drop until the vitamin Dz has passed through the adsorbent column. I n the authors' experience this separation is complete when the lowest visible band reaches the bottom of trhe column. The filtrate from the column is then evaporated to dryness, using suction and a hot water bath (about 50' C.). The residue is taken up in absolute alcohol and the extinction at 265 mp is measured on the Beckman uarta spectrophotometer. The complete absorption curve of L i s alcoholic solution should have a maximum a t 265 mp and be similar to that exhibited by pure calciferol (Figure 1).

Table I.

w

240 250

'

Antimony Trichloride Colorimetric Method. The residue from the ether extract is taken up in 10 ml. of the purified chloroform. T o 1 ml. of this solution, 10 ml. of the antimony trichloride reagent are added. After 30 seconds' swirling, a 1-em. cell is filled and the extinction a t 500 mp is measured in exactly 3 minutes from the time the reagent was first added, using the Bausch & Lomb visual spectrophotometer. The potency of the original oil in vitamin DSunits per gram is then determined by calculating the from the extinction a t 500 m'p and multiplying by the factor 19,300 as determined by Ewing et al. (6). For the purpose of this publication this conversion factor is used, althoush it is being made the subject of further investigation. RESULTS

The first part of Table I1 gives the potencies of various irradiated ergosterols in corn oil as determined by the chromatographic ultraviolet absorption curve method. In every case the absorption curves obtained are similar to that of pure calciferol, exhibiting a maximum a t 265 mp, and the rkspective potencies, calculated from these curves, agree fairly well with the bioassay values, when the U.S.P. procedure is used. Animal tests 1.0

I

I

I

I

I

I.--Oil

.9

KEY

w after

saponification and

I

I I

Extinction Ratios of Crystalline Calciferol and Chromatographed Sample in Ethanol '

Wave Length,

'

Comparison of the absorption curves of ethanol solutions of crystalline calciferol and of the chromatographed sample is iwll shown in Table I. The extinction ratios for given xave lengths are tabulated according to the method of Oser, Melnick, and Pader (8). The potency of the original oil in vitamin DPunits per gram is then determined by calculating the of the sample from the extinction obtained a t 265 mp and multiplying it by 86,960. This factor is obtained by dividing the number of vitamin D Punits per gram of the standard calciferol (40,000,000) a t 265 mp, which is 460. Since this work was by the done, Arnold (1) has indicated that calciferol contains 49,000,000 units per gram. Further studies are being made in this laboratory on the conversion factor.

260 270 280 290 300

Extinction Ratios (265 ms) Test material Calciferol 0.76 0.89 0.98 0.95 0.75 0.47 0.20

0.67 0.85 0.98 0.98 0.73 0.43 0.23

I

i

319

V O L U M E 20, NO. 4, A P R I L 1 9 4 8 Table 11. Potencies of Irradiated Ergosterols as Determined by Chromatographic Ultraviolet Absorption Curve at 265 Mp Sample KO.

El%

1 cm., 265 ml.r

Calculated, Dz Units/G.

Bioassay, U.S.P. Dz Units/G.

Table 111. Potencies of Irradiated Ergosterols as Determined by Antimony Trichloride Colorimetric Method Sample NO.

High-Potency Samples in Corn Oil ,468,000 ,555,000 .,318,000 ,164,000 ,,221.000 ,,033,000 472,000 479,000 407,000 430,000 437,500 371,000 370,000 357,000 448,000 529,000 495,000 479,000 389,000 509,000 438,000 404,000 473,500 446,500 407,000 439,000 476,000 444,000 513,000 398,000 395,000 466.000 280,000 268.500 239,000 281,000 257,000 234,000 226,000 238,000 198,000 195,500 226,000 211,000 157,500 2 19,000 226,000 216,000 214,000

.

2.57 2.42 2.32 2.97 2.02

223,000 210,500 202,000 258,300 175,800

jo0 mp

Calculated,

Bioassay, G.S.P. DPUnits/G

Dz UnitdG.

High-Potency Samples in Corn Oil 1,200,000 1,200,000 1,200,000 1,200,000 1,000,000 1,000,000 600,000 526,000 525,000 525,000 525,000 525,000 525,000 525,000 525,000 525,000 525,000 525,000 525,000 525,000 525,000 525.000 500,000 600,000 475,000 400,000 450,000 450,000 400,000 450,000 450,000 450,000 330,000 330,000 300,000 275,000 275,000 250,000 250,000 250,000 250,000 250,000 250,000 250,000 250,000 204,500 204,500 200,000 200,000

High-Potency Irradiated Ergosterols in Fish Liver Oil 74,254 66,844 71,064 20,206 25,646

Average ':%n.,

250,000 200,000 200,000 300,000 200,000

nrre run at two or three lcvels, 15 to 20% apart, and the U.S.P. reference oil was used as the standard. As no attempt was made t o iriterpolatc, between the bioassay levels, some of the discrepancies between the physical chemical and the biological data may he due t o the 15 to 20% range a t which the samples were tested. The values obtained from the animal tests represent the highr i t biological potency that could be obtained from the levels at which the samples xere tested. For example, the data of a aample tested a t 1,200,000 and 1,000,000 units per gram might indicate the material to be slightly less than 1,200,000 units per gram but above 1,000,000 units per gram. I n this case the assay vould be reported at 1,000,000 units per gram, although the material might actually contain 1,100,000 or 1,150,000 units per gram. Of 49 irradiated ergosterols in corn oil assaved by the chromatographic ultraviolet absorption curve method, only 8 oils differed from the bioassav values by more than 257,. The maximum per cent difference from the bioassay figures was 36.9, and the average variation of all the oils run by this method was 14.570. The results obtained when using the chromatographic method for five high-potency irradiated ergosterols, in fish liver oil containing vitamin A, are tabulated in the second part of Table 11. The close agreement of these results Tvith the bioassay values indicated that the chromatographic method might also be applicable t o fish liver oils fortified with irradiated ergosterol. However,

87,114 88,875 97,195 1,015 1,735 7,305 89.025 94,435 23,446 92.345 25,476 25.456 6.105 4,985 7,705 19,656 21,226 21,976 21,986 23,196 91,09.5 25,496 8,645 78,174 84,614 77.424 80,434 79,404 83,024 64,184 64,824 65,104 67,784 68,564 69,934 84.944 5,30.5 80,904 84,174 65,464 70,514 74,294 73.534 42,943 38,133 75,584 18,546 19.196 20,826 24,646 24,876

71.28 58.08 70.10 56.76 52.80 50,49 16.72 23 76 25.4.5 18..i3 24.86 25.82 23.10 22.22 20.57 22,60 23 .75 23.10 26.10 25.10 23.76 21.34 15.40 14.11 11.22 9.46 12.98 13.97 13.42 13.64 10.12 12.32 9.46 9.68 10.01 11.oo

10,23 9.35 10,34 10.18 10.56 10.85 10.56 6.09 6.19 8.14 22 70 20.45 8.03 23.10 22.45

1,200,000 1,200,000 1,200.000 1,000,000

1,376,000 1,359,000 1,121,000 1,095,000 1,019,000 974,000 323,000 459,500 492,000 376,000 480,000 498,000 446,000 429,000 397,000 436,000 459,000 446,000 504,000 484,000 458,500 412,000 297,500 275,000 216,500 182,000 251.500 270,000 259,000 263,500 195,500 238,000 182,500 187,000 193,000 212,500 197,500 180,500 199,500 198,500 204,000 209,500 204,000 117.000 100,000 157,000 438,000 395.000 154,800 446,000 433,000

1,200,000 600,000 600,000 525,000 525,000 5 15,000 500,000 500,000 600,000 600,000 526,000 525,000 526.000 ;2.5,000 n25,000 400,000 450,000 325,000 330,000 330,000 300,000 3on.000 275,000 275,000 250.000 230.000 250 000 250,000 250,000 250,000 250,000 250,000 204,500 204,500 200,000 200,000 200,000 180,000 160,000 125,000 100,000 400,000 450,000 250,000 450,000 450,000

,

Low-Potency Samples in Corn Oil 0,855 0,865 2.985 0,905 74,334 89,545

0.594 0.550 0,605 0.418 0,450 0.594

11,500 11,000 12,000 8,000 9,000 11,500

.

14,000 14,000 14,000 10,000 10,000 10,000

not enough oils of this type were tested to recommend using this method for them. Table I11 gives the potencies of various irradiated ergosterols in corn oil as determined by the antimony trichloride colorimetric method. The first part of Table I11 is made up of values obtained for high-potency irradiated ergosterols in corn oil, and the second part consists of values for low-potency samples in corn oil or around 10,000 units per gram. These were obtained on the open market. Out of the 51 high-potency oils assayed by the antimony trichloride colorimetric method, 10 oils showed a difference from the bioassay of more than 257,. The mayimum per cent difference shorn-n by this method was 57.0, n-hile the average variation of all the oils tested was 15.Spo. The maximum variation shonn when testing six low-potency oils of about 10,000 vitamin Dg units per gram by this method was 21.4y0. The average per cent difference was 16.4. The potency values of an oil d e t e r m i n d by each of the two methods agree very closely and usually fall on the same side of the bioassay value. I n almost every case where the difference from the bioassay value was appreciable, the two physicalchemical values agreed with each other very well.

ANALYTICAL CHEMISTRY

320

DISCUSSION

Table IT.

Reproducibility for Irradiated Ergosterols in Corn Oil

El% 1cm.

Calculated, Da Units/G.

Bioassay Dz Units 'G.

Chromatographic Ultraviolet Absorption Curve Method, Oil 65,464 2.28 2.09 2.12 2.02 2.10

187,000 182,000 184,500 176,000 183,000

200,000

.I\..182,500 Antimony Trichloride Colorimetric JIethod, Oil 25,466 25.52 25.30 24.42 25.96 25.96 26.18 26.40 25.74 25.96 26.40

500,000

Av. 497,700

Table

V. Effect of Amount of Sample upon Accuracy of Antimony Trichloride Colorimetric Method

Wt, of Sample, G. 1.000 0,4992 0.2514 0.1262 0.0640 0.0372 0.0144

Calculated Dz Units in Sample (Based on Bioassay) 14,000 7,000 3,520 1,769 897 52 1 202

El% 1 cm.' 500 mp 0.606 0.473 0.542 0.634 0,676 0.537 0.389

Calculated Potency, Dz Units/G. 11,690 9,130 10,480 12,220 13,030 10,370 7,510

Results obtained by both physical chemical methods can be reproduced very easily, as shown in Table IV. Five separate samples of oil 65,464 were run by the chromatographic ultraviolet absorption curve method and the maximum deviation from the average value was 8.7%. Ten different determinations for oil 25,456 by the antimony trichloride colorimetric method showed a maximum deviation from the average of 1.6%. Reliable determinations were obtained by the antimony trichloride colorimetric method with oils containing as low as 10,000 vitamin D? units-per gram and the indications are that oils of much lower potency can be evaluated successfully. I n order t o determine how small an amount of sample may be employed and still an accurate potency determination be obtained by the antimony trichloride colorimetric method, various amounts of an oil containing about 10,000 vitamin D1 units per gram were put through the procedure. The results, as shown in Table T', indicate that, samples containing as low as 500 vitamin Dz units can be assayed n i t h a fair degree of reliability. However, when working rvith very small amounts of the sample, it is necessary t o add the reagent directly to the ether estract residue. Ordinarily, in a large sample, the residue is taken up in chloroform and a 1-nil, aliquot of this solution is mixed with 10 nil. of reagent. Thus, the concentration of the reagent in the solution cell is slightly more dilute than that in the solvent cell. This difference cannot be detected on the visual spectrophotometer at 500 nip, r, and no appreciable error is introduced by adding the reagent directly t o the dry residue. Although very small amounts of vitamin DSmay be measured as indicated by Table V, the antimony trichloride colorimetric method is not recommended for oils containing below 10,000 vitamin DS units per gram. Corn oil alone also exhibits, t o a slight extent, the same color reaction with the antimony trichloride reagent as vitamin D?, thus introducing another error in the determination. At the present time more work is in progress t o determine the extent and possible ways of correcting for the error due to a high concentration of corn oil. ,/, t

For testing oil solutions containing less than 50,000 vitamin Dz units per gram the antimony trichloride colorimetric method iq preferred, since the residue in the solvents n-hich have been used as developers of the Superfiltrol column may shon- a considerable amount of absorption in the ultraviolet region. The absorption, due t o a residue picked up from the Superfiltrol, may be caused by dissolved impurities. This can, hon-ever, be reduced to a minimum by first xvashing the column with 40 to 60 ml. of the developer. The last portion of the wash solution will have a n extinction of about 0.01 v-hen referred t o the original developing solution. This small amount of absorption, however, will introduce a considerable error in the determination of vitamin DP oils containing less than 50,000 units per gram when I-gram samples are used. Taking larger samples will increase the accuracy when working nit h lon--potency oils. The method of packing and making the chromatogram is very similar t o that used by Ewing et al. (6). The main differences include an essential prewashing of the column, the use of a longer column, and the substitution of hesane for Skellysolve in t,he developing solution. The Skellysolve used by En-ing et al. ( 5 ) cannot be employed successfully in this method because of its absorption in the ultraviolet, Redistilled hesane does not have this absorption and is very satisfactory. CONCLUSION s

Two phy4cal-chcniical methods for determining the vitamin D? content of samples of irradiated ergosterol in corn oil have been developed. One method, using tht, ultraviolet absorption curve of the nonsaponifiable fraction of thc oil sample which had first been chromatographed t o separate the impurities, gave an average variation from the bi0aasa.y value of 14.5vcwhen 49 different oils were tested. This method is recommended for oils containing 50,000 or more vitamin D2 units per pram, when 1-pram samples are used. Aicolorimetric method, using the color reaction of vitamin DS obtained by adding an antimony trichloride reagent t o the nonsaponifiable fraction of the oil sample, pave' an average variation of 15,Syc from the bioassay values of 51 high-potrncy oils and a n averacr difference of 1 6 . 4 7 from the bioassay values of 6 lowpotcncy oils. This method is recomnicndrd for oil solutions of irradiated ergosterol, having a potency of 10,000 vitamin DI units or more per gram. -4pplications of these two methods to oil wlutinns of lower potencies than rccomniended may he possible if interfering materials introduced by the chromatographic procedure and those present in corn oil arc c~liminatcd. Furthc LITER4TURE CITED

(1) Arnold, -4.. Proc. SOC.E t p f . B i d . X e d . , 63, 230 (1946). (2) Baker, D. H., 113.thesis, Michigan State College. 1944. (3) Brockrnann, H., a n d Chen, Y., Z. physiol. Chem., 241, 104 (1936). (4) D e K t t , J. B., a n d Sullivan, >X., I. ISD.E ~ GCHEM., . ANAL. ED.,18, 117 (1946). and Emmett, (5) Ewing, D. T., Kingsley, G. \-., B r o w i , I:. -1.. -1.D.. Ihid., 15, 301 (1943). (6) Hage, J.. M.S.thesis, Michigan S t a t e College. 1943. (7) Yield. C., Russell, W,, arid Zirnmerli, h.,J . B i d . Chem., 136, 73 (1940). (8) Oser, B . L..Melnick. D., a n d Pader, l f . , IXD. ESG. CHEM., A x ~ LED., . 15, 717 (1943). (9) Powell, M .J., M.S. thesis. Michigan S t a t e College, 1946. (10) Shanta, E. XI.,ISD.ENG.CHEM.,ANAL.ED.,16, 179 (1944). (11) Young, R., P h . D . thesis, Michigan State College, 1943. (12) Zirnmerli, A , , Yield, C., a n d Russell, K., J . B i d . Chem., 148, 245 (1943). RECEIVED April 8, 1947.

Presented before the Division of Biological Chern. ~ \ I E R I C A S CHEJIICAL SOCIETY,Chicago, 111.

istry at t h e 110th lleeting of t h e