902 (44)
V O L U M E 19, NO. 1 1 Reed, G. B., Rice, C. E., and Sinclair, R. J., Contrib. Can. Biol. Fisheries, 4, 229-56 (1929).
(45) Sadikov,
V. S.,Shoshin, A. F., Starukhina. K. M.,and Livshita, 11.I., Compt. rend. acad. s c i . C'. S . S . R., 3, 39-41 (1934). (46) Sharp, J. G., Biochem. J . . 29, 850-3 (1935). (47) Shewan, J. M., Dept. Sei. Ind. Research ( B r i t . ) ,Rept. Food Invest. Board, 1937, 75-8 (1937). (48) Sidaway, E. O., Fisheries Research Board Can., Progress Rept. Pacific Stas., 49, 3-5 (1941). (49) Sigurdsson, G. J., and Wood, A. J., J . Fisheries Research Board Canada, 6, 45-52 (1942). (50) Siser, I. W.,and Josephson, E . S.,Food Research, 7, 201-9 (1942). (51) Stansby, M.E., J . Assoc. Oficial Agr. Chem., 18, 616-20 (1936). (52) Stansby, M. E., and Lemon, J. M., IND. ESG. CHEM.,ANAL. ED., 5 , 208-11 (1933). (53) Stansby, J f . E., and Lemon, J. M.,U. S. Fish and Wildlife Service, Rept. X , 1, 1-46 (1941).
(54) Strohecker, R., Vaubel, R., and Kirchberg, H., 2. anal. Chem., 110, 1-11 (1937). (55) Tarr, H. L. A . , J . Fisheries Research Board Can., 4, 367-77 (1940). 156) Ibid.. 5. 187-96 (1940). (57) Waksman, D. TV , and Lomanits, S.,J . Agr. Research, 30, 2638 1 119251 ( 6 8 ) Watson, D. W., J . Fisheries Research Boa,d C a n , 4, 252-66 1192Q)
(59) ILzh;.l4, 267-80 (1939). (60) Weaver, R. H., Am. E x p t . Sta., Michigan State Coll. Tech. Bull., 79, 1-28 (1927). (61) Kood, -4.J.. Sigurdsson, G. J., and Dyer, W.J., J . Fisheries Research Board Can., 6, 53-62 (1942).
RECEIVED October 17, 1946. The work reported in this paper was done i n 1944, though the author's absence f r o m the country and other circumstances have delayed publication.
DELTA-TOCOPHEROL Assay of Total Tocopherols in Mixtures Containing Delta-Tocopherol 3IAX H. STERR AND JAJIES G. BAXTER Research Laboratories, Distillation Products, Inc., Rochester 13, S. Y . A modification of the Emmerie and Engel method is described for the assay of total tocopherols i n mixtures containing 6-tocopherol. Ethanol is used as the solFent for the reagent. -4 .procedure in which the reagent is prepared in acetic acid v a s inbestigated and found unsuitable for the determination. RIived tocopherol concentrates prepared by the molecular distillation of \egetable oils may be assayed directly by the ethanol procedure. Low-
potency preparations, such as vegetable oils, usually require purification prior to assay i n order to remove carotenoids and other pigments. -4 correction by the Kaunitz and Beaver procedure is also needed for inhibition of color formation. An improved method, which has been developed for the assay of total tocopherols in soj-bean oil, includes purification by the Parker-RIcFarlane reagent and application of the correction for inhibition.
ECEKTLT a newly identified tocopherol, called &tocopherol, was isolated from soybean oil and structural evidencc obtained xvhich indicates that it is 8-methyltocol (17).
BEHAVIOR O F PURE TOCOPHEROLS WITH FERRIC CHLORIDE-BIPYRIDINE REAGENTS
R
The reagent of Emmerie and Engel has become the one most widely employed for the estimation of mixtures of tocopherols, but a single procedure for its preparation and use has not been accepted. R a d i n g s prepared the reagent n-it'h alcohol as the solvent (15)) as did the originators of the method; Devlin and llattill (5)preferred to use acetic acid as the solvent, a procedure
&Tocopherol This tocopherol \vas also separated from wheat germ oil ( I ? ) and an analytical procedure indicated that it is present in cottonseed and peanut oils (18). The possible occurrence of d-tocopherol in a variety of vitamin E preparations of natural origin must, therefore, be considered by the analyst. &Tocopherol \vas distinguished from a-,,@-, and r-tocopherols by the fact that it gives more color on a molecular basis with the ferric chloride-2,2'-bipyridine reagent of Emmerie and Engel (61, so that its presence in a mixture of tocopherols results in assay values which are too high. This paper is concerned with the proposal of an improved procedure for the estimation of total tocopherols in mixtures containing &tocopherol. Principal attention is given to the assay of commercial tocopherol concentrates prepared a t Distillation Products, Inc., by the molecular distillation .of mixed vegetable oils (called distilled concentrates, Type I11 or IV) and to the assay of vegetable oils. However, the principles involved are applicable to any mixed tocopherol preparation.
I 2 34 5
7
IO
I
I
15
20
I
30
40
T I M E (MIN.)
Figure 1. 1,2,3,
a-,y-,
Rate of Color Formation for Pure Tocopherols in Acetic Acid
and 8-tocopherols. Concentration, 30.8, 59.3, and 90.0 micrograms per 25 ml., respectively
N O V E M B E R 1947
.
903
developed in the Merck Laboratories (the ferric chloride reagent prepared in ethanol and in acetic acid is referred to below as t,he ethanol reagent and the acetic acid reagent, respectively). The time of interaction of tocopherols and reagent before the red color is measured has been varied Tyithin wide limit,s (5,8, 14, 15). Baxter et al. ( 1 ) examined the modifications of the Emmerie and Engel method then in practice and concluded that the acetic acid reagent is unsuitable for the assay of mixtures of tocopherols. A modified assay method using the ethanol reagent was proposed. This review (1) Tras written a t a time when &tocopherol xas unknonn and it has thus become necessary to revise the conclusions reached in it. Assay in Acetic Acid. The assay of mixtures of tocopherols by the acetic acid reagent \\-as found unsatisfactory because the intei1sii)- of color produced with the individual tocopherols \vas not a linear funct ion of the tocopherol concentration, Linearity is not wsential in assaying samples containing a single tocop!ierol but a lack of linearity makes it difficult to jtandardize a metliod which is t o be applied to mixtures in Ti-hich the proportion of the various tocopherols is unknowi. .Inother disadvantage of the acetic acid reagent is indicated in Figure 1, rvhich gives a plot of the color intcnsity produced with pure CY-, y-, and &tocopherols as a function of the reaction time. The intensity is expressed as the L value ( 4 ) and was measured with an Evc,l!-ri colorimeter and a No. 520 filter. B-Tocopherol r a s riot included in the Study because it is osidized by ferric chloriilc at a rate intermediate bet\\-cen that of a- and y-tocopherols ( 2 ) . Substantially complete oxidation of a-tocopherol n-as effected in 15 minutes. -,-Tocopherol, honever, reacted more slon-ly and com:)lete osidntion ivas not obtained in 40 minutes. 6-Tocophern1 rcquired a still longer period. .I timr could not be selected in xhich all the tocopherols gave approsimately the same color intensity n-ith the acetic acid reagent, so that it nould he difficult to use it for assaying mixtures of tocopherols, except in the special case n-hew the composition of the tocopherols v-as kno5v-n and it, n-as possible to calibrate the method with pure tocopherols having the same composition. Thus, the Tyidely differing oxidation rates of the tocopherols n-ith the acetic acid reagent is an additional reason for using it only on the individual tocopherols and not on their mixtures. RIETHOD
The procedure varied from ( 5 ) only in minor det,ails. The reagent, was prepared containing ferric chloride (FeCI,.GH20, 0.250 gram) and 2,2’-bipyridine (0.500 gram) in glacial acetic acid (1 liter). -In Evelyn colorimeter Tyas used Rith a KO.520 filter. The instrument was set t o give a galvanometer reading of 100 for a blank containing petroleum ether (Skellysolve B, 5 ml., purified as described below) and reagent (20 ml.). A 5-ml. sample containing 30 to 90 micrograms of the proper tocopherol was then added t o a colorimet,er tube, followed by 20 ml. of reagent. The tube was quickly shaken and put in the inst,rument. After the indicated periods of time t’he galvanometer readings, G, were determined and the L values were calculated. , Assay in Ethanol. It had been concluded ( 1 ) that the ethanol reagent is preferable for assaying mixtures of tocopherols because the intensity of color formed is a linear function of the tocopherol concentration over a wide range of concentration, Furthermore, the rate of oxidation of a-,0-,and y-tocopherols with the reagent was found to be rapid and the intensity of color produced was substantially the same, allowing for differences in molecular weight. On the basis of the oxidation curves a modified procedure for the assay of mixtures of tocopherols with the ethanol reagent was proposed in which a 2-minute reaction time was used prior t o making the color measurements. A comparison of the oxidation properties of &tocopherol with those of the other three tocopherols (Figure 2) has necessitated a change in the reaction time taken when &tocopherol is present. It is evident from the curves that color formation for e-, p-, and
ytocopherols is substantially complete in 2 minutes, mith only a small increase, or “rise” occurring when the reaction time is extended to 10 minutes. Apparently these tocopherols are oxidized rapidly to the p-quinones and then the reaction ceases. If only these three tocopherols occurred naturally, any reaction period from 2 to 10 minutes might be taken for the assay.
0
1
2
3
4
5
7
IO
TIME (MIN.1
Figure 2. 1,2,3,4,
8-,
OL-,
Rate of Color Formation for Pure Tocopherols in Ethanol
and 8-tocopherols. Concentration, 106, 131, 142, and 116 micrograms per 25 ml., respectively
Y-
The rise in the case of pure &tocopherol, h o w v c r , is about 30% and the magnitude of the L values indicates t,hat,this is possibly due to oxidation past the p-quinone stage. Since this t,ocopherol gives a greater intensity of color on a molecular basis than the other tocopherols if the reaction time is prolonged, this time becomes a critical factor in the assay of distilled concentrates or vegetable oils ryhich contain &tocopherol. From the curves it is clear that for mixtures of tocopherols whose composition is unknown the best r e a d o n time is 2.5 minutes, since color formation for three of the tocopherols is substantially complete t,lien, and the color intensities for all four are most nearly the same. If this time is taken as the basis of a n assay procedure for mixtures containing &tocopherol, the assay may be standardized either with pure natural a-tocnpherol ‘or preferably with a 50 t o 50 mixture of a- and y-tocopherols (pure a- and y-t,ocopherols are sold for the purpose by Distillation Products. Inc.). REAGENTS AND SOLVENTS
Reagents. Separate solutions of ferric chloride (0.1 gram per 100 ml., FeC13.6Hz0) and 2,2’-bipyridine (0.25 gram per 100 ml.) in purified absolute ethanol are prepared and stored in amber glass bott’les (preferably painted black). Fresh preparations should be made every 2 weeks. Purified Ethanol. Absolute ethanol was purified by distilling a commercial grade (U. S. Industrial Chemicals, Inc., 5 gallons) containing potassium permanganate ( 10 grams) and potassium hydroxide (20 grams) through a Vigreux column. Purified Petroleum Ether. Skellysolve B was purified by shaking with concentrated sulfuric acid, washing with dilute sodium hydroxide solution, and distilling. PROCEDURE
X 1-ml. aliquot of the sample containing 50 t o 150 micrograms of tocopherol in purified ethanol (for concentrates) or petroleum ether (for low-potency preparations containing glycerides) is added t o a 2-ounce glass-stoppered bottle painted black, followed in order by 1 ml. of bipyridine reagent, 1 ml. of ferric chloride reagent, and 22 ml. of purified ethanol. The last is added from a 50-ml. buret. When 17 ml. of ethanol have been added a timer
V O L U M E 19, NO. 1 1
904 equipped with a second hand is started. This operation can be completed in the time necessary for the remainder of the 22 ml. to run into the reaction bottle. The stopper is inserted, the solytion is swirled gently, and the bottle is set aside for 2.5 minutes. A blank previously prepared (1 ml. of ethanol, 1 ml. of each reagent then 22 ml. of ethanol) is used to adjust the galvanometer of the Evelyn colorimeter to read 100 using a No. 520 filter. Approximately 10 seconds before the completion of the 2.5-minute period, the test solution is poured into an Evelyn tube in the colorimeter; the tube is covered with a black cap; the galvanometer reading, G, is determined a t 2.5 minutes; and L (2-log G ) is calculated. The tocopherol content is determined with a calibration curve (relating L to the tocopherol concentration) which is prepared with pure natural a-tocopherol or with a 50 to 50 mixture of natural a- and y-tocopherols. W t h i n galvanometer readings of 25 to 70, L is a linear function of the tocopherol concentration (15). Lower galvanometer readings should be avoided since they indicate an insufficient excess of ferric chloride. Precautions. Excessive exposure to light of either the blank or the test sample must be avoided because this causes the formation of spurious color. Painting the reagent bottles black and putting a black cap over the tube while it is in the galvanometer will prevent this in part. Use of a low-wattage bulb in the colorimeter (the Evelyn instrument uses only a 2-watt bulb) is of further help. Spurious color formation due to impure ethanol and petroleum ether was avoided by the purification methods described. Using these precautions not more than a one-quarter division change should be noticed in the galvanometer reading for the blank in the course of 10 minutes. Precision. To test the precision of the method, employing the 2.5-minute time interval, mixtures of a-,y-, and 6-tocopherols In the proportions, respectively, of 45:40:15, 10:60:30, 10:45:45, and 10:0:90, were prepared and the percentage recovery of total tocopherols was determined. The recoveries averaged 100.1% with a spread from 98.5 to 101.0%. This error is not significantly different from that found in assaying individual tocopherol preparations by the ferric chloride-bipyridine method. Application. The method is the one considered most suitable for assaying mixed tocopherol preparations where the proportion of the individual tocopherols is unknon-n. This is the case with many vegetable oils and tocopherol concentrates prepared from them by molecular distillation. Distilled concentrates (Types I11 and Is’) are assayed somewhat differently. I n such concentrates, the kind of tocopherols present (CY-, y-, 6-) and the proportion of each have been determined (18). Because of this, the Products Control Department of this laboratory has found it more convenient to use the 10minute reaction interval recommended for the ethanol reagent in the piocedure of Rawlings (15),standardizing the method with a mixture of pure tocopherols having the same percentage composition as the concentrate. A more recent innovation has been to use a-tocopherol as the standard and to multiply the apparent assay value a t 10 minutes by a factor (currently, this is 0.95 for Type I11 and Type IV concentrates and represents the ratio of the slope of the a-tocopherol calibration curve to that of the CY-, y-, &tocopherol curve) to correct for &tocopherol. Reproducibility is somewhat improved by using a reaction period of 10 minutes, since color formation from &tocopherol is substantially complete then and small changes in the reaction time have little effect on the assay. I n the experimental section only the “2.5minute” method is described because of the necessarily limited applicability of the “10-minute” procedure. Assays by the two methods should agree within *2%. Direct application of the ethanol reagent is usually possible only with tocopherol concentrates. I n assaying low-potency preparations, such as vegetable oils, interference must be considered and a correction applied for it. ISTERFERENCE
Inhibition. A major source of interference in the assay of vegetable oils is the inhibition of color formation with the ferric chloride reagent. This effect was studied by Kaunitz and Beaver
Table I .
Bubstrate
Sesame oil h‘lineral oil Sesame oil Sesame oil Distilled concentrate (Type 111)
Inhibition with Ethanol and Acetic Acid Reagents Initial Tocopherols
Weight Taken
Added Tocopherol
Recovery (2.5-Min. Method)
%
Mo.
Y
%
A. 0.06 0.00 0.06 0.06 27.8
Ethanol Reagent 40 Synthetic a- 96 20 Synthetica- 49 30 Natural y - 118 40 Satural 6- 86 0,176 Satural a- 60 0.176 Katural y - 58 0.176 Natural 6- 60
91.5 100 93.5 89.9 99.5 99.0 98.0
Acetic Acid Reagent (10-Min. Reading) 0.04 90 Synthetic 107 0.00 90 Synthetic a- 107
80.5 99.5
B.
Sesame oil Mineral oil
(I-
(9, 10) who observed that when known amounts of synthetic a-tocopherol were added to a number of oils, such as sesame oil, and even to mineral oil, in low concentrations-e.g., 0.1%recoveries as small as 15% were obtained using the acetic acid reagent. These authors suggested that in the case of glyceride oils the inhibition might be due to a complex formed between ferrous ion and fat which retarded the formation of the colored complex between ferrous ion and bipyridine. To correct for inhibition Kaunitz and Beaver determined the extinction of a given weight of oil before and after the addition of a known amount of pure a-tocopherol. By proportion a corrected value for the tocopherol content of the oil was obtained. The authors have studied the inhibition in distilled concentrates with the ethanol reagent by determining the percentage recovery of added pure a-, y-, and &tocopherols. The percentage recovery of pure tocopherols added to sesame oil and to mineral oil was also determined with the ethanol and acetic acid reagents in order to compare the inhibition observed (Table I). For a distilled concentrate (Type 111), having a potency of about 27%, the inhibition amounted to 1%for CY- and 7-tocopherols and 2% for &tocopherol. The suppression was so small that it was concluded that the Kaunitz and Beaver effect can be neglected in the assay of either Type I11 or IV concentrates. An inhibition amounting to 6 to 10% TTith the ethanol rcagent was observed for CY-, y-, and &tocopherols added to sebame oil in concentrations of 0.2 to 0.47,. An inhibition of 15% was previously reported for 7-tocopherol added to soybean oil (1). Thus, a correction using the Kaunitz and Beaver procedure should be applied when assaying tocopherol preparations of loiv potency. In making this correction it appears desirable, TI herever possible, to use a mixture of pure or concentrated tocopherols having approximately the same composition as those in the sample being analyzed rather than to use a pure single tocophcrol. I n this way differences in the degree of inhibition for the individual tocopherols are avoided. The inhibition was greater with the acetic reagent (20%) than Jvith the ethanol reagent (8yo)when synthetic a-tocopherol was added to sesame oil. The experiment was complicated by the fact that the assay of the sesame oil itself was lower with the acetic acid reagent (0.04%) than Rith the ethanol reagent (0.06%) and a larger oil sample was required. This evidence of increased inhibition with the acetic acid reagent is an additional reason why the authors prefer the ethanol reagent for the assay of tocopherols in natural products. I n contrast to the findings of Kaunitz and Beaver the authors observed no suppression with the acetic acid reagent (or the ethanol reagent) for a-tocopherol added to a mineral oil sample (Kaydol, extra heavy white mineral oil, L. Sonneborn Sons, Inc.). It would therefore appear that the inhibition observed by Kaunitz and Beaver is not characteristic of all highly purified hydrocarbons.
*
90s
N O V E M B E R 1947 Method. The recovery data in Table I were obtained in the following way: A 1-ml. aliquot containing the indicated weights of pure tocopherols in ethanol was added t o a reaction bottle folio\\ cd by bipyridine solution, ferric chloride solution, and ethanol, in the usual way. The value of L was determined at. 2.5 minutes. rinother I-ml. aliquot was evaporated t o dryness in a stream of nitrogen and a I-ml. aliquot containing the indicated weights of concentrate, sesame, or mineral oil, was added. Solution 11as effected by swirling and L was redetermined. Finally, the value of L for a 1-ml. aliquot of the concentrate or oil was determined. If Lt, L,, and L, represent the values of L for the pule tocopherols, the substrate, and the combination of the two, thc peicentage recovery of tocopherol is lOOLt/L, - L,. The amount of sample was limited to contain 60 to 70 micrograms of tocopherol and that added to 60 to 120 micrograms t o avoid having the total tocopherol content much in excess of 150 micrograms. When too much tocopherol was present 6-tocopherol was incompletely oxidized. The recovery data x i t h the acetic acid reagent were determined In similar fashion, using the procedure described. The cause of inhibition is still obscure. I n two vegetable oils it \vas found to be associated with the unsaponifiable matter. This was determined by separating soybean and corn oils into the high-boiling glyceride fraction and the lower-boiling unsaponifiable fraction by distillation in a molecular still. Substantially 100% recovery of added pure 7-tocopherol was obtained ivith the glyceride fraction in each case, while the unsaponifiable fraction exhibited inhibition. This inhibition was largely removed by acetylation; hence the authors concluded that it was hydroxylic in nature. It is probable t h a t no single substance will be found responsible for inhibition but a number of unsaponifiable constituents in oils and possibly certain glycerides will possess the property. The mechanism of inhibition needs further study. Pigments. Pigments, including carotenoids and vitamin A, cause interference due either to their intrinsic color or to their reducing activity which produces color with the ferric chloridebipyridine reagent. One microgram of @-caroteneand vitamin 4 . was cquivalent to 1.8 and 0.04 micrograms, respectively, of tocopherol in the 2.5-minute assay method (16). The interference due to pigments is usually eliminated as far as possible by preliminary purification of the sample by mol&ular distillation (S, I S ) , 85% by weight aqueous sulfuric acid (the Parker-McFarlane method, 11), hydrogenation (12, 14), and selective adsorption (7, 8). The advantages and disadvantages of cach of these procedures have been discussed (1 ). Tests were made on several distilled concentrates (Types 111 and IIr) to determine whethei sufficient pigments were present to interfere with the assay. The test was done by assaying t h e , Concentrates before and after applying the technique of Quaife and Harris ( l a , 1 4 ) which removes carotenoid and pigment interferencc by hydrogenating the double bonds present. K O change in thc assay value occurred after hydrogenation. Since there is evidently too little pigment present in distilled concentrates to intei fcrc n ith the tocopherol estimation, preliminary purification by 11) drogcnation, 85% sulfuric acid, or adsorption is unnecessar: . The relative efficiency of the available purification procedures for the separation of pigments and other reducing substances from lom-potency tocopherol preparations, such as vegetable oils, has been difficult to determine. A criterion proposed (1) for cstimating the efficiency consistcd in determining the percentage increase in the apparent potency of the sample or rise when the reaction time was increased from 2 to 10 minutes with the ethanol reagent. The method which gave a purified sample having the smallest rise was considered to be best on the ground that it removed most effectively those substances, such as the carotenoids, which are oxidized more slowly than the tocopherols. This criterion was applied to the development of an improved method for the assay of soybean oil which consisted in treating the sample by the Parker-McFarlane procedure to remove pig-
ments and applying the Kaunitz and Beaver correction for inhibition. The usefulness of this procedure for the assay of soybean oil has been confirmed by Xlr. Kuhrt of this laboratory. Since no experimental details were given in the earlier paper it seems desirable to record them here. IMPROYED METHOD FOR SOYBEAN OIL
Assay of Soybean Oil. A sample of crude soybean oil prepared by the solvent extraction process (apparent tocopherol assay 0.165%, 227, rise) was degummed by stirring a t 40" with 27q by weight of water. The sludge was allowed to separate overnight and the oil was filtered through a bed of sodium sulfate to remove traces of water (assay 0.169%, 20y0 rise). The best evidenre indicates that no loss of tocopherol occurs in this procwlure, whereas some loss occurs in alkali refining. A 10-ml. aliquot of purified petroleum ether (Skellysolve B) containing approximately 0.1 gram of degummed oil mas added to a conical glass-stoppered centrifuge tube. Sulfuric acid (2 nil, 85% by weight) was added. The tube was inverted five times and then centrifuged. The supernatant layer was decanted into a clean tube and 5 ml. of 1% potassium hydroxide solution were added. The tube was shaken and again centrifuged. B 3-ml. aliquot containing approximately 60 micrograms of tocopherol was added to a reaction bottle and the solvent evaporated in a stream of nitrogen. T o the residue were added Skellysolve B (1 ml.), bipyridine solution (1 ml.), ferric chloride solution (1 ml.), and ethanol (22 ml.). The value of L was determined by the procedure described in ( f ) , which is essentially the same as that described in this paper except that a 2-minute reaction time was used. Reference t o a calibration curve prepared from a 50 t o 50 mixture of 01- and y-tocopherols gave the partially corrected value 0.176y0 for the total tocopherol content of the water-refined oil. A correction was made for inhibition using the Kaunitz and Beaver method in a manner similar to that described. A 3-ml. aliquot, containing approximately 60 micrograms of tocopherol, of the petroleum ether solution obtained after Parker-McFarlane purification was used. Pure natural 7-tocopherol was employed to determine the recovery. The value 0.176% was then corrected for the inhibition, giving the value of 0.190% for the degummed soybean oil. Thus the appirent assay of 0.169% had been in error by about 12%. Support for this conclusion was obtained by distilling the oil in a molecular still to obtain the tocopherols in concentrated form. The value of 0.190% for the original oil gave a recovery of 99% in the distillate. Had the value of 0.169% been taken the recovery would have been well over 100%. The more recent work with &tocopherol suggests that this assay procedure would be improved by the use of the 2.5-minute reaction time in the assay and by the use of a 70 to 30 mixture of y- and &tocopherols (18) in the inhibition studies. LITERATURE CITED
W.,Hove, E. L., Quaife, M. L., \Yewler, Leonard, and Stern, M. H., Bid. Symposia, 12, 484 (1947). Baxter, J. G., Robeson, C. D., Taylor, J. D., and Lehman, R W., J . Am. Chem. Soc., 65, 918 (1943). Dam, H., Glavind, J., Prange, I., and Ottesen, J., K Q ~Danskr . Videnakab. Selskab Bwl. M e d d . , 16, No. 7 (1941). Dam, W. J., and Evelyn, K. A., Biochem. J., 32, 1008 (1938). Devlin, H. B., and Mattill, H. A., J . Bid. Chem.. 146, 123 (1942) Emmerie, A., and Engel, C., Rec. trav. chim., 57, 1351 (1938). Ibid., 58, 283 (1939). Hines, L. R., and Mattill, H. A., J . B i d . Chem., 149, 549 (19431 Kaunitz, H., and Beaver, J. J., Ibid.,156, 653 (1944).
(1) Baxter, J. G., Lehman, R.
(2)
(3) (4) (5) (6)
(7) (8) (9)
(10) Zbid., 156, 661 (1944). (11) Parker, W. E., and McFarlane, W. D., Can. J . Research, B18, 405 (1940). (12) Quaife, M. L., and Biehler, R. M., J . Biol. Chem., 159, 663 (1945). (13) Quaife, M. L., and Harris, P. L., IND. ENG.CHEM.,ANAL.ED., 18, 707 (1946). (14) Quaife, M. L., and Harris, P. L., J . BioZ. Chem., 1 5 6 , 4 9 9 (1944) (15) Rawlings, H. W., Oil & Soap, 21, 257 (1944). (16) Robeson, C. D., unpublished work from this laboratory.
(17) Stern, M . H., Robeson, C. D., Weisler, Leonard, and Baxter. J. G.. J . Am. C h m . Soc.. 69.869 (1947). (18) Weisler, Leonard, Robeson, C. D.; and Baxter, J. G., ANAL. CHEX, 19, 906 (1947). RECEIVED December 6, 1948. Presented before the Division of Analytical and Microchemistry a t the 110th Meeting of the AMERICANC H E m c A L SOCIETY, Chicago, Ill.
