544

T H E J O U R N A L OF THE; A M E R I C A N

Acknowledgment

0IL

CtIEMISTS'

VOL. 34

SOCIETY

REFERENCES

2. Bailey, A. E., "Cottonseed and Cottonseed Products," Interscience, New York, 1948, p. 380. 3. Bailey, A. E., ed., "Industrial Oil and Fat Products," 2rid ed., Interscience, New York, 1951, pp. 646--649. 4. Deehary, J. M., Kupperman, R. P., Thurber, F. H., and O'Connor, 1~. T., J. Am. Oil Chemists' Soc., 31, 4 2 0 - 4 2 4 (1954). 4a. Earle, F. R., and Milner, R. T., Oil and Soap, 17, 106-108 (1940). 5. Eaves, P. H., Spadaro, J. $., D'Aquin, E. L., Crovetto, A. J., Cirino, V. 0., and Stansbury, M. F., J. Am. Oil Chemists' Soc., 33, 639--645 (1956). 6. Jancik, V., Pokorny, J., and Mares, E., Prumysl Potravin, 7, 213--215 ( 1 9 5 6 ) ; C. A. 50, 10430. 7. K a u f m a n n , It. P., Fette u. Seifen, 48, 53-59 (1941). 8. Keith, F. W. dr., Blachly, F. E., and Sadler, F. S., J. Am. Oil Chemists' Soc., 31, 298-302 (1954). 9. Keith, F. W. Jr., Bell, V. G., and Smith, F. tt., J. Am. Oil Chemists' Soc., 32, 517-519 (1955). 10. Lambou, M. G., and Dollear, F. G., Oil and Soap, Z2, 226-232 (1945). 11. Linteris, L., and Handschumaker, E., J. Am. Oil Chemists' Soc., 27, 260--264 ( 1 9 5 0 ) . 12. National Cottonseed Products Association, " T r a d i n g Rules, 1 9 5 6 57," Memphis, Tenn., 228 pp. 13. Pack, F. C., and Goldblatt, L. A., J. Am. 0il Chemists' Soc., 32, 551--553 (1955). 14. Pons~ W. A. Jr., Murray, ~r D., LeBlanc, M. F. I-I. Jr., and Castillon, Leah E., J. Am. Oil Chemists' Soc., 30, 128-132 (1953). 15. Ports, W. A. Jr., Thurber, F. H., and Itoffpauir, C. L., 3. Am. Oil Chemists' Soc., 32, 98-103 (1955). 16. Ports, W. A. Jr., Mitcham, D., O'Connor, R. T., and Stansbury, M. F., J. Am. Oil Chemists' Soc., 33, 324-330 (1956). 17. Pons, W. A. Jr., Stansbury, M. F.~ and ttoffpauir, C. L., J. Assoc. Offic. Agr. Chemists, 36, 492-504 (1953). 18. Royce, H. D., and Lindsey, F. A. Jr., Ind. and Eng. Chem., 25, 1047--1050 (1933). 19. Stansbury, M. F., and Hoffpauir, C. L., J. Am. Oil Chemists' Soc., 29, 53-55 (1952). 20. Wurster, O. H., Govan, W. 5. Jr., and Stockmann, G. 5., in "Cottonseed and Cottonseed Products," A. E. Bailey, ed., Interscience, New York, 1948, pp. 814-816.

1. American Oil Chemists' Society, "Official and Tentative Methods," 2rid ed., rev. to 1955, Chicago, 1946-1955.

[ R e c e i v e d A p r i l 29, 1 9 5 7 ]

The authors gratefully acknowledge the cooperation of Buckeye Cotton Oil Company, Jackson, Miss., and Memphis, Tenn. ; Dothan Oil Mill Company, Dothan, Ala. ; Kershaw Oil Mill, Kershaw, S. C. ; Lever Bros. Company, Edgewater, N. J., Hammond, Ind., and Los Angeles, Calif. ; Lookout Oil and Refining Company, a division of A r m o u r and Company, Chattanooga, Tenn.; Mrs. Tucker's Products, Sherman, Tex.; Opelousas Oil Refinery, Opclousas, La.; Procter and Gamble M a n u f a c t u r i n g Company, Macon, Ga., and Portsmouth, Va.; Ranchers Cotton Oil, F r e s n o , C a l i f . ; S o u t h e r n C o t t o n Oil C o m p a n y , Gretna, La. ; South Texas Cotton Oil Company, Houston, Tex. ; and Wilson and Company Inc., Oklahoma City, Okla., in supplying acidulated soapstocks of known processing histories. The a u t h o r s are also indebted to T. H. H o p p e r for direction and guidance, to R. M. H. Kullman, Julian F. Jurgens, James A. Harris, and Voyce P. Whitley for many of the chemical analyses reported in this paper, and to Elsie F. DuPr~ and Dorothy C. Heinzelman for spectrophotometric measurements.

Determination of Tocopherol in Oxidized Fats 1 E. N. FRANKEL, C. D. EVANS, and J. C. COWAN, Northern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture, Peoria, Illinois MODIFICATIONS Of the Emmerie-Engel method (2) for the determination of tocopherol in fats have been developed to remove substances which interfere with the ferrous-bipyridine color reaction (1, 2, 7, 8). Fats were reported to interfere with this color reaction (4, 6). Kaunitz and Beaver (6) introduced a proportionality factor to correct for the color-depressing effect observed in the presence of increasing concentrations of fat. However Gupta and Basu (3) found that this correction factor did not apply to oxidized groundnut oil. They showed that this oil when free of peroxides did not inhibit the color development in the EmmerieEngel procedure. Therefore this interference of fats with the color reaction may be caused by peroxides in the fats. Lips (7) used the sulfuric-acid treatment of P a r k e r and McFarlane (9) and saponification at room temperature to remove interfering substances in autoxidized methyl esters of f a t t y acids. This paper describes a simple method for tocopherol determination in oxidized fats where peroxides are removed by thermal destruction since the presence of peroxides gives erroneously low values. The method is currently being used in a study of the fate of tocopherols in oxidizing fats.

s

EVERAL

vations showed that soybean oil immediately after deodorization at 210~ did not interfere with the Emmerie-Engel color reaction for tocopherol. When a series of determinations was carried out with different concentrations of freshly deodorized oil (20 to 140 rag. per 10-ml. solution), the color developed was proportional to the sample weight. However, when allowed to oxidize, the oils interfered with the determination of tocopherol (Figure 1). These results are in agreement wih those of Gupta and Basu (3) in showing the interference of fat peroxides with the Emmerie-Engel color reaction for tocopherol. A study was made of the effect of deodorization on the tocopherol and peroxide contents of soybean oil and lard. The fats were heated at 210~ under reduced pressure (less than 1 ram. Hg.) in 50-ml., round-bottom flasks immersed in a thermostatically 1.00 PEROXIDE VALUE

.

'

"--0 0 ~

o

J#'~"

,/

u~

- ~ "

........"~..... ...............

< ~90-50

Experimental The method of Stern a n d Baxter (11) was used for tocopherol, except 10 rain. were allowed for color development instead o f 2.5 rain. Preliminary obserz Presented at annual meeting of American Oil Chemists' Society, New Orleans, La., April 2 8 - M a y 1, 1957.

0.0(

50 100 OIL CONCENTRATION,

150 MG. PER 10 ML.

200

FIG. 1. E f f e c t o f p e r o x i d e s i n s o y b e a n oil o n t h e f e r r o u s b i p y r i d i n e color r e a c t i o n for toeopherol.

Nov.

1957

FRANKEL

/

'

0~1

I J J

'

TOCOPHEROL

gJJ~l~lllllllllllll

~.25t

E T AL. :

i

D E T E ; R M I N A T I O N OF T O C O P H E R O L

.

0 IIIIIII IIIIII ~o Iiii iii iiii iiI1~ iiiiiiiiiiiii1~111

.........................

I ~ 50 ~ ~'0 75 ; i ~

I~,

-o

o...,.,.,F RES H LARD "1" TOCOPHEROL ,,--,FRESH SOYBEAN OIL D~m.OXIDIZED LARD A, . . . . OXIDIZED SOYBEAN OIL

~o

PEROX,DE ;

5

I

10 15 20 25 TIME OF HEATING, MINUTES

30

35

F r o . 2. E f f e c t o f h e a t i n g a t 2 1 0 ~ in vaeuo on the tocopherol content of fresh fats (initial tocopherol concentration: 1,500 microgram per gram) and the peroxide value of oxidized fats (initial peroxide value: 22).

controlled oil-bath; agitation was provided by a fine stream of nitrogen. A f t e r heating, the oils were immediately cooled to room t e m p e r a t u r e , and the v a c u u m was broken with nitrogen. The results represented in F i g u r e 2 show t h a t heating soybean oil and lard at 210~ for 10 and 15 min., respectively, is sufficient to remove all peroxides f r o m the oxidized fats. This heat t r e a t m e n t caused a loss of 1 - 2 % of the tocopherol in fresh fats. I t is interesting to note t h a t the loss of both tocopherol and peroxides b y heat is more r a p i d in soybean oil t h a n in lard. The more r a p i d destruction of peroxides in oxidized soybean oil would be in agreement with the finding of P r i v e t t and Quackenbush (10) t h a t tocopherol caused a n increase in the rate of t h e r m a l destruction of fat-peroxides. To determine the recovery of toeopherol in oxidized fats, fresh lard containing a known concentration of added a-toeopheroI was mixed in different proportions with oxidized lard (peroxide value, 23.8) containing no added tocopherol. The tocopherol was determined in these mixtures a f t e r heating at 210~ for 10 min. in wacuo. The linear relation obtained

' j j~ 1500

,

,

,

,

,

,

,

,

, j

o,"

~ 1000 u

between toeopherol and the extent o f dilution (Figure 3) demonstrates t h a t the heat t r e a t m e n t does not affect the tocopherol content in oxidized fat. I n another experiment the heat t r e a t m e n t (210~ for 10 min.) was applied to a series of soybean oil samples containing different concentrations of d-atocopherol. These samples were p r e p a r e d b y first removing the n a t u r a l tocopherols f r o m the oil by shaking with carbon black in pentane and then addment with carbon black was carried out as follows. A solution of 500 g. of oil in 1 liter of redistilled pening different a m o u n t s of d-a-tocopherol. The treattane was shaken mechanically with 100 g. each of carbon black (Darco G-602) and 100 g. of Celite in a 3-1. bottle for 30 min. The oil suspension was filtered through a thin layer of Celite, and the adsorbent was washed thoroughly with pentane. The volume of the oil solution was reduced to 1.5 liters with a rotating evaporator. This t r e a t m e n t with carbon black-Celite was repeated three or four times to remove the tocopherols completely f r o m the oil. The results (Table I ) show t h a t no loss of tocopherol resulted f r o m this heat treatment. The heat-treatment procedure was compared with other methods used to remove i n t e r f e r i n g peroxides ( a n d / o r substances) prior to the determination of toeopherol in oxidized fats. These methods include t r e a t m e n t with 80% sulfuric acid and a hot and a cold saponification. The sulfuric acid t r e a t m e n t and the cold saponification were carried out by the method of Lips (7). The hot saponification was carried out as follows. Ten grams of oil were saponified with 20 ml. of 3.5 N alcoholic K O H in the presence of 5 ml. of 5% alcoholic pyrogallol b y refluxing for 10 rain. A f t e r saponification 40 ml. of distilled w a t e r were added to the saponification mixture, and the solution was immediately cooled to room t e m p e r a t u r e . The unsaponifiable m a t t e r was extracted in the d a r k with peroxide-free ethyl ether according to the procedure described b y Zseheile et al. (13). The results given in Table I I show t h a t a greater recovery of tocopherol is obtained in the fats with the heat t r e a t m e n t t h a n with the acid or saponification treatments. The cold saponification resulted in considerable loss of tocopherol in soybean oil while the hot saponification method yielded a recovery equivalent to t h a t obtained with the sulfuric-acid treatment. The interference of peroxides with the E m m e r i e - E n g e l color reaction is evident f r o m the low values obtained with the control oxidized-fats. The possibility that toeopherol m a y be regenerated in the oxidized oils b y the heat t r e a t m e n t at 210~ i n v,acuo was investigated. Two lots of fresh soybean oil were oxidized at 60~ with a stream of oxygen for four days to a peroxide value of 60 and 72, res Since the D e p a r t m e n t of A g r i c u l t u r e does not recommend the products of one c o m p a n y over those of another, these names a r e furnished for information only.

500

TABLE I Effect of H e a t Treatment on Toeopherol Contents Of Soybean Oil

o o

Oil samples a 0

545

,

,

,

,

,

0 FRESHLARD-I-TOCOPHEROL 50 100 OXIDPZED LARD 50

,

,

,

100 0

COMPOSITION, PERCENT FIG. 3. T o c o p h e r o l c o n t e n t o f m i x t u r e s o f f r e s h l a r d q- 1 , 5 0 0 microgram per grain a-tocopherol with oxidized lard (peroxide value, 23.8) after heating at 210~ f o r 10 r a i n . i n v a c u o .

Tocopherol Before h e a t treatment b

After h e a t treatment b

,ag./g. l~g./g. 1 ........................................................ 662 661 1,052 1,050 2 ........................................................ 1,220 1,250 3 ........................................................ 4 ........................................................ 1,330 1,310 1,770 1,710 5 ........................................................ Treated with carbon black q- various concentrations of d-a-tocopherol. b 2 1 0 o 0 , in vacua, 10 min.

546

THE

TABLE

JOURNAL

OF T H E

AMERICAN

II

Effect of Different P e r o x i d e - R e m o v a l T r e a t m e n t s on Tocophero] Contents of F a t s Tocopherol Fats

Contro] not treated

Heat treatment a

Sulfuric acid

Cold

Hot

tcg./g.

Itg./g.

Itg./g.

p.g./g.

,q,g./g.

1,530

1,460

1,260

841

1,264

414

1,210

1,140

633

1,115

1,615

1,592

1,352

719

1,375

887

1,294

1,164

--

5OO

483

442

--

--

1,540

1,513

1,497

1,302

1,324

3O8

37.9

373

--

--

F r e s h soybean oil A ........................ Oxidized soybean oil A ........................ F r e s h soybean oil B ........................ Oxidized soybean oil B ........................ F r e s h l a r d plus d-a-tocopherol ......... Fresh lard plus d-a-toeopherol ......... Oxidized l a r d plus d-a-tocopherol ..........

Saponification

--

a 210oc., in vaeuo, 1 0 - 1 5 m m .

spectively. The tocopherol content of the fresh oil was compared with that of the oxidized oils before and after heating at 210~ for 10 rain. under vacuum. Tocopherol was determined in the oils directly and in their unsaponifiable matter, which was obtained by the hot saponification method. The data (Table I I I ) show that toeopherol is not regenerated in the oxidized oils by the heat treatment since this treatment had essentially no effect on the tocopherol concentration in their unsaponifiable matter. It is interesting to note the relatively small loss of tocopherol incurred in the oxidized oils which have been taken far beyond the induction period. The loss of tocopherol observed in different oxidized soybean oils at the end of the induction period was less than 10%. Peroxides were also effectively removed from oxidized oils by adsorption chromatography on silicic acid. The procedure used was to pass 100 ml. of a 10% solution of soybean oil in benzene through a column of sillcic acid (1.9 x 10 cm., 100 mesh) under nitrogen pressure to obtain a rate of approximately 3 ml. per minute. The column was then washed with 100 ml. of benzene. This procedure did not affect the tocopherol content of fresh soybean oil, and the toeopherol values in oxidized oils corresponded to those obtained with the heat treatment. In a representative experiment the concentration of tocopherol of an oxidized soybean oil sample (peroxide value ----22.1) was 1,412 micrograms per gram after passage through a silicic-acid column and 1,426 micrograms per gram after heating in vacuo at 210~ for 10 min. The agreement between the two methods is additional evidence that tocopherol is not regenerated in oxidized oils by the heat treatment. T A B L E III Effect of I-Ieat T r e a t m e n t on Tocopherol Contents of F r e s h a n d Oxidized Soybean Oil a n d T h e i r U n s a p o n i f i a b l e Matter Tocopherol inthe

Oil samples

F r e s h oil A, Oxidized oil Oxidized oil F r e s h oil B, Oxidized oil Oxidized oil

heated a ........................ A, b heated a ................. A, b not heated ............. heated ~................. :...... B, e heated 9 ................. B, e not heated .............

a 210oc., in vacuo, 10 min. b P e r o x i d e v a l u e ---- 60. c F e r o x i d e v a l u e ---- 72.

Oil

Unsaponifiable m a t t e r

~g./~.

~g./g.

1,530 1,320 314 1,583 1,495 465

1,264 1,043 1,115 1,375 1,239 1,273

OIL

CHEMISTS'

SOCIETY

VOL. 34

Ultraviolet spectrophotometric measurements were made on soybean oil and its unsaponifiable matter in petroleum ether and ethyl ether solutions, respectively. With the unsaponifiable matter prepared by the hot saponification method, the absorption at 298 mt~ gave values for tocopherol concentration of the same order of magnitude as the values obtained by the colorimetric method. With the oils, other substances interfered with the absorption of tocopherols. The unsaponifiable matter prepared by the cold saponification method of Lips (7) showed absorption at 258 m~ but not at 298 mr,. Since the absorption at 258 ln:~ would be due to tocopheryl-p-quinone (5), it would appear that the cold saponification method of Lips causes oxidation of tocopherol in soybean oil, thus explaining the low tocopherol values obtained by this method. The unsaponifiable matter obtained by hot saponification from oxidized soybean oil (pe r oxide value of 72) showed no absorption at 258 m~ before or after the heat treatment (210oc., in vacuo, 10 rain.). Therefore tocopheryl-p-quinone does not appear to form in soybean oil at this level of oxidation. This observation is consistent with the relatively small decrease in tocopherol observed in the oxidized soybean oils. Absorption measurements in the visible range showed an increase in general absorption between 400 and 500 m~ in oxidized soybean oil (peroxide value of 72), which may be attributed to browning. No absorption maximum was evident at 470 m~ which is characteristic of the red chroman-5,6-quinone. Therefore, unlike cottonseed oil, chroman-5,6-quinone does not appear to form in autoxidized soybean oil (12).

Summary Heating oxidized fats to 210~ under reduced pressure for 10 to 15 min. removed peroxides without a f f e c t i n g the tocopherol content. This simple heating method yielded a higher recovery of toeopherol in oxidized fats than other modifications of the EmmerieEngel method used to remove interfering substances. The data indicate that tocopherol is not regenerated in the oxidized oils by the heat treatment. The loss of tocopherol in soybean oil which had been oxidized beyond the induction period was relatively small.

Acknowledgment The authors wish to acknowledge the help of E. H. Melvin and Jean Mallan for the spectrophotometric analyses and their interpretation. REFERENCES 1. Baxter, J. G., Biol. Symposia, 12, 4 8 4 - 5 0 7 ( 1 9 4 7 ) . 2. E m m e r i e , A., a n d Engel, C., Rec. Tray. Chim., 57, 1 3 5 1 - 1 3 5 5 ( 1 9 3 8 ) ; 58, 2 8 3 - 2 8 9 ( 1 9 3 9 ) . 3. G u p t a , M. L. S., a n d Basu, U. P., J'. I n d i a n Chem. Soc., I n d . a n d News Ed., 17, 1 7 1 - 1 7 6 ( 1 9 5 4 ) . . Hove, E. L., a n d Hove, Z., J. Biol. Chem., 156, 6 0 1 - 6 1 0 ( 1 9 4 4 ) . 9 K a r r e r , P., a n d Geiger, A., Helv. Chim. Acta, 23, 4 5 5 - 4 5 9 (1940). 6. K a u n i t z , M., a n d B e a v e r , J. J., J. Biol. Chem., 156, 6 5 3 - 6 5 9 (1944). 7. Lips, H. J., J. Am. Oil Chemists' Soc., 33, 4 2 6 - 4 2 8 ( 1 9 5 6 ) . 8. Mattill, H . A., "The V i t a m i n s , " vol. 3, pp. 5 0 6 - 5 0 9 , New York, Academic P r e s s Inc., 1954. 9. P a r k e r , W. E., a n d M c F a r l a n e , W. n . , Can. J . l%esearch, B18,

405-409 (1940).

10. P r i v e t t , O. S., a n d Q u a c k e n b u s h , F. W., J . Am. Oil Chemists' Soc. 31, 2 8 1 - 2 8 3 ( 1 9 5 4 ) . 11. Stern, M. I-L, a n d B a x t e r , J. G., Analyt. Chem., 19, 9 0 2 - 9 0 5 (1947). 12. Swift, C. E. Mann, G. E., a n d Fisher, G. S., Oil and Soap, 21, 317-320 (1944). ' 13. Zscheile, F. P., Nash, M. A., H e n r y , R. L., a n d Green, L. F., I n d . Eng. Chem., Anal. Ed., 16, 8 3 - 8 5 ( 1 9 4 4 ) .

[Received May 13, 1957]