and many helpful suggestions given prior to the design and construction of our apparatus. LITERATURE CITED
(1) Farquhar,
J. W., Insull, William, Jr., Rosen, Paul, Stoffel, Wilhelm, Ahrens, E. H., Jr., Nutrition Revs. 17, 8 (Suppl.) (1960).
(2) Haahti, E., Nikkari, T., Kulonen, E., J . Chromatog. 3, 372 (l9Go). (3) Lipsky, s. R., Landowne, R. A.8 Ann* Rev. Biochem. 29, 649 (1960). Chromatog. 1, 35 (4) Lovelock, J. E..
(1958).
(5) Lovelock, J. E. “Argon Detectors,”
Third Gas Chromatography Sympo-
sium, 8-10 June 1960, edited by R. P. W.
Scott, Butterworths Scientific Publications, London. RECEIVEDfor review October 31, 1960. Accepted March 9, 1961. Work supported in part by Research Grant H-1882 (C5) from the National Heart Institute, Public Health Service, Bethesda, Md., and by the U. S. Atomic Energy Commission, Washington, D. C.
An Improved Spectrophotometric Method for the Determination of Tocopherols Using 4,7-Diphenyl-l ,I 0-Phenanthroline C. C. TSEN Grain Research Laboratory, Board of Grain Commissioners for Canada, Winnipeg 2, Man., Canada An improved method for the determination of tocopherols, based on the method of Emmerie and Engel, is proposed. 4,7 Diphenyl 1,lOphenanthroline (bathophenanthroline), selected from a group of the iron(l1) reagents, is recommended as the reagent in the place of 2,2’-bipyridine for the determination of tocopherols. It can increase the sensitivity of the original method by 2l/2-fold. The maximal color intensity for tocopherols can be attained in 15 seconds with bathophenanthroline compared with 4 minutes when 2,2’-bipyridine is used as the reagent. The faster rate of the color development can simplify the analytical work. Orthophosphoric acid is used to combine with the residual iron to form Fe(P04)2-3 and to prevent the photochemical reduction, thus facilitating the determination. By using bathophenanthroline or 2,4,6tripyridyl-s-triazine as the reagent, the results of measuring the absorbances at different concentrations of a-,P-, 7-, and 6-tocopherols conform to Beer’s law.
-
T
HE
CHEMICAL
-
DETERMINATION
Of
tocopherols in food or other materials is rather complicated. The general procedure includes: the extraction of the lipide containing tocopherols, the removal of interfering substances, and the determination of tocopherols. The Emmerie-Engel method (6) has been considered to be most suitable for the determination of purified tocopherols ( I , 6). The method depends on the subsequent determination of the amount of ferrous ions thus formed by the color reaction with 2,2’-bipyridine. Any iron(I1) reagent can be used in the place of 2,2‘-bipyridine if it is as sensitive and as specific as the bipyridine. I n recent years, a number of reagents have been reported to be more sensitive than 2,2‘-
bipyridine for the determination of iron(I1). The best known are 1 , l O phenanthroline (IO), 2,2’,2“-terpyridine (IO), o-nitrosoresorcinol monomethyl ether (NRME) ( l a ) , 4,7diphenyl-lJ10phenanthroline (bathophenanthroline) (9),and 2,4,6-tripyridyl-s-triazine (TPTZ) (3). By substituting one of these reagents for 2,2’-bipyridine, the sensitivity and precision of the EmmerieEngel method could be improved. Under standardized conditions, the Emmerie-Engel method gives a high degree of precision with pure tocopherols. However, it suffers from the following drawback: ferric chloride is readily converted to ferrous state photochemically as well as by tocopherols (1,d). Unless every precaution is taken to exclude light, exaggerated values may result. An attempt was also made in the present study to select a suitable reagent that would combine with the residual ferric ions to prevent the photochemical reduction. REAGENTS
All reagents were of analytical grade. 2,2’,2”-Terpyridine, bathophenanthroline, and TPTZ were purchased from the G. Frederick Smith Chemical Co.; 2,2’-bipyridine and 1,10-phenanthroline from the British Drug Houses, Ltd.; D,OL- tocopherol, D,B - tocopherol, D,Ytocopherol, and D,G-tocopherol from the Distillation Products Industries, Division of Eastman Kodak Co.; DL, a-tocopherol from the Nutritional Biochemicals Co. NRME was synthesized and purified according to the method of Tetsuya (IS). Ethanol (absolute alcohol) was purified by distilling from 0,02’% by weight of potassium permanganate and potassium hydroxide. Iron Reagents (6.0 X 10-3M). All iron reagents except NRME were dissolved in ethyl alcohol a t the concentration of 6.0 X 10-3M. The solutions were then stored in amber bottles in a refrigerator.
Ferric Chloride ’ Solution (1 X lO-3M). The ethanolic solution was prepared fresh before use. To avoid photochemical reduction, the solution was prepared directly in an amber bottle. Standard Tocopherol Solution. 0.5% (w./v.) tocopherol was prepared with ethyl alcohol. It was refrigerated in an amber bottle. The standard solution was prepared by the appropriate dilution of the solution to the desired concentration with ethyl alcohol. Orthophosphoric acid solution (4 X 10-2M) was prepared in ethyl alcohol. Saturated NRME solution was prepared ’by saturating redistilled water with NRME. The solution was filtered before use. Sodium Acetate Buffer (pH 3.78). 42.5 ml. of IN HC1 was added to 50 ml. of 1N sodium acetate. GENERAL PROCEDURES
A desired amount of the standard tocopherol solution was pipetted into a 10-ml. amber bottle. The iron reagent (0.5 ml. of 6.0 X 10-3M) was added and the volume was made up to 4 ml. with ethyl alcohol. The bottle was gently swirled for a few seconds. Immediately after the mixing of 0.5 ml. of 1 x 1 0 - ~ J 4 ferric chloride solution with the above solution, the reaction was timed with a stop watch. At the end of a selected period, 0.5 ml. of 4 X l O - * X phosphoric acid solution was introduced. The absorbance of the solution was measured with a Beckman Model DU spectrophotometer a t the wave length selected for each iron reagent against a blank. The blank was prepared in the same manner, except that ethyl alcohol was used in place of the standard tocopherol solution. The amount of the tocopherol was expressed in micrograms per milliliter based on the volume of the final mixture. A different procedure was used with the NRME reagent: 1 ml. of a desired concentration of the standard tocopherol solution was pipetted into a 10-ml. VOL. 33, NO. 7, JUNE 1961
849
,150
TRlPY R lDYL - S- TRlAZ INE
WAVE LENGTH,rnp.
Figure 1. A.
B.
Absorption spectra
Fe(bothophenonthroline)a+* Fe(TPTZ)Z+2
t
.450
amber bottle followed by 1 ml. of 1 x 10-3M ferric chloride solution. The contents were then thoroughly mixed with 4 ml. each of the sodium acetate buffer and the saturated NRME solution. Thereafter the procedure of Tetsuya ( l a ) was followed. During the early stage of the development of this improved method, all operations, unless otherwise stated, were carried out in a laboratory darkened by drawing the window shades and turning off artificial lights. The results reported are averages of duplicate determinations. EXPERIMENTAL
Selection of Iron Reagents. The sensitivities of the iron(I1) reagents were compared by determining their absorptivities according to the general procedure when D, a-tocopherol was used. Results in Table I clearly show t h a t all reagents tested are more sensitive than 2,2'-bipyridine. Among these reagents bathophenanthroline and TPTZ were 2.56 and 2.48 times as sensitive as the reagent used in the original method. The same absorptivities of the iron reagents were obtained when DL, a-tocopherol was used instead of D, a-tocopherol. Figure 1 presents the absorption spectra of the iron(I1)-bathophenanthroline derivative and of the iron(I1)-TPTZ derivative in the presence of the oxidized tocopherol and phosphoric acid in ethyl alcohol. The final concentration of D,
.150
t
BATH0 PHENANTHROLINE.
2
Figure 2. Q
A
ANALYTICAL CHEMISTRY
8
10
a-tocopherol in each solution was 2.8 fig. per ml. Almost identical spectra with the same maximal peak region were obtained for D,B-tocopherol, D,y-tocopherol and D,G-tocopherol when bathophenanrethroline : 3 TPTZ were used agents. Rate of, Color Development. Since bathophenanthroline and T P T Z were more sensitive, a further study was made t o measure the rates of color development for the determination of each tocopherol, as compared with 2,2'-bipyridine. The tocopherols used were a-, p-, y-, and &tocopherols. The general procedure was followed. However, the phosphoric acid solution was added a t specified times after the addition of the ferric chloride solution in the amber bottle in a darkened laboratory as indicated in Figure 2. The results show that the maximal color intensity for tocopherols can be attained in 15seconds, 60 seconds, and 4 minutes, n -
~
1050 (534mp)
2.56
Rote of color development
D,a-Tocopherol D,y-Tocopherol
Absorptivities of Different Iron Reagents Used for D,a-Tocopherol Determination Iron Reagent E;%% Sensitivity 2,2'-Bipyridine 407 (520 mp) 1.00 1,10-Phenanthroline 511 (508 mp) 1.26 2,2',2"-Terpyridine 584 (552 mp) 1.43 o-Nitrosoresorcinol monomethyl ether 880 (700 mp) 2.16 2,4,6-Tripyridyl-s-triazine(TPTZ) 1010 (593 mp) 2.48
850
6
TIME, min.
Table 1.
Bathophenanthroline
4
0
D,B-Tocopherol
A D,G-Tocopherol
when bathophenanthroline, TPTZ, and 2,2'-bipyridine are used as reagents, respectively. The faster rate of color development by using bathophenanthroline or TPTZ can simplify the analytical work. However, the intensity for 6tocopherol increages slightly as the reaction period is prolonged; this is especially so with 2,2'-bipyridine (Figure 2 ) . The rate of color development obviously depends on the rate of the reduction of ferric iron by each tocopherol and on the rate of combination between ferrous iron and the iron reagent in ethyl alcohol. Figure 2 presents the data showing that with the same iron reagent the rates of the color development are slightly different for each tocopherol. This illustrates that the rate of the reduction changes with different tocopherols. The result is in accord with the findings of others ( 4 , 7 , 1 1 ) . It also agrees with the expectation that the extent, and the rate of the reduction of ferric iron by each tocopherol will be different because of a structural variation both in the number and in the position of the methyl group in tocol among the tocopherols (1). With the same tocopherol, the difference in the rates of the color development for the three reagents, as shown in Figure 2, could then be attributed to their different rates of combining with ferrous iron. I t appears that the fast rate of the combination favors the reduction toward
the completion and results in the faster color development. Prevention of Photochemical Reduction of Ferric Ions. T h e EmmerieEngel method is based on the qiiantitative reduction of ferric ions by the tocopherols, and the excess ferric ions remain after the completion of the reduction. The photochemical reduction of these residual ferric ions takes place readily. Because of this reduction, i t has been suggested t h a t opaque flasks be used in the determination (6). However, some reduction is still likely t o occur during the transfer of the solution to spectrophotometric cells. It has also been proposed that part of the determination should be done under dim artificial light in a darkened room (1). Besides excluding light to prevent the photochemical reduction, an alternative would be t o inactivate the rcsidual iron by combining it with a suitable reagent. Schulek and F16derer observed that the addition of orthophosphoric acid could stabilize the ratio of ferrous to ferric ion for the determination of ferrous iron by the 2,2’bipyridine method ( 8 ) . I n addition t o orthophosphoric acid, the following reagents were tested: 8-quinolinol, (ethylencdinitri1o)tetraacetic acid, citric acid, metaphosphoric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, and acetic acid. However, orthophosphoric acid was found to be the most suitable for combining with the residual iron to prevent the photochemical reduction in the determination of tocopherols. Identical absorbanccs were obtained for the two sets of solutions (2, 4, and 6 pg. of D,a-tocopherol per ml.) with the addition of 0.5 ml. of 4 X 10-2hf phosphoric acid. One set of solutions was storcd in transparent glass bottles and exposrd t o the light condition of a n ordinary laboratory; the other sct was storcd in amber bottles in a darkened laboratory. Thc time of storage was 60 minutes. The general procedure was followed, and 2,2’-bipyridine or bathophenanthroline was used as the iron reagcnt. The order of the addition of these reagents is very important. When phosphoric arid solution was introduccd beforc the addition of the ferric chloride solution, no color was produced. To ascertain the form of combination of ferric ions and phosphate ions, the following study was made. One half milliliter of 6 X 10-SM bathophenanthroline and 0.5 ml. of 1 X I O - S M FeCIs were mixed with 3.5 ml. of ethyl alcohol in a transparent glass flask. One half milliliter of the phosphoric acid solution, at the various concentration levels as indicated in Figure 3, was addcd to the mixture and allowed to stand for 1 hour undrr the light condition of an ordinary l:ihora-
Table II. Calibration Data Using bathophenanthroline as reagent for determination of tocopherols ConcenAbsorbance, 534 mp tration D,a-‘rocophcrol D,@-Tocopherol qy-Tocopherol D,G-Tocopherd M./M1. 1 2 3 4 5 6 7 8
0.116 0.232 0.350 0.464 0.570 0.690 0.810 0.920 Using TPTZ as reagent for determination of tocopherols Absorbance, 593 mp
1
2 3 4 6 8
0.105 0.210 0.315 0.420 0.526 0.632 0.735 0.840
0.107 0.214 0.322 0.428 0.535 0.643 0.749 0.857
0.101 0.201 0.303 0.402 0.600 0.802
0.102 0.203 0.304 0.407 0.605 0.809
tory. The absorbance was then measured. Typical results are plotted on semilogarithmic paper, as shown in Figure 3. These results indicate that the ferric ions combine with the phosphate ions t o form Fe(PO&-s, as the absorbance curve reaches its minimum level at ratio of 1 to 2 for ferric and phosphate ions. Determination of Tocopherols with The the Recommended Method. details of the recommended method are the same as those described in the present paper under general procedure. The time for color development-i.e., the time between the additions of the ferric chloride solution and the phosphoric acid solution-can now be specified t o be 15 seconds for bathophenanthroline and 60 seconds for TPTZ. The changes in absorbance due to the photochemical reduction during this short time for color development were 0.001 to 0.005. For most work, the determination need not be carried out in a darkened room. Different concentrations (1 to 8 pg. per ml.) of each tocopherol were determined with bathophenanthroline or T P T Z as the reagent according to the recommended method. The results in Table I1 show that the color developed with these two reagents follows Beer’s
05
a
1 H,PO,.
LO M
so
LOO
.Id’
Figure 3. Effect of orthophosphoric acid concentration on photochemical reduction of ferric ions
0.120 0.240 0.360 0.480 0.600 0.720 0.840 0.958
0.104 0,209 0.312 0.416 0.630 0.838
0.111 0.220 0.330 0.438 0.655 0.875
law. Although different tocopherols do not result in the same absorbances on a weight or molar basis, the values in Table I1 are much closer than those that can be obtained from the EmmrrieEngel method. ACKNOWLEDGMENT
The author thanks Isydore Hlynka and J. A. Anderson for their advice throughout the work and for their assistance in the preparation of the manuscript. Part of the work was done at the University of Alberta during the author’s tenure of a Postdoctorate Fellowship from the National Research Council of Canada. The technical assistance of R. A. Stein and the advice of H. B, Collier arc gratefully acknowledged. LITERATURE CITED
(1) Analytical Methods Coiiimittee, Apalust 84. 356 (1950). (2) Baxter, J . G., Biol. Symposia 12, 484 (1947). (3) Collins, P. F., Diehl, H.,Smit!l, ci. F., ANAL.CHEM.31, 1862 (1859). (4) Eggitt, P. W. R., Norris, F. W., J . Sci. Food Agr. 7, 493 (1956). (5) Emmerie, A,, Engel, C., Rec. Irav. chim. 58, 283 ( I 939). (6) Lehman, It. “Methods of Biochemictd AnalysiR,” Vol. 11, pp. 153-87, Interscicnce, Ncw York, 1955. ( 7 ) Quaife, M. J,., Uarrih, P. L., ANAL. CHEM.20, 1221 (1!)48). (8) Schulck, I(;.. Y16dcrer, I., 2. anal. Chem. 117, 170 (1!33!3). (9) Smith, G. I?., McCurdy, W. H., Jr., Diehl, €I., Analyst 77, 418 (1952). (10) Snell, F. I)., Snell, C. T., “Colorimet,ric Methods of Analysis,” Vol. 11, p. 316, Van Kofltrnnd, Ncw York, 1949. (11) Stern, M. H., Bnxter, J. G., ANAL. CHEM.19, 902 (1947). (12) ‘l’etsuya, T., J. Chem. Soc. Japan, Pure Chem. Sect. 76,336 (1955). (13) Tetmya, T., Ibid., 76, 328 (1955).
W.,
RECEIVUI, for review October 24, 1960. Acceptcd March I , 1961. Paper No. 195 of the Grain Rcwnrrh Laborntory Board of Grain CommiwionrrR for danada, Winnipeg, Man., Canada. VOL. 33, NO. 7, JUNE 1961
851
