Determination of Sesamol, Sesamolin, and Sesamin in Sesamin Concentrates and Oils CARLOS SCAREZ C., R. T. O'CONNOR, E. T. FIELD, AND W. G. BICKFOKD
Southern Regional Research Laboratory, Set0 Orleans 19, La.
A preiiouslj described method for the determination of free and bound sesamol and sesamin i n sesame oil was found to be inapplicable in the analysis of sesamin concentrates, because of the l o w solubilitj of the sesamin concentrate in isooctane and the presence of an iron-containing substance in the sesamin concentrate. The method was modified by using a mixed solbent (chloroformiso-octane) to dissol\e the sesamin concentrate and by centrifugation in the phasic separation of free and bound sesamol to remoie substances t h a t interfere with normal color detelopment in the \ illatecchia reaction. The accurac? of the spectrophoto-
metric and photocolorimetric procedures was established by analyzing sesamin concentrates and sesame oils to which known amounts of pure sesamol and sesamin had been added. Analytical data are reported for the free and bound sesamol contents of five samples of commercial sesamin concentrates and five sesame oils. The spectrophotometric method was also applied to the determination of sesamin in these samples. The improved method not only permits accurate determination of free and bound sesamol in sesamin concentrates, but is preferred for the analysis of crude sesame oil because it eliminates interfering substances.
S
in 100 nil. of ethyl alcohol; and potassium hydroxide solution prepared hy dissolving 10 grams of reagent grade pellets in 80 ml. of distilled water and adding 20 ml. of ethyl alcohol (99%). A slightlJ- yellow color in the furfural solution is not objectionable. The solution is stahle if stored in a refrigerator. T h r following apparatus is required: a Beckman spectrophotometer. Model DU, and I-cni. absorption cells, Evelyn colorimeter, ~ncchanicalshaker, and centrifuge.
ES.\hIOL occurs free and in bound form (sesamolin) in sesame oil. I n the form of sesamolin, sesaniol is combined n-ith an unidentified substance, samin (Cl3HI4Os),from which it, is liberated by acid hydrolJ-sis and hydroyeno11 (5). In addition to sesamol (C€I?O~CJI,OH)and sesamolin (CH,O,C,H,OC,,Il,,O,), sesame oil contains sesaniin (CZOH1S06), 71-hich has as part of its stmc.ture the sanie meth!-lrnediox!?,henyl radical as the other two nieinbers of this group. Recently, these substances have assumed considerable practical importance by virtue of their specific chemical artivities (2). Sesaniol, for esample, possesses marked antioxidant activity in lard and various vegetable oils ( 1 . 8, 9 ) and sesamin exerts similarly marked synergistic activity with pyrethrin insecticides (9). Concentratrs of sesainin are n o x produced commercially for use in the nianufacture of such insecticides. These concentrates contain, besides sesamin, variable amounts of sesaniol and sesaniolin. Until the present,, no satisfactory method has been available for the quantitative determination of the relative aniounts of each of these subetancrs in commercial preparations. h quantitative method, based on a modification of the Villavecchia test, \vas described by Budowski et al. (4)for the determination of free and bound sesaniol (sesamolin) in sesame oil. When applied t o the analysis of sesamin concentrates, the method proved unsatisfactory because the concentrates were not completely soluble in iso-octane (2,2,4-triniet,hylpentane) and an unknown substance was found to be present which interfered with the development of the color in the Villavecchia test. Gravenhorst (6) and Honig ( 7 ) have reported t'hat rancid sesame oils contain cert,ain alkali-soluble substances which interfere with the Tillavecchia test. Spectrophotometric and photocolorimetric procedures which overcome the foregoing difficulties have been developed and are described herein. The determination of sesamin is carried out in conjunction with the determinat,ion of free and bound sesamol in a manner ent,irely analogous to that previously reported ( 5 ) . The modified method is applicable to sesame oils as well as to concentrates, and is particularly rerommended for the analysis of crude oils.
SPECTROPHOTOMETRIC METHOD
-1wriyhed portion of sesaniin concentrate or sesame oil is dissolved in chloroforni-iso-octane (1 to 4) and made up to 100 ml. with t h e same solvent. This solution serves for t,he determination of free and bound sesltniol. For sesame oil a concentrat'ion of approximately 100 grams per litcr is rerommended, hut for sesamin concentrates 2.5 grams per liter is preferahle. These concentrations are hereafter referred to as the concentration of the original solution, GO. If the material dissolves uith ciiFicultj- in the mixed solvent. it should be dissolved in chloroform and then diluted with isooctanp t o produce a final chloiofoim-iso-octane ratio of 1 t o 4. Determination of Free Sesamol. A 50-ml. portion of the original solution is pipetted into a 250 ml. centrifuge bottle and 10 nil. of potassium hydroxide reagent are added. The bottle is capped and shaken vigorously for 3 minutes, and the mixture is centrifuged at 2000 r.p.m. foi 10 minutes to separate the alkaline layer from the chloroform-iso-octane layer. The loxer alkaline layer is filtered and used foi the determination of free sesamol.
Y 3
2 J
REAGENTS AND APPARATUS MILLIGRAMS
The following reagents are required for the quant,itative determination of free and bound sesamol: optically pure iso-octane; optically pure chloroform; optically pure ethyl alcohol (99%); sulfuric acid, specific gravity 1.37 at 15' C.; 2% solution of furfural prepared by dissolving 2 grams of freshly redistilled furfural
OF
SESAMOL
Figure 1. Calibration Curve for Quantitative Determination of Sesamol A.
B.
668
Beokman quartz spectrophotometer E v e l ~ ncolorimeter
V O L U M E 2 4 , N O . 4, A P R I L 1 9 5 2
669
The residual chloroform-iswxtane layer is reserved for the determination of bound sesamol. A 50-ml. volumetric flaskis filled t.0 the mark uith the sulfuric acid reagent and 1 ml. of the furfural reagent is added, followed by 0.6 ml. of the alkaline extract. Only a small amount of the alkaline solution is used, so t h a t the iniensity of the developed color will not be affected by slight changes in the concentration of the sulfuric scid. The flask ~. is .stonnnrrd n d invert.wl ~ ~ s~.~~.~... ~ . . ~ SPVWSI . ~ times to mix the contents. A 1-em. absarntion cell is filled with a portion of t h e colored solution and the op&ca,l density is moasured at 518 mp within 50 to 75 minutes after mixing, using a blank prepared under similar conditions with ethyl alcohol instead of furfural reagent.
T h e percentage of hound sesamol in t h e sample is calculated using Equation 1, where S is now the percentage of bound sesamol in the original sample. If the ahove procedure for hound sesamol has been followed exactly, the concentration can again be expressed in terms of the s:~mplein the original solution in grams per liter:
C
~~~
=
2Co/51 = 0.03922 Go
(4)
where C and Cohave the same meanings as before. Equation 1 for bound sesamol can then be simplified as follows:
S = 15.9375 D / C 0
(5)
-
Figure 2.
Emission Spectra of Red Gelatinous Substance ( A and C) Showing Preponderance of Iron Lines ( B Iron)
When determinations are made with t h e Beokman spectrophotometer a t 518 mp the extinction coefficient, 160, is used in the Beer-Lambert equation:
S = (100 D/Cl)/160 (1) where S is the ercentage of free sesamol in the original sample, D is the observe$ optical density, C i s the concentration (in grams per liter) of t,he original sample iu t h e final aliquot a t time of reading, and 1 is the cell length in centimeters.
If an instrument other than the Beckman spectrophotometer is used, it will again be necessary t.o canstmct a calibration curve. The bound sesamol may be expressed as sesamolin by multiplying its value by 2.68, the ratio of the molecular weights of sesamolin to sesamol. Determination of Sesamin. The procedure for the determination of sesamin is essentially that previously. described (6), but modified by the use of the mired chloroform-iso-octane solvent.
The extinction coefficient, 160, was obtained by measuring the slope of a curve ( A , Figure 1) oonstructed from measurements of the optical densities of the red color developed u,ith a series of different concentrations of pure sesamol in the chlorafarm-isooctane solution. The straight line whioh was obtained verified the applicability of the Beer-Lambert law to this color System. Equation 1 may not hold if an instrument other than the Beck, man spectrophotometer is used. I n such a case a calibration curve for the instrument used must be constructed. If solutions and aliquots are prepared exactly &E outlined in the procedure ahove, the concentration, C, may be expressed in terms of the sample in the original solution (in grams per liter), C,, as follows:
T h e alkali-treated chloroform-iso-octane solution used far the determination of bound sesainol is diluted, if necessary, with thc mired solvent (chloroform-is-octane 1to 4) and measured in the ultraviolet region with the Beckmsn spectropbot.ometer against a, blank of the mixed solvents in the I-em. cells a t 287 to 288, 255, and 320 mp. I n practice, t h e extinction coefficients are determined for the actual maximum and minimum (at about 287 to 288 and 255 mp, respectively) and the third wave length, a t about 320 mp, is then selected so that the measurement is made a t t h e same distance ahove t h e maximum a s the minimum is helow this maximum. Extinction coefficient curve8 throughout the ultraviolet region of the spectra have been published (4, 6). The percentage of sesamin in the sample is obtained from the simplified equation given by Budowski et a?. (5):
C = Co X 50 X 0.6/10 X 1/51.6 = 0.05814 Co
where C, equals the percentage concentration of sesamolin and the K’s are the observed extinction coefficients a t the indicated wave lengths. This equation is valid because the extinction coefficients a t the wave lengths of maximum and minimum absorption of pure sesamin and sesamolin in chloroform-iso-octane solution are identical with those previously reported (6) for these substances in pure iso-octane. These values are 21.75 and 1.54 lor the extinction coefficients of pure sesamolin a t 288 and 255 mp, respectively; and 23.03 and 2.02 for the corresponding values for sesamin. T h e extinetion coefficients a t 320 mp for both pure sesamolin and pure sesamin are essentially Zero (5).
(2)
and Equation 1 may be simplified:
S
= 10.750
D/Co
(3)
Where m y changes in the procedure are required, the percentage of free sesamol must be calculated from Equation 1. Determination of Bound Sesamol. The residual (alkalitreated) chloroform-isa-octane solution, which was separated as described under the determination of free sesamol, is filtered to remove any turbidity and the bound sesamol is determined as follows: A 50-ml. portion of the sulfuric acid reagent is pipetted into a 125-ml. glass-stoppered Erlenmeyer h s k . A 1-ml. portion of furfural reagent is added, followed by 2 ml. of the residual (alkalitreated) chloroiorm-iso-oetane solution. T h e flask is tightly stoppered and shaken on a mechanioal shaker for 30 minutes. The contents of the flask are then poured into a 100-ml. separatory funnel, and the layers are allowed to separate. T h e acid layer may initially be slightlyturhid, but it will clear up gradually, and a t the time the color is measured no significant turbidity should be observed. A 1-cm. absorption cell is filled with a portion of the colored acid layer and its optical density a t 518 m9 is read between 50 and 75 minutes after shaking was started, using a blank obtained under similar conditions with 1 ml. of ethyl alcohol instead of furfural reagent. A rea ent blank consisting of sulfuric acid and furfural need not be use$, as it has no appreciable absorption when measured against pure ethyl alcohol or water at 518 rnn,
% scsamin
=
4.541 KS,,- 0.953 CI- 2.271 ( I L
+ &o)
(6)
PHOTOCOLORIMETRIC PROCEDURE
Free and bound sesamol may also be determined in sesamin concentrates and sesame oils by the use of a photoelectric eolorimeter. An Evelyn colorimeter, equipped with Evelyn filter No. 515, and 7 X ’/e inch round-bottomed test tubes were used. Solutions of the analytical samples and the blanks are prepared exactly as described for the spectrophotometric procedure. ,The per cent transmittance of a solution of the analytical sample is read against the blank, the Evelyn L value computed, and the concentrat.ion of sesamol read from the calibration curve (B, Figure 1). I n the present instance the calibration curve was constructed from computed L values for t h e observed transmittances for a series of solutions of different concentrations of ~ u r sesamol e in chloroform-iso-octane. The percentage of sesamol (free or bound) in the oil or in the sesamin concentrate is obtsined by use of t h e general formula:
S = ( m / W ) 100
(7)
A N A L Y T I C A L CHEMISTRY
670 Table I. Material Commercial concentrate A" (1947)
B (1950)
Added, mg./100 6. 0.0 3046,6 5017.6 0.0 3013.2 5022.0
Spectrophotometric Method Found, Recovered, Recovered, mg./100 g. mg./100 g . % 0.0 0.0 34.2 97.7 3009.9 2975.7 94.8 4845.8 4811.6 0.0 I52 0 0.0 2787 0 92.6 2939.0 4729.4 4577.4 91.1
Sesame oils 0.0 Refined, 201.1 bleached 402. '2 Refined, 0.0 bleached, 50.1 desesami100.3 nized a Letters refer to source of sesaniin
Table 11.
by the change in solvent. Pure sesamin, melting point Colorimetric Method . 122.5' C %as obtained b>Found, Recovered. Recovered, recrystallizing the commermg./lOO g. mg./100 6 . % cial pentaneinsoluble fraction ... of sesamin concentrate from , . 95% ethyl alcohol. 0 . 0 158.9 0.0 A sample of pure sesamol, 91.3 2908.8 2749.9 90.9 4723.8 4564.9 melting point 64.4' C., synthesized by oxidizing piper84.9 0.0 0.0 195.6 97 2 280,s onal with commercial per366.8 91.2 451.7 acetic acid ( 1 ) and purified 5.9 0.0 0.0 by crystallization and subli.55.9 50.0 99 8 105.3 99.4 99.1 mation, was used in preparing t h e calibration curves shown in Fimre 1. This sam~__ ple was apparently somewhat purer than the sesamol previously used ( 6 ) , as indicated by its higher melting point and more intense color developmpnt in the Villavecrhia test. Application of the procedure ( 4 ) for determination of free, bound, and total sesamol in sesamin concent,rates dissolved in chloroform-iso-octane gave lower values for total sesaniol than for free plus bound sesamol. This anomaly was found to be caused by the presence of a substance interfering with the develop ment of the red color in the Villavecchia test. The interfering substance was also observed in very small amounts in crude sesame oils, but if present a t all in refined or in desesaminised sesame oils, it was inappreciable. This interfering substance, possibly the same as that described in the literature (6, 7 ) ,was not completely identified. It occurred as a gelatinous red precipitate a t the interface during the phasic separation of free and bound sesamol. This red precipitate was shown by emission spectra analysis to consist mainly of iron, with traces of magnesium and sodium. The emission spectra are shown in Figure 2. By adding the isolated precipitate to a sesamol solution, it, n-as demonstrated that i t partially inhibited the \?llavecchia color reaction. During the separation of free and bound sevamol by treatment of the chloroform-iso-octane solution of the sample of sesaniin concentrate or sesame oil with alkali, the interfering subst'ance could be removed by centrifugation, which also served to destroy any emulsions that had formed. .is inclusion of alkali treatment and centrifugation is necessary for the removal of this substance, the direct determination of total sesamol in either sesamin concentrates or crude sesame oils may be subject to error; and i t is preferable, therefore, t,o calculate the total sesamol from the determination of free and bound sesamol by t.he procedures herein described.
Recovery of Sesamol Added to Sesamin Concentrates and Sesame Oils
84.7 280.3 466.4 5.9 56.6 104.5
0.0 195.6 381.7
0 0 97.3 94.9
0.0 50.:
0.0 ioi.2 98.3
98.6
Commercial concentrates A a (1949) A-1 (1950)
sesame oils Refined, bleached, desesaminized Indian, crude
Added. nig.:100 g.
Spectrophotometric Method Found, Recovered, Recuyered, ing./lOO g. nig./lOO g. 4x
0 0
14,560 26,208 37.815
11,648 23,255
0.0 2,984 5,968
12,567 15,415 18,329
2,848 5,762
11,969 23,937
0.0
0 0
97.3 97.2
0.0
0.0 95.4 96 6
0.0
139.6 324.9 523.3
0.0 185 3 383.7
0.0 92.8 96.1
0.0 199.7 399.4
898.8 1,091.4 1,280.9
0.0 192.6 382.1
0.0 96.4 95.7
;!",I
Letters refer to source of sesamin concentrates,
where S is the percentage of free or bound sesainol in the original sample, rn is the weight of sesamol in milligrams obtained from the measured L value p-ith the aid of the calibration curve, and W is the total number of milligrams of oil or sesamin concentrate in the aliquot of the color solution measured.
If the procedure is follon ed exactly as described, thc following simplified equations for free and bound sesamol can be used:
yofree sesamol = 0.1728 ( L value) 0.0296 CO % bound sesamol = 0.1728 ( L valu~)/O0200 CO
(8) (9)
where the factor 0.1728 is the reciprocal of the slope of the calibration curve and COis the concentration of the original solution. DISCUSSION
~
-
concentrate.
Recovery of Sesamin Added t o Sesamin Concentrates and Sesame Oils
Material
-
The procedures of Budom ski et nl. ( 4 , 6 )for the determination of sesamin and of free and bound sesanlol could not be applied to commercial sesamin conTable 111. Sesamol, Sesamolin, and Sesamin in Sesarnin Concentrates and Sesame Oils centrates because the latter Spectrophotometric Method Colorimetric Method \\ere insoluble in iso-octane. Sesamolin, Sesamolin, It u a s found that a mixSesamol,Bound % bound x Sesamol, % bound X Sesamin, Free 2.68. X ture of 1 part of chloroform Material Free Bound 2.68. R % and 4 parts of iso-octane was Coniinercial concentrates Aa (1947) 0.04 1.01 2.71 8.43 ... 0.95 2.53 an efficient solvent for the 0.08 2.03 5.44 15.05 ... 1.99 5.33 A (1949) A-1 (19.50) 0.23 1.16 3.10 12.16 0.22 1.14 3.0.5 commercial concentrates, Use 6.29 19.23 0.10 2.29 6.14 4 - 2 (1950) 0.08 2.30 of this mixed solvent necB (1950) 0 15 0.90 2.41 5.13 0.17 0.87 2.33 essitated redetermination of Sesame oils Crude 0.001 0.12 0.33 0.77 0.002 0.12 0.32 the extinction coefficients of Refined bleached 0.08 0.002 0.004 0.82 0.08 0.002 0.004 sesaniolin and sesamin in the Refined: bleached, desesaminized 0,006 0,0002 O.OOO5 0.14 0.006 0.0006 0.W2 chloroform-iso-octane solvent. Indian, crude 0.002 0.13 0.34 0.86 0.002 0.12 0.32 Colombian, crude 0.0003 0.13 0.35 1.06 ... .. ... However, the values for the evtinction coefficients of these a Letters refer t o sources of sesamin concentrates. compounde were unaffected
V O L U M E 2 4 , N O . 4, A P R I L 1 9 5 2
67 1
RESULTS
The accuracy of the methods was verified by determining the sesaniol and sesamiri constants of sesaniin concentrates and sesame oils before and after the addition of known amounts of these substances. The results of the recovery tests with added sesamol as determined by the spectrophotonietric and photocolorimetric procedures are shon.11 iri Table I, while those for added sesamin as determined spectrophotonietrically are shown in Table 11. I t is seen from these tables that the recoveries of added sesaniol and sesaniin in sesamin concentrates and sesanie oil vary from 90 to 10lyo,which may be considered satisfactory. The spectrophotometric and photocolorimetric procedures were applied to a series of commercial sesamiri concentrates and t o sesame oils, as indicated in Table 111. The sesamin concentrates ranged from 0.04 to 0.23% free sesamol, 0.90 to 2.35% bound sesamol, and 5.13 to 19.23% sesaniin, respertivcly, n-hereas the oils ranged from 0.0003 to 0.08% free
seeaniol, 0.0002 to 0.13% bound seeamol, and 0.14 t o 1.06% sesaniin. LITERATURE CITED
( I ) Budowski, P., J . Am. Oil Chemists’ Soc.. 27, 264-7 (1950). (2) Budowski, P., and Markley, K. S., Chcm. Rei).,48, 125-51 (1951). (3) Budowski, P., Meneees, F. G. T., arid Dollear, F. G., J . Am. Oil Chemists’ Soc., 27, 377-80 (1950). (1) Budowski, P., O’Connor, R. T . , and Field, E. T., I h i d . , 27,307-10 (19501. --, ~~
(5) (6) (7) (8)
Ihid., 28, 51-4 (1951). Gravenhorst, C . O., I n d . Eng. Chon., 16, 47-8 (1924).
Honig, P., Chena. Weekhlad, 22, 509-12 (1925). 3Iooi.e. R.S . ,and Bickford, K. G., J . L 4 7 ~Oil ~ . Chemists’ Soc., 29, 1-4 (1952). 1%. S., and Mattill, H. .i..(.’hem Rev., 29, 257-6s (1941).
(!>) Olcott,
R~~CF:IVE for : D review October I?, 1951. Accepted J a n u a r y 17, 1932. Presented a t the 25th fall meeting oi the American Oil Chemists’ Society, Chicago, Ill., October 8 t o 10. 1951. S o . 9 of a series of communications on sesame oil. Instriiments are named as part of t h e exact experimental conditions. This does not constitiite a recommendation oi t h e Department of A g r i c u l t i i r e of these instrunients over those uf a n y other manufacturer.
The Dead-Stop End Point I-ticreduction at the cathode. An>-titration w-hich can be arranged so that a change from an electrolytic redox couple to onlr one process or vice yersu orcurs at the equivalence point can be adapted to a dead-stop end point.
F
(4)origiiially proposed the dead-atop method using tivo platinum electrodes and a small applied potential. The end point in a titration !vas shown by either the disappearance or appearance of a current flowing between the electrodes. The phenomenon was explained by assuming that hydrogen aiid oxygrn xere adporbed on the surface of the cathodicali7. and anodically charged electrodes and the removal of one of t h r w changed the system. Bottger and Forche ( 1 ) suggested that the 1,Fniv. applied potential used by Foulk and Baxden was not sufficient for hydrogen formation on the cathode, and showed that when the iodine-thiosulfate reaction ivas rarried out in divided cells, the electrode potential varied tyith the concentration. Delahay ( 2 )reported that the “dead-stop” end point depend8 011 the change from a reversible redos couple to an irreversihlr redox couple or vice versa at the equivalence point. These observations suggest that thc dead-stop phenomenon is not a polarization effect based on gas adsorption on the electrode surface, but at least in part an electrochemical phenomenon based on oxidation a t the anode and reduction at the cathode. If this is so, then hy applying suitable potentials and using appropriate electrodes any redox titration may be followed using the deadstop method. In order to test this theory, dead-stop titrations were carried out with various sJ-stenis, and electrode potentials and currents !yere measured. OULK and Bawden
0.3
- 0 I(0
0,’s
O(5
0.;
IlO
~
ML. I, Figure 1. 0.1
Electrode Potential and Current between Electrodes N thiosulfate titrated with 0.1 N potassium iodideiodine with 50
mv.
applied
EXPERI;\I E S T A L
Apparatus. A systein such as that described by Kernimont and Hopkinson (8) is convenient for applying the potential t o the electrodes and measuring the current which flows. A Fisher
