I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY PUBLISHED BY THE A M E R I C A N C H E M I C A L S O C I E T Y W A L T E R J. M U R P H Y , EDITOR ~~
Studies on the Chemistry Absorption Spectra Analysis of
OF
the Fatty Acids Conjugation in Fatty Acid
WALLACE R. BRODE, JOHN W. PATTERSON, J. B. BROWN, AND JEROME FRANKEL The O h i o State University, Columbus, O h i o
A method of determining the amount of two, three, and four double bond conjugation in the presence of nonconjugated unsaturated fatty acids has been used i n the analysis of samples of linoleic acid. The results indicate that the recrystallization method of purification gives a product more nearly free from conjugation than is obtained b y debromination procedures. The conjugation rearrangement is probably caused by the zinc bromide formed during the debromination, and the 6 form of the acid contains a comparatively high percentage of conjugated material.
at each of the three characteristic frequencies was set at 100 and by direct proportion other values were converted to the same scale, thus giving per cent. In both methods, however, the assumption has been made that all the absorption in the portion of the spectrum characteristic of a given combination of ethenylene linkages was due entirely to the arrangement of double bonds in the mixture being analyzed. Since the pure conjugated acids have absorption bands that overlap, some error is introduced by the above assumption, and hence the method presented in this paper has been prepared to correct for this error.
SAMPLES
of linoleic acid of high purity have recently been prepared by both the classical method of debromination of the tetrabromide (6) and the fractional crystallization of the free acid from corn oil (4, 6). Since these samples were known t o contain small amounts of conjugated unsaturated fatty acids, an absorption spectra study was made to obtain a more accurate estimate of the amount of these impurities. This knowledge, in turn, should help to determine the method of preparation that will lead to the purest product. The purpose of this investigation has been not only to present the data observed on linoleic acid, but also to indicate a more accurate method of analyzing the absorption spectra of the conjugated unsaturated fatty acids. Spectroscopic methods have been previously used to indicate the amount of taro, three, and four double bond conjugation in samples of fatty acids. By one of these procedures (12, 15), the amount of conjugation was indicated by comparing the values found for the extinction of a 1% solution in a 1-cm. cell a t each of the three frequencies characterized by the most intense band in unsaturated acid samples containing two, three, and four conjugated ethylene groups. In another procedure ( I ) , the highest value of
Table Sample
1.
EXPERIMENTAL
All the samples of linoleic acid used were prepared by Frankel and Brown (6, 6),with the exception of the first which was prepared by Matthew, Brode, and Brown ( 1 1 ) . The analytical constants of the acids are summarized in Table I. The form of the acid, indicated as CY or p, does not have a structural significance, but indicates whether the tetrabromide used to form the acid was insoluble (CY),or soluble (a), in petroleum ether. I n all cases the soluble tetrabromide used in preparing the 8-acid was prepared from an a: form comparable in purity to that of sample 3. To make the ultraviolet absorption measurements, a Bausch & Lomb medium quartz spectrograph, with a modified Hilger spectrophotometer attached, was used in conjunction with a hydrogen arc. The detailed procedure followed was similar to that already reported (21. Examples of absorption spectra curves are shown in the accompanying illustrations. Figure 1 shows the curve obtained for the first sample and indicates no resolution of the fine structure. Although sample 4 (Figure 2) has a smaller percentage of total conjugation, the bands caused by the conjugation of three double bonds are clearly resolved. The reason for this is obvious
Analytical Constants of Linoleic A c i d Samples
Form of Acid
Method ,of Preparation
Solvent Used in Debromination
a
Debromination, 12 reoryatallizations
hlethyl alcohol
M.P.
Iodine Value
Tetrabromide No.
c. 1
b
(11)
%
181.0
...
Debrominationb .......... .... Debromination Methyl alcohol -7.0 180.9 Crystallization 180.8 -5.4 (4. 6 ) Debromination Methyl alcohol -20.2 167.8 B Debromination Pyridine -2.0 173.5 8 Debromination -22.2 Isopropyl ether 166.3 7 B Debromination Diethyl ether -21.8 169.0 8 B Debromination Acetic aaid 1.0 89.8 9 B Debromination Dioxane -22.2 174.1 10 B Based on difference between % of linoleic found from iodine value and tetrabromide number. Mixture of aeveral specimens of a-acid, propared by debromination in various solvents (6). 2 3 4 5 6
0
-5.2 to -5 n
..aa
77
Linoleic Acid from Iodine Tetrabrovalue mide No.
102.9
...
100
...
... 88
90.6 100.6 52.4 21.3 43.6 44.6
100 100 85 91 83 87
47.7
94
...
%
...
...
Y8
51 21 42 43
... 46
Isomario Acid.
% 0.0
..
12 2 34 70 41 44
..
48
78 Table II.
. -.... Observed for Linoleic 4
Frequency (f.) Wave Number (cm. -1) Wave Length (mr)
938 31,250 320
Sample 1 2 3 4 5 6 7 8 9
0.0725" 0.0617 0.960 0,725 0.417 0.589 0.537 0.562 0.0725 0.612
10 a
Vol. 16, No. 2
INDUSTRIAL AND ENGINEERING CHEMISTRY A c i d Samples
Number of Conjugated Ethenylene Linkages 4 4 3 3 3 977 1028 1071 1110 1160 38,650 32.550 34,250 35,660 37,000 307 292 280 270 258
....
0.135 1.32
.,,,, .....
.. .. ....
0.35:" 3.63
1.01Q 0.89Ia 0.145 1.00'
3:47; 12.9& 0.47ga 6.17'
....
0.835' 0.780 3.39 2.19 10.1 10.1 18.6 30.2 1.29 21.8
0.316
0.87Za 1.01 3.72 2.48 13.2 13.8 24.1 37.8 1.66 25.4
Inflection.
2 1290 43,000 233
conjugation of four double bonds. Sample 2 gives a curve similar to that shown in Figure 2, while all the p forms of the acid are best illustrated by Figure 4. The data from the ultraviolet absorption spectra are marized in Table 11.
13.50 12.7a
METHOD OF ANALYSIS
'
.,. ...
3.09 12.16 0.2 18,4 11.88
8.75a
Sinre [ E : ? ~ . ]a~t any frequency, v, is directly proportional to the concentration, it is possible to calculate the percentage of a 32.8 52.2 ... 39.8 substance present in a sample by dividing the 22.0 40.7' observed value of [E:?mn.]v by that of the pure substance, provided the absorption is caused entirely by one substance. If the absorDtion is due to more than one substance. the-observed value of will be equai to the sum of the individual contributions of each of the components, or in a three-component system: 13.72a 4.0 25.2 37 ,2
[E;7m.]
when the intensities of the maxima caused by two conjugated double bonds are compared in the two graphs. Figure 3 is included to show t,he resolution of the bands produced by the
,
,
I
I
+ [t"
[~Mlv
[E:7m.]vof each of the
where L, ;If, and N are the values of components in the pure state a t frequency
1.50 -
1.00
+
100 [E:Fmn.'Jv = [$LIP
I
Y
and
5,y,
and z rep-
-
t -W
m
0.00
-
-0.50-
-1.00
1
i
- 1.50900
30,000 33 3
Figure 1.
r200
40,000 250
1600
50,000 c m,-l 200 m p .
Absorption Spectra of Linoleic A c i d E m p l e 1 in 95% ethyl alcohol
- I. 609 0I 0 30,000 333
Figure 2.
I
1200 40,000 25 0
I
1600 f . 60,000 c m,-l
200 m p .
Absorption Spectra of Crystallization Linoleic Acid, Smple 4
ANALYTICAL EDITION
February, 1944
T
I
I
I
1
tions. This is also true of the coeflicients of I3 and C in the third equation. For instance, in the first of these equations the coefficient of A is only l/lm that of C, and since the value of A never exceeds that of C in any of the samples studied, the term containing A is negligible. I n the same way, it can be shown that the last term in the second e uation and the last two terms in the third contribute an insignhant amount to the values of g and E, respectively. When these terms are omitted the equations become:
I
1
I
79
I .oo
x = 0.0833C y = 0.0758B z = 0.05OOA
- 0.00706B - 0.0269A
Having obtained these equations, the percentage of each type of conjugation is found by substituting in the appropriate equation and solving for P, y, or z. Several samples of linoleic acid have been analyzed in this manner and the results are compared with others, calculated as in the earlier method ( I ) , in Table IV. Inasmuch as the percentage difference in some cases is small, it should be noted that it increases as the ratio of the higher to the lower types of conjugation increases and will become very large if, for instance, the amount of three and four double bond conjugation is equal. DISCUSSION OF RESULTS
900 30,000 333 Figure 3.
1500 6 50,000 c ,.-I 200 m p ,
1200 40DOO 250
Absorption Spectra of Linoleic A c i d , Sample 3
resent the per rent of each oi the substances in the mixture. The ! , and N a t any selected frequency can be obtained values of L, k from the absorption npectra of the pure substances and [E:Zrn.Iv, x, y, and z remain to be determined. The exy , must be measured at three different tinction value, frequencies in order to provide three equations, which can be solved simultaneously to determine the per cent of earh of the three components.
A comparison of Tables I and IV indicates some intereRting results concerning the best method of purification of linoleic acid in order to keep the percentage of conjugation at a minimum. The fact that the lowest total conjugation is found in sample 4 is a strong recommendation for crystallization methods of purification (6). It is conceivable that this total might be further reduced by recrystallization, which should remove the three and four double bond portions.
I
I
I
I
I
1.50 -
In order to apply this method to the determination of the amount of two, three, and four conjugated double bonds in fatty acids, it is necessary that the absorption curves of the pure material containing each of these resonating bystems he available. The data used for these standards aw taken from the literature end sumniarized in Table 111.
-
1.00 Table Ill.
E
Valuer of Standard Curves
Conjugated Ethenylenv T,inl
