Analysis of Fat Acid Oxidation Product Countercurrent Distribution Methods Model Compounds AND H. J. UUTTON Regional Research Laborutory, Peoriu, 111.
K. T. ZILCH .\brthern
Countercurrent distribution techniques are currently providing valuable information in the study of fat acid oxidations. However, interpretation of results obtained by this new procedure of fractionation is contingent upon a knowledge of t h e influence of the functional groups upon t h e partition coefficient and t h e resulting w-eight distribution rurve. >lode1 compounds chofen for study and for distribution between hexane and 80% ethyl alcohol included methyl stearate, methyl hydroxy stearate, methyl dihydroxy stearate, methyl epoxy stearate, m e t h ) . keto stearate, methyl oleate, methyl ricinoleate, stearic acid, hydroxy stearic acid, azelaic acid,
heptenal, nonenal, and mono- and digly cerides. The compounds investigated t h u s far behave nearly ideally in the concentration employed. Their weight distribution curves are predictable from their partition coefficient through use of the binomial theorem, and are little influenced by the presence of other dissol\ed solutes. The partition coefficients serve for qualitative identification and description of compounds; in addition, partition coefficients are useful because from them the degree of separation of compounds and t h e number of transfers t h a t are required in t h e countercurrent distribution apparatus may be calculated.
H E statement “every scientific advance is an advancr ill method,” finds support in numerous fields of research. Onr method recently introduced to the lipide field and applied with considerable success is Craig’s countercurrent distribution procedure ( 2 ) . However, interpretation of the results obtained on oxidized methyl linolenate requires a knowledge of the behavior of siinplrr systems.
Autoxidations of ethylenic c,onipounds have been variously reported to introduce peroxidic, hydroxyl, keto, and oxirane groups as well as to promote polymerization. Further oxidation yields cleavage products including mono- and dicarboxylic acids, aldehydes, and various combinations of theae functional groups. The behavior on countercurrent distrihution of model compounds, some of which have been postulated as oxidation interrnediaks, containing these functional groups is the mbjext of this paper. METHODS I K I ) \IATERIAL
12
I U
K
I;
h 25-tube Craig countercurrent distribution apparatus was used for fractionating the model compounds ( 2 ) . This equipment consists, in principle, of a series of separatory funnels, each containing equal volumes of immiscible solvents. In the actual apparatus, the separatory funnels are formed by drilling a series of holes (called tubes) in a cylindrical stainless steel block. This block is cut near the middle, perpendicular to its axis. When the interface t)etween the two immiscihle solvents is adjusted to the level of the cut, the upper phase of each tube may readily be transferred to the adjacent tutx by rotat,ing the upper section, thus achieving countercurrent movenicnt of solvents. Mxing the phases in all thc tubes, simultaneously, is accomplished hy rocking the whole apparatus to and fro a half turn. Fractionation of the material is effected by introducing the mixture into one of the tubes containing the immiscible solvents. Mixing and separation of the phases are alternat,ed with transfer of thr upper phase t,o nest adjacent tuhes until the upper section has made a complete revolution. -1more coniplrtc description of t h r apparatus, the theory of operation, and some of its applications to lipidos are included in thc literature (!3, 14, 16, 17, 18). In the experiments described herein, SO’% ethyl alcohol and hexane-pentane (35” to 60” C.) were used a3 the immiscible solvent pair. The weight of material fractionated ranged from 17 to 250 mg. Purificat,ion of the niodrl compounds was generally necessary and was accomplished h>-recrystallization or countercurrent distribution procedure. The contents of each tube were withdrawn into weighed flasks aft,er count,ercurrent distrihution, numhrrcd corresponding to the tubes, plitced in a vacuum oven, mess, and again weighed. The weight of residue i n as thrn plotted against the tube number.
I
The partition coefficient, K , of each compound W M calculated from the weights of each compound in pairs of tub% ( T r and T , - chosen near the maximum in the weight curve by use of the equation ($0)
Figure 1. Countercurrent Distribution Curve of Dihydroxy Methyl Stearate, Hydroxy 3Iethy.l Stearate, and Rlethyl Stearate
7 75
ANALYTICAL CHEMISTRY
116 Results of a t least three calculations from three pairs of points were averaged to obtain the coefficient. The theoretical weight distribution curve can also be calculated by use of this equation and the experimentally determined partition coefficient. This calculated curve is designated by a broken line in the figures. In a few instances, which will be discussed later, a 24-transfer distribution n as insuffirient to effect a satisfactory separation. However, the number of transfers necessary can be calculated by use of the equation )L =
t2
y-a
2KaKblz
KgK b-i-Ka
I
I
1
I
I I l I I ! l I I l
(2)
where n = number of transfers; K. = partition coefficient of compound a; Kb = partition coefficient of compound 6; and t = a statistical coefficient for which the percentage impurity may be found in standard probability tables ( 4 , I d ) . RESULTS AND DISCUSSION
The results obtained by countercurrent fractionation of model compounds are shown in the following series of curves. Figure 1gives the weight distribution curve of hydroxy methyl stearates. These compounds are of interest since the decomposition of perosides leads to the formation of hydroxy acids and esters (5-10, 19), The data shoa- that in fractionating an oxidation mixture, the maxima for the weight curves of monohydroxy esters and dihydroxy esters of (318 acids would appear in the vicinity of tube 12 and tube 4, respectively. for a 24transfer distribution. The curve shows that the more polar the compound the lower the tube number in which the material will be found. To obtain dihydroxy and monohydroxy methyl stearate with 2% of material impurity. 44 transfers are calculated to be necessary.
Figure 3.
Countercurrent Distribution Curve of Epoxy Methyl Stearate and Methyl Stearate
the maximum is found in tube 12, whereas for the dimer hydroxy ester the maximum is found in tube 22. Therefore, the presence of an additional aliphatic group shifts the curve toward higher tube numbers. Figure 3 gives the fractionation of epoxy methyl stearate and methyl stearate. A more complete (2% impurity) separatibn, by calculation, would require the application of 100 plates. Since an epoxy group is less polar than a hydroxyl group, the maximum for epoxy methyl stearate is expected and found in a higher tube number, 20. If additional functional groups, such as a hydroxy group, were present in the dimeric compounds described in the literature (1,19), the maximum would occur toward tube numbers lower than 18. The weight distribution curve for methyl oleate hydroperoxide, which is believed to be the first reaction product in the oxidation
Figure 2. Countercurrent Distribution Curve of the Mono- and Diglycerides of Cottonseed Oil
The weight distribution curve for the mono- and diglycerides of cottonseed oil is shown in Figure 2. Polymer formation has been postulated (12, 16)and found to occur as a product in the oxidation of fat acids. If one considers the diglyceride as a model compound for a dimer with one hydroxyl group and the monoglyceride as a monomer with two hydroxyl groups, these data indicate that dimeric material containing one functional group would appear toward the higher number tubes where relatively nonpolar substances are found. The relative importance of polar and nonpolar groups in determining the partition ratio i s illustrated by the monomer hydroxy ester of Figure 1 for which
4
TUBE NUMEER
Figure
4.
Countercurrent Distribution Curve Methyl Oleate Hydroperoxide
of
V O L U M E 2 3 , NO. 5, M A Y 1 9 5 1
777
of methyl oleate (IO), is given in Figure 4. The hydroperoxide group, being more polar than the hydroxyl group, has shifted the curve toward lower tube numbers (maximum in tube 9). Figure 5 shows the fractionation of 12-hydroxy stearic acid and methyl stearate. Replacement of the ester group in hydroxy methyl stearate by a carboxyl group has markedly shifted the curve toward a Ion-er tube number. The weight distribution curve of azelaic acid, a short chitin dibasic acid postulated as one of the scission products in the oxidation of fat acids ( I I ) , is given in Figure 6. Owing to the presence of two carboxyl groups and a short carbon chain, this highly polar acid appears in the lower tube numbers. The presence of a carbonyl group in methyl stearate shifts the curve tonard lower tube numbeis, as shown in Figure 7 , for a mixture of 9 and 10 keto methyl stearate. Since experimental and theoretical curves agree closely, the position of the keto group on the rarbon chain would appear to have little effect on the
partition coefficient. LIethyl keto stearate is not as polar as the monohydroxy stearate but is more polar than methyl epoxy stearate. Figure 8 s h o w the effect on the w i g h t distribution curve of the addition of a hydroxyl group to methyl oleate. The peaks for methyl ricinoleate and methyl oleate appear, in the same tubes, as those for hydroxy methyl stearate and methyl stearate, respectively. This indicates that the double hontl has little upon the polarity of the compound.
60
I
I
I
' I I "
1
50-
40-
I
i
30-
20-
10-
0
4
8 12 16 TUBE NUMBER
18
1
Figure 5. Countercurrent Distribution Curve of 12Hydroxy Stearic Acid and Methyl Stearate
TUBE
NUMBER
Figure 8. Countercurrent Distribution Curve of Methyl Ricinoleate and Methyl Stearate
TUBE NUMBER
Figure 6. Countercurrent Distribution Curve of Azelaic Acid and Methyl Stearate
The weight distribution curves for or-heptenal and e-nonenal, shown in Figures 9 and 10:respectively, are of particular interest because the former aldehyde has been isolated from revertetl soybean oil ( 3 ) . The maximum for the more polar a-heptenal occurred in tube 16 and the peak for or-nonenal was found in tulle 18. An 85-transfer distribution would be necessary to separate a mixture of these two aldehydes (9Syo purity).
ANALYTICAL CHEMISTRY
778 The weight in each tube was determined spectrophotometrically by measuring the absorption a t a wave length of 2400 A. This wave length does not occur a t the maximum but was selected in consideration of the solvent transparency and intensity of absorptlon. The absolute weight values given are relative since the distrhutlon data show that impurities were present in the preparation. a t the time they were used for calibration and dlstribution.
2-
4
4
0 Figure 9.
12 16 TUBE NUMBER
24
20
8
Countercurrent Distribu tioti Curve of a-Heptenal
Table I gives a summary of the compounds fractionated, their partition coefficients, the tube number of the maximum in a 24transfer distribution, and the important functional groups present. It becomes apparent that the partition coefficient may well assume the importance of other physical constants such a'i, melting point, refractive index, and optical rotation. Sot only do the coefficients serve for qualitative identification and description of compounds but, in addition, they have other useful features. If B pair of partition coefficients are given, the degree of separation of the compounds for any given number of transfers may be calculated or, conversely, the number of transfers necessary to achieve a desired purity or degree of separation may be calculated. The compounds investigated thus far behave almost ideally in the concentration employed. Their weight distribution curve?
Table I.
Partition Coefficients and Positions of Maxima for Various Model Compounds
Compoiind Azelaic acid Dihydroxy methyl stearate 12-HydroxS xtranc acid Monoglyceride of cottonseed oil Methyl oleate bydroyeroxide Hydroxy methyl ztearate Methyl ricinoleate Heptenal Keto methyl stearute Sonenal Stearic acid Epoxy methyl stearate
Partition Coefficient
Tube" Number
0.05 O,l'i_to 0 21 0 20
1 3, 4
0 28 0 57 0 97 t o 1.01 1.m 2.05 2.22 2.96 3.38 4.40
Fiinctional Groups di-COOH di-01%and COOR
4
OHandCOOH
5
di-OH (nionomer)
9 11, 12
OOHandCOOR O H aud COOR
12 16 17 18 18 20
OH.--.COOH. = a n d C=O -R&=O and COOH = and C=O
COOH
C--C \
Methyl oleate Diglyceride of cottonseed oil Methyl stearate *-
and COOR
/
5.15
20
0 = a n d COOR
10.76 19.75 t o 19.31
22 23
OH (dimer)
COOR
Distribution (24-transfer) between hexane and 80% ethyl alcohol.
Figure 10. Countercurrent Distribution Curve of m-yonenal
are predictable from their partition coefficient and the binomial theorem, and are little influenced by the presence of other dissolved solutes. ACKhOWLEDGMEYT
The authors are indebted to H. AI. Teeter for the dihydroxy methyl stearate and keto methyl stearate used in these studies, a r i d to B. F Daubert for the a-heptenal and a-nonrnal LITERATURE CITED
(1) Bolam, T. R., and Sim, W. S., J . Soc. Chem. Irrd. L o i t d u r i , 60, 50 (1941). ( 2 ) Craig, L. C., and Post, O., ANAL.Cmnr., 21, 500 (1949). (3) Daubert, B. F., J. Am. Oil Chemists' Soc., 27,367 (1950). 14) Dutton, H. J., Lancaster, C. R.. and Brekke, 0. L., Ihid., 27, 25 (1950). (6) Ellis, G. W., Biochem. J., 30, 763 (1936). (b) Fahrion, W.? Chem. Umschau Gebiete Fette, ole, Wachse u Harze, 27, 158, 201 (1920); 28, 5, 20 (1921). (7) Farmer, E. H., Bloomfield, G. F., Sundralingam, A , , and Suttoii, D. A., T T U Faraday ~. SOC.,38, 348 (1942). (8) Farnier, E. H., K o c h , H. P., and Putton, D. A, .I. Clteiri. Soc., 1943, 541. (9) Farmer. E. H., and Michael, 8. E., I W . , 1942, 513. (10) Farmer, E. H., and Sutton, D. A., I M . , 1942, 139; 1943, 119. 122. (11) French. R. B., Olcott, H. P.,and Mattill, H. 9., I n d . Eng. Chem., 27, 724 (1935). (12) Goldschmidt, S.,and Freudenberg, K., Ber., 67, 1589 (1934). (13) Hamilton, L. A., and Olcott, H. S., I d . Eng. Chem., 29, 217 119371 \---.,.
c. R., Lancaster, E. B.. Dutton, H. J.. J . A m . Oil Chemists'Soc., 27, 386 (1950). (15) Miller, A. U . , and Claxton, E., Ind. Eny. Chem., 20, 43 (1928). Barry, G. T., and Craig, I,. C., J. Biol. Ciieni.. 170, 501 (16) Sato, P.,
(14) Lanoaster,
11949). (17) Scholfield. C. R., and Dutton. H. J.,J . Am. Oil Chemists' Soc., 27, 352 (1950). (15) Soholfield. C. R., Dutton. H. J . , Tanner, F. I\'., and Cowan, J. C., Ibid., 25, 368 (194s). (19) Skellon, J. H., J . SOC.Chenl. I d . , L h n , 50, 3825 (1931). (20) Williamson, B., and Cmig, L. C., J . B i d . C b . ,168,687 (1947). RECEIVED Sovember 2, 1950. Presented before the Division of .4gricultural and Food Cheinistry a t the 118th hleeting of the AXBRICANCHEMICAL SOCIETY,Chicago, Ill. Report of 2% Study made under the Research and 1Inrketing . i c t of 1946.
