R. J. Hamilton and M. Y. Raie
The Polytechnic Liverpool 3, England
I
I
Oxidation of Fats A study using glc
Experimentsinvolvinglipidsare normally too time-consuming to be incorporated into undergraduate teaching. However, if the high resolution and sensitive detection of gas-liquid chromatography can be used, much smaller quantities of lipids can be reacted with consequent reduction in the time of reaction. This point is emphasized in the following experiment which enables the position of the ethylenic bond in a fatty acid to he determined, illustrates the use of potassium permanganate-periodate mixtures for oxidation and can, if required, he adapted for use in an analytical course where statistical methods can be applied to results of standard fatty acid analyses. The most abundant unsaturated fatty acids' in nature have ethylenic bonds a t either the 9, 12, or 15 positions, i.e., olive oil is very rich in oleic acid (9), cottonseed oil in linoleic acid (9, 18), and linseed oil, in linolenic acid (9, 18, 15). Oxidation of these oils will result in cleavage of the fatty acid at the double bond with consequent liberation of a short-chain fatty acid, Cs, Cs, or Ca, respectively. It shouldbe remembered, however, that each of these oils contains other unsaturated acids which will liberate a mixture of short-chain fatty acids after oxidation. Normally, the isolation of such short-chain acids is a problem since they are appreciably water soluble. We have recently shown2 that shortchain free fatty acids up to decanoic acid in aqueous solution can he analyzed by glc with a flame ionization detector which does not respond to water molecules. Potassium permanganate has been used for many
decades by lipid chemists to hydroxylate3 and then cleave unsaturated fatty acids, but some overoxidation of the short-chain fatty acid can occur. Von Rudloff4 made use of the fact that, a t pH range 7-8, potassium periodate can reoxidize mauganate ions to potassium permauganate and that the over-oxidation of the shortchain fatty acids is minimized. The reaction is believed to be
IOPI 4
R'COOH
MnO.
+ R'CHO + R'CHO + lteCOOH
+ R'COOH
+R'COOH
In the experiment, t.he readily available oils (olive linseed, and cottonseed) are used where the fatty acids are esterified to glycerol with the result that the oxidation liberates only the mouoearboxylic acid while the other end of the molecule is attached to glycerol as the azeleo glyceride.
-
CHI(CHz)ICH=CH(CHn)l CO OCHn CHdCHs),COOH I
CHa(CH1)ICH=CH(CH.), COO b~
1
+
CHa(CH&20 0 CHn HOOC (CH2)C00CH2 HOOC (C&)rCOO &H
1
1 GUNSTONE, F. D., "An Introduction to the Chemistry and Biochemistry of Fatty Acids and their Glyce"des," Chapman hnd Hall, London, 1967. HAMILTON, R. J., and Rars, M. Y., Chem. & Ind., 1228 (1971). 3 H r ~ n n c T. ~ , P., A N D WILLIAMS, P. N., "Chemical Constitution of Natural Fats," (4th Ed.), Chapman & Hall, 1964. 'YON RUDLOFF, E., J. Amw. Chem. Sac., 33, 126 (1956).
CH,OCO(CH&CHI
The azeleoglyceride does not interfere with the analysis of the free fatty acids. A weighed mixture of short-chain fatty acids (CaCo) is analyzed on Porapalc Q with temperature programing (Fig. 1). From their retention times it is possible to
Volume 49, Number 7, July 1972
/
507
&
3'6
$0
2'4
MINUTES
i8
1'2
6
o
Figure 1. GlC molysir of propioni~butyric, n-vderic, hexanoic, heptmoic, and nononoic ocids on Poropak Q with temperotvro program from 1 2 5 to 240°C.
plot a graph of retention time against chain length (Fig. 2) for each fatty acid. Thc peak areas can be measured by triangulation and the percentage composition based on areas can be compared with the known percentages by weight. The figures obtained by analyzing the same mixture three times can then he treated statistically to determine the standard deviation and coefficient of variation for each component of the mixturc. Olive oil is oxidized under von Rudloff conditions with mechanical shaking for 1 hr. The short-chain fatty acids which are liberated can then be analyzed by injecting an aliquot from the aqueous reaction mixture directly on to glc column undcr conditions similar to those used for the standard mixture above. Olive oil will liberate a mixture of acids with nonanoic acid Co at the major component, while cottonseed yields hexanoic acid Ceand linseed oil propionic acid CI. Experimental
CHAIN
LENGTH
Figure 2. Groph of retention time against number of corbon atoms each fatty acid in standard mixture.
for
of samples of aqueous standard fatty acids. The Pye 104 series instrument with a flame ionization detector was used. Purification of f-BufonolS
&Butand (6.50 ml) was refluxed with potassium hydroxide (2 g) and potassium permangmtnate (2 g) for 2 hr and then disbilled through a 6-in. column packed with glass helices. Von Rudloff Oxidonf5
Stock oxidant solution was prepared by mixing sodium periodate (0.2 M ) and potassium permanganate (0.005 M ) solutions in a. ratio of 1:1. Modified Von Rudloff Oxidafion
A sample of olive oil (10 mg) was dissolved in rs mixture of purified t-hutanol (2 ml) and stock oxidant solution (4 ml) and the reaction mixture placed on a. meehmicel shaker for 1 hr. was removed from the reaction vessel and An aliquot (40 injected directly on to the Porapak Q column a t 10O0C. When the solvent had eluted, the temperature was increased from 100-230T a t a rate of 6' min.
Gas Liquid Chromdography5
Analyses of free fatty acids were performed on a. short column Porapak Q (80-1W mesh) (18 X V t sin.) which had been preconditioned by heating a t 230°C for 24 hr with regular injections
508 / Journal of Chemical Educofion
Each of these steps can be performed in advance by the technic$ staff in preparetion for the class.
