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Heat induced synthesis of linolenic acid isomers-a reference standard.


Edible oils contain saturated and unsaturated fatty acids in varying quantities in the form of tri-glycerides. Among unsaturated fatty acids, fatty acids containing 18 carbon atoms can reach up to 95% or more in certain edible oils. The unsaturated fatty acids include mono-unsaturated oleic acid, poly unsaturated linoleic and linolenic acids. All these unsaturated fatty acids are found in c/5-configurations in nature. All these fatty acids undergo isomerization, oxidation, polymerization and decomposition during heating and the degree of transformation through these processes depend on the stability of the unsaturated fatty acids in the glyceride molecules [1-5].

The trans isomers produced during frying process are unhealthy and has been linked to the risk of coronary heart disease [6-11]. The double bonds in all the naturally occurring unsaturated fatty acids are in cis configurations. During isomerization oleic acid with one double bond forms one trans isomer, linoleic acid with two double bonds forms three trans isomers and linolenic acid with three double bonds forms 7 trans isomers. Identifying and quantifying different isomers formed in research work involving fatty acids is not an easy task for an analytical chemist. Accurate identification and quantification of the isomers of fatty acids need reference standards containing all the different isomers of the unsaturated fatty acids to confirm or eliminate the presence or formation of one or more of the fatty acid isomers.

My intention in this article is to study the cis-trans isomerization and isomers formed during the thermal induction of methyl linolenate at 275[degrees]C. The study would give an idea the conditions needed to synthesize a mixture with all the linoleic acid isomers that can be used as a reference standard.

2. Experimental:

Methyl linolenate (99% purity) was purchased from Sigma and used as received. A FAME (Fatty Acid Methyl Esters) reference standard of linolenic acid (100 mg) isomers inmethylated form was also bought from Sigma. The reference standard was dissolved in 1ml GC quality heptane.

Laboratory made glass ampoules were used in thermally inducing methyl linolenate sample. Ampoules were of 4 cm long, 1.55 mm internal diameter and 1 mm wall thickness. The ampoules were sealed at one end using propane, oxygen and air flame. Small amounts of methyl linolenate (250 [micro]l) were injected in the ampoules using a plastic syringe with needle. The open ends of the ampoules were sealed. A total of fifteen ampoules were placed in a glass beaker and placed in a gas chromatographic oven set at 275[degrees]C. The ampoules were then removed at regular time intervals until all the 15 were exhausted. The ampoules were then cut open and the contents were dissolved in 1.5 ml heptane in gas chromatographic vials.

The GC analysis was carried out by using Perkin Elmer Auto system XL gas chromatograph. A 120 m capillary column with 0.25 mm internal diameter coated with 0.25 [micro]m thick, 70% cyanopropyl(equiv) polysilphenylene-siloxane stationary phase was used. A temperature program with initial temperature of 150 0C with 2 minute equilibration time, a temperature gradient of 0.5[degrees]C /min up to 170[degrees]C with 50 minutes holding time, then a temperature gradient of 1[degrees]C /min up to 190[degrees]C with 10 minutes holding time was used for the GC analysis. The total running time was 122 minutes. The peak identification was carried out using the literature references [12] and the Sigma reference standard.


A gas chromatogram of the isomers of methyl linolenate is shown in Fig. 1. The concentrations of the isomers in the standard mixture are shown in Table1.

Three chromatograms of the heat induced methyl linolenate sample are shown in Fig 2. The figure clearly shows the progress of the formation of different isomers.

A plot of the concentrations of isomers in different samples is shown in Fig. 3. It is easy to identify the sample where all the isomers formed during the heating process. The sample heated at 275[degrees]C for 6-10 hours contain all the isomers of linolenic acid in the mixture. The concentration of ttt isomer is very low compared to all the other isomers and not shown in the plot.

The preparation of an analytical reference sample can be made by dissolving a heat treated sample containing all the isomers in a heptane solution containing methyl margarate as reference standard. It appears that that peak heights in the chromatograms of the reference standard in Fig. 1 correspond to the proportions of the concentrations of the different isomers in the sample. Therefore quantification of a particular isomer in an unknown sample can be determined by peak heights comparison.


I have shown in this paper an easy an inexpensive method to synthesize a mixture of linolenic acid isomers for the purpose of identifying different isomers in unknown samples. All one needs is a sample of methyl linolenate and glass ampoules for thermal induction. The newly formed mixture of isomers of linolenic acid can be used as a reference standard and eliminate the need of purchasing expensive reference standard from vendors.


[1.] Moreira, R.G., M.E. Castel-Perez, M.A. Barufet, 1999. Deep-fat frying. In: Fundamentals and Applications. Aspen Publishers, Inc., Gaithursburg, Maryland.

[2.] Rojo, J.A., E.G. Perkins, 1987. Cyclic and fatty acid monomer formation in frying fats. J. Am. Oil Chem. Soc., 64: 414-421.

[3.] Fullana, A., A.A. Carbonell-barrachina, S. Sidhu, 2004. Volatile aldehyde emissions from heated cooking oils. J. Sci. Food Agric., 84: 2015-2021.

[4.] Umano, K., T. Shibamoto, 2001. Analysis of cooking oil fumes by ultraviolet spectroscopy and gas chromatography-mass spectrometry. J. Sci. Food Agric. 49: 4790-4794.

[5.] Fujisaki, M., Y. Endo, K. Fujimoto, 2002. Retardation of volatile aldehyde formation in the exhaust of frying oil by heating under low oxygen atmospheres. J. Am. Oil Chem. Soc., 79: 909-914.

[6.] Mensink, R.P., M.B. Katan, 1990. Effect of dietary trans fatty acids on high-density and low density lipoprotein cholesterol levels in healthy subjects. New Engl. J. Med., 323: 439-445.

[7.] Zock, P.L., M.B. Katan, 1992. Hydrogenation alternatives: effects of trans fatty acids and stearic acid versus linoleic on serum lipids and lipoproteins in humans. J. Lipid Res., 33: 399-410.

[8.] Daush, J.G., 2002. Trans-fatty acids: a regulatory update. J. Am. Diet. Assoc. 102: 18-20.

[9.] Stender, S., J. Dyerberg, 2004. Influence of trans-fatty acids on health. Ann. Nutr. Metab., 48: 61-66.

[10.] Judd, J.T., B.A. Clevidence, R.A. Muesing, J. Wittes, M.E. Sunkin, J.J. Podczasy, 1994. Dietary trans fatty acids: effects on plasma lipids and lipoproteins of healthy men and women. Am. J. Clin. Nutr., 59: 861-888.

[11.] Christy, A.A., Z. Xu, P.B. Harrington, 2009. Thermal degradation and Isomerization Kinetics of Triolein studied by Infrared Spectrometry and GC-MS combined with Chemometrics. Chem phys Lipids, 158: 2231

[12.] Mjes, S.A., 2004. Quantification of linolenic acid isomers by gas chromatography-mass spectrometry and deconvolution of overlapping chromatographic peaks. Eur. J. Lipid Sci. Technol., 106: 307-318.

Alfred A. Christy

Department of Science, Faculty of Engineering and Science, University of Agder, Serviceboks 422, 4604 Kristiansand, Norway

Received 2 September 2016; Accepted 2 December 2016; Published 31 December 2016

Address For Correspondence:

Alfred A. Christy, Department of Science, Faculty of Engineering and Science, University of Agder, Serviceboks 422, 4604 Kristiansand, Norway

Caption: Fig. 1: GC chromatogram of linolenic acid isomers (in methylated form)

Caption: Fig. 2: Gas chromatograms of heat induced sample at 275[degrees]C

Caption: Fig. 3: Concentrations of the methyl linolenate isomers with heat induction times
Table 1: Composition of the methyl linolenate isomers

Compound   Composition (%)

9c12c15c   3
9t12c15c   7
9c12t15c   7
9c12c15t   7
9t12t15c   15
9t12c15t   15
9c12t15t   15
9t12t15t   30
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Author:Christy, Alfred A.
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Date:Dec 1, 2016
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