Regiospecific Analysis of Triacylglycerols

13C-NMR Spectroscopy

Fatty acid analysis is the starting point for most analytical studies of lipids and is generally carried out by gas chromatography of methyl esters obtained from the lipid by methanolysis. Sometimes information is needed about the fatty acids attached to the α (sn-1/3) and the β (sn-2) hydroxyl groups of glycerol. Methods of getting these data involve lipases, chemical reactions and several chromatographic procedures. These can be extended to distinguish between the sn-1 and -3 positions. However, besides requiring skilled effort, these methods fail with acids having double bonds close to the carboxyl group. 13C-NMR spectroscopy provides an alternative way of distinguishing between acids in the α and β chains though it will not distinguish between the two α chains (but see McNeill). The method is normally based on the clusters of signals for the acyl (C1) carbon atom. To use the NMR spectrum for regiospecific analysis, two requirements must be met.

  1. It is first necessary to distinguish two clusters of signals related to acids in the α chain and the β chain. This is not a problem as there are usually clusters corresponding to these two environments and separated from each other by about 0.4 ppm.

  2. Within each of these two clusters, there must be separate signals for each acid or group of acids. This criterion is most easily met when the acids have unsaturation close to the carboxyl group, and acids with Δ4, Δ5, or Δ6 unsaturation are readily handled in this way. Some acids falling into this category are of considerable significance and include docosahexaenoic acid (DHA Δ4,7,10,13,16,19), eicosapentaenoic acid (EPA Δ5,8,11,14,17), arachidonic acid (AA Δ5,8,11,14), γ-linolenic acid (GLA Δ6,9,12), and petroselinic acid (Δ6). The C1 signal will not normally distinguish between acids from the same double bond group such as EPA and AA both of which are Δ5 acids. The method can be applied to oils with the more common unsaturated acids having Δ9 unsaturation such as oleic and linoleic acids, but only with the best NMR spectrometers and with data acquisition over periods of time longer than usual. Vlahov (1998) has described simpler procedures for obtaining this spectroscopic information for common vegetable oils.

Examples:

The absolute value chemical shifts vary slightly in different laboratories, but the differences between the α and β shifts remain constant. More important are the different chemical shifts between the different acids in the α chain or in the β chain. Values in the Table show, for example, why it is harder to distinguish oleic, linoleic, and linolenic acyl groups from each other than, say, EPA and DHA. Examples of the ways in which this procedure is applied are given in the papers from which these chemical shifts are taken. They include DHA, EPA, GLA, petroselinic acid, and Δ-5 non-methylene interrupted polyenoic acids.

Redden et al. (1996) compared the NMR method of sn-2 analysis for oils containing GLA with alternative Grignard deacylation procedures and demonstrated good agreement between all the results. Some of the C1 chemical shifts for acids or esters containing Δ4, Δ5, and Δ6 acids are listed in the Table.

Using values of 173.10 and 172.70 ppm obtained from tripalmitin, 173.07 and 172.67 from triolein, and 173.06 and 172.66 from trilinolein and an improved procedure for collecting the spectra (proton decoupled spectra with full NOE, relaxation delay of 15 sec, 128 scans), Vlahov (1998) reported a regiospecific analyses of six vegetable oils (sunflower, peanut, grapestone, corn, hazel, and olive fruit).

Table 1
AcidUnsaturationαβRef.
18:0 - 173.186 172.778 Bergana and Lee (1996)
18:1 Δ9 173.133 172.728 Bergana and Lee (1996)
18:2 Δ9,12 173.111 172.706 Bergana and Lee (1996)
18:3 Δ9,12,15 173.107 172.702 Bergana and Lee (1996)
18:3 Δ6,9,12 172.959 172.563 Bergana and Lee (1996)
18:1 Δ6 173.098 172.071 Lie Ken Jie et al. (1996)
20:4 Δ5,8,11,14 172.893 172.510 Bergana and Lee (1996)
Nmip# Δ5 173.050 - Lie Ken Jie et al. (1996)
Nmip# Δ5 173.03 - Gunstone
20:5 Δ5,8,11,14,17 172.896 172.513 Bergana and Lee (1996)
20:5 Δ5,8,11,14,17 172.97 172.58 Gunstone
20:5 Δ5,8,11,14,17 172.98 172.59 Aursand et al. (1995)
22:6 Δ4,7,10,13,16,19 172.403 172.025 Bergana and Lee (1996)
22:6 Δ4,7,10,13,16,19 172.50 172.10 Gunstone
22:6 Δ4,7,10,13,16,19 172.51 172.11 Aursand et al. (1995)
 
Saturated - 173.247 - Blaise et al. (1997)
Erucic 13-22:1 173.240 172.830 Blaise et al. (1997)
Eicosenoic 11-20:1 173.233 - Blaise et al. (1997)
Oleic 9-18:1 173.215 172.807 Blaise et al. (1997)
Conj triene* 9,11,13-18:3 173.206 172.799 Blaise et al. (1997)
Sciadonic 5,11,14-20:3 173.054 - Blaise et al., (1997)
Taxoleic 5,9-18:2 173.042 - Blaise et al. (1997)
Pinolenic 5,9,12-18:3 173.030 - Blaise et al. (1997)
Lin/linolenic 9,12 and 9,12,15 173.204 172.796 Blaise et al. (1997)

# Non-methylene-interrupted polyenes.
* Triene acids with conjugated unsaturation.
 

 

References

  • Aursand, M., Jorgensen, L. and Grasdalen, H. Positional distribution of ω-3 fatty acids in marine lipid triacylglycerols by high-resolution 13C nuclear magnetic resonance spectroscopy. J. Am. Oil Chem. Soc., 72, 293-297 (1995).
  • Bergana, M.M. and Lee, T.W. Structure determination of long-chain polyunsaturated triacylglycerols by high-resolution 13C nuclear magnetic resonance. J. Am. Oil Chem. Soc., 73, 551-556 (1996).
  • Blaise, P., Wolff, R. and Farines, M. Regiospecific study of vegetable oil triacylglycerols by chemical cleavage and high-resolution NMR 13C. Oleagineux Corps gras Lipides, 4, 135-141 (1997).
  • Gunstone, F.D. and Seth, S. A study of the distribution of eicosapentaenoic acid and docosahexaenoic acid between the α and β glycerol chains in fish oils by 13C-NMR spectroscopy. Chem. Phys. Lipids, 72, 119-126 (1994).
  • Gunstone, F.D., Seth, S. and Wolff, R.L. The distribution of Δ5 polyene acids in some pine seed oils between the α- and β-chains by 13C-NMR spectroscopy. Chem. Phys. Lipids, 78, 89-96 (1995).
  • Lie Ken Jie, M.S.F., Lam, C.C. and Pasha, M.K. 13C nuclear magnetic resonance spectroscopic analysis of the triacylglycerol composition of Biota orientalis and carrot seed oil. J. Am. Oil Chem. Soc., 73, 557-562 (1996).
  • Redden, P.R., Lin, X. and Horrobin, D.F. Comparison of the Grignard deacylation TLC amd HPLC methods and high resolution 13C-NMR for the sn-2 positional analysis of triacylglycerols containing γ-linolenic acid. Chem. Phys. Lipids, 79, 9-19 (1996).
  • Vlahov, G. Regiospecific analysis of natural mixtures of triglycerides using quantitative 13C nuclear magnetic resonance of acyl chain carbonyl carbons. Mag. Res. Chem., 36, 359-362 (1998).
  • Vlahov, G., Schiavone, C. and Simone, N. Triacylglycerols of the olive fruit (Olea europa L.): characterisation of mesocarp and seed triacylglycerols in different cultivars by liquid chromatography and 13C-NMR spectroscopy. Fett/Lipid, 101, 146-150 (1999).

Updated: February 1, 2007