NMR

Nuclear Magnetic Resonance (NMR)
Spectroscopy applied to the analysis of
Unsaturated Fat content in Seed Oils
Background

Application Note 2

The constituents of plant derived oil fall under the general class of chemicals defined
as lipids. Prof. William Christie classifies them as “fatty acids and their derivatives,and substances related biosynthetically or functionally to these compounds”. Withfurther clarification of the term fatty acid as “compounds synthesised in nature via
condensation of malonyl coenzyme A units by a fatty acid synthase complex. They
usually contain even numbers of carbon atoms in straight chains (commonly C14 toC24), and may be saturated or unsaturated; they can also contain other substituent
groups” [1]. See figure 1 below. Although a variety of lipids and starch comprisethe storage tissues of many food plants the major constituents of oil seeds are the triacylglycerol lipids; three fatty acid chainsattached as esters to a glycerol back bone. The fatty acid composition of oils is commercially important, e.g., high oleic acidcontent, as found in olive oils, has been implicated in a reduced risk of heart disease and linoleic acid, found in sunflower oils, isan essential nutritional fatty acid. High levels of these fatty acids can increase the commercial value of an oil whereas excessive
amount of linolenic acid can decrease the value since it is more susceptible to oxidation and problems with rancidity.
While most plant oils are produced for food and feed, up to 15% of some oils, e.g., soy and rapeseed, and 100% of certain
commodity oils, e.g., castor and tung, have non-food related industrial applications. Most oils comprise five primary fatty acids,palmitic, stearic, oleic, linoleic and linolenic that can be utilised in the production of lubricants, surfactants, polymers and inks aswell as their used in food/feed applications. The fatty acids of commodity oils tend to have little or no nutrition value, however
their unusual chemical properties make them ideal for particular industrial applications. The most well known example is thelaurate fatty acid derived from coconut oil which has excellent foaming properties that makes it useful in the production ofanionic surfactants.
Figure 1: Schematic
representation of a typicaltriacylglycerol. R1, R2 and R3 arealiphatic chains that may or may
not contain double bonds.

OH2CO
R2 COO

CH
H2CC

R1OO

C

R3

Nuclear Magnetic Resonance (NMR)
Spectroscopy applied to the analysis of
Unsaturated Fat content in Seed Oils
Glycerolipids

H

The class of substances described as lipids covers a broad
range of chemicals including fats, waxes, sterols and thefat soluble vitamins whose primary functions include
energy storage, cell membrane structure and biochemicalsignalling. Industrially the most common lipids are theglycerolipids. The glycerolipids are mainly composed ofmono, di and tri-substituted glycerol (shown in figure2). Of these the triacylglycerols, also referred to as
triglycerides, are by far the most common.
Triacylglycerols

C OHC H
2C OH
3
HH
HO
H

1

In the triacylglycerols (TAG) each of the three hydroxyl
groups of the glycerol backbone are esterified with a fatty
acid. Typically the TAG will contain three different fatty

Figure 2: Chemical structure of glycerol

acids. There are many different types of TAG with theirlevel of saturation being dependent on the constituent
fatty acid chains. Figure 3 shows a triglyceride comprising

The different chemical groups give rise to specific signals inthree different fatty acid chains; a saturated C16 chain fatty

an NMR spectrum, which at high field, typically 9.4 T, provide

acid (palmitic), a monounsaturated C18 chain (oleic) and aa characteristic fingerprint or fatty acid profile that can bepolyunsaturated C18 chain fatty acid (linonelic). Different

used to identify an oil [2]. In this report it is demonstrated thatseed oils have different levels of the fatty acid chains anddifferent chemical groups are still clearly identifiable in the 1H

this composition determines their physical and chemical

NMR spectrum even at lower field, i.e., 1.4 T corresponding to aproperties, which in turn determines their commercial use.

proton Larmor frequency of 60 MHz.

O
H2COO

HC

OO

H2CO αFigure 3: Example of a TAG, with glycerol backbone on the right andfrom top to bottom the fatty acid chains, palmitic, oleic and α-linolenic.

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Analysis
Anatomy of the 60 MHz, 1H Spectrum of Triacylglycerols
The 1H spectrum of glyceryl trilineolate (figure 4), a triglyceride where all the fatty acid chains derive from linoleic acid, is shownin figure 5. It is possible to identify various resonances with specific chemical groups in the molecule. Resonance A originatesfrom the four 1H nuclei attached to carbon 1 and 3 on the glycerol backbone. This is a particularly useful signal because it is ameasure of the number of glyceride molecules in the sample and as such can be used as a reference signal. The signal from thefifth 1H nuclei lies at about 5.3 ppm, directly below signal C. This must be taken into account when integrating the peaks.
Resonance C can be assigned to the 1H nuclei attached to carbons with a double bond, usually referred to as olefinic. This
peak is a measure of the total level of unsaturation in a triglyceride, regardless of whether it is in a monounsaturated or
polyunsaturated chain. Resonance D originates from a methylene group (-CH2-) sandwiched between two double bonds,
referred to chemically as bis-allyllic. This is specifically a measure of polyunsaturation.

CCDFigure 4: Chemical structureof glyceryl trilinoleate

AOOOGGAFEBCOO O FGE

FEGGD

DCGCCResonance E can be assigned to the terminal methyl group (-CH3) for each chain. Resonance F comes from the methylene groupon the fatty acid chain closest to the ester coupling with the glycerol back bone and resonance G comes from a methylenegroup with a neighbouring double bond, often referred to chemically as an allylic group.

The large peak at approximately 1.2 ppm originates from the remaining methylene groups in the fatty acid chains. The integralof resonances A, C and D can be used to quantify the level of unsaturation in a seed oil. The ratio of integral C and A gives
the average number of olefinic 1H nuclei per triglyceride and as such is a measure of overall unsaturated character. The ratio ofintegral D and A provides an estimate of the number of bis-allyllic 1H nuclei, which is a measure of polyunsaturation.

NMRApplication Note 2Figure 5: 1H spectrum of glyceryl trilinoleate obtained at 60 MHz.

To illustrate these estimates thespectra of glyceryl trilinoleate(bottom), glyceryl trioleate (middle)
and glyceryl tristearate (top) are
shown in figure 6. As shown infigure 4 glyceryl trilinoleate iscomprised of a glyceryl backbone
with three linoleic fatty acid chains.
The spectrum shows both a large
olefinic resonance and a bis-allyllic
resonance.

C: -CH = CHF: -00C-CH2-

G: =CH - CH2-

E: - CH3

Glyceryl trioleate comprises a glycerol
D: =CH - CH2 - CH=

backbone with three oleic acid
A: Glyceride

chains. Oleic acid has a single doublebond in each chain and as can beseen from the spectrum, there is asignificant olefinic resonances, but
no bis-allyllic resonance. The allyllic
resonance is clear in the spectrum ofboth glyceryl trilinoleate and glyceryl
trioleate.

Figure 6: Comparison of the 60MHz 1H spectrum of glyceryl
tristearate, glyceryl trioleate and glyceryl trilinoleate

Glyceryl tristearate comprises a
glycerol backbone with three stearic

Glyceryl Tristearateacid chains. Stearic acid is a fully
saturated C18 chain and, as can beseen from the spectrum there is nobis-allyllic signal, no allyllic signal.

Glyceryl Trioleate

While there appears to be a rather
small olefinic resonance at ~5.3 ppm,
this is in fact the signal from the 1H
nuclei attached to carbon 2 on theglycerol backbone.

The integral ratios are shown intable 1 and it can be seen thatthese are commensurate withthe unsaturated character ofthe molecule

Glyceryl Trilinoleate

NMR

Nuclear Magnetic Resonance (NMR)
Spectroscopy applied to the analysis of
Unsaturated Fat content in Seed Oils
Analysis of 60 MHz 1H Spectrum of Seed Oil
5 samples of 10 different seed oils were dissolved in chloroform

Application Note 2

Table 1: Average number of olefinic and bis-allyllic 1H nuclei estimatedby peak integration. The colours in brackets after the name of the seed
oil is the colour used to represent the data for each oil in figure 7.

in a 1:1 ratio by volume. 1 mL of each sample was transferred
Seed Oil

Olefinic(1H per triglyceride)

Bis-allyllic(1H per triglyceride)

glyceryl tristearate (grey)--

set to 4 corresponding to 4 1H nuclei per glycerol backbone andglyceryl trioleate (grey)

6.17 ± 0.07

-the other integrals were calculated as a ratio with respect to thisglyceryl trilinoleate (grey)

12.1 ± 0.1

5.9 ± 0.2

value. Providing estimates for the average number of olefinic (C)Rapeseed (canola, blue)

8.4 ± 0.1

1.4 ± 0.1

and bis-allyllic H nuclei per triglyceride molecule. The results wereSunflower (black)

9.9 ± 0.1

2.6 ± 0.1

Olive (green)

6.08 ± 0.05

0.29 ± 0.04

Corn (maize, red)

9.9 ± 0.2

2.68 ± 0.09

Peanut (groundnut,purple)

7.0 ± 0.1

Rice bran (orange)

7.5 ± 0.2

1.5 ± 0.2

Walnut (magenta)

11.5 ± 0.3

5.0 ± 0.2

Linseed (brown)

12.5 ± 0.2

6.6 ± 0.2

plot of the two most significant principal components and illustrated

Coconut (cyan)

0.31 ± 0.05

-the separation between the different oils. While it is convenient toPalm (pink)

3.58 ± 0.06

0.35 ± 0.02

to 5 mm NMR tubes. A 60 MHz 1H NMR spectrum was acquired
for each of the samples. The area under the resonances A, C andD was estimated by peak integration. The value of integral A was1averaged across the five samples for each oil and are summarisedin table 1 along with the results for similar samples of pure
triglycerides, glyceryl tristearate, glyceryl trioleate and glyceryl
trilinoleate described previously.

The complete spectra can also be analysed using principal componentanalysis (PCA). The principal components can then be used asclassifiers to distinguish between different oils. Figure 7 shows the0.96 ± 0.04

illustrate this classification with a two dimensional plot, statisticallymore than two principal components can be used as discriminators.

Figure 7: Classification of seed oils using principal component analysis of thefull spectrum.

RSummary
In conclusion, 60MHz NMR spectroscopy provides asuitable tool for the study of seed oils. The chemical
specificity of the spectral peaks can be used as markers toestimate the levels different types of the fatty acid chains.
The analysis of the spectra could be extended using aline fitting package. This could be used to estimate signal
amplitudes from overlapping resonances, especiallythose associated with the methylene and methyl groups.
The integral of this region of the spectrum provides
information about the average fatty acid chain length.
The analysis of the whole spectrum using chemometricmethods such as principal component analysis provides
a suitable framework for classification of different typesof oil. This work could be extended beyond simple
classification to provide a method for the detection ofadulterants or monitoring seasonal changes in oil crops.
[1] http://lipidlibrary.aocs.org/Lipids/whatlip/index.htm[2] Guillen M. D. and Ruiz A. Eur. J. Lipid Sci Technol.
105 (2003) 501-507

visit nmr.oxinst.com/x–pulse for more information or email magres@oxinst.com

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