NMRFatty Acid Compositionof Triglycerides

Application Note 5Background
The fatty acid composition of oils and fats is important for health (forexample, high levels of polyunsaturated fats and omega-3 oils are generally
considered to be good for health; high levels of saturated fats are consideredunhealthy). It will become mandatory in the EU at the end of 2016 to display
levels of saturated fats on processed food labels; this is already mandatory in
the USA, and other countries can be expected to follow. With this minimum
requirement in place, food producers may choose to add further information
where health benefits can be promoted through high levels of polyunsaturates
and omega-3 oils. It is therefore important to have rapid, simple, and reliable
methods for the measurement of fatty acid composition.
The traditional method of analysis
The traditional method for measurement of fatty acid composition ofproviding a means to separate chains of different length and different

triglycerides is AOAC Method 996.06, which is a hydrolytic extraction

numbers of double bonds per chain (i.e. by degree of unsaturation).

gas chromatographic (GC) method. Under this method, fat is firstOften, information on chain length is ignored and the relevant peaks

extracted from the sample into ether and then methylated to fatty

are simply summed to get a total of saturated, mono- andacid methyl esters (FAME) using boron trifluoride before runningpoly-unsaturated components.

through the GC. Individual FAMEs elute in the GC at different times

Glyceride backbone

Saturated fatty acid

Monounsaturated fatty acid (MUFA)

Polyunsaturated fatty acid (PUFA)

A typical triglyceride

X-PulseX-Pulse is a high performance benchtop Nuclear Magnetic Resonance (NMR)
spectrometer operating at 60MHz proton frequency. It uses a permanent magnet,
so does not require liquid nitrogen, liquid helium, or compressed gasses, and it has
no special health and safety requirements. It can be operated in a normal analyticallaboratory by non-NMR expert laboratory technicians.

Fatty Acid Composition of Triglycerides

The X-Pulse method
The X-Pulse method has been developed in collaboration
with QIB Extra Ltd in Norwich, UK. The method works
by analysing the high resolution 60MHz NMR spectrumof an oil or fat sample, either in liquid form or extracted
from a food sample using chloroform. The numbersof individual and multiple double bonds per chain are

Indicators of total
unsaturation
Indicator ofpolyunsaturation

Measure ofomega-3

determined using a specially developed algorithm and thiscan be related to the relative concentrations of saturated,mono- and polyunsaturated oils in the sample. In addition,
components having double bonds in the omega-3
position can also be identified, leading to quantificationof the omega-3 oil content as well. All measurements arenormalised to the signal from the four alpha protons on
the glyceride backbone, so no calibration is required.

60MHz NMR spectrum of a typical unsaturated triglyceride

Measurement process
For a liquid sample, an aliquot of about 0.3ml is taken and shaken in a vial with 0.7ml chloroform to dissolve it. The mixture isthen transferred into a 5mm NMR tube using a glass pipette. Fat samples, such as lard, can be dissolved directly in chloroform.
Solid food samples are first homogenised, then a representative sub-sample of about 3 - 4g is shaken in a vial with 1.5ml
chloroform. The mixture is then filtered, and pipetted into a 5mm NMR tube. The tube is then inserted into the X-Pulse
instrument and the special applications software run.
Measurement time
The total measurement time, including
sample preparation, is typically less than
5 minutes for most samples, but could
be up to an hour for very lean samples.
Results
The graphs opposite show thecorrelation between the X-Pulse
method and the standard GC
method for the measurement ofmonunsaturated, polyunsaturated andomega-3 fatty acids, together with thetotal saturated fat content, in a varietyof nut, seed and vegetable oils.

NMR
Application Note 5PUFAOmega-3

MUFA

SFA

X-Pulse results are presented as a final summary of the triglyceride components, as shown below:

Summary
X-Pulse provides a fast, convenient and reliable method of determining fatty acid compositions. The method does not requirecalibration, and it presents results clearly and directly. X-Pulse does not require liquid helium, liquid nitrogen, or compressedgases, and can be operated in a normal laboratory environment without any special health and safety considerations.
This work received support from the UK’s Technology Strategy Board project no. 101250.

Also available:
X-Pulse for meat speciation
The fatty acid composition can befurther used to verify the species of apiece of raw or frozen meat for positive
identification within the food chain.

MQC+ benchtop NMR analysers
for fast and easy measurementof total oil or fat. Applications include:• Fat in food• Solid Fat Content (SFC)• Oil in snack food
• Fat in chocolate• Oil & moisture in seeds

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

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