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LAAN-A-FT-E035

Spectrophotometric Analysis

No.A430

Quantitative Analysis of Trans Fatty Acid by FTIR

Trans fatty acids are a kind of fat that is found in


foods. Excessive ingestion of this type of fat is
associated with elevated LDL cholesterol (low density
lipoprotein cholesterol) levels, which increases the risk
of heart disease 1). In Japan, there is currently, no
obligation to display trans fatty acid content on food
labels, nor is there a specified content limit in Japan.
However, food entrepreneurs have been taking
measures for some time by independently developing
low trans fatty acid foods and selling them on the
market. On the other hand, nations such as the United

States and Denmark are obligated to display the trans


fatty acid content on food labels and to adhere to
content restrictions on trans fatty acids.
Here we introduce a quantitative analysis study we
conducted for trans fatty acids by the ATR and
transmission methods. Measurement was conducted
according to the official method2) as prescribed by The
American Oil Chemists' Society (AOCS). Measurement
was also conducted by the transmission method in
cases where the trans fatty acid content was assumed
to be very low.

n Measurement of Trans Fatty Acid by Single Reflection ATR Spectroscopy


According to the AOCS method, analysis of trans fatty
acid content by FTIR is to be conducted by the
transmission method3) and the ATR method. Here we
used the latter method, which features easier
operation. Fig. 1 shows the MIRacle A (ZnSe prism)
single reflectance ATR (Attenuated Total Reflectance)
accessory that was used to conduct measurement of
the trans fatty acid content. A temperature controller
was used during measurement to maintain the prism
temperature at 65 C (2 C).
Unsaturated fatty acids are characterized as being
either a cis type or trans type of isomer, as shown in
Fig. 2. The samples used for analysis consisted of
triolein (Glyceryl Trioleate, Sigma Corp.), with a single
cis double bond, and trielaidin (Tokyo Chemical
Industry, Co., Ltd.), having a trans type double bond.
Table 1 shows the analytical conditions used, and Fig. 3
shows the infrared spectra of triolein and trielaidin. In
the infrared spectrum of trielaidin, the characteristic
trans-vinylene peak of the trans-type unsaturated fatty
acid is seen at 966 cm-1.
Next, using triolein as a base, we prepared sample
solutions spiked with trielaidin at concentrations of 0.1,
0.2, 0.5, 1.0, 2.0, 5.0, and 10.0 %, respectively, to
generate a trans fatty acid calibration curve.
The infrared spectra were measured similarly as
described above. Fig. 4 shows the trans fatty acid
peak in the vicinity of 966 cm-1 which was used to
generate the calibration curve. An excellent correlation
coefficient of 0.9999 was obtained with respect to the
calibration curve generated from this, as shown in Fig. 5.

Cis Unsaturated Fatty Acid

Trans Unsaturated Fatty Acid

H2
H

C
C

H2

C
H2

H2

H2

H2

H2

H2
Fig. 2 Structures of Cis and Trans Fatty Acids

Table 1 Analytical Conditions


Analytical Instrument : IRPrestige-21, MIRacle A (heating plate)
: 4cm-1
Resolution
: 45
Accumulation
: DLATGS
Detector

Triolein
Trielaidin

0.14
Abs
0.12
0.1
0.08
0.06
0.04
0.02
0
4000

Fig. 1 PIKE MIRacle A ATR Accessory with Optional Heated Crystal Plate

3000

2000

1500

1000 750
1/cm

Fig. 3 Infrared Spectra of Triolein and Trielaidin

No.A430

Trielaidin 10.0 % content

Abs

Trielaidin (non-spiked)
Trielaidin (1.0% spiked)

2
Abs
1.5
1
0.5

Trielaidin 0.1 % content


1000

990

980

970

0
960

950

940

930
1/cm

Fig. 4 Trans Fatty Acid Peak Region Selected for Calibration Curve

4000

3000

2000

1500

1000 750 500


1/cm

Fig. 6 Infrared Spectra of Unspiked Olive Oil and Olive Oil Spiked with Trielaidin

Corr. Height
0.25

Trielaidin (1.0% spiked)

Abs

0.2
0.15
0.1
0.05

Trielaidin (non-spiked)
0

1.5

4.5

r = 0.9999

7.5

990

Concentration %

Fig. 5 Calibration Curve of Trans Fatty Acid by Single Reflection ATR Spectroscopy

n Measurement of Trans Fatty Acid by Transmission Spectroscopy

We also conducted measurement by the transmission


method to allow quantitation of trans fatty acid at even
lower concentrations. Detection of low absorption is
possible by selecting a fixed thickness cell with an
appropriate wavelength. Here we used a 0.1 mm-fixed
thickness cell (window plate material: KBr).
This time, using as a base a commercially available
olive oil in which the principle substance is a cis
isomer, trielaidin-spiked samples were prepared at
concentrations of 0.01, 0.05, 0.1, 0.2, 0.5, and 1.0 %,
respectively. Measurement was conducted by the ATR
method using the same conditions as shown in Table 1.
The infrared spectra obtained from measurement of
unspiked olive oil and 1 % trielaidin-spiked olive oil are
shown in Fig. 6. Fig. 7 shows the trans fatty acid peak
in the vicinity of 966 cm-1 which was used to generate
the calibration curve. An excellent correlation coefficient
of 0.9999 was obtained with respect to the calibration
curve generated from this, as shown in Fig. 8.

References:
1) Information on Trans Fatty Acids, Ministry of Agriculture, Forestry
and Fisheries Web Page
http://www.maff.go.jp/j/syouan/seisaku/trans_fat/
2) AOCS Official Method Cd 14d-99
Rapid Determination of Isolated trans Geometric Isomers in Fats and
Oils by Attenuated Total Reflection Infrared Spectroscopy
3) AOCS Official Method Cd 14-95
Isolated trans Isomers Infrared Spectrometric Method

SHIMADZU CORPORATION. International Marketing Division

1000

980

970

960

950

940

930
1/cm

Fig. 7 Trans Fatty Acid Peak Region Selected for Calibration Curve
Corr. Height
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1


r = 0.9999
Concentration %

Fig. 8 Calibration Curve of Trans Fatty Acid by Transmission Spectroscopy

n Conclusion

A calibration curve was generated using the simpleoperation ATR method for trielaidin-spiked samples
prepared at concentrations from 0.1 % to 10.0 %. In
addition, assuming a situation involving even smaller
concentrations of trans fatty acid content, transmission
method was conducted to generate a calibration curve
with trielaidin-spiked sample at concentrations from
0.01 % to 1.0 %. Using either method, an excellent
correlation coefficient of 0.9999 was obtained,
confirming that these methods are effective for
quantitative analysis of trans fatty acids.

3. Kanda-Nishikicho 1-chome, Chiyoda-ku, Tokyo 101-8448, Japan Phone: 81(3)3219-5641 Fax. 81(3)3219-5710