LAAN-A-FT-E035

No.A430Spectrophotometr ic Analys is

Quantitative Analysis of Trans Fatty Acid by FTIRTrans 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 disease1). 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 SpectroscopyAccording 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.

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

Cis Unsaturated Fatty Acid Trans Unsaturated Fatty Acid

H2

H2 H2H

H2 H2H

C

C C C

C C C

CC C

C

CC

H

H

H2

H2

H2

Fig. 2 Structures of Cis and Trans Fatty Acids

750100015002000300040001/cm

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14Abs

Triolein ―Trielaidin ―

Fig. 3 Infrared Spectra of Triolein and Trielaidin

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

Accumulation : 45Detector : DLATGS

Table 1 Analytical Conditions

SHIMADZU CORPORATION. International Marketing Division3. Kanda-Nishikicho 1-chome, Chiyoda-ku, Tokyo 101-8448, Japan Phone: 81(3)3219-5641 Fax. 81(3)3219-5710

No.A430

1/cm

AbsTrielaidin 10.0 % content

Trielaidin 0.1 % content

9309409509609709809901000

0 1.5 3 4.5 6 7.5 9r = 0.9999 Concentration�%

0.05

0.1

0.15

0.2

0.25

Corr. Height

5007501500 10002000300040001/cm

0

0.5

1

1.5

2

Abs

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

9309409509609709809901/cm

Abs Trielaidin (1.0�% spiked)

Trielaidin (non-spiked)

1000

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

0.05

0.1

0.15

0.2

0.25

0.3

0.35Corr. Height

r = 0.9999 Concentration｠%

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

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

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

Fig. 7 Trans Fatty Acid Peak Region Selected for Calibration Curve

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

n Measurement of Trans Fatty Acid by Transmission SpectroscopyWe 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.

n ConclusionA calibration curve was generated using the simple-operation 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.

References:1) Information on Trans Fatty Acids, Ministry of Agriculture, Forestry

and Fisheries Web Pagehttp://www.maff.go.jp/j/syouan/seisaku/trans_fat/

2) AOCS Official Method Cd 14d-99Rapid Determination of Isolated trans Geometric Isomers in Fats andOils by Attenuated Total Reflection Infrared Spectroscopy

3) AOCS Official Method Cd 14-95Isolated trans Isomers Infrared Spectrometric Method