There are three main classes of triglycerides—saturated fats, and unsaturated trans-fats and cis-fats. Unsaturation indicates the triglyceride contains one or more carbon–carbon double bonds. Most natural oils like vegetable oil consist of cis-fats and are poly-unsaturated, meaning they contain more than one double bond. The cis nature prevents solidification of the fat; trans-fats solidify more readily, which can lead to blockages in the bloodstream. Fourier transform infrared (FT-IR) spectroscopy functions well to analyse trans-fats.
The vibrational absorptions of fats show C–H bands around 2800–3000 cm–1 and the C=O band between 1600 cm–1 and 1800 cm–1. Critically, intra- and intermolecular interactions shift the peaks, allowing IR to distinguish between cis and trans (and saturated) fats.1,2 Several IR-based methods for trans-fat analysis are currently used in the American Oil Chemistry Society (AOCS).3,4
- Nicolet Series FT-IR spectrometer equipped with a standard mid-IR source
- Heated Smart™ MIRacle™ (PIKE Technologies) using a ZnSe single-bounce ATR crystal operating at 65°C was placed in the sample compartment
- Standards using the triglycerides trielaidin and tripalmitin (Nu-Chek Prep) were prepared gravimetrically giving trans-fat concentrations of 0–20%.
The pure fats were heated and then delivered to glass vials. Heating the entire vial in a warm water bath allowed homogenisation.
The spectrometer and accessory were purged with dry air and the Smart MIRacle was allowed to thermally equilibrate at 70°C. The standards were liquefied in a hot water bath and carefully homogenised before each measurement. A micropipette with the tip heated was used to extract and deliver 10–30 µL of the fat to the ATR.
The infrared spectra were converted to second derivatives, and then multiplied by –1 to invert the peak of interest (“–2D”). The trans fat shows a unique peak relative to the cis fats at 966 cm–1. A series of raw and “–2D” spectra are shown in Figure 1. The peak height of the “–2D” spectrum was measured and entered into a spreadsheet, along with the trans-fat concentrations. The resulting linear regression calibration plot is shown in Figure 2.
Discussion and conclusion
The spectra shown in Figure 1 clearly show that FT-IR can distinguish the cis and trans-fats. The process of taking a second derivative can be troublesome in the presence of noise. The high signal-to-noise seen in the raw spectra, and in the “–2D” spectra, reveals the reliability of the spectrometer and accessory combination. The good correlation in Figure 2 indicates the data collected corresponds directly to the trans-fat concentration.
A series of natural oils were analysed using the “–2D” protocol. Figure 3 shows some of the spectra. Most of the samples showed the same high degree of separation for the trans-fat signal, even with their natural origin.
The Nicolet Series FT-IR and Smart MIRacle coupled with the OMNIC software provides the tools for a rapid and complete analysis of trans-fat content. The key benefit to FT-IR is the time savings relative to other methods. The preparation of standards by quantitative means requires no complex skill set, and the calibration is straightforward.
- Curve Fitting in Raman and IR Spectroscopy: Basic Theory of Line Shapes and Applications. Thermo Scientific Application Note AN 50733.
- D.W. Oxtoby, Adv. Chemical Physics Vol. XL. Wiley, New York, pp. 1–48 (1979).
- Rapid determination of isolated trans geometric isomers in fats and oils by attenuated total reflection infrared spectroscopy. AOCS Official Method Cd14d-99.
- Determination of total isolated trans unsaturated fatty acids in fats and oils, ATR-FT-IR spectroscopy. AOAC Official method 2000.10.