Improved Analysis of FAMEs using GC-VUV
The determination of the fatty acid composition of edible oils and fats (as their methyl esters or fatty acid methyl esters [FAMEs]) is routinely performed in food laboratories globally. The level of detail that is required depends on the intended use of the data. For label claim purposes a distinction between saturated, mono-unsaturated, and poly-unsaturated fatty acids generally suffices. Studies on the health impact of fat consumption, on the other hand, require detailed information on chain length distributions and number and positions of double bonds, as well as double bond orientation (cis versus trans). Trans levels have attracted increased attention in recent years because of adverse health impacts associated with such acids, such as increased cholesterol levels and an increased risk of coronary hearth diseases.
The standard method for trans-FAME analysis is capillary gas chromatography (GC) on a 50 m to 100 m highly polar cyanopropyl column. With this technique trans analysis is possible in relatively “simple” samples such as untreated or mildly processed vegetable oils and fish oils. For more complex samples, more sophisticated methods such as combined high performance liquid chromatography (HPLC) on silver-ion columns (Ag+-HPLC) and GC are needed. These techniques provide a good separation, but at the expense of an increased complexity and longer analysis times.
GC with VUV (vacuum ultraviolet) detection offers numerous advantages compared to these traditional techniques. The GC–VUV analysis separates saturated FAMEs from the unsaturated cis/trans FAMEs, and also identifies the number and position of the double bonds.
The GC–VUV system used for this study consists of a gas chromatograph configured with an automatic liquid sampler (Agilent Technologies), a split/splitless injector hyphenated to a vacuum ultraviolet detector (VUV Analytics). The separation of the FAMEs was performed using a 50 m x 0.25 mm, 0.2-µm CP-Sil 88 capillary column (Varian), isothermally at 176 °C. Hydrogen was used as the carrier gas (1 mL/min) and the make-up gas for the flow cell was nitrogen. Data was acquired in the range of 120–240 nm using controlling software (VUV Analytics).
The absorption spectra of the saturated and unsaturated fatty acids possess distinct characteristics, which enable the simple classification of the eluted peaks into two groups. All saturated FAMEs absorb in the low wavelength range of 125-160 nm; all unsaturated FAMEs exhibit an additional strong absorbance at longer wavelengths (170–220 nm).
The difference in spectral response between saturated and unsaturated FAMEs allows for the use of spectral filters to exclude the unsaturated FAMEs from the chromatogram.
Level of Unsaturation
The two absorbance maxima, 125–160 nm and 170–200 nm, can also be used to determine the level of unsaturation. The higher the number of double bonds in an unsaturated FAME, the higher the absorbance in the 170–200 nm wavelength region, that is, a higher peak area in the 170–200 nm region compared to the 125–160 nm region.
By calculating the relative response factor (RRF) of these two absorbance regions, the level of unsaturation can be determined. The RRF is the ratio of the total peak absorbance of the 170–200 nm region and the total peak absorbance of the 125–160 nm region.
GC–VUV can be applied for a detailed analysis of FAMEs. VUV absorption spectra are unique and with the use of spectral filters the unsaturated FAMEs can be easily distinguished from the saturated FAME.
The number of double bonds present in an unsaturated FAME can be determined based on peak area of the two different wavelength areas. In addition, cis/trans isomer identification is possible with VUV detection, based on their specific spectrum and deconvolution software.