200 Meter GC Columns for Detailed Analysis of cis/trans FAME Isomers

Over the last half of the previous century, the use of partially hydrogenated vegetable oil (PHVO) replaced the use of animal fats for baking purposes in most western countries. Initially developed for supply/demand and economic reasons, it was discovered that the use of PHVO could increase a food’s shelf-life and/or increase its taste. It was also suggested that the unsaturated fatty acids in PHVO were healthier than the saturated fatty acids in animal fat.

In nature, the overwhelming majority of unsaturated fatty acids occur in the cis orientation. As such, humans evolved metabolic pathways to break down cis fatty acids. However, the process to make PHVO converts cis fatty acids into trans fatty acids. Scientific research over the last decade has shown that this situation (the increased intake of trans fatty acids coupled with our inability to properly metabolize them) can increase the risk of coronary disease. This is most evident by the proliferation of this disease in countries where the use of PHVO has replaced the use of animal fats. To help combat this trend, in June 2015 the US FDA mandated that food manufacturers must eliminate the use of all artificial trans fats (i.e. they can no longer use PHVO) within three years¹.

The qualitative and quantitative testing ofcis/trans fatty acids is best accomplished using gas chromatography (GC) after conversion of the fatty acids to fatty acid methyl esters (FAMEs). To assist with this testing, Supelco recently developed two new capillary GC columns. These 200 m versions of SP™-2560 and SLB^®-IL111 are specifically designed for and specially tested for the detailed analysis of cis/trans FAME isomers. Specifications for both columns are shown in Table 1. This article will show the suitability of these columns for analysis of cis/trans FAME isomers as well as other FAME isomer applications.

C18 FAME Isomer Mix

Some of the most studied fatty acids are the C18 family. A custom mixture was made by combining a C18:0 FAME standard, a custom C18:1 PHVO sample (containing multiple C18:1 FAME isomers), a 4-component C18:2 FAME isomer standard, and an 8-component C18:3 FAME isomer standard. This mixture was injected on each column, and run conditions were adjusted to achieve maximize resolution. The optimized chromatograms are shown in Figure 1. Peak identification was assigned based on previous work. While neither column can separate every isomer, both columns provide a high degree of separation of trans FAME isomers fromcis FAME isomers. Of interest is that with SLB-IL111, no trans C18:1 FAME isomer co-elutes with C18:1Δ9c, one of the most abundant naturally occurring unsaturated fatty acids. It often results in a very large peak area when analyzing food extracts. This is significant because this entire peak area must be considered as being contributed by the trans FAME if there is a co-elution, resulting in trans fat values that are biased high.

GC Conditions

columns: 1) SP-2560, 200 m × 0.25 mm I.D., 0.20 µm (Product No. 29688-U); 2) SLB-IL111, 200 m × 0.25 mm I.D., 0.20 µm (Product No. 29689-U); oven: 175 °C (SP-2560), 150 °C (SLB-IL111); inj. temp.: 250 °C; carrier gas: hydrogen; detector: FID, 250 °C; injection: 1.0 µL, 50:1 split; liner: 4 mm I.D., split type, cup design; sample: Mix of C18:0, C18:1 (from partially hydrogenated vegetable oil [PHVO]), C18:2, and C18:3 FAME isomers

CLA FAME Isomer Mix

The stomachs of several mamalian species have four compartments. These mammals are known as ruminants, and include cows, sheep, goats, and deer. Ruminant fat contains conjugated linoleic acid (CLA) isomers, which are C18:2 fatty acids in which a single carbon-carbon bond separates the two double bonds. A custom mixture containing four CLA FAME isomers was prepared and injected on each column. Run conditions were adjusted to achieve maximize resolution. The optimized chromatograms are shown in Figure 2. Peak identification was assigned by injecting each isomer individually. Both columns were able to provide resolution, although with slightly different elution patterns.

GC Conditions

columns: 1) SP-2560, 200 m × 0.25 mm I.D., 0.20 µm (Product No. 29688-U); 2) SLB-IL111, 200 m × 0.25 mm I.D., 0.20 µm (Product No. 29689-U); oven: 175 °C (SP-2560), 150 °C (SLB-IL111); inj. temp.: 250 °C; carrier gas: hydrogen; detector: FID, 250 °C; injection: 1.0 µL, 50:1 split; liner: 4 mm I.D., split type, cup design; sample: 4-component CLA FAME isomer mix, 0.5 mg/mL in methylene chloride

Rapeseed Oil FAMEs with CLA FAME Isomers

Rapeseed oil is a simple vegetable oil that contains a series of saturated and unsaturated fatty acids ranging from C14 through C24 in carbon number. A custom mixture containing rapeseed oil FAMEs plus four CLA FAME isomers was prepared and injected on each column. Run conditions were identical to those previously used. The resulting chromatograms are shown in Figure 3. Peak identification was assigned based on previous work.

Monitoring the elution locations of the polyunsaturated C18 FAME isomers (peaks 5-10) relative to the saturated and monounsaturated FAME isomers is an indication of a column’s ability to undergo dipole-induced dipole interactions. Both columns exhibited great relative retention of these isomers. In fact, the SLB-IL111 retained the CLA FAME isomers (C18:2 species) after C22:0. Also of note is that the co-elutions on one column are fully resolved on the other, providing complementary data.

GC Conditions

columns: 1) SP-2560, 200 m × 0.25 mm I.D., 0.20 µm (Product No. 29688-U); 2) SLB-IL111, 200 m × 0.25 mm I.D., 0.20 µm (Product No. 29689-U); oven: 175 °C (SP-2560), 150 °C (SLB-IL111); inj. temp.: 250 °C; carrier gas: hydrogen; detector: FID, 250 °C; injection: 1.0 µL, 50:1 split; liner: 4 mm I.D., split type, cup design; sample: Mix of rapeseed oil FAME isomers and CLA FAME isomers

38-Component FAME Isomer Mix

Determining the degree of fatty acid unsaturation of a food product is difficult because foods can contain a complex mixture of saturated, monounsaturated, and polyunsaturated fatty acids with a variety of carbon chain lengths.

The Supelco 37-Component FAME Mix contains methyl esters of fatty acids ranging from C4 to C24, including key monounsaturated and polyunsaturated fatty acids, making this standard very useful to food analysts since it can be used to identify fatty acids in many different types of foods. A custom standard comprised of this mix plus C22:5n3 FAME was prepared, and analyzed on each column under identical conditions. Figure 4 shows the chromatograms obtained from both columns.

GC Conditions

columns: 1) SP-2560, 200 m × 0.25 mm I.D., 0.20 µm (Product No. 29688-U); 2) SLB-IL111, 200 m × 0.25 mm I.D., 0.20 µm (Product No. 29689-U); oven: 100 °C (13 min), 4 °C/min to 240 °C (30 min); inj. temp.: 250 °C; carrier gas: hydrogen, 27 cm/sec; detector: FID, 250 °C; injection: 1.0 µL, 50:1 split; liner: 4 mm I.D., split type, cup design

Discussion

The SP-2560/SLB-IL111 pairing allows the most comprehensive fatty acid composition information possible, able to provide accurate results (qualitative and quantitative) for both saturated and trans fatty acids. Observations include:

• While not shown, increased resolution was achieved when comparing chromatograms from 200 m versions to chromatograms from 100 m versions.
• Analytes tend to elute from the SLB-IL111 at a lower oven temperature (Figures 1-4)
• SLB-IL111 provides resolution of C18:1Δ9c (one of the most abundant naturally occurring unsaturated fatty acids) from all trans FAMEs (Figure 1).
• SP-2560 and SLB-IL111 provide different elution patterns for the CLA FAME isomers analyzed (Figures 2 and 3)
• SLB-IL111 provides increased retention of unsaturated FAME isomers (Figures 3 and 4).
• SP-2560 provides better resolution of saturated FAME isomers from unsaturated FAME isomers (Figure 4).

Conclusion

The SP-2560 chemistry was first introduced in 1983, and the SLB-IL111 chemistry was first introduced in 2010. The new 200 m versions of these chemistries indicate our commitment to remain at the forefront of detailed analysis ofcis/trans FAME isomers. The SP-2560/SLB-IL111 pairing allows the most comprehensive fatty acid composition information possible.