Ionic Liquid GC Column Option for the Analysis of Omega 3 and Omega 6 Fatty Acids

Essential fatty acids are nutrients that must be obtained from the diet because humans lack the anabolic processes for their synthesis. Two closely related groups of essential fatty acids are the omega 3 and omega 6 fatty acids. These unsaturated fatty acids contain the initial double bond located directly after the third (omega 3) or the sixth (omega 6) carbon atom, as measured from the methyl end of the compound.

Omega 3 and omega 6 fatty acids are typically analyzed using gas chromatography (GC) after their conversion to fatty acid methyl esters (FAMEs). Columns described in promulgated methods^1,2 contain a stationary phase based on polyethylene glycol (PEG), and often have ‘wax’ in the name.

In this work, a new ionic liquid GC column, SLB^®-IL60, is evaluated against an Omegawax^® column for its suitability for this application. The SLB-IL60 column has selectivity similar to ‘wax’ columns, but is different enough to provide a unique elution pattern. It is not based on a PEG phase. Instead, it has various functional groups that allow for an increased number of interaction mechanisms compared to a PEG phase. Specifications for both columns are:

Omegawax

• Phase: Bonded; Poly(ethylene glycol)
• Temp. Limits: 50 °C to 280 °C (isothermal or programmed)

SLB-IL60

• Phase: Non-bonded; 1,12-Di(tripropylphosphonium)dodecane bis(trifluoromethylsulfonyl)imide
• Temp. Limits: 35 °C to 300 °C (isothermal or programmed)

Experimental

The GC conditions used are from the AOAC^® 991.39 and AOCS^® Ce 1i-07 methods. This was possible as both methods share the same set of run conditions. Two standard mixes were initially analyzed to gauge elution patterns. An individual FAME was also analyzed to confirm identification. Overall, the following standards were used:

1. Supelco 37-Component FAME Mix
2. PUFA No. 3 (from menhaden oil), diluted in 1 mL of hexane
3. C22:5n3 methyl ester

C4-C24 FAME Mix

A custom 38-component mix was prepared by the addition of C22:5n3 FAME to the stock 37-component FAME mix. The resulting mix was then analyzed on both columns under identical conditions. Resulting chromatograms are shown in Figure 1. Observations are that the SLB-IL60 column provides:

• Resolution of C18:1n9c (peak 17) and C18:1n9t (peak 18)
• Elution of C18:1n9t (peak 18) before C18:1n9c (peak 17)
• Elution of C18:2n6t (peak 20) before C18:2n6c (peak 19)
• Elution of C20:3n3 (peak 28) before C20:3n6 (peak 26)
• Elution of C22:6n3 (peak 37) before C22:5n3 (peak 35) – confirmed by analysis of an individual C22:5n3 standard

GC Conditions

column 1: Omegawax, 30 m x 0.25 mm I.D., 0.25 µm (Product No. 24136); column 2: SLB-IL60, 30 m x 0.25 mm I.D., 0.20 µm (Product No. 29505-U); oven: 170 °C, 1 °C/min to 225 °C; inj. temp.: 250 °C; carrier gas: helium, 1.2 mL/min; det.: FID, 260 °C; injection: 1 µL, 100:1 split; liner: 4 mm I.D., split/splitless type, single taper wool packed FocusLiner™ design; sample: Supelco 37-Component FAME Mix + C22:5n3, in methylene chloride (Product No. 17269)

Peak Identification

1. C4:0 2. C6:0 3. C8:0 4. C10:0 5. C11:0 6. C12:0 7. C13:0 8. C14:0 9. C14:1 10. C15:0 11. C15:1 12. C16:0 13. C16:1 14. C17:0 15. C17:1 16. C18:0 17. C18:1n9c 18. C18:1n9t 19. C18:2n6c

20. C18:2n6t 21. C18:3n6 22. C18:3n3 23. C20:0 24. C20:1n9 25. C20:2 26. C20:3n6 27. C21:0 28. C20:3n3 29. C20:4n6 30. C20:5n3 31. C22:0 32. C22:1n9 33. C22:2 34. C23:0 35. C22:5n3 36. C24:0 37. C22:6n3 38. C24:1n9

PUFA No. 3 Mix

A qualitative polyunsaturated fatty acid (PUFA) methyl ester standard (made from menhaden oil) was also analyzed on both columns under identical conditions. Figure 2 shows both chromatograms. Use of the SLB-IL60 column resulted in:

• An overall faster elution (31 minutes compared to 52 minutes)
• Coelution of C20:4n3 and C20:5n3 (peaks 16 and 17)
• Elution of C22:6n3 (peak 19) before C22:5n3 (peak 18) – confirmed by analysis of an individual C22:5n3 standard

GC Conditions

column 1: Omegawax, 30 m x 0.25 mm I.D., 0.25 µm (Product No. 24136); column 2: SLB-IL60, 30 m x 0.25 mm I.D., 0.20 µm (Product No. 29505-U); oven: 170 °C, 1 °C/min to 225 °C; inj. temp.: 250 °C; carrier gas: helium, 1.2 mL/min; det.: FID, 260 °C; injection: 1 µL, 100:1 split; liner: 4 mm I.D., split/splitless type, single taper wool packed FocusLiner™ design; sample: PUFA No. 3 Mix (Product No. 47085-U), diluted in 1 mL of hexane

Peak Identification

1. C14:0 2. C16:0 3. C16:1n7 4. C16:2n4 5. C16:3n4 6. C18:0 7. C18:1n9 8. C18:1n7 9. C18:2n6 10. C18:3n4

11. C18:3n3 12. C18:4n3 13. C20:1n9 14. C20:3n3 15. C20:4n6 16. C20:4n3 17. C20:5n3 18. C22:5n3 19. C22:6n3

Conclusion

Compared to existing ‘wax’ columns, the SLB-IL60 column offers unique selectivity, some cis/trans resolution, and the faster elution of analytes. The elution of trans isomers before cis isomers of the same carbon chain length, plus degree and position of unsaturation, demonstrates the analyte-stationary phase mechanism difference of the SLB-IL60. These differences make the SLB-IL60 a good column choice for the analysis of omega 3 and omega 6 fatty acid methyl esters.