Fractionation of FAMEs Using Silver-Ion SPE

When silver ions are anchored onto strong cation exchange (SCX) solid phase extraction (SPE) functional groups as counter-ions through electrostatic interaction, they have the ability to form polar complexes with the double bonds of unsaturated FAMEs under normal-phase conditions. More specifically, pi electrons of the FAME double bonds act as electron donors and silver-ions act as electron acceptors. The strength of the interactions between FAMEs and the silver counter-ions varies depending on the structure of the FAME:
  • Saturated FAMEs (no double bonds) have no interactions. Therefore, they are poorly retained.
  • Double bonds in the cis orientation offer more steric accessibility than their trans counter parts, and therefore form stronger polar complexes. As a result, cis fatty acids are more strongly retained than trans fatty acids.
  • FAMEs with a greater number of double bonds have stronger interactions than those with fewer double bonds. Trienes are retained stronger than dienes, which are retained stronger than monoenes.

The differences in the strengths of these polar complexes between classes of FAMEs and the silver counter-ions can be exploited, allowing for fractionation of cis and trans isomers by adjusting the elution solvent strength. These fractions can then be analyzed by GC to yield very detailed information concerning the cis and trans fatty acid make-up.

A typical procedure for a 750 mg, 6 mL silver-ion SPE tube is:
  • Derivatize the fatty acids in a sample to FAMEs, using hexane as the final solvent.
  • Condition the SPE tube with 4 mL acetone followed by 4 mL hexane.
  • Add 1 mL of the sample extract (in hexane).
  • Elute fraction 1 using 6 mL hexane:acetone (96:4).
  • Elute fraction 2 using 4 mL hexane:acetone (90:10).
  • Elute fraction 3 using 4 mL acetone.
  • If necessary, fractions can be concentrated using a gentle stream of nitrogen to 1 mL to achieve lower levels of detection.
  • Analyze each fraction separately by GC.

Fractionation of cis/trans FAMEs (strength of the interaction is greater for cis FAMEs than for trans FAMEs) and also for the fractionation of FAMEs by degree of unsaturation (strength of the interaction increases with increasing number of double bonds) is typically:
  • Fraction 1 = contains 100% of C18:0, 100% of the C18:1 trans, and 2% of the C18:1 cis analytes.
  • Fraction 2 = contains 98% of the C18:1 cis analytes.
  • Fraction 3 = contains 100% of the C18:2 cis/cis analytes.