A practical guide to structural analysis of carbohydrates

Fatty acid analysis using methyl esters

The total content of fatty acids and the distribution over different types of fatty acids present in a lipopolysaccharide can be investigated using a combined procedure with hydrolysis followed by methanolysis and GLC analysis of the methyl esters. The fatty acids can be liberated both through acidic and alkaline hydrolysis. The latter liberates all fatty acids as the sodium salts which must be derivatized before they can be analysed by GLC. Ester-bound fatty acids can be liberated e.g. by NaOMe or mild hydrazinolysis. Strong alkaline hydrolysis or strong hydrazinolysis removes both the ester- and amide bound fatty acids. Alkaline hydrolysis has, however, side reactions. A substantial amount of α,β-unsaturated fatty acids are formed by β-elimination of 3-acyloxyacyl residues.


  • 4M HCl
  • Chloroform
  • Na2SO4 for drying
  • Methanol
  • Acetyl chloride
  • Trifluoroacetic anhydride (TFAA)
  • Acetonitrile
  • Heptadecanoic acid C17 (6 mg/mL CHCl3 )
  • Half saturated NaCl solution


  1. 1-2 mg of LPS is weighed carefully in a screw cap tube and 20 µL of the C17 solution is added (=120 µg)
  2. Evaporate to dryness, add 0.5 mL 4 M HCl and keep at 100 °C for 4h.
  3. Dilute the reaction mixture with 5 mL water and partition between an equal volume of chlororform, keep the bottom chloroform phase. Repeat once.
  4. Add Na2SO4 (200 mg) to the pooled chloroform phases and stir in a sealed flask. Filter or centrifuge after 1 h, concentrate to 1 mL, transfer to screw cap tube and blow down to dryness.
  5. Dissolve in 0.5 mL MeOH, add 70 µL acetyl chloride (2M), keep at 85 °C for 16 h
  6. Add 1 mL of the half saturated NaCl solution, extract with 3 mL CHCl3 (bottom phase). Repeat once. Combine the CHCl3 phases and blow down to dryness.
  7. Add 50 µL acetonitrile and 50 µL TFAA,keep at 100°C for 2 min. After cooling add 50 µL acetonitrile. Inject on DB5 on GLC.


  • C17 does not occur in natural products and should therefore hopefully not co-elute with any of the sample peaks.
  • The prehydrolysis is necessary for the amide cleavage.
  • Leave an hour for drying because water may destroy the reaction.