A practical guide to structural analysis of carbohydrates

Sugar analysis using methyl glycosides

For some sugars it is either impossible or inconvenient to use the alditol acetate method. Thus acidic sugars like uronic acids are not detected by that method. There is a but however, if the hydrolysate is carefully evaporated the uronic acids will lactonise and the lactone will be reduced to the alditol. The analysis of uronic acids are preferably done with authentic references. Sugars like Kdo and NeuAc can preferentially be analysed as their methyl glycoside methyl esters. When acetamido sugars are present re-N-acetylation is necessary. The disadvantage with this method is that it gives more than one peak per sugar (α- and β-pyranosides and α- and β-furanosides and possibly lactones and esters) compared to the alditol acetate method which gives only a single peak for each sugar.

Flow scheme

Solvolysis right arrow Re-N-acetylation right arrow Acetylation / Trimethylsilylation


  • Methanol
  • Acetyl chloride
  • Hydrogen chloride in methanol (1 M)
  • Pyridine
  • Acetic anhydride
  • Sil-A (commercial silylating reagent from Sigma, pyridine, hexametyldisilazane and trimethylchlorosilane 9:3:1)
  • Silver carbonate, Ag2CO3


  1. Transfer 0.2 mg of dry polysaccharide to a 13 x 100 mm screw-cap tube.
  2. Add 0.5 mL dry methanol. Add 35 µL of acetyl chloride (1 M), and blow nitrogen through the tube and seal it.
    Alternatively: add 0.5 mL 1 M HCl in methanol.
  3. Leave the sample for 24 h at 85°C. (Alternatively 12 h see comments)
  4. Evaporate the solution to dryness with a stream of air. Add 0.5 mL of MeOH and evaporate to dryness.
  5. If trimethylsilyation is desired and the polysaccharide contains amino sugars, re-N-acetylate with 25 µL of acetic anhydride for 4 h at 20°C. Evaporate to dryness with a stream of air.
  6. Acetylate by adding 100 µL Ac2O and 100 µ pyridine 100°C 20 min. Evaporate to dryness with a stream of air. Dissolve in EtOAc and analyse by GLC.
    Trimethylsilylate at 20°C for 30 min with 100 µL of Sigma SilA solution.
    Evaporate the solution with a stream of air and redissolve the sample in n-hexane. Filter the solution through glass down to a sample tube before analysis by GLC.


  • Dry material minimises the number of extra peaks in the GLC chromatogram.
  • The polysaccharide need not necessarily dissolve immediately but will gradually do so during the reaction.
  • For some sugars it takes time to establish equilibrium conditions (pyranosides, furanosides, α- and β-configuration, lactones etc.) and 24 h is therefore recommended. The solvolysis is most certainly finished earlier. Especially uronic acids have several peaks in their chromatograms and should be allowed to equilibrate properly.
  • The last traces of acid are difficult to get rid of and may optionally be disposed of by Ag2CO3 addition and centrifugation, but this is not recommended.
  • If starting with 2-acetamido-2-deoxy-sugars, amide groups will be solvolysed to amino groups as a competing reaction and if trimethylsilylated derivatives are wanted, these amino groups must be re-N-acetylated before GLC-analysis.
  • The choice of OH-protecting groups is arbitrary, and acetates and TMS-ethers are equally good. In rare cases the time for acetylation must be prolonged to ensure complete derivatisation, try 1 h 100°C.