Table of Contents

Sugar analysis using methyl glycosides

Introduction

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 (a- and b-pyranosides and a- and b-furanosides and possibly lactones and esters) compared to the alditol acetate method which gives only a single peak for each sugar.

Flow scheme

Solvolysis Re-N-acetylation Acetylation / Trimethylsilylation

Reagents

Full list of chemicals

Procedure

  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.
    Or…
    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.

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