Table of Contents

Sugar analysis using alditol acetates


The hydrolysis of glycosides and polysaccharides to reducing sugars and the concomitant conversion to alditol acetates (borohydride reduction and acetylation) is a standard method to analyse polysaccharides containing aldoses, ketoses, deoxyaldoses, and acetamidohexoses and other related sugars. Sugars that cannot be observed with this analysis are uronic acids, ulosonic acids (e.g. Kdo), 4-aminosugars, or charged species like phosphorylated sugars which may arise in a hydrolysate. The acids are not observed because the sodium salt of the acid that is formed on addition of NaBH4 is not volatile. Of special concern is the difference in rate of hydrolysis between sugars. Thus, uronic acids are seldom completely hydrolysed and the sugar that the uronic acid is linked to is underrepresented. 2-Acetamidohexoses undergo partial N-deacetylation and the resulting 2-aminosugars are not hydrolysed at all and both the 2-aminosugar and the sugar to which it is linked are underrepresented. Normally, yields of 60-80% are obtained. 4-acetamido sugars are hydrolysed to pyrrole derivatives which are polymerised and such sugars are therefore not observed. Methanolysis is however possible with 4-acetamido sugars.

Note that all ketoses on reduction are expected to yield approximately equal amounts of both the R- and the S-isomer, which may or may not separate on GLC analysis. Thus, fructose gives an equimolar mixture of glucitol and mannitol.

fructose glucitol mannitol
Fructose Glucitol

A number of acids may be used for the hydrolysis, the most common are trifluoroacetic acid, (TFA) sulfuric acid, and hydrochloric acid. Trifluoroacetic acid is somewhat weaker than the others but is fairly easy to evaporate.

Flow scheme

Hydrolysis Reduction Acetylation


Full list of chemicals


  1. Transfer sample (ca 0.2 mg, ~1 µmol) to a 13 x 100 mm screw cap tube.
    Option: Add internal standard, 50 µg of xylose or suitable sugar.
  2. Hydrolyse in ~0.3 mL 2M TFA at 120° for 2h or in 0.5M TFA at 100° 12-16 h.
  3. Evaporate the solution to dryness by a stream of compressed air, add 0.5mL MeOH, and evaporate. Repeat once.
  4. Reduce with 0.3 mL fresh solution of NaBH4 in NH3 for 30 min at 20°
  5. Quench with 0.5 mL 10% HOAc in MeOH, evaporate to dryness. Add 0.5 mL 10% HOAc in MeOH and evaporate to dryness. Repeat once or twice. Add 0.5mL MeOH and evaporate to dryness. Repeat once or twice.
  6. Acetylate with 0.1 mL Ac2O and 0.1 mL pyridine 100° 20 min. Add 50 µL of water if problems.
  7. Evaporate the solution and add 0.5 mL toluene, evaporate to dryness. Repeat once.
  8. Partition between 0.5 mL H2O and 0.5 mL EtOAc by stirring fast but not violently using a triangular magnetic rod in a conical vial (Reacti-vial type) for a couple of minutes. Transfer the upper EtOAc phase to the old rinsed tube. Add another 0.5 mL EtOAc and extract. Repeat a third time. Concentrate to dryness dissolve in ca 0.2 mL EtOAc, transfer into sample tube and concentrate to 25-50 µL.


Test substances, Standards

A mixture of Rha, Fuc, Ara, Rib, Xyl, Man, Gal, Glc, GlcNAc, GalNAc. (Take 0.5 mg each dissolve in 1 mL of H2O, take 0.2 mL reduce and acetylate as described above. Freeze-dry the remainder. Dissolve in 50 µL EtOAc.