Table of Contents

Sugar analysis using anhydrous hydrogen fluoride

Introduction

An alternative to hydrolysis of polysaccharides with dilute carboxylic acids in order to get monosaccharides, is solvolysis (reaction with solvent), with anhydrous hydrogen fluoride. The result of the solvolysis, after evaporation of the solvent, is a glycosyl fluoride which most often is directly hydrolysed to the monosaccharide upon addition of water as the last amount of HF is most difficult to evaporate. If, however, the hydrogen fluoride is completely removed the resultant glycosyl fluorides must be hydrolysed with acid, normally an hour reflux with 0.5 M TFA. The rest of the reaction sequence i.e. BH4-reduction, and acetylation, are as described for TFA hydrolysis. As hydrogen fluoride reacts with glass the solvolysis is often performed in a specially designed reaction apparatus made of Kel-F, a trifluorochloro-ethylene polymer. Teflon can also be used but the amount of liquid in the vessels can hardly be observed without a strong lamp. In Kel-F it can easily be observed, however. The use of anhydrous hydrogen fluoride is extremely hazardous and should normally be performed in the specially designed apparatus which is completely sealed. For complete solvolysis to the monomers usually room temperature reactions are used.
The main advantage with anhydrous hydrogen fluoride is that amides are not cleaved but glycosidic linkages are, i.e. the glycosidic linkage of a 2-acetamido-2-deoxy-hexoside can be quantitatively cleaved. When TFA is used amide hydrolysis is a competing reaction and the glycosidic linkage of the liberated 2-aminohexoside is not possible to cleave at all.

Flow scheme

Solvolysis Hydrolysis Reduction Acetylation

Reagents

Full list of chemicals

Procedure

  1. Transfer dried sample (ca 0.2 mg) to the HF reaction vessel together with a dry mini-size magnetic rod.
    Option: Add internal standard, 50 µg of xylose or suitable sugar.
  2. Cool with CO2+ethanol or liquid N2, the reaction vessel and heat, with a hair dryer, the HF supply vessel (Kel-F) until the required volume has distilled over (ca 0.5 mL). Leave for 3h at room temperature.
  3. Evaporate solvent by applying vacuum slowly to the reaction vessel and when it looks dry add 1 mL MeOH, evaporate. Transfer the reaction mixture with 0.5 mL 0.5M TFA and reflux for 1h. Evaporate to dryness, add 0.5 mL MeOH and evaporate again.
    Optional: No hydrolysis.
  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 0.5 mL 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 by 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.

Comments

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 200 µL reduce and acetylate as described above. Freeze-dry the remainder.) Dissolve in 50 µL EtOAc.

Detection and Quantification

GLC with e.g. DB1 or DB225 fused silica columns. (100% methyl silicone or 95% methyl/5% phenyl silicone)