A practical guide to structural analysis of carbohydrates

Hydrolysis with trifluoroacetic acid and morpholinoborane

Acid labile sugars often degrade completely during hydrolysis. For that reason conditions have been worked out in which you can reduce during the hydrolysis to get the corresponding alditols. Two reagents are available, dicyanoborohydride and 4-methylmorpholinoborane. Only the latter is commercially available but is unfortunately easily degraded during the hydrolysis, especially if the hydrolysis is performed in heat. If acid labile sugars as well as "normal" sugars are present in a saccharide, a two step reduction is recommended using sodium borohydride in the second step.
Note! Kdo gives a 1:1 mixture of 3-deoxy-D-glycero-D-galacto- and 3-deoxy-D-glycero-D-talo-octono-1,4-lactones.

Flow scheme

Hydrolysis/Reduction right arrow BH4- -reduction right arrow Acetylation


  • Trifluoroacetic acid (TFA), 0.5 or 2 M
  • Methanol
  • Borane-4-methylmorpholine complex
  • Sodium borohydride, 0.25 M in 1 M NH4OH
  • Ammonia 1 M
  • Acetic acid, 10% in methanol
  • Acetic anhydride


  1. Reduction of the acid labile sugar. The sugar (1 equivalent) and morpholinoborane (10 eq.) in 0.5 M TFA is kept at 100° for 2h.
    E.g. 1 mg hexose (6 µmol) requires 7 mg morpholinoborane (60 µmol).
  2. The solution is evaporated to dryness and after addition of methanol again evaporated to dryness.
  3. Reduce with 0.3 mL fresh solution of NaBH4 in NH4OH for 30 min at 20°.
  4. 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.
  5. Acetylate with TFA/Ac2O (0.2+0.2 mL) 10 min 100°.
  6. Partition between dichloromethane and water.

Test substances, Standards

Test your reagents with stachyose, αDGal(1->6)αDGal(1->6)αDGlc(1<->2)βDFruf, which should give galactose, glucose and mannose (glucose and mannose from Fruf).