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
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.
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.
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.
- Trifluoroacetic acid (TFA), 0.5 or 2 M
- Sodium borohydride, 0.25 M in 1 M NH4OH
- Ammonia 1 M
- Acetic acid 10% in methanol
- Acetic anhydride
- 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.
- Hydrolyse in ~0.3 mL 2M TFA at 120° for 2h or in 0.5M TFA at
100° 12-16 h.
- Evaporate the solution to dryness by a stream of compressed air,
add 0.5mL MeOH, and evaporate. Repeat once.
- Reduce with 0.3 mL fresh solution of NaBH4
for 30 min at 20°
- 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.
- Acetylate with 0.1 mL Ac2O
and 0.1 mL pyridine 100° 20 min.
Add 50 µL of water if problems.
- Evaporate the solution and add 0.5 mL toluene, evaporate to dryness.
- 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.
- For an exact weight of a sample of ca 0.2 mg take 1-2 mg and
dissolve in 1 mL of water and take out appropriate volume.
- Too little liquid will give only drops on the wall and little
in the bottom, so use >0.3 mL.
- Use blow down equipment throughout, normally compressed air is dry
enough to be used, if not use N2
- Trace amounts of acid are almost impossible to get rid of but will not
harm this step. Bubbles of hydrogen on the bottom normally indicate
but in ammonia they may be scarce.
The solution of NaBH4
in ammonia easily lasts a week.
If not enough BH4
- is present cyclic acetates will be formed which
are fast-moving on the GLC column and with m/z 115 and 157 as
prominent peaks in the mass spectrum.
-reduction, which is
performed as described above except that NaBD4
is used instead of
, and will give CHDOAc as the top fragment.
- Acetic acid will convert the borohydride to boric acid which must be
removed by acidic methanol as its methyl ester i.e. methyl borate.
The procedure must be repeated. If boric acid is left it may form
complexes with the alditols and cause underacetylation.
- Be careful so that no water is removed with the organic phase.
Use holder for the Reacti-vial. Alternatively partition may be
effected with the pasteur pipette.
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.