For a general introduction to hydrolysis of glycosides see
Sugar analysis using alditol acetates.
This procedure gives another type of alditol derivative, trifluoroacetates, suitable for
the separation on chiral columns. The derivative is more labile than acetates and
samples that have been standing may require reacylation.
Flow scheme
| Hydrolysis |
 |
Reduction |
|
Trifluoroacetylation |
- Trifluoroacetic acid, TFA 0.5 or 2 M
- Methanol
- Sodium borohydride, 0.25 M in 1 M NH3(aq)
- Acetic acid 10% in methanol
- Acetic acid
- Trifluoroacetic anhydride
- Acetonitrile
Procedure
- Transfer sample (ca 0.2 mg, ~1 mmol) to a 13 x 100 mm screw cap tube.
- Hydrolyse in ~0.3 mL 2M TFA at 120 °C for 2h or in 0.5M TFA at 100 °C 12-16 h.
- Evaporate the solution to dryness by a stream of compressed air, add 0.5 mL MeOH, and evaporate. Repeat once.
- Reduce with 0.3 mL fresh solution of NaBH4
in NH3
for 30 min at 20 °C.
- 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.
- Trifluoroacetylate with 50 µL Ac2O
and 50 µL acetonitrile 80 °C 4 min. Allow to cool for 10 minutes. Alt. If the peaks on GLC look bad evaporate to dryness and dissolve in dry CH2Cl2
.
- Inject the whole reaction mixture on GLC
Comment
- 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 excess NaBH4
but in ammonia they may be scarce. The solution of NaBH4
in NH3
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. NaBD4
-reduction, which is performed as described above except that NaBD4
is used instead of NaBH4
, 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 thus other things than alditol acetates.
|
|
