Pw. Needs et Rr. Selvendran, A CRITICAL-ASSESSMENT OF A ONE-TUBE PROCEDURE FOR THE LINKAGE ANALYSIS OF POLYSACCHARIDES AS PARTIALLY METHYLATED ALDITOL ACETATES, Carbohydrate research, 254, 1994, pp. 229-244
Methylation analysis data for the exudate gum from Actinidia deliciosa
, obtained by a one-tube procedure, were compared with published data.
Several discrepancies were apparent, which were subsequently attribut
ed to differences in the partially methylated alditol acetate (PMAA) d
erivatisations used. These deficiencies were also noted during methyla
tion analyses of pectic polysaccharides from potato, xyloglucan from t
amarind seed, and amylase-resistant starches from maize and pea. Under
estimation of terminal galactosyl and glucosyl groups was traced to de
gradation of their partially methylated alditol derivatives in the per
chloric acid-catalysed acetylation procedure used as the final stage o
f PMAA preparation. In addition, extended acid-catalysed reactions res
ulted in low values for terminal arabinosyl and fucosyl groups. Triflu
oromethanesulfonic (triflic) acid, adopted in these reactions as a saf
er, non-oxidative alternative to perchloric acid, gave the same losses
although it catalysed acetylation of non-terminal derivatives success
fully. Underestimation of terminal arabinosyl groups was traced to ina
dequate extraction of the corresponding PMAAs into dichloromethane. Su
bsequently, other highly methylated sugar derivatives, such as those o
btained from the alditol end groups of reduced oligosaccharides, prove
d highly susceptible to this loss; in addition, their tendency to evap
orative loss, particularly during the early stages of their derivatisa
tion (i.e., after hydrolysis and reduction rather than after acetylati
on), was demonstrated. A method which minimises these losses is descri
bed. Long-term instability of samples for gas chromatography was preve
nted by washing them with water prior to storage to remove residual ac
etic acid.