Polysaccharide determination in protein/polysaccharide mixtures for phase-diagram construction

The effect of protein removal from protein–polysaccharide mixtures on the determination of total
sugar concentration for phase-diagram construction was explored by response surface methodology.
Phenol–sulphuric acid method was used to estimate the total sugar concentration whereas phase separation
and microstructure of biopolymers in the mixtures were probed by means of fluorescent microscopy.
All the factors studied (trifluoroacetic acid (TFA) volume, protein concentration, precipitation time) had
significant influence on the polysaccharide recovery from the mixtures. The overall pattern of protein
precipitation with TFA was also found to highly depend on protein–time interactions. Images taken at different
mixture concentrations and with two different filter sets revealed a phase-separated system with
the guar gum domains surrounded by a continuous protein phase. Experimental measurements as well as
microscopical evidence suggests that, for greater than 60% polysaccharide recovery (±6% error), the protein
content in the mixtures should be <∼5.5% (w/v) with less than 0.5mL TFA combined with more than
30 minrest under the influence of TFA. The results of the present investigationshowedthatwhenattempting
to construct phase diagram of mixed biopolymer systems the equilibrium phases must be diluted to
a protein content <∼5% (w/v) before TFA treatment if the maximum polysaccharide concentration is to
be determined.