Analysis of carrageenans using capillary electrophoresis.
This thesis reports the use of capillary electrophoresis (CE) for the analysis of carrageenans,
anionic polysaccharides extracted from red seaweeds and widely used in the food industry
for their gelling and thickening properties. The three main types, kappa, iota and lambda,
differ in the number of sulfate groups and the presence or absence of a 3,6-anhydro bridge
in the disaccharide residue repeat unit. CE separates analytes according to their charge to
frictional coefficient ratios, therefore it is suitable to separate these biopolymers. In order
to detect polysaccharides in CE, our approach consisted in derivatising the reducing ends
of the saccharides by reductive arnination with a fluorophore, l-arninopyrene-3,6,8-
trisulfonate (APTS). This allowed sensitive detection by laser induced fluorescence.
Method development gave optimal conditions for separation using a polyvinyl alcohol
coated capillary and a 25 mM ammonium acetate, pH 8.0 background electrolyte. The
effects of changes of both instrumental parameters (temperature, injection mode, field
strength) and, the composition of the BGE (concentration and pH) are reported, and
explained in terms of the physical chemistry of the BGE and the biopolymers.
The conditions of the derivatisation reaction were studied in order to minimise degradation
due in particular to acid catalysis and to reduction of the reacting sites occurring in
competition with derivatisation. Characterisation of the derivatised carrageenans by SEC-MALLS-
RI was performed and showed that the extent of degradation occurring during the
labelling reaction was a maximum of 40 % for kappa and 20 % for iota and lambda. The
presence of the label APTS in excess and its reaction with the reagents during the labelling
reaction produces peaks interfering with those from the carrageenan. A sample clean-up was
therefore required before injection onto CEo A comparison was made of a range of clean-up
procedures (centrifugation, dialysis, preparative SEC) to remove side products of the
reaction and salts and to concentrate the carrageenans.
Various seaweed extracts were analysed, including standards of carrageenans not available
commercially. This study revealed that carrageenans are complex structures, and often
occurring as hybrids between sUb-types. CE has the ability to characterise these hybrids,
unlike spectroscopic methods which detect individual residues.
When using actual food products, preliminary steps such as defatting and dialysis were
found to be necessary to allow satisfactory detection of carrageenans. Finally the strategy
for sample purification, derivatisation, clean-up and separation was successfully applied to
additive mixtures used as raw materials in the food industry and to finished products (jelly,
dairy products). CE has proved to be a fast and sensitive method to identify and provide
semi-quantitative information on carrageenans present in such mixtures.