Periodate Oxidized Polysaccharides : Degradation Patterns and Analysis of Residual Periodate

The main goal of this thesis was to study and compare the degradation patterns of the periodate oxidized polysaccharides mannuronan, pullulan and hyaluronan in both alkali and acid. Oxidized polysaccharides were reduced prior to degradation in acid. Analysis were made by SEC-MALLS. The analyses confirmed that 20-60 % of the oxidized residues in mannuronan were resistant towards alkaline β-elimination, and that 30-45 % of the oxidized residues in pullulan were not cleaved by alkali, which can be attributed to the double oxidation of α-1,6 linked glucose residues in pullulan. The percentage of cleaved residues exceeded the percentage of assumed oxidized residues in hyaluronan (120-130 %), probably because of several oxidized residues than estimated. The experimental conditions of acid hydrolysis were considered as too mild since the acid hydrolysis was not completed during the time of analysis. The SEC-columns did not separate the smallest fragments adequately. Another goal of this thesis was to study the kinetics of periodate oxidation of polysaccharides, and to investigate if all periodate is consumed for low P0 (degree of added periodate). Periodate oxidation of higher concentrations of Alginate LF 10/60 was studied by titration with thiosulphate, and the results showed an increase in the rate of periodate oxidation with increasing concentrations. A method for detecting accurate residual periodate in periodate oxidized polysaccharides by RP-HPLC with a C-18 column was developed. Periodate oxidation (2, 8 and 20 %) at 4 °C of Alginate LF 10/60 and pullulan (both 4.44 mg/ml) was completed within 4 hours, whereas complete periodate oxidation (2 and 8 %) of mannuronan (4.44 mg/ml) did not occur until after 24-48 hours. The RP-HPLC with the C-18 column was considered as a more accurate method for detecting residual periodate than the thiosulphate titration.