Impact of HILIC Amino-Based Column Equilibration Conditions on the Analysis of Chitooligosaccharides

Obtaining well-defined Chitooligosaccharides (COS) structures is very important as their physicochemical and biological properties depends strongly on their degree of polymerization (DP) and degree of N-acetylation (DA). Hydrophilic Interaction Liquid Chromatography (HILIC), that is commonly used for COS analyses, suffers from several drawbacks, related to their complex structure, leading to broadening/splitting of the chromatogram signals. We investigated, herein, on the role of the equilibration step of an amino-based HILIC column on the separation of COS DP <= 5. We demonstrated that COS could be separated according to their DP after equilibrating the column by a NaHCO3 buffer solution (100 mM, pH = 10) and mild elution chromatographic parameters (neutral mobile phase, r.t.); or according to their DA after equilibrating the column by an NH4Ac buffer solution (50 mM, pH = 4.5). Also, the nature of the counterion of the column stationary phase was found to affect both retention times and signal profiles of analyzed COS.