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Vaccimonitor
Print version ISSN 1025-028XOn-line version ISSN 1025-0298
Abstract
CUEVAS, Matilde et al. Evaluation of Different Chromatographic Matrixes for Molecular Size Determination of the Neisseria meningitidis Polysaccharide C by HPLC. Vaccimonitor [online]. 2001, vol.10, n.2, pp.25-28. ISSN 1025-028X.
The immunological properties of N. meningitidis Polysacharide C are closely related to its molecular size, a physicochemical parameter determined by conventional Gel filtration chromatography (GFC) as recommended by the World Health Organization (WHO). Nevertheless, the distribution coefficient (Kd) determination by this method is timeconsuming and requires a relatively large quantity of sample. The High Performance Liquid Chromatography (HPLC) is more efficient, so it could substitute the conventional method. For that purpose, we evaluated different chromatographic columns and run conditions to determine Kd by (GFC) HPLC. In all cases the void and the total volumes, and also the capacity factor, the column efficiency, the peak asymmetry and the repeatability were determined. The mobile phase was 0,2 mol/L NaCl. The Sepharose CL 4B (Pharmacia, Sweden) resulted the best choice, because it had more capacity to retain the sample; although it was less efficient than the TSK G3000PW (Tosohaas, Japan) column, it showed the same peak asymmetry, the best repeatability and it was cheaper. Different flow rates, sample concentrations and sample volumes were studied to determine the best analysis conditions, which resulted in: an injections of 100 μl of sample (0,1 mg) with a 0,3 mL/min of flow.
Keywords : Polysacharide C; HPLC; Kd.