4.4. Monosaccharide analysis by HPAEC-PAD chromatography

Polysaccharide hydroxylates (10 μL) were injected onto a Dionex^® ICS-5000^® HPAEC-PAD system and the monosaccharide analysis was done using an 8 mM NaOH (Fisher^® HPLC grade) isocratic gradient (0.25 mL/min) for 40 min followed by a 6 min wash step (100 mM NaOH) and a 15 min re-equilibration step. Detection was achieved using pulsed amperometric detection (PAD). Spiking experiments for identification of 6-deoxy-manno-heptopyranose and d-mannopyranose were performed on a monosaccharide by monosaccharide basis. This was due to the impact of the addition of a monosaccharide on the retention times of the monosaccharide mixture. The polysaccharide hydroxylate samples were supplemented with monosaccharide standards (10/25 μM final concentration) and injected onto the column (10 μL).