Hpaec system

One milligram of polysaccharide was hydrolyzed with 6N HCl at 80 °C in a heating block for 6–8 h. The mixture was cooled and evaporated to remove the acid, resuspended in milli-Q water and passed through a Millipore-GX nylon membrane before analysis. Monosaccharides of polysaccharide hydrolysates were separated on a high-performance anion-exchange chromatographic (HPAEC) system (Dionex, Sunnyvale, CA, USA) and an anion-exchange column (Carbopac PA-10, 4.6250 mm). The analysis of monosaccharides was carried out at an isocratic NaOH concentration of 18 mM at ambient temperature.

Monosaccharide compositions were analyzed using the method reported by Singthong et al. [9 (link)]. Yanang gum was hydrolyzed in 1 M H₂SO₄ for 2 h at temperature 100 °C to provide the constituent monosaccharides. The hydrolysate was cooled down, diluted and filtrated before being injected into an HPAEC column (Dionex HPAEC system composed of a pulsed amperometric detector (PAD), Dionex Canada Ltd., Oakville, ON, Canada).

Prior to analysis, samples collected from the TIM model were thawed, centrifuged and diluted 200- to 2000-fold. Glucose and maltose concentrations were analysed using a Dionex HPAEC system (Dionex, Sunnyvale, CA, USA) consisting of a CarboPak PA-1 ion exchange column (4 × 250 mm with guard 4 × 50 mm) coupled with a pulsed amperometric detector. The mobile phase was run isocratically with a mixture of 95% 150 mM NaOH (A) and 5% 150 mM NaOH with 500 mM NaOAc (B) for 8 min for elution of glucose and maltose, and then increased to 100% B and maintained at 100% B for 6 min for elution of soluble carbohydrates of higher molecular weight. The mobile phase mixture was then reset to 95% A. The flow rate was 1 ml/min and total run time for each sample 20 min. Quality control samples run with each batch gave within-batch and between-batch variation of 1.4–3.6 and 4.4%, respectively. The sum of glucose and maltose recalculated to glucose was used for statistical analysis and is referred to as glucose in the text.