Resolution and signal-to-noise in analysis of carbohydrate isomers by graphitised carbon chromatography with charged aerosol detection

The effects of co-eluents and additives on separation and signal-to–noise ratio in analyses of monosaccharides by graphitised carbon chromatography (GCC) in combination with charged aerosol detection were studied. Design of experiments was used to model and predict the elution of two monosaccharide isomers, galactose and glucose and the corresponding amine at varying isocratic conditions, including concentration of water-soluble co-eluent, flow and temperature. The study confirmed the well-known order of eluent strength of the co-eluents investigated but showed that the eluent strength of MeOH was significantly lower than that of ACN, and at co-eluent concentrations ≥5% (v/v) IPA approached that of THF. Addition of NH ₃ increased retention and improved both peak shape and separation but the detector response decreased with increasing NH ₃ concentration lowering the signal-to-noise ratio. The best combination of response, retention and separation was obtained at 0.1% NH ₃. Increasing column temperature in the range of 15–50 °C resulted in decreased retention times and resolution. The corresponding Van't Hoff correlations showed negative adsorption enthalpies indicating an exothermic adsorption process driven by a decrease in entropy minimising the surface energy of the system. Isocratic elution with MeOH as co-eluent offered limited possibilities for optimisation of resolution due to the opposite effects of changes in co-eluent concentration and changes in flow rate. Elution with acetonitrile as co-eluent showed possibilities for optimisation of the resolution within the range of flow rates of 0.6-0.95 mL/min and co-eluent concentrations of 0.1–0.3%, with the highest resolution predicted at 0.1% acetonitrile and a flow rate of 0.81 mL/min. Saccharides in the size range DP1–4, including amino, acetamido, and deoxy sugars, were separated using a binary gradient method. Higher retention was observed for increasing degree of polymerisation (DP) and N-acetylated saccharides were retained longer than non-substituted saccharides of corresponding DP. Partial resolution of two lacto-N-tetrasaccharide positional isomers was obtained.