1 Department of Chemistry and Bioscience, The Faculty of Engineering and Science, Aalborg University, VBN2 Section of Biotechnology, The Faculty of Engineering and Science, Aalborg University, VBN3 The Faculty of Engineering and Science (ENG), Aalborg University, VBN4 Microbial Communities, The Faculty of Engineering and Science, Aalborg University, VBN
A broad range of elution strategies for RP-HPLC analysis of sucrose alkanoate regioisomers with CAD was systematically evaluated. The HPLC analyses were investigated using design-of-experiments methodology and analysed by analysis of variance (ANOVA) and regression modelling. Isocratic elutions, isocratic elutions with increased flow, and gradient elutions with step-down profiles and step-up profiles were performed and the chromatographic parameters of the different elution strategies were described by suitable variables. Based on peak resolutions general resolution deviation for multiple peaks (RDm) was developed for sample-independent evaluation of separation of any number of peaks in chromatographic analysis. Isocratic elutions of sucrose alkanoates showed similar relationships between eluent acetonitrile concentration and retention time for all regioisomers of sucrose caprate and sucrose laurate, as confirmed by evaluation of the curvatures using approximate second derivatives and Kendall rank correlation coefficients. Regression modelling and statistical analysis showed that acetonitrile concentration and flow rate were highly significant for both average adjusted retention time and RDm for sucrose laurate. For both responses the effect of changes in acetonitrile concentration was larger than the effect of changes in flow rate, over the ranges studied. Regression modelling of the step-down gradient profiles for the sucrose alkanoates showed that the eluent acetonitrile concentrations were the overall most significant variables for retention time and separation. The models for average adjusted retention time of sucrose caprate and sucrose laurate showed only a few differences in the significance levels of terms, while the models for RDm showed larger differences between the sucrose alkanoates, in both the number of terms and their significance. Efficiency evaluation of elution strategies, in terms of RDm and analysis time, showed that the best results were offered by step-down gradient elution for sucrose caprate and isocratic elution with increased flow for sucrose laurate. Step-down gradient elution of sucrose caprate offered improvements in separation at similar analysis time compared to isocratic elution, with the most efficient elutions achieved with elution profile acetonitrile concentrations at 32.5% and 25%, resulting in reduction of RDm by 13–38% and reduction of analysis time by 3–9%. For sucrose laurate, isocratic elution with increased flow showed improvements in separation and reductions in analysis time compared to isocratic elution, such as elution at 37% with flow 2.0 mL/min resulting in reduction of analysis time by 34% and equal RDm, while elution at 35% with flow 2.0 mL/min reduced RDm by 29% and analysis time by 6%, compared to isocratic elution at 38% acetonitrile with standard flow (1.0 mL/min).
Journal of Chromatography a, 2013, Vol 1311, p. 127-133