Improved chromatographic response function in HILIC analysis: Application to mixture of antidepressants

This paper presents exploration of chromatographic behavior in HILIC system by experimental design and improved chromatographic response function denoted as NCRF*. As a model mixture six antidepressants were chosen: selegiline, mianserine, sertraline, moclobemide, fluoxetine and maprotiline. Due to complexity of retention mechanisms in HILIC system, detailed examination of experimental space assessing the influence of important factors (acetonitrile content in the mobile phase, buffer concentration and pH of the mobile phase) and their interactions was done by applying 33 experimental design. NCRF* is developed and designed to be the only output of the system which simultaneously measures the separation of all the examined substances, the chromatographic run duration and the quality of the obtained peaks shape. It allowed objective estimation of overall chromatogram quality and excluded the arbitrary judgment in ambiguous situations. The applied function highlighted the influence of investigated factors on entire mixture and enabled identification of experimental regions where the chromatographic behavior was satisfactory. Applied experimental design strategy combined with NCRF* proved to be valuable assistance in HILIC separation of complex mixtures.