Improving HPLC post-column derivatisation assays via In-Column Derivatisation

Abstract

In column derivatisation (ICD) has been applied to antioxidants in food and agricultural matrices. ICD’s ability to reduce the post-column reaction loop volume is critical to investigate chromatographically before future applications are established. Due to the large number of post-column derivatisation (PCD) assays, this is out of the scope of this study to cover in one article. Hence, in this communication, we strictly focused on implementing ICD and its ability to reduce the post-column volume associated to the PCD’s reaction loop. Specifically, reversed phase (RP) separations where we take three different reaction schemes: (i) amino acids derivatised with ninhydrin, (ii) amino acids derivatised with o-phthalaldehyde (OPA), and (iii) sulfonamides derivatised with fluorescamine to exemplify three different case scenarios. This communication can be used as a practical guide for analysts. In the order of the workflow’s largest reduction in the reaction loop volume: (i) the amino acids derivatised with OPA, the ICD enabled the post-column volume to be reduced significantly from 500 µL down to 80 µL. This drastic reduction resulted in the chromatographic gain in performance – number of theoretical plates (N) (55–167 %) and peak height sensitivity (35–181 %). For the sulfonamides derivatised with fluorescamine, the ICD setup reduced the post-column volume from 500 µL down to 200 µL, and demonstrated significant improvements in efficiency – N (33–324 %), and peak height sensitivity (4–33 %). The amino acids derivatised with ninhydrin, the PCD reaction loop could not be decreased, and ICD demonstrated decreased signal to noise (S/N).