Investigation on the retention and separation of glucosinolates with a mixed-mode reversed-phase/weak anion-exchange column

Authors: LI, YANFANG - University Of Maryland; Chen, Pei; ZHANG, MENGLIANG - Ohio University; Sun, Jianghao

Submitted to: Journal of Separation Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/14/2024
Publication Date: 11/7/2024
Citation: Li, Y., Chen, P., Zhang, M., Sun, J. 2024. Investigation on the retention and separation of glucosinolates with a mixed-mode reversed-phase/weak anion-exchange column. Journal of Separation Science. 47(21). Article 70007. https://doi.org/10.1002/jssc.70007.
DOI: https://doi.org/10.1002/jssc.70007

Interpretive Summary: Glucosinolates are a group of important secondary metabolites with various health beneficial effects, which are abundant in the Brassica vegetables. Due to their structural diversity, high polarity, and anionic nature, retain and separation of glucosinolates using conventional single mode C18 column is difficult. Mixed-mode chromatography becomes a powerful tool for separation of complex mixtures, especially polar and charged analytes, for more than one separation mechanism was used. Mixed-mode columns were designed to obtain improved separation selectivity that otherwise be unretained on conventional single mode columns. A promising way to separate polar and ionic compounds is to use a mixed-mode RP anion/cation-exchange column. The additional contribution of ionic interactions could provide an alternative selectivity for polar and ionic compounds than common RP column. The recently introduced Atlantis Premier BEH C18 AX column is a mixed-mode reversed-phase/weak anion-exchange column, which the stationary phase contains not only C18 moieties, but also tertiary alkylamine groups on the surface. Therefore, the BEH C18 AX column could be an alternative option to traditional single mode C18 column for glucosinolates investigation. However, the application of BEH C18 AX column on the retention and separation of glucosinolate has not been reported yet. To address this issues,the present study examined the effect of a mixed-mode RP/weak anion exchange column (BEH C18 AX column) on glucosinolate retention and separation, as well as compare its chromatographic properties with a conventional RP C18 column. Effects of mobile phase concentration and pH on the retention and separation of glucosinolates were also investigated. Finally, the applicability of the mixed-mode reversed-phase/weak anion-exchange column for the analysis of glucosinolates in Brassica samples was demonstrated through studies on broccoli and red cabbage. The results demonstrated that the mixed-mode reversed-phase/weak anion-exchange column can be used as an alternative or complementary tool to traditional single mode column for investigating glucosinolates with higher retention and better separation ability.

Technical Abstract: Glucosinolates, a crucial group of secondary metabolites in Brassica vegetables, present significant chromatographic separation challenges due to their anionic form, structure diversities, and co-existence of other phenolic compounds. This study comparatively investigated the retention and separation of seven glucosinolates using a mixed-mode reversed-phase/weak anion-exchange column and a conventional reversed-phase C18 column. Separation factors for each glucosinolate with its adjacent peaks were over 1.0 on the mixed-mode column, while co-eluting was observed on the C18 column. The effects of mobile phase additives and pH on the separation and retention of glucosinolates were also investigated. Results showed that glucosinolate retention was inversely related to both buffer concentration and pH. The optimized method for the mix-mode column was applied to the complex Brassica vegetable samples. In addition to the 17 well-resolved glucosinolate peaks, 34 peaks for phenolic compounds were identified in broccoli microgreen, suggesting the successful application scenarios for qualitative analysis in comparison with the single mode reverse phase C18 column. This study demonstrates that the mixed-mode reversed-phase/weak anion-exchange column can be used as a promising separation tool for organic anions in a complex sample matrix.