, Volume 82, Issue 1, pp 181–196 | Cite as

Application of Kinetically Optimised Online HILIC × RP-LC Methods Hyphenated to High Resolution MS for the Analysis of Natural Phenolics

  • Magriet Muller
  • Andreas G. J. Tredoux
  • André de VilliersEmail author
Part of the following topical collections:
  1. 50th Anniversary Commemorative Issue


Phenolics are a large group of secondary plant metabolites that are of interest because of their proposed health benefits. The analysis of plant phenolics is challenging due to their extreme structural diversity. Comprehensive two-dimensional liquid chromatography (LC × LC) coupled to high-resolution mass spectrometry (HR-MS) offers a powerful analytical tool for the analysis of such complex mixtures. Especially, the combination of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RP-LC) is attractive for phenolic analysis due to the orthogonal group-type separations attainable. However, online hyphenation of HILIC and RP-LC is complicated by the relative elution strengths of the mobile phases used in both dimensions. Coupled to the inherent complexity of method development in LC × LC, this hampers the more widespread application of HILIC × RP-LC. In this study, a generic HILIC × RP-LC‒DAD-MS methodology for phenolic analysis utilising dilution of the first dimension flow and large volume injection in the second dimension is derived by kinetic optimisation of experimental parameters to provide maximum performance. The scope of the experimental configuration is demonstrated by its application to the analysis of rooibos tea, wine and grape samples containing a range of different flavonoid and non-flavonoid phenolic classes. Using this approach, excellent chromatographic performance was obtained, and a total of 149 phenolic compounds were tentatively identified in the investigated samples based on retention data in two dimensions, UV–Vis spectral as well as high- and low collision energy HR-MS data (72 in grape seeds, 32 in rooibos tea and 45 in wine and grapes) with minimal method development time. The results confirm the applicability of the proposed methodology for the detailed screening of phenolic constituents in natural products.

Graphical Abstract


Comprehensive two-dimensional liquid chromatography (LC × LC) Hydrophilic interaction chromatography (HILIC) Reversed phase liquid chromatography (RP-LC) High resolution mass spectrometry (HR-MS) Online Phenolics Flavonoids 



The authors would like to acknowledge financial support from Sasol (Collaborative Grant to AdV) and the National Research Foundation of South Africa (Grants 98897 to AdV, 91436 to AGJT and post-graduate bursary to MM). The authors gratefully acknowledge Agilent Technologies (University Relations & External Research) for the donation of some of the instrumentation used in this work (Research Gift #3888 to AdV). Maria A. Stander is thanked for providing the rooibos samples, and Wessel J. Du Toit for the grape and wine samples.


This study was funded by Sasol (Collaborative Grant to AdV), the National Research Foundation of South Africa (Grants 98897 to AdV, 91436 to AGJT and bursary to MM), Agilent Technologies (Research Gift #3888 to AdV).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10337_2018_3662_MOESM1_ESM.pdf (931 kb)
Supplementary material 1 (PDF 931 KB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Chemistry and Polymer ScienceStellenbosch UniversityMatielandSouth Africa

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