Phenolic profiling of rooibos using off-line comprehensive normal phase countercurrent chromatography × reversed phase liquid chromatography

- An off-line CCC × LC method for rooibos phenolics was optimised for peak capacity.
- CCC and RP-LC provided highly orthogonal (ca 80%) separations.
- Contour plots enabled comparison of complexity of green and fermented rooibos.
- 18 of 39 compounds were tentatively identified for the first time in rooibos.

Limited performance of one dimensional chromatographic methods provides the incentive for the development of multidimensional chromatographic techniques for the analysis of complex mixtures. Rooibos (Aspalathus linearis) represents such a complex sample, containing a range of phenolic compounds that cannot be separated and identified using a single chromatographic technique within a reasonable run time. By implementing NP high performance countercurrent chromatography (NP-HPCCC) in the first dimension (1D) and RP ultra-high pressure LC (RP-UHPLC) in the second dimension (2D), a highly orthogonal (∼80%) off-line comprehensive two-dimensional separation of rooibos phenolic compounds was achieved in a total analysis time of 17 h. The use of a gradient for the 1D HPCCC separation ensured a good spread of relatively polar flavonoid di-C-glycosides and less polar mono- and di-O-glycosides, while the highly efficient UHPLC method was able to separate compounds eluting in the same 1D fraction. Analysis of green (“unfermented”) and traditional "fermented" rooibos samples enabled tentative identification of 39 phenolic compounds based on UV-vis and MS characteristics, of which 18 have not previously been reported in rooibos. Scolymoside (a flavone), hesperidin (a flavanone) and phloretin-3ʹ,5ʹ-di-C-β-d-glucopyranoside (a dihydrochalcone) were identified for the first time in rooibos by comparison with authentic reference standards.