title: Phenolic profiling of rooibos using off-line comprehensive normal phase countercurrent chromatography×reversed phase liquid chromatography.
abstract: 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 17h. The use of a gradient for the1D 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 same1D 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-3',5'-di-C-β-d-glucopyranoside (a dihydrochalcone) were identified for the first time in rooibos by comparison with authentic reference standards.
authors.affiliation: Department of Food Science, Stellenbosch University, Private Bag X1, Matieland, Stellenbosch, 7602, South Africa; Plant Bioactives Group, Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC), Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch, 7599, South Africa.
authors.forename: Nico A
authors.initials: NA
authors.lastname: Walters
authors.affiliation: Department of Chemistry and Polymer Science, Stellenbosch University, Private Bag X1, Matieland, Stellenbosch, 7602, South Africa.
authors.forename: André
authors.initials: A
authors.lastname: de Villiers
authors.affiliation: Department of Food Science, Stellenbosch University, Private Bag X1, Matieland, Stellenbosch, 7602, South Africa; Plant Bioactives Group, Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC), Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch, 7599, South Africa.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Joubert
authors.affiliation: Department of Food Science, Stellenbosch University, Private Bag X1, Matieland, Stellenbosch, 7602, South Africa; Plant Bioactives Group, Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC), Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch, 7599, South Africa. Electronic address: DBeerD@arc.agric.za.
authors.forename: Dalene
authors.initials: D
authors.lastname: de Beer
mesh_terms: D036903:Aspalathus / Q000737:chemistry*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D003377:Countercurrent Distribution / Q000379:methods*; D005419:Flavonoids / Q000032:analysis*; D006027:Glycosides / Q000032:analysis*
chemical_list: D005419:Flavonoids; D006027:Glycosides
keywords: Aspalathus linearis; Countercurrent chromatography; Mass spectrometry; Phenolic compounds; Rooibos; Ultra-high pressure liquid chromatography