Browsing by Author "Venter, Pieter"
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- ItemAnalysis of complex tannins by multidimensional techniques(Stellenbosch : Stellenbosch University, 2019-03) Venter, Pieter; De Villiers, Andre J.; Pasch, Harald; Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science.ENGLISH ABSTRACT: Hydrolysable tannins are plant derived (poly)phenolics which for centuries have been used in tanning, the transformation of animal hides into leather. In addition to their industrial applications, epidemiological evidence suggests that the consumption of hydrolysable tannins can be linked to disease prevention and treatment, although their mode of action at a molecular level is still unknown. In order to investigate their biological and chemical properties, the determination of the molecular composition of hydrolysable tannins in plant material is a critical step. Liquid chromatography hyphenated to mass spectrometry (LC-MS) is the preferred analytical approach to obtain this information, although conventional methods often provide insufficient performance for highly complex mixtures of hydrolysable tannins. The goal of the work reported in this thesis was, therefore, to develop improved multidimensional methods for the analysis of hydrolysable tannins. In the first part of the study, a methodology for the comprehensive analysis of ellagitannins and gallotannins in chestnut and tara using reversed phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) combined with ultraviolet (UV), ion mobility (IM) spectrometry and high-resolution mass spectrometry (HR-MS) detection is reported. Good chromatographic performance was achieved using both HILIC and RP-LC for chestnut tannins, with the complementary separation mechanisms proving useful for particular compound classes also pointing to the potential of the combination of HILIC and RP-LC in a comprehensive two-dimensional separation. In contrast, RP-LC provided much better separation of tara gallotannins than HILIC. Incorporation of IM into the LC-MS workflows offered several advantages for such non-targeted screening analyses, including improved mass spectral quality by filtering MS data according to IM arrival time, the availability of an additional identification criterion in the form of collisional cross section (CCS) values obtained from arrival times, and an improved MS duty cycle and, therefore improved sensitivity. Furthermore, additional isomeric species were resolved by IM and, in combination with UV spectral data, IM provided a simple methodology to differentiate between positional isomers. In the second part of the study, an on-line comprehensive two-dimensional HILIC×RPLC method was developed for the analysis of hydrolysable tannins by kinetic optimisation of the relevant experimental parameters. The use of a weak make-up flow to dilute the HILIC effluent and avoid second dimension injection band broadening resulted in excellent performance, with practical peak capacities in excess of 1000 determined for both samples. Despite on-column dilution and the short second dimension analysis times ultimately limiting the number of compounds detected in chestnut and tara, the group-type separations obtained show promise for the fast fingerprint analysis of hydrolysable tannins. Building on previous findings, the final part of the work evaluated the potential of IM spectrometry as a third dimension in a comprehensive three-dimensional HILIC×RPLC×IM configuration. Apart from the inherent advantages of IM alluded to above, the additional separation step offered by IM contributed to an increase in practical peak capacity by a factor of 13 when integrated into the LC×LC workflow. Certain instrumental constraints which limit the ultimate performance of LC×LC×IM-HR-MS are highlighted. Despite these, this approach shows significant promise for the high resolution separation of highly complex mixtures.