Flavonoids chromatographic techniques

Various countercurrent chromatographic techniques have been successfully employed for the separation of flavonoids. Countercurrent chromatography is a separation technique that relies on the partition of a sample between two immiscible solvents, the relative proportions of solute passing into each of the two phases determined by the partition coefficients of the components of the solute. It is an all-liquid method that is characterized by the absence of a solid support, and thus has the following advantages over other chromatographic techniques ... 

The coupling of chromatographic techniques such as HPLC with NMR, LC-NMR can, in principle, provide the molecular structures of compounds in mixtures (extracts) in just one online experiment. The use of LC-NMR in the flavonoid field has been reviewed by... 

In addition to the molecular techniques, technical advances both in chromatographic techniques and in identification tools, particularly the diverse forms of mass spectrometry, has allowed successful challenges to the separation and characterization of compounds of increasing complexity, poor stability, and low abundance [Whiting, 2001]. Information generated utilizing these techniques has resulted in characterization of a plethora of complex secondary metabolites that, in conjunction with the characterization of the enzymatic steps, has permitted the complete or partial elucidation of the flavonoid and the phenolic pathways present in many plants (Figs. 1.35 and 1.36). 

Spectrophotometric methods are sensitive but not really suitable for estimating saponins in crude plant extracts since the reactions are not specific and colored products may form with accompanying compounds such as phytosterols and flavonoids. Similarly, hemolytic methods also suffer from a lack of specificity. Consequently, alternative ways of analyzing for saponins using different chromatographic techniques have been developed. 

MS has proved to be a very powerful technique in the analysis of flavonoids and phenolic acids mainly due to its high sensitivity and the possibility of coupling with different chromatographic techniques such as gas chromatography (GC-MS), capillary electrophoresis (CE-MS), and especially LC-MS. Nowadays, techniques such as LC-DAD-MS, and particularly LC-DAD-electrospray ionization (ESI)/MS, are regarded as necessary tools for the analysis of phenolics in natural matrices. ... 

However, antioxidant screening in complex mixtures of plant origin requires simple and rapid in vitro models for a possible combination with chromatographic techniques such as TLC, HPLC, HSCCC, or CPC. The reduction of the stable free-radical DPPH (l,l-diphenyl-2-picryUiydrazyl) by antioxidant substances is currently the most widely used chemical test for the screening of plant extracts. Only a few bioassay-guided fractionation processes have also evaluated the antimicrobial, antibacterial activity, or cytotoxicity of fractions enriched in specific flavonoids by using HSCCC or CPC liquid-liquid systems [30, 35]. 

The role of advances in chromatographic techniques has been a step point in the development of phytochemistry [67]. Because of the complexity of crude herbal extracts, various online hyphenated techniques have been developed for the analysis of the complex mixtures. These techniques include liquid chromatography (LC), mass spectrometry (MS), LC nuclear magnetic resonance (NMR), and LC-NMR-MS [68]. They facilitate the structure determination of unknown constituents in crude extracts. For example, they are of great applicability in the analysis of flavonoids and other phenolic compounds [69, 70]. 

The separation of complex flavonoid mixtures demands in many cases the use of several chromatographic techniques. Thus, a comparison of the HPTLC and high-performance liquid chromatographic (HPLC) behavior of twenty-six flavonoids, and a method for establishing HPLC gradient elution conditions by using TLC data (23) is useful. 

MS has proven to be a very powerful technique for flavonoids analysis because of its high sensitivity and the possibility to combine with different chromatographic techniques, for example, GC/MS, CE/MS, and specially LC/MS, as a stand-alone instmment allowing both qualitative and quantitative determinations. 

As the separation characteristics of liquid chromatographic and electrophoretic techniques markedly differ from each other, combined methods using the advantages of both procedures have been successfully used for the analysis of flavonoids. Thus, the use of CZE-UV, HPTLC-UV and GC-MS for the measurement of flavonoids in seeds and root exudates of Lotus pedunculatus has been reported. The rooting solution and seed exudate were passed through cellulose acetate filters to bind the flavonoids. After extraction,... 

Finally, since many natural product compounds have been investigated with various chromatographic modes and detection techniques, a selection of examples has been summarized in this chapter. This information has been compiled in the form of tables for well-researched classes of secondary metabolites selected from the major subgroups of isoprenoids (mono-, sesqui-, di-, and triterpenes iridoids and secoiridoids carotenoids saponins and ecdysteroids), of phenolics (coumarins, flavonoids, and isoflavonoids), and of alkaloids. 

For separation and determination of phenolic acids and flavonoids, HPLC is the established technique (Nave et al., 2007 Rodriguez-Delgado et al., 2001 Spranger et al., 2004 Vitrac et al., 2002). The chromatographic conditions include the use of, almost exclusively, a reversed phase C18 column UV-vis diode array detector, and a binary solvent system containing acidified water and a polar organic solvent (Tsao Deng, 2004). 

Molni -Perl, I. Fuzfai, Z. Chromatographic, capillary electrophoretic and capillary electrochromatographic techniques in the analysis of flavonoids. J. Chromatogr. A, 2005,1073 (1-2), 201-227. 

The most common techniques used for separation of flavonoids are chromatographic and electrophoretic methods. 

High-speed countercurrent chromatography (HSCCC) is a relatively new technique. It is a liquid-liquid partitioning chromatographic method in which the stationary phase is immobilized by a centrifugal force. HSCCC is the most advanced form of CCC in terms of partition efficiency and separation time. As solvent system, a mixture in different ratio is usually used, depending on the flavonoids nature ethyl acetate/... 

A common drawback to LC-NMR analysis derives from the interference of the resonances of the mobile phase, usually much higher than those of the analyte itself. To overcome this problem, the mobile phase coming from the liquid chromatograph is efficiently suppressed with techniques such as water-suppression enhanced-through Ti effects (WET). This allows the separation of analytes in typical RP-HPLC conditions using common solvents such as MeOH or acetonitrile, yet usually replacing water by D2O to achieve better quality spectra however, in the case of analyte signals coincident with the suppressed solvent resonances, those of the analytes will also be suppressed. On the other hand, in spite of the usefulness of LC-NMR for stmcmral elucidation of flavonoids, this is not a sensitive LC detection technique. Therefore, to improve sensitivity, transients can be accumulated by operation in two different modes the on-flow and stop-flow modes. In both cases, the analytes from an LC column pass into an NMR microflow probe that typically has an active volume of 60-120 p.L, comparable with the conventional NMR sample size of 500-600 p,L. 

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