Phenols/polyphenols fractionation

Dissociation of the Protein-Poly phenolic Complex and Characterization of the Polyphenolic Fraction. Since Indulin ATR is almost completely soluble in THF while the APPL s are quite insoluble in this solvent, but are soluble in DMF, a sequence of different percentage mixtures of these two solvents was used in order to dissociate the protein-lignin complexes for further analyses of the lignin part. 

Zaprometov, M. N., N. V. Zagoskina and T. F. Koretskaya. Effect of some precursors on the formation of phenolic compounds in tea plant tissue cultures. Fiziol Rast 1976 23 1274. Higashi-Okai, K., S. Otani and Y. Okai. Potent suppressive activity of pheophytin A and B from the non-polyphenolic fraction of green tea (Camellia sinensis) against tumor promotion in mouse skin. Cancer Lett 1998 129(2) 223-228. 

In a polyphenolic extract, anthocyanins can interfere with other polyphenolics such as pro-cyanidins during HPLC analysis and hence should be removed prior to analysis. Anthocyanins from crude polyphenolic extracts can be removed as described in Basic Protocol 2. The ethyl acetate used for elution of phenolic compounds other than anthocyanins is removed using a rotary evaporator at 20°C. The non-anthocyanin polyphenolics are dissolved in deionized distilled water and the pH is adjusted to 7.0 with NaOH as described in Alternate Protocol 2 or the method developed by Oszmianski and Lee (1990a). In the latter method, polyphenolics were fractionated into three groups neutral fraction A (flavanols and other polar phenolics), neutral fraction B (flavonols), and acidic phenolics. Polyphenolic extracts were adjusted to pH 7.0 with NaOH... 

The rather simple solvent classification schemes yield complex fractions of botanochemicals. Their detailed composition depends not only on the species but also on maturity of the plant and the method of extraction (1 5, ). The polar fraction isolated by acetone extraction and readily soluble in 87.5% aqueous ethanol, termed "polyphenol" by Buchanan and coworkers (11,12), no doubt consists of phenolics and a wide variety of other substances. For plants of high tannin content, (e.g., Rhus g/aubra) the polyphenol fraction might well be called tannin (24)... 

Methods used for the detection of PAs in cmde or partially purified extracts can also be adapted for post-column analysis after fractionation (see below). Direct quantitative analysis of PAs in crude grape phenolic extracts is often impossible due to the complex sample matrix. Thus, fractionation or purification is often necessary before analysis. The Folin-Ciocalteu and Pmssian Blue assays are widely used for the quantification of total polyphenols in plants [27,28]. These methods are not specific for PAs due to the reaction of other phenolic compounds with these reagents. 

In earlier times, thin-layer chromatography (TLC), polyamide chromatography, and paper electrophoresis were the major separation techniques for phenolics. Of these methods, TLC is still the workhorse of flavonoid analysis. It is used as a rapid, simple, and versatile method for following polyphenolics in plant extracts and in fractionation work. However, the majority of published work now refers to qualitative and quantitative applications of high-performance liquid chromatography (HPLC) for analysis. Llavonoids can be separated. 

In this protocol, polyphenolics are fractionated into neutral and acidic fractions to prevent interference among polyphenolics in HPLC analysis. Phenolic acids are completely ionized at pH 7.0 and un-ionized at pH 2.0. This property allows for fractionation of neutral polyphenolics at pH 7.0 and acidic polyphenolics at pH 2.0. Two individually preconditioned Cl8 cartridges, one for neutral polyphenolics and the other for acidic polyphenolics, are used for this separation (Figure 11.2.2). 

Retention times of the peaks are subject to the particular type of column. The acidic fraction from solid-phase extraction consists of phenolic acids such as cis-coutaric, trans-coutaric, and trans-caftaric acids. Isocratic elution is suitable because of the limited number of compounds found in the acidic fraction. Analysis of the acidic fraction is completed within 30 min. See Figure 11.3.1 for an HPLC chromatogram of the acidic polyphenolics isolated from Niagara grapes. 

The determination of polyphenolics may result in interference due to co-elution of phenolic acids and procyanidins. This problem can be eliminated by fractionation of polyphenolics into acidic and neutral polyphenolics prior to sample injection into the HPLC system. Because the fractionation techniques effectively improve the resolution of many polyphenolic peaks in the reversed-phase HPLC system, it is suggested that further characterization and identification of unknown peaks be conducted by additional methods such as mass spectrometry and nuclear magnetic resonance. 

Svedstrom, U. Vuorela, H. Kostiainen, R. Laakso, L Hiltunen, R. 2006. Fractionation of polyphenols in hawthorn into polymeric procyanidins, phenolic acid, and flavonoids prior to high-performance liquid chromatographic analysis. J. Chromatogr. A. 1112 103-111. 

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