Phenols/polyphenols analysis

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... 

Paganga, G. et al.. The polyphenolic content of fruit and vegetables and their antioxidant activities what does a serving constitute Free Radical Res., 30, 153, 1999. Maatta, K.R. et al.. High-performance liquid chromatography (HPLC) analysis of phenolic compounds in berries with diode array and electrospray ionization mass spectrometric (MS) detection Rihes species, J. Agric. Food Chem., 51, 6736, 2003. 

The differences between castoreum from different species and sources still need to be investigated. As suggested by Tang et al [69] it is quite likely that these differences could be diet related because the phenols are most likely derived from the diet of these animals. Indeed, already in 1949 Lederer [73] recognized this possibility and pointed out that 2, 3"-dihydroxydibenz-2-pyrone and 4,4 -dihydroxydiphenic acid dilactone 12 (Fig. 3) are closely related to ellagic acid, which is abundant in the bark of trees. A comprehensive chemical analysis of the polyphenols present in the preferred diet of the beavers and a comparison of these compounds with the phenols present in castoreum from animals fed exclusively on this diet could shed some light on this unresolved problem. 

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. 

Gutes, A., Cespedes, F., Alegret, S., and del Valle, M. (2005). Determination of phenolic compounds by a polyphenol oxidase amperometric biosensor and artificial neural network analysis. Biosens. Bioelectron. 20(8), 1668-1673. 

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. 

An accurate sample weight before extraction and the amount of final extract after sample cleanup should be known for an accurate quantification of phenolic compounds extracted from plant materials by HPLC analysis. The characteristic wavelengths for detection of polyphenolics can be selected at the discretion of the experimenter. The solvent gradients described in the Basic and Alternate Protocols can be modified for better resolution. 

Many excellent discussions of natural occurrence, structure, characterization, and analysis of phenolic compounds are available in the literature, and a series of books devoted to flavonoid chemistry has also been published. Detailed discussions on various chromatographic modes, including HPLC, GC, column chromatography (CC), capillary electrophoresis (CE), PC, and TLC, of simple phenolics and polyphenols are also presented in the recent book, Handbook of Food Analysis, volume 1, edited by Nollet (1). Due to their diversity and the chemical complexity of phenolic compounds, this chapter is limited to phenolic compounds that are considered to be important to foods and the food industry. 

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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