Reverse phase chromatography polyphenols

Two different sample preparation methods by size-exclusion and reverse-phase chromatography were proposed for analysis of PCs and PAs in wine. In the former, a volume of 5 mL of dealcoholized wine is passed through a Fractogel Toyopearl TSK gel HW-50 (F) (12 x 120 mm) column. The stationary phase is washed with 25 mL of water and the simple polyphenols are eluted with 50 mL of an ethanol/water/trifluo-roacetic acid 55 45 0.005 (v/v/v) solution. The polymeric fraction is recovered with 50mL of acetone/H20 60 40 (v/v). Figure 6.19 shows the LC/ESI-MS extracted ion chromatograms of dimers and trimers in a wine (Fulcrand et al., 1999). 

Sample preparation by reverse-phase chromatography can be performed using a Ci8 SPE cartridge 30 mL of dealcoholized wine are loaded onto the cartridge, after rinsing with 40 mL of water PAs are recovered with lOmL of acetone/water/acetic acid 70 29.5 0.5 (v/v/v) (Lazarus et al., 1999). A method for fractionation of polyphenols in wine by reverse-phase chromatography is reported in the flow diagram in Fig. 6.20 (Sun et al., 2006). 

In this unit, methods for reversed-phase high-performance liquid chromatography (HPLC) are described for the analysis of polyphenolics. HPLC analysis can be employed in an easy and fast manner to obtain an accurate elucidation and quantification of individual polyphenolic compounds found in plant-based materials. The separation of each polyphenolic is based on the polarity differences among polyphenolics with structural similarities and uses various combinations of mobile and stationary phases. 

Because polyphenolics show chemical complexities and similar structures, isolation and quantification of the individual polyphenolic compounds have been challenging. Many traditional techniques (paper chromatography, thin-layer chromatography, column chromatography) have been used. HPLC, with its merits of exacting resolution, ease of use, and short analysis time, has the further advantage that separation and quantification occur simultaneously. A reversed-phase HPLC apparatus equipped with a diode array detector makes possible the easy isolation and separation of many polyphenolics. For enhanced performance of HPLC separation, the polyphenolics should first be isolated into several fractions to effectively separate the individual polyphenolics (Jaworski and Lee, 1987 Oszmianski and Lee, 1990). 

Cooper K.A. Campos-Gimenez E. Alvarez D.J. Nagy K. Donovan J.L. Williamson G. 2007. Rapid reversed phase ultra-performance liquid chromatography analysis of the major cocoa polyphenols and inter-relationships of their concentrations in ehoeolate. J. Agric. Food Chem. 55 2841-2847. 

Lee et al. (2005) proposed a method for analysis of flavonols in grape by performing berry extraction with acidified methanol (0.01% of 12 N HC1). After filtration, the solvent is removed under vacuum and the residue is dissolved in a 0.1 M citric acid buffer with pH 3.5. First, polyphenols are fractionated on a reverse-phase Ci8 cartridge (e.g., Sep-Pak 5g), then on a Sephadex LH-20 3-g cartridge (a cross-linked dextran-based stationary phase used for gel permeation, normal-phase partition, and adsorption chromatography). Four fractions finally are recovered by ethyl acetate and methanol, as shown in the f >w diagram Fig. 6.5. 

Liquid chromatography Separation and quantitative analysis of flavanols and polyphenols may be conducted by Cig reversed-phase LC methods, using either isocratic or gradient modes. In one example of the isocratic separation method, the solvent system used consists of acetic acid-methanol-dimethylform-amide-water (1 2 40 157 v/v/v/v). Eluting peaks are monitored by UV absorption at 254 nm. 

Huck, C.W. Bonn, G.K. Evaluation of detection methods for the reversed-phase HPLC determination of 3, 4, 5 -tri-methoxyflavone in different phytopharmaceutical products and in human serum. Phytochem. Anal. 2001,12,104—109. Huck, C.W. Huber, C.G. Ongania, K.H. Bonn, G.K. Isolation and characterization of methoxylated flavones in the flowers of Primula veris hy Uquid chromatography and mass spectrometry. J. Chromatogr. A, 2000, 870,453-462. Milhury, P.E. Analysis of complex mixtures of flavonoids and polyphenols hy high-performance hquid chromatography electrochemical detection methods. Methods Enzymol. 2001, 335, 15-26. 

Momar, A. Medic-Saric, M. Jasprica, I. ADME data for polyphenols characterized by reversed-phase thin layer chromatography. J. Planar Chromatogr.-Mod. TLC 2006, 19,409-417. 

Ding M, Yang H, Xiao S (1999) Rapid, direct determination of polyphenols in tea by reversed-phase column liquid chromatography. J Chromatogr A 849 637-640... 

Zhang L, Wang Y, Wu D, Xu M, Chen J (2011) Mioowave-assisted extraction of polyphenols from camellia oleifera fruit hull. Molecules 16(6) 4428-4437 Sharma UK, Sharma K, Sharma N, Sharma A, Singh HP, Sinha AK (2007) Microwave-assisted efficient extraction of different parts of Hippophae rhamnoides for the comparative evaluation of antioxidant activity and quantification of its phenolic crmstituents by reverse-phase high-performance liquid chromatography (RP-HPLC)t. J AgricFood Chem 56(2) 374-379... 

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