Phenolic compounds column chromatography

SEC in combination with multidimensional liquid chromatography (LC-LC) may be used to carry out polymer/additive analysis. In this approach, the sample is dissolved before injection into the SEC system for prefractionation of the polymer fractions. High-MW components are separated from the additives. The additive fraction is collected, concentrated by evaporation, and injected to a multidimensional RPLC system consisting of two columns of different selectivity. The first column is used for sample prefractionation and cleanup, after which the additive fraction is transferred to the analytical column for the final separation. The total method (SEC, LC-LC) has been used for the analysis of the main phenolic compounds in complex pyrolysis oils with minimal sample preparation [974]. The identification is reliable because three analytical steps (SEC, RPLC and RPLC) with different selectivities are employed. The complexity of pyrolysis oils makes their analysis a demanding task, and careful sample preparation is typically required. 

High-performance liquid chromatography (HPLC) techniques are widely used for separation of phenolic compounds. Both reverse- and normal-phase HPLC methods have been used to separate and quantify PAs but have enjoyed only limited success. In reverse-phase HPLC, PAs smaller than trimers are well separated, while higher oligomers and polymers are co-eluted as a broad unresolved peak [8,13,37]. For our reverse-phase analyses, HPLC separation was achieved using a reverse phase. Cl8, 5 (Jtm 4.6 X 250 mm column (J. T. Baker, http //www.mallbaker.com/). Samples were eluted with a water/acetonitrile gradient, 95 5 to 30 70 in 65 min, at a flow rate of 0.8 mL/min. The water was adjusted with acetic acid to a final concentration of 0.1%. All mass spectra were acquired using a Bruker Esquire LC-MS equipped with an electrospray ionization source in the positive mode. 

Hostettmann, K. et al., On-line high-performance liquid chromatography ultraviolet-visible spectroscopy of phenolic compounds in plant extracts using post-column derivatization, J. Chromatogr., 283, 137, 1984. 

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. 

Reversed-phase chromatography is the most popular mode of analytical liquid chromatography for phenolic compounds. In most cases, the reported systems for the separation of phenolics and their glycosides in foods are carried out on reversed-phase chromatography on silica-based Cl8 bonded-phase columns. Occasionally, silica columns bonded with C8 were applied in the analysis of phenolic acid standards and coumarins (7), and C6 columns for the analysis of ferulic acid in wheat straw (8). 

A mixture of phenol 1 (10 mmol) and a,/ -unsaturated carboxylic derivative 2, 4 or ethyl acetoacetate 6 (15-20 mmol) supported on 1 g of solid acid support (by dissolving the mixture in 5 mL of diethyl ether followed by evaporation of the solvent) was exposed to microwave irradiation in a focused microwave reactor (Prolabo MX350). For isolation of the compounds, the solid support was removed by extraction with ethanol (for reactants 2 and 6) or acetonitrile (reactions of 4). After solvent evaporation, the products were purified by column chromatography on silica gel (hexane-ethyl acetate, 3 1). 

Castillo, M., D. Puig, and D. Barcelo. 1997. Determination of priority phenolic compounds in water and industrial effluents by polymeric liquid-solid extraction cartridges using automated sample preparation with extraction columns and liquid chromatography Use of liquid-sohd extraction cartridges for stabilization of phenols. J. Chromatogr. A 778 301-311. 

De Ruiter et al. [4] observed that photochemical decomposition by ultraviolet irradiation of dansyl derivatives of chlorinated phenolic compounds in methanol-water mixtures led to the formation of highly fluorescent dansyl-OH and dansyl-OH3 species. The optimal irradiation time was 5.5s. This reaction was utilised in a post-column photochemical reactor in the high performance liquid chromatography determination of highly chlorinated phenols in river water. The method calibration curve (for dansylated pentachlorophenol) was linear over three orders of magnitude. 

In a dry nitrogen flushed 100 ml flask sealed with a septum were placed the phenol (5 mmol) and dry methanol (10 ml). A solution of DIB (1.61 g, 5 mmol) in methanol (25 ml) was transferred via a double-ended needle to the stirred phenolic solution at room temperature over 40 min. After 40 min, the reaction mixture was concentrated and the residue was purified by column chromatography (silica gel, light petroleum to dichloromethane) to afford the title compounds in 65-99% yield. 

Dry clean tanshen rhizomes were powdered and extracted with hexane for three days at room temperature. The hexane solution was kept overnight and then filtered. After removal of the solvent a residue was obtained which was separated into seven colored fractions by column chromatography with silica gel. Miltirone was isolated by preparative tic from fraction 1 (light red) using hexane ethyl acetate (4 1) followed by benzene-acetone (20 1). The product obtained was recrystallized from ethylacetate, m.p. 100-101°C. Its structure was confirmed by mass spectrum, NMR, IR and UV spectra which agree quite closely with those of Ho et al [76], Miltirone showed antioxidant behavior comparable to that of the commonly used phenolics BHT and BEA [77], The antioxidant activity of miltirone in lard at 100°C was determined with a Rancimat. Miltirone and other related compounds may have the potential of being used as natural antioxidants in food and cosmetics. 

XAD-7 resin as the column material. Additionally, procyanidins have been separated from sugars and other interfering phenolic compounds by SPE using Sephadex LH-20 as the sorbent (Prior et ah, 2001). SPE has also been used to isolate phytoestrogens - including isoflavones, coumestans, and lignins from foods prior to further separation by liquid chromatography mass spectrometry (LC-MS) (Kuhnle et al, 2007). 

The adventitious roots, cultured in MS liquid medium containing 2 mg/1 NAA for 4 weeks, were lyophilized and extracted with 80% aqueous acetone. The extract, after concentration, was subjected to Sephadex LH-20 column chromatographies using EtOH and 60 % MeOH to give a phenolic compound 2 which is one of the structural elements of rhatannins. 

The axenic plants obtained above (see 2.5.2) were transferred and cultivated under hydroponics for 3 months. The mature plants were harvested and separated into two parts (one was the leaf and stem portion while the other was the root portion). These parts were separately extracted with 80% aqueous acetone (leaf and stem) or MeOH (root) and subjected to the same column chromatographies mentioned above. From the extract of the leaf and stem portion, five phenolics, 1, 12, 13, 1,2,4,6-tetra-O-galloyl-P-D-glucose (35) (40) and phyllanthusiin D (36) (94, 95) (Fig. 14) were isolated. On the other hand, from the extract of the root portion, five compounds 3 and 28-31 all of which were the flavan-3-ols produced in the hairy roots (Pn-H-6) were isolated. 

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