Solid-phase extraction polyphenols

Suarez B, Picinelli A and Mangas JJ. 1996. Solid-phase extraction and high-performance liquid chromatographic determination of polyphenols in apple musts and ciders. J Chromatogr A 727(2) 203-209. 

A description of methods for isolating polyphenolics and anthocyanins from grapes by solid-phase extraction. 

Since considerable amounts of potential interfering materials can be extracted along with the polyphenolics, an isolation/purification step is often required to eliminate components that may interfere with analysis. The fractionation techniques presented in Basic Protocol 2 and Alternate Protocol 2, using solid-phase extraction to minimize the effects of sample preparation/cleanup on the integrity of the extract, will make possible the identification and quantification of individual polyphenolics by HPLC (unit ii j), MS, and NMR. 

Besides solid-phase extraction, column chromatography is also often used for cleanup and purification of polyphenolics from plant material. Ionic adsorbants (polyvinylpyrrolidone or PVP, polyamides, and Sephadex LH-20) and Amberlite XAD-2 resin have been used to isolate and purify polyphenolics from crude extracts. For the separation of polyphenolics from plant material, column chromatography using Sephadex LH-20, a gel-filtration matrix, is often used with various eluting solvents (Park and Lee, 1996). The most widely used solvents for column chromatography are aqueous methanol and aqueous ethanol. 

C18 solid-phase extraction is used to fractionate polyphenolics for their identification and characterization. This technique can eliminate interfering chemicals from crude extracts and produce desirable results for HPLC or other analytical procedures. To obtain a sufficient volume for all analyses, several separations by solid-phase extraction may be performed. The individual fractions need to be combined and dissolved in solvents appropriate for HPLC analysis. In Basic Protocol 2, the application of a current of nitrogen gas for the removal of water from the C18 cartridge is an important step in the selective fractionation of polyphenolics into non-anthocy-anin and anthocyanin fractions. After the collection of non-anthocyanin polyphenolics, no additional work is necessary to elute anthocyanins bound to the C18 solid phase if anthocyanins are not to be determined. 

The Basic Protocol describes the reversed-phase HPLC analysis of polyphenolic compounds isolated into nonanthocyanin and anthocyanin fractions by solid-phase extraction. The Alternate Protocol describes the HPLC separation of acidic and neutral polyphenolic fractions. Fractionated samples are used because significant amounts of interfering compounds are extracted along with polyphenolics from plant materials. Solid-phase extraction with C18 Sep-Pak cartridges (vnitu.2) is used to selectively eliminate undesired components from crude extracts, and may minimize the effects of sample cleanup or preparation on the integrity of polyphenolics. The isolation and purification step using solid-phase extraction of polyphenolics will make possible the efficient analysis of individual polyphenolics by reversed-phase HPLC. 

Additional reagents and equipment for fractionating crude polyphenolics by solid-phase extraction into anthocyanin and nonanthocyanin fractions unit 11.2)... 

This fractionation step may be optional. Some samples can be directly analyzed by HPLC after filtration (step 2) without solid-phase extraction. Anthocyanins that can be detected at 280 nm can interfere with the separation of some polyphenolics. If the analyst is interested in nonanthocyanin polyphenolics, and especially if plant materials containing high levels of anthocyanins are being analyzed, this fractionation technique should be utilized. 

Phenolic acids are ionized at pH 7.0 and are un-ionized at pH 2.0. This property allows for solid-phase extraction of neutral polyphenolics at pH 7.0 and acidic polyphenolics at pH 2.0 to prevent interference. In this protocol, polyphenolics isolated as neutral and acidic fractions using pH adjustments are analyzed by reversed-phase HPLC in a thermostatically controlled environment. 

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. 

Dvorakova M, Hulin P, Karabin M, Dostalek P. 2007. Determination of polyphenols in beer by an effective method based on solid-phase extraction and high performance liquid chromatography with diode-array detection. Czech J. Food Sci 25 182-188. 

Figure 3.12 Flow-diagram of fractionation polyphenols in red wine. PA, proan-thocyanidins (Reprinted from Journal of Chromatography A 1128, Sun et al., Fractionation of red wine polyphenols by solid phase extraction and liquid chromatography, p. 29, Copyright 2006, with permission from Elsevier)...

Recently, Romani et al. [200] published a sophisticated solid phase extraction procedure for the qualitative analysis of grape skins. In a first extraction on Extrelut 20 (kieselguhr, Merck) polyphenols are separated from anthocyanins. The polyphenols are subsequently fractionated on a C-18 cartridge (Bond Elut , Varian) into phenolic acids and esters, procyanidins, flavonol glycosides and acylated anthocyanins. 

Alonso Garcia A, Cancho Grande B, Simal Gandara J (2004) Development of a rapid method based on solid-phase extraction and liquid chromatography with ultraviolet absorbance detection for the determination of polyphenols in alcohol-free beers. J Chromatogr A 1054 175-180... 

The extraction solvents are then evaporated in the rotary evaporator under vacuum condition to concentrate the extract, to which other solvents can be added for purification and separation. Solubihty of compounds in different solvents and pH are important criteria used for the separation and purification of polyphenolic compounds. Separation of phenolic compotmds from the mixtures of compounds can be done using solvent separation methods and various chromatographic methods (including solid-phase extraction). 

Lalaguna F (1993) Purification of fl-esh cassava root polyphenols by solid-phase extraction with Amberlite XAD-8 resin. J Chromatogr A 657 445-449... 

The isolation and pre-concentration of polyphenolic compounds from biological fluids is usually carried out on solid phase extraction (SPE) columns of different packing materials (24, 25, 33-36). These compounds are usually extracted from urine and plasma samples by means of the Oasis HLB columns (26-30). The sorbents of this column are a copolymer of w-divinylbenzene and A -vinylpyrrolidone, which determines its broad uses. The application of hydrophile-lipophile balance (HLB) sorbents obtains high recoveries for a number of polyphenolic compounds with different physicochemical properties. The preparation of plasma samples for the analysis was also carried out by liquid-liquid extraction (LLE) where ethyl acetate was used as an extraction solvent (22,31). 

Disposable screen-printed electrodes are useful single-use sensors for routine analysis. Method of screen printing allows one to construct the whole-electrode systems. Screen-printed carbon thick-film electrodes were used to measure 2,4,6 trinitrotoluene by square wave voltammetry in as little as 50 pL sample volume. This assay was coupled with a solid-phase extraction and the preconcentration factor of 1 500 yielded a 100-fold higher sensitivity resulting in a calibration range of 2 ppb to 2 ppm for 2,4,6-trinitrotoluene. A polyphenol-coated... 

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