Anthocyanins solid-phase extraction

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. 

Betalains are vacuolar plant pigments. Hence their hydrophilic nature is comprehensible. Although they are slightly soluble in ethanol and methanol, water is the best snited solvent both for stability and solnbility reasons. In contrast to the antho-cyanins, the betalains are even more polar as can be demonstrated by shorter retention times in RP-HPLC and lower solubilities in alcoholic solutions. The varying polarities may also be beneficially used to separate anthocyanins from betalains on an RP-18 solid-phase extraction cartridge (Stintzing, unpublished data). 

Kraemer-Schafhalter, A., Fnchs, H., and Pfannhauser, W., Solid-phase extraction (SPE) a comparison of 16 materials for the purification of anthocyanins from Aronw melanocarpa var Nero, J. Sci Food Agr., 78, 435, 1999. 

The solid-phase extractant will become colored. Stop loading sample if excessive color is passing through the cartridge (see Critical Parameters and Troubleshooting, discussion of anthocyanin purification). 

The volume of extract applied to the cartridge will depend mainly on sample quantity, its anthocyanin content, and the amount of sorbent packing in the column. Usually a sample volume of 5 to 10 ml is used for a Cl 8 solid-phase extraction cartridge containing 360 mg sorbent. 

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

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. 

Ultrasound-assisted extraction provides efficient extraction in a shorter processing time than is needed for conventional extraction. The aid of ultrasound will result in a higher extraction yield and reduce solvent consumption. The extract may exhibit a wide range of colors (pale yellow, brown, red). For anthocyanin extraction in an acidic environment, the extract will be deep red, pink, or purple. The extract may contain considerable amounts of lipophilic compounds (e.g., chlorophyll, carotenoids, lipids). Prior to solid-phase extraction, those compounds can be eliminated from the extracts using liquid-liquid extraction. 

A method for fractionation of polyphenoiics into non-anthocyanin and anthocyanin fractions from red grapes by solid-phase extraction using disposable C18 cartridges. 

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)... 

FIGURE 3.11 HPLC chromatogram (a) and ESMS spectrum (b) of anthocyanins in highbush blueberry (Vaccinium corymbosum var. Bluecrop) extracts. The anthocyanins were isolated by solid-phase extraction of C-18 cartridges with acidified methanol—cartridges had previously been washed with acidified water and by ethyl acetate. Peak identities for both the HPLC chromatogram and ESMS spectrum correspond to numbers in Table 3.5. [Source Skrede et al. (2000a), with permission.]... 

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. 

Based on our own experience, solid phase extraction with 500 mg C18 SEP-Pak disposable cartridges works satisfactory in terms of extraction yield and repeatability of results. We use methanol/formic acid = 9/1 (v/v) for anthocyanin extraction, followed by evaporation to dryness and reconstitution in water/formic acid = 9/1 (v/v). This solution is brought onto cartridges equilibrated with water/formic acid = 9/1 (v/v). The samples are then washed with the same solvent and eluted with methanol/formic acid =9/1 (v/v) and again evaporated to dryness. After reconstitution in an appropriate solvent, the samples are ready for use. On average, the recovery of anthocyanins is better than 95 %. Repeated extraction (n=15) of a reference standard (en-3-O-gle) gave a scattering of less than 7 % (relative standard deviation). 

Before analysis anthocyanins must be extracted from the source material with mixtures of organic solvents/water/acids. Further purification/enrichment involves usually automated Solid Phase Extraction. The necessity of further purification steps depends on the analytical requests as the UV-absorption maxima of anthocyanins with >500 nm is selective enough for most analytical procedures without fiuther purification. 

The use of ethyl acetate was suggested by Oszmianski and Lee (1990) to wash out phenolics other than anthocyanins. Finally, a relatively pure anthocyanin extract can be removed from the colnmn with acidified methanol (0.1% HCl). Anthocyanin extracts can be enriched in this way by use of solid phase purification, which is especially helpful for diluted samples such as biological samples. Two factors in the nse of these purification techniques are the stability of anthocyanins to the conditions nsed and the ease of anthocyanin recovery from the column. ... 

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