Purification anthocyanins

Supercritical fluid extraction (SEE) is another modern separation technology usually employed to extract lipophilic compounds such as cranberry seed oil, lycopene, coumarins, and other seed oils. Anthocyanins generally and glycosylated anthocyanins in particular were considered unsuitable for SEE due to their hydrophilic properties, since SEE is applicable for non-polar analytes. However, a small amount of methanol was applied as co-solvent to increase CO2 polarity in anthocyanin extraction from grape pomace. New applications of SEE for anthocyanin purification have been reported for cosmetic applications from red fruits. ... 

Support Protocol Sample Preparation for Anthocyanin Purification FI. 1.4... 

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). 

Anthocyanin purification steps are important for anthocyanin characterization. Removal of interfering compounds allows for more reliable HPLC separation, spectral information, mass spectra, and NMR spectra during the identification of anthocyanins in plant extracts. 

Degenhardt, A., Knapp, H., and Winterhalter, P, Separation and purification of anthocyanins by high-speed countercurrent chromatography and screening for antioxidant activity, J. Agric. Food Chem., 48, 338, 2000. 

To purify, concentrate, and recover different pigments (flavonoids or anthocya-nins), various ion-changing resins were used. Recent screenings of 13 commercial resins [acrylic or styrene-divinylbenzene (SDVB)] for the purification and specific absorption of anthocyanins - used ethanol, methanol, and water mixtures as eluents at pH 3.5. DDVB resins (EXA-118 and EXA-90) were found most suitable using a mixture of methanol and water (1 1) for elution. The other routinely used resins like XAD-7 showed low efficiency. 

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. 

Purification of anthocyanin-containing extracts is often necessary for further structural identification. Since none of the solvents used for extraction is specific for anthocyanins, considerable amounts of other compounds may be also extracted and concentrated. The variety and concentration of other compounds will depend on the solvent and methodologies used. The presence of extraneous materials could affect the stability and/or analysis of anthocyanins. Therefore, the next step toward anthocyanin characterization is the purification of those extracts. 

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. ... 

ESI-MS has emerged as a powerful technique for the characterization of biomolecules, and is the most versatile ionization technique in existence today. This highly sensitive and soft ionization technique allows mass spectrometric analysis of thermolabile, non-volatile, and polar compounds and produces intact ions from large and complex species in solution. In addition, it has the ability to introduce liquid samples to a mass detector with minimum manipulation. Volatile acids (such as formic acid and acetic acid) are often added to the mobile phase as well to protonate anthocyanins. A chromatogram with only the base peak for every mass spectrum provides more readily interpretable data because of fewer interference peaks. Cleaner mass spectra are achieved if anthocyanins are isolated from other phenolics by the use of C18 solid phase purification. - ... 

Rodriguez-Saona, L.E. and Wrolstad, R.E., Extraction, isolation, and purification of anthocyanins, in Current Protocols in Food Analytical Chemistry, Wrolstad, R.E., et al., Eds., John Wiley Sons, New York, 2001, 1. 

Fiorini, M., Preparative high-performance liquid chromatography for the purification of natural anthocyanins, J. Chromatogr. A, 692, 213, 1995. 

Absorption spectra have also been used in the reexamination of pH-dependent color and structural transformations in aqueous solutions of some nonacylated anthocyanins and synthetic flavylium salts." ° In a recent study, the UV-Vis spectra of flower extracts of Hibiscus rosasinensis have been measured between 240 and 748 nm at pH values ranging from 1.1 to 13.0." Deconvolution of these spectra using the parallel factor analysis (PARAFAC) model permitted the study of anthocyanin systems without isolation and purification of the individual species (Figure 2.21). The model allowed identification of seven anthocyanin equilibrium forms, namely the flavylium cation, carbinol, quinoidal base, and E- and Z-chalcone and their ionized forms, as well as their relative concentrations as a function of pH. The spectral profiles recovered were in agreement with previous models of equilibrium forms reported in literature, based on studies of pure pigments. 

Ordaz-Galindo, A. et al.. Purification and identification of Capulin (Prunus serotina) anthocyanins, Food Chem., 65, 201, 1999. 

Figure F1.1.1 Solid-phase (C18) purification of anthocyanins. The sample components (represented by differentially shaded circles) are resolved by subsequent wash steps as indicated. The last wash, with acidified methanol, elutes anthocyanins. Acidified methanol and/or water should be used as solvents if electrospray mass spectrometry is to be carried out subsequently.

Different resins have been used to clean up or prefractionate anthocyanins prior to isolation or characterization, including ion-ex-change resins, polyamide powders, and gel materials. Chromatography on Dowex or Amber-lite ion-exchange resins, as well as polyamide powders such as polyvinylpyrrolidone (PVP), have been used to isolate polar nonphenolic compounds from crude anthocyanin extracts. Column chromatography on Sephadex LH-20 can be used for fractionation of crude extracts and is also particularly useful for purification... 

A key factor in the use of these purification techniques is the stability of anthocyanins under the conditions used as well as the ease of anthocyanin recovery from the column (Strack and Wray, 1989). 

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