Anthocyanins solid phase 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. ... 

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. - ... 

Only for dietary supplements or processed food samples, extraction of anthoeyanins followed by solid phase purification with subsequent analysis by HPLC with UVA/ IS detection is performed as first level analysis. The matrix in those samples is complex and may include synthetic colorants in accordance with applicable food legislation, hence simple UVA IS analysis would yield most certainly erroneousness results. At this stage of analysis a decision is necessary on whether or not HPLC/MS analysis has to be performed. HPLC/ MS is powerful for the confirmation of anthocyanin structures but seldom useful for quantification as the calibration is complicated and robustness is low. 

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. 

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.

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 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. 

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. 

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