Anthocyanins thin-layer chromatography

Paper chromatography (PC) and thin layer chromatography (TLC) have been used since the 1940s. Preparative PC on Whatman 3 paper, analytical PC on Whatman 1 paper, and analytical TLC on microcrystalline cellulose, silica gel, or polyamide have been applied with a variety of solvents and the behaviors of anthocyanins have been similar in all media. Two-dimensional TLC allows the separation of several compounds and has been nsed to clarify the anthocyanin compositions of different commodities. ... 

Quantification of anthocyanins/anthocyanidins is usually performed either by UVA/ IS-spectroscopy or by HPLC with UV-detection. For qualification and structural elucidation HPLC coupled to mass-spectrometry is used. Other techniques include thin layer chromatography, gas chromatography and capillary zone electrophoresis (Figure 17). 

Under routine conditions, enrichment and isolation of single anthocyanins followed by classical structural elucidation (mass spectrometry, elemental analysis, UVA IS absorbance evaluation using shift reagents, NMR, thin layer chromatography of sugars and anthocyanidins) is rarely necessary, hence will not be discussed in detail. Routine analysis focuses on the quantification and the distribution of known anthocyanins to confirm the content and source of anthocyanins. 

Although neither paper nor high performance- or thin-layer chromatography plays a major role for routine analysis of beny anthocyanins, the value of these methods for research oriented work should never be underestimated. 

Paper and thin layer chromatography have been used since decades for flavonoid analysis. As anthocyanins are colored substances, separation can be directly monitored and may serve as a quick, qualitative test for the anthocyanin distribution in a given sample. Classical paper chromatography involves two-dimensional development followed by treatment with selective spraying reagents or exposure to ammonia vapor and allocation of spots in visible or UV-light [90]. 

Paper chromatography may be used as one step for separation and/or isolation of anthocyanins from complex samples. Thin-layer chromatography, performed on cellulose or silica gel plates, was in the first period of use a convenient extension of paper chromatography. The introduction of new stationary phases and the development of instrumentation offering the possibility of quantitative analysis provided new possibilities for method developments. The advantages of TLC are the simplicity in terms of instrumentation, sample preparation and data evaluation. 

Kirchner (1978) has discussed the utility of thin-layer chromatography for the following natural pigments chlorophylls, carotenoids, xanthophylls, flavonoids, anthocyanins, porphyrins, and bile. From the standpoint of practical TLC, the most important of these pigments are the chlorophylls, carotenoids, xanthophylls, and anthocyanins, and it is these pigments that are considered further in this chapter. For practical TLC of the flavonoids, consult Harbome (1984, 1992). For information on the TLC of porphyrins, see Doss (1972), Dolphin (1983), and Jacob (1992). Jain (1996) has provided useful information on the examination of porphyrins (in studies on clinical porphyrias) by TLC in clinical chemistry. 

Asen, S. (1965). Preparative thin-layer chromatography of anthocyanins. J. Chromatogr. 18 602-603. 

Matysik, G. (1992). Thin-layer chromatography of anthocyanins with stepwise gradient elution. J. Planar Chromatogr.—Mod. TLC 52 146-148. 

Matysik, G., and Benes, M. (1991). Thin-layer chromatography and densitometry of anthocyanins in the petals of red poppy during development of the flowers. Chromato-graphia 32 19-22. 

Also Raman mapping combined with cluster analysis was successfully applied to discriminate flavonoids, anthocyanins, and carotenoids occurring in differently colored flower petals of various pansy cultivars Viola X wittrockiana) [27]. Extracts of the viola petals were separated by thin-layer chromatography (TLC) in order to get the Raman data of the individual pure substances. Applying hierarchical cluster analysis (HCA) of the spectra taken from the flower petals and from the pure TLC extracts, flavonols, anthocyanins, and carotenoids could... 

In the last years new developments in the field of plate chromatography have led to high performance thin layer chromatographic (HPTLC) systems. HPTLC offer simultaneous and cost-efficient screening of anthocyanin samples and provides the possibility for quantitative analysis. 

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