Anthocyanins column chromatography

The anthocyanin profile of the flowers of Vanda (Orchidaceae) was investigated with a similar technique. Flowers (2 kg) were extracted with 101 of methanol-acetic acid-water (9 l 10,v/v) at ambient temperature for 24 h. The extract was purified by column chromatography, paper chromatography, TLC and preparative RP-HPLC. Analytical HPLC was carried out in an ODS column (250 X 4.6 mm, i.d.) at 40°C. Gradient conditions were from 40 per cent to 85 per cent B in 30 min (solvent A 1.5 per cent H3P04 in water solvent B 1.5 per cent H3P04, 20 per cent acetic acid and 25 per cent ACN in water). The flow rate was 1 ml/min and analytes were detected at 530 nm. The chemical structures of acylated anthocyanins present in the flowers are compiled in Table 2.90. The relative concentrations of anthocyanins in the flower extracts are listed in Table 2.91. It can be concluded from the results that the complex separation and identification methods (TLC, HPLC, UV-vis and II NMR spectroscopy, FAB-MS) allow the separation, quantitative determination and identification of anthocyanins in orchid flowers [262],... 

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... 

Johnson, T.V. and Morris, J.R. 1996. Separation of anthocyanin pigments in wine by low pressure column chromatography. J. Food Sci. 61, 109-111. 

For isolation of single anthocyanins semi-preparative column chromatography has been successfully applied [67, 132-134]. Gradient or stepwise elution of anthocyanins with collection of fractions is in most cases followed by evaporation of the individual fractions and collection of the dry residue (in most cases as crystalline Cl -salt) for further analysis steps. 

Strack D, Mansell RL (1975) Polyamide column chromatography for resolution of complex mixtures of anthocyanins. J Chromatogr A 109(2) 325-331... 

Flavonol glycosides were suggested as a suitable indicator of the adulteration of black currant products with red currants (74) and with blackberries (75). The black currant methanolic extract is purified using polyamide columns and analyzed for flavonoids by HPLC. Anthocyanins are removed by chromatography on Dowex 50W-X4, eluting with 70% methanol (74). Quercetin-3-0-gluccuronide was a suitable indicator to detect adulteration of blackcurrant products with blackberries (75). 

Reversed-phase high-performance liquid chromatography (RP-HPLC) is the usual method of choice for the separation of anthocyanins combined with an ultraviolet-visible (UV-Vis) or diode-array detector (DAD)(Hebrero et al., 1988 Hong et ah, 1990). With reversed-phase columns the elution pattern of anthocyanins is mainly dependent on the partition coefficients between the mobile phase and the Cjg stationary phase, and on the polarity of the analytes. The mobile phase consists normally of an aqueous solvent (water/carboxylic acid) and an organic solvent (methanol or acetonitrile/carboxylic acid). Typically the amount of carboxylic acid has been up to 10%, but with the addition of a mass spectrometer as a detector, the amount of acid has been decreased to as low as 1 % with a shift from trifluoroacetic acid to formic acid to prevent quenching of the ionization process that may occur with trifluoroacetic acid. The acidic media allows for the complete displacement of the equilibrium to the fiavylium cation, resulting in better resolution and a characteristic absorbance between 515 and 540 nm. HPLC separation methods, combined with electrochemical or DAD, are effective tools for anthocyanin analysis. The weakness of these detection methods is a lack of structural information and some nonspecificity leading to misattribution of peaks, particularly with electrochemical... 

Vergara, C. Mardones, C. Hermosin-Gutierrez, 1. von Baer, D. 2010. Comparison of high-performance liquid chromatography separation of red wine anthocyanins on a mixed-mode ion-exchange reversed-phase and on a reversed-phase column. J. Chromatogr. A. 1217 5710-5717. 

Separation of the anthocyanins by partition chromatography was reported by Spaeth and Rosenblatt (1949, 1950), who utilized columns of silicic acid with 10% phosphoric acid as the immobile aqueous phase and a mixture of phenol and toluene as the nonaqueous phase. Following separation the anthocyanidins were quantitatively determined by means of optical density measurements. Mathers (1951) purified prior to chromatographing. He used calcium chloride-methyl alcohol to precipitate the color, dissolving in phosphoric acid-isopropyl alcohol, adding ether, and passing the solution through a silicic acid column. 

A series of HPLC methods for quantitative anthocyanin analysis have been published in the last years (Table 11). Most methods are comprised of reversed-phase chromatography (Cl8 columns, standard dimensions) at 0.5-2.0 mL/min and 25-45 °C for native anthocyanin separation with subsequent UV-detection at 520-530 nm and quantification against external anthocyanin standards. 

The fundamental components of any modern-day HPLC system are a solvent delivery system, a sample injector, a column, a detector, and a computer with the appropriate data acquisition and processing software. There are numerous HPLC methods described in the literature for isoflavones [13-25] and for the common anthocyanins, each method invokes different combinations of solvent systems, columns, and detectors. HPLC has been interfaced with a variety of detection methods such as ultraviolet/visible (UV/vis) spectrocopy and hquid chromatography-mass spectrometry (LC-MS) [21,22]. In this chapter, however, discussion is restricted to the most commonly used pairing in flavonoid analysis, that of a reverse-phase (RP-18) column and a UV/visible detector. 

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