Anthocyanins reversed-phase chromatography

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. 

Kozminski R and Brett AMO, Reversed-phase high-performance liquid chromatography with electrochemical detection of anthocyanins. Anal Lett 39 2687-2697 (2006). 

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. 

After a progressive decline in anthocyanin concentration, the presence of a broad unresolved peak is typically observed from aged wine when analyzed by reversed-phase HPLC (23) or gel permeation chromatography (7). Historically this hump was considered to be composed primarily of pigmented polymers, although until recently (16) evidence for this was based upon chromatographic evidence. 

Downey MO, Rochfort S (2008) Simultaneous separation by reversed-phase high-performance liquid chromatography and mass spectral identification of anthocyanins and flavonols in Shiraz grape skin. J Chromatogr A 1201 43—47... 

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. 

Casteele, K.V., Gieger, H., De Loose, R., Van Sumere, C.F., Separation of some anthocya-nidins, anthocyanins, proanthocyanidins and related substances by reversed-phase high-performance Uquid chromatography, J. Chromatogr., 1983, 259, 291-300. 

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