Table 817

Separation and Purification Methodologies on Betalain Studies

Methodology

Electrophoresis

Capillary zone electrophoresis

Thin layer chromatography

Ion exchange chromatography

High-performance liquid chromatography (HPLC)

Characteristics

Solid support: paper

Solvents: pyridine, formic, or acetic acid

Voltage: 5.6 V/cm

Temperature: 4°C

Solid support: fused silica

Voltage: 22 kV

Temperature: 18°C

Separation of isomeric forms such as betanin and isobetanin as well as the aglycones Solid support: cellulose-coated plate

Solvents: isopropanol-ethanol-water-acetic acid 6:7:6:1 v/v and 11:4:4:1 v/v

The acid makes the separation easy because protonated betalains have high mobility Solid support: diethylaminoethyl cellulose Solvent: isopropanol-water-acetic acid 13.4:1 v/v Good separation of betaxanthins

Solid support: Dowex, 50W-X2, Merck I, DEAE-Sephadex, and A-25, among others Solid support: reverse-phase C8 or C18 (Nucleosil™, LiChrosorb™,

^Bondapack™, among other columns) Solvents: water-methanol Water-acetonitrile

Mixtures are acidified with acetic, formic, or phosphoric acid Paired-ion systems using tetrabutylammonium The elution order is betanin, betanidin, isobetanin, and isobetanidin

Source: Adapted from Delgado-Vargas et al. (2000).17

Other important evaluations are used to characterize betalains (Table 8.19).17 They, as pigments, have maximum absorption in the visible region and this characterizes them; it is clear that structural modifications can be followed by using UV-visible spectroscopy.83,120,121 Acylated betalains exhibit a second maximum absorption in the UV region of 260 to 320 nm and the ratio of maximum visible region to maximum UV region is used as a measurement of the number of the acyl groups in the structure. In addition, pigment quantitation has been calculated by using mathematical methods associated with UV-visible spectroscopy. In particular, nonlinear curve fitting of the spectrum has been used with a predicted function of the individual pigments (betanin, betalamic acid, and vulgaxanthin-I, among others). The procedure is rapid and accurate, and interestingly, betalain separation is not required (Table 8.19).122 Analytical methodologies have shown impressive progress, and today HPLC methodology is coupled with DAD, MS, and NMR. Thus, separation and

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