BETANINE SEPARATION AND QUANTIFICATION BY CHROMATOGRAPHY ON GELS

ABSTRACT Published methods for separation and quantification of betalaine pigments from plant extracts are based on electrophoretic and spectro‐photometric techniques. These methods are either time consuming, or lack accuracy if interfering substances are present; therefore, a more rapid and accurate method for betalaine separation and betanine quantification is needed. Chromatography on gel filtration supports (Sephadex or Bio‐Gel polyacrylamide) is shown to be a rapid and efficient method of separating betalaines from raw beet juice. Raw and fermented beet juice ( Beta vulgaris ) directly applied to a column of polyacrylamide gel (Bio‐Gel P‐6) resulted in the detection of numerous pigment bands. Observed average distribution coefficients (K av ) for betanine on Sephadex G‐25 or Bio‐Gel P‐6 ranged from 0.8–2.0 at a pH value of 4.0–2.0, respectively. These data suggest that the major mechanism of retention of the pigments on gel supports can be adsorption rather than gel‐filtration. Resolution of betanine and betanidme was greater on columns packed with Sephadex G‐25 compared to columns packed with Bio‐Gel P‐6. Loading capacity (based on reduced plate height) was greater on columns packed with Bio‐Gel P‐6 when comparing the two column packings. When Bio‐Gel P‐6 was chosen as the support for the separation of pigments, excellent separation was obtained using a phosphate buffer at pH 3.0. Elution patterns of betanine were recorded by measuring the absorption at the maximum wavelength. Peak areas obtained were related to standard concentrations of betanine.