Abstract
This study was undertaken to evaluate the possibility that an analytical ion-pair HPLC procedure used to determine phytic acid (IP6) and its degradation products (IP3–IP5) can be transformed to a preparative purification method. A commercial phytic acid (CPA) preparation was separated into its component fractions of IP3, IP4, IP5, and IP6 on two C 18 columns (1.8 and 4.2 ml) using 51% methanol containing 0.6–1% tetrabutylammonium hydroxide as ion-pair reagent and 0–0.025 M formic acid (pH 4.3) as eluent at 1.7 and 3.0 cm/min linear velocity, respectively, and 40°C. Elution was monitored at 40°C by a refractive index detector. Reproducible separation of CPA into four well-resolved peaks on these columns was possible after optimizing method variables, particularly the concentration of ion-pair reagent in the injected sample (>1.5%). The same separations were obtained after CPA loads were scaled up 25 times on a steel column (15 cm×19 mm I.D.), packed with Ethyl C 2 sorbent (10 μm) and on a 25 cm×21.2 mm I.D. C 18 column, but at a reduced linear velocity to increase the resolution. Therefore, this optimization of separation not only is useful for analysis of phytic acid and its degradation products but also it provides key parameters for scale up for further fractionation and characterization.
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