Abstract
Determination of correct amount (concentration) of phytic acid is of vital importance when dealing with protonation and/or metal complexation equilibria. A novel approach for precise and reliable assay of phytic acid, based on the difference between end points by potentiometric titration, has been presented. Twelve phytic acid protons are classified into three groups of acidity, which enables detection of 2 to 3 distinct equivalent points (EPs) depending on experimental conditions, e.g. counter-ion concentration. Using the differences between individual EPs enables correct phytate determination as well as identification of potential contamination and/or determination of initial protonation degree. Impact of uncertainty of phytate amount on the calculation of protonation constants has been evaluated using computer simulation program (Hyperquad2013). With the analysis of titration curves different binding sites on phytate ligand have been proposed for complexation of Ca2+ and Fe3+ ions.
Highlights
Myo-inositol 1,2,3,4,5,6-hexakis(dihydrogen phosphate), known as phytic acid (H12Phy), exhibits six acidic phosphate groups around a cyclohexane ring (Fig. 1)
The general protonation equilibrium of phytate (Phy12–) can be given by the reaction where Phy12– represents completely deprotonated form of phytic acid H12Phy and index i is the number of protonation step, 0 ≤ i ≤ 12
In the region before first EP1 a group of strong acidic protons exists with an average dissociation constants pKa around 2.6 ± 0.1, the second group appears between EP1 and EP2 with an average pKa of 7.2 ± 0.2, and a third group of protons can be predicted at an average pKa of 11.5 ± 0.5
Summary
Myo-inositol 1,2,3,4,5,6-hexakis(dihydrogen phosphate), known as phytic acid (H12Phy), exhibits six acidic phosphate groups around a cyclohexane ring (Fig. 1). The magnitude of the interaction between metal ions and chelating agents is defined by the corresponding equilibrium constants, and nowadays most of them are determined experimentally by spectrophotometry, potentiometry and other techniques (NMR) in combination with computational analysis of data.[18] Protonation of phytates and/or determination of stability constants with metallic ions are the subject of numerous publications.[1,12,19] With help of computerized programs determination of such data becomes easier and published constants are nowadays of high precision, which was not the case by data published in the classical monographs on stability constants,[20,21] where data frequently scatter between different methods and sources for more orders of magnitude
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