Introduction

1. Study On Tannin–Metal Interactions in Aqueous Solution Using Spectrophotometric Titration and Micelle-Mediated Separation/Inductively Coupled Plasma. Ruiqiang Liu1, Michael A. Schmidt1, Steven R. Tindall1, Ann E. Hagerman1, Jonathan J. Halvorson2 and Javier M. Gonzalez2, (1)Chemistry & Biochemistry, Miami Univ., Oxford, OH (2)USDA-ARS-AFSRC, Beaver, WV Methods Introduction Methyl gallate and Fe(III) complexation – 1:2 and 3:2 metal: ligand complexes formed at pH 4 (Fig 3) and pH 6 (not shown) • Spectrophotometric titration • add 990 uL acetate buffer solution (pH 4 or 6) to the cuvette (2 mL capacity) • add 10 uL of 1 mM tannin solution (in 50% methanol ) • titrate with 1 mM metal solution using successive 2 uL aliquots • record the spectrum after each addition (200-800 nm) • Micelle mediated separation • add 300 uM iron(III) to 0-20 uM PGG • add 1 mL of surfactant mixture (60% Tx-114, 40% Tx-45) • heat above the micelle temperature for 30 min • cool on ice for 60 min and centrifuge at 3,000 g for 60 min (PGG:Fe complex partitions into the micelle leaving free iron in supernatant) • measure Fe in the supernatant using ICP • Tannins are the fourth most abundant biochemical produced by vascular plants after cellulose, hemicellulose, and lignin • Tannins are polymeric polyphenols and are classified as hydrolysable or condensed tannins (see below) • Tannin-metal interactions in soil may be important in: • a. podzolization of soils • b. mobilization of phosphorus & nitrogen • c. formation of humic substances • d. solubilization and toxicity of metals • Complexation between tannins and metals is involved in metal/tannin dissolution/precipitation, sorption/desorption, & reduction/oxidation • Most earlier studies focused on small nontannin phenolics or on poorly defined mixtures of tannins • Our laboratory uses structurally defined polymeric polyphenolics and their subunits as model compounds to understand tannins in the environment Interactions of Al(III) , Fe(III) & Ca(II) with other tannins and their monomeric units Table 1. Stoichiometry of some tannin-metal complexes as determined by UV-Vis spectrophotometric titration Fig 3a. UV titration spectra of Fe(III)/MeGallate/pH4 • Hydrolysable tannin • ( β-pentagalloyl-D-glucose, PGG) Results PGG and Fe(III) complexation at pH 6 – 1:1 Fe(III): PGG complex formed (Fig 1) b. Hydrolysable tannin (Fireweed tannin) * Metal : Tannin ratio * *Sorghum tannin molar ratio in catechin equivalent Fig 3b. UV Absorbance of Fe(III) /Methyl gallate /pH4@ 308nm Fig 3c. 1:2 (top) and 3:2 (bottom) Fe(III) / Methyl gallate complexes Fig 1a. UV titration spectra of Fe(III)/PGG/pH6 Micelle Mediated Separation with ICP detection Over the concentration range of PGG used, the stoichiometic ratio of Fe to PGG was 5.9 +/- 0.9. Unlike the UV/Vis method, the micelle-mediated method did not employ a buffer—so there was no competition between buffer and PGG for metal binding. Other differences between the methods may include time allowed for binding (instantaneous in UV/Vis, long equilibration in micelle-mediated), ratio of metal to phenolic in the reaction mixture, and direct vs. indirect measurement of the complex. Fig 3. 1:2 and 3:2 Fe(III) – methyl gallate complexes formed at pH 4 d. Methyl gallate PGG and Al(III) complexation– the PGG/Al(III) complex has 1:1 stoichiometry at both pH 4 and pH 6 c. Condensed tannin (Sorghum tannin, n=15) e. Catechin Summary • UV-Vis spectrophotometric titration and micelle mediated separation allowed us to determine the stoichiometric ratios of complexes between tannins and metals such as Fe(III) and Al(III) • Different tannins bind metals with different stoichiometries • pH influences stoichiometry, but the molecular basis for the pH-dependence has not been established • UV-Vis indicated that although tannins are polyphenolic, not every phenolic site binds metal • In contrast, micelle mediated separation suggested that every phenolic subunit of PGG does bind metal • In the immediate future we will establish the mechanistic basis for differences between the two methods, and will determine which method provides more useful data for understanding tannin-metal ion interactions Fig 1b. UV Absorbance of Fe(III) /PGG /pH6 @ 315nm Fig 1c. 1:1 Fe(III)-PGG complex Fig 1. 1:1 Fe(III) – PGG complex formed at pH 6 PGG and Fe(III) complexation at pH 4 – 2:1 Fe(III): PGG complex formed (Fig 2, below) Fig 4. 1:1 Al(III) – PGG complex formed at pH 4 & 6 • Ca(II) and Mn(II) complexation with PGG • Spectral changes reveal that PGG complexes Ca(II) or Mn(II) • 3:1 Ca(II)-PGG complex formed at pH 6 (Fig 5) • The spectral changes for Ca-PGG at pH 4, Mn-PGG at pH 4 & pH 6 were too small for quantitative analysis Fig 2a. UV titration spectra of Fe(III) /PGG /pH4 Objectives To characterize tannin complexation with Fe(III), Al(III), Ca(II) and Mn(II) using UV-Vis spectrophotometric titration To determine the stoichiometry of the tannin-metal complexes by UV-Vis spectroscopy and micelle mediated separation with ICP detection To evaluate the effect of pH on tannin-metal interactions Acknowledgement This project was funded by ARS Specific Cooperative Agreement Number 58-1932-6-634 with Miami University Fig 5. 3:1 Ca(II) – PGG complex formed at pH 6 Fig 2b. UV Absorbance of Fe(III) /PGG /pH6 @ 315nm Fig 2c. 2:1 Fe(III) – PGG complex