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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

Introduction

1. Tannins are the fourth most abundant biochemical produced by vascular plants after cellulose, hemicellulose, and lignin

2. Tannins are polymeric polyphenols and are classified as hydrolysable or condensed tannins (see below)

1. 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

1. Complexation between tannins and metals is involved in metal/tannin dissolution/precipitation, sorption/desorption, & reduction/oxidation

2. Most earlier studies focused on small nontannin phenolics or on poorly defined mixtures of tannins

3. Our laboratory uses structurally defined polymeric polyphenolics and their subunits as model compounds to understand tannins in the environment

Methods

Results

1. PGG and Fe(III) complexation at pH 6 – 1:1 Fe(III): PGG complex formed (Fig 1)

Summary

Acknowledgement

1. 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)

2. Different tannins bind metals with different stoichiometries

3. pH influences stoichiometry, but the molecular basis for the pH-dependence has not been established

4. UV-Vis indicated that although tannins are polyphenolic, not every phenolic site binds metal

5. In contrast, micelle mediated separation suggested that every phenolic subunit of PGG does bind metal

6. 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

a. Hydrolysable tannin ( β-pentagalloyl-D-glucose, PGG)

b. Hydrolysable tannin (Fireweed tannin)

c. Condensed tannin (Sorghum tannin, n=15)

Objectives

1. Spectrophotometric titrationa. add 990 uL acetate buffer solution (pH 4 or 6) to the cuvette (2 mL

capacity)b. add 10 uL of 1 mM tannin solution (in 50% methanol )c. titrate with 1 mM metal solution using successive 2 uL aliquotsd. record the spectrum after each addition (200-800 nm)

2. Micelle mediated separation

a. add 300 uM iron(III) to 0-20 uM PGGb. add 1 mL of surfactant mixture (60% Tx-114, 40% Tx-45)c. heat above the micelle temperature for 30 mind. 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)

e. measure Fe in the supernatant using ICP

4. PGG and Al(III) complexation– the PGG/Al(III) complex has 1:1 stoichiometry at both pH 4 and pH 6

6. 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

* Metal : Tannin ratio* *Sorghum tannin molar ratio in catechin equivalent

This project was funded by ARS Specific Cooperative Agreement Number 58-1932-6-634 with Miami University

1. To characterize tannin complexation with Fe(III), Al(III), Ca(II) and Mn(II) using UV-Vis spectrophotometric titration

2. To determine the stoichiometry of the tannin-metal complexes by UV-Vis spectroscopy and micelle mediated separation with ICP detection

3. To evaluate the effect of pH on tannin-metal interactions

2. PGG and Fe(III) complexation at pH 4 – 2:1 Fe(III): PGG complex formed (Fig 2, below)

Tannins

Fe(III) Al(III) Ca(II)

pH4 pH6 pH4 pH6 pH6

PGG 2:1* 1:1 1:1 1:1 3:1Methyl Gallate

1:2; 3:2 1:2; 3:2 1:1 1:2 1:1

Catechin 1:1 1:1 1:1 1:1 Not clear

Fireweedtannin

1:2 1:1 1:1 1:1 1:1

Sorghum tannin*

3:2 1:2; 3:2 Not clear 1:2; 1:1 1:1

d. Methyl gallate

e. Catechin

Fig 1. 1:1 Fe(III) – PGG complex formed at pH 6

Fig 1c. 1:1 Fe(III)-PGG complex

Fig 2c. 2:1 Fe(III) – PGG complex

Fig 4. 1:1 Al(III) – PGG complex formed at pH 4 & 6

Fig 5. 3:1 Ca(II) – PGG complex formed at pH 6

3. 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)

Fig 3. 1:2 and 3:2 Fe(III) – methyl gallate complexes formed at pH 4

Fig 3c. 1:2 (top) and 3:2 (bottom) Fe(III) / Methyl gallate complexes

7. 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 1b. UV Absorbance of Fe(III) /PGG /pH6 @ 315nm

Fig 2b. UV Absorbance of Fe(III) /PGG /pH6 @ 315nm

Fig 3b. UV Absorbance of Fe(III) /Methyl gallate /pH4@ 308nm

5. Ca(II) and Mn(II) complexation with PGG a. Spectral changes reveal that PGG complexes Ca(II) or Mn(II) b. 3:1 Ca(II)-PGG complex formed at pH 6 (Fig 5)c. The spectral changes for Ca-PGG at pH 4, Mn-PGG at pH 4 &

pH 6 were too small for quantitative analysis

Fig 1a. UV titration spectra of

Fe(III)/PGG/pH6

Fig 3a. UV titration spectra of

Fe(III)/MeGallate/pH4

Fig 2a. UV titration spectra of

Fe(III) /PGG /pH4