Introduction. Introduction. Properties of Tannins. Chemical Structures of Humic and Fulvic Acids. Chemical Structures of Phenolic Acids/Tannins

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1 Isolation and Analysis of Humic, Fulvic and Tannic Acids from Savannah, GA Marsh Soils and their Binding apacity for Metal Ions. Eugenia S. Narh Advisor: Delana Nivens Department of hemistry and Physics Armstrong Atlantic State University Savannah, GA Introduction Humic, fulvic, and tannic acids are complex organic molecules produced when plants, fats, excrement and organisms decompose oxidatively in the environment. Fulvic acid has the lowest molecular weight in the humic group and solubility over the entire ph range. Humic acids have higher molecular weight but are soluble only above ph. These materials have been shown previously to affect the ph of natural waters, trace metal aquatic chemistry, bioavailability, and the degradation and transport of hydrophobic organic materials. As a consequence of acid rain and other environmental processes, many metal ions are increasingly prevalent in aquatic environments. Studying these acids from natural environments can provide valuable information about their interaction with biologically hazardous metals. hemical Structures of Humic and Fulvic Acids Introduction Tannins are phenolic compounds composed of a very diverse group of oligomers and polymers found in plants parts including the leaves, roots and fruits. They precipitate proteins and also complex with starch, cellulose, and minerals. Tannins are usually subdivided into two groups: hydrolyzable tannins (HT) and condensed tannins. HTs include gallic acid (gallotannins) and ellagic acid (ellagitannins), and are usually present in low amounts in plants. These substances are environmentally important because they are water soluble at most ph s and they tend to bind and sequester toxic metal ions which reduces bioavailability. Properties of Tannins Hydrolyzable Tannins (HT) hydrolyzed by mild acids or mild bases to yield carbohydrate and phenolic acids Under the same conditions, proanthocyanidins (condensed tannins) do not hydrolyze HTs are also hydrolyzed by hot water or enzymes Tannins core of D-glucose carbohydrate esterified with phenolic groups Gallic acid Most famous source of gallotannins is tannic acid obtained from the twigs galls of Rhus semialata Murray plant Ellagic acid Molecular weight range: The phenolic groups consist of hexahydroxydiphenic acid, which spontaneously dehydrates to the lactone form, ellagic acid ondensed Tannins polymers of flavonoid units hemical Structures of Phenolic Acids/Tannins H H Ellagic acid H H H H H H H H H H H H H H H Tannic acid H H H H H H Gallic acid H H H H H H H Flavone 1

2 Experimental Details ollection and Preparation of Samples ollection and preparation of samples Five samples each of Spartina grass and marsh soil were obtained along the Savannah marsh Experimental Details Extraction of Humic, Fulvic acids and Tannins Humic and fulvic acids were extracted from the marsh soil through a process that employed the differences in ph of the humic and fulvic acids with the use of several solvents Tannins were extracted with an aqueous organic solvent consisting of 7% acetone and 3% water from the leaves and roots of the grass samples Extraction of polyphenolics Polyamide mini-column chromatography was utilized to separate flavanols and ellagic acid derivatives Experimental Details Total phenolics determination ondensed Tannin Determination with Vanillin- Hl HPL analysis Fluorescence titration analysis G-Derivitization Total Phenolic Determination Total Phenolics Determination Total phenolics were determined with tannic acid standards equivalents as described by Siriwoharn and Wrolstad. To an aqueous solution of tannin extract and a series of tannic acid solutions was added % Na 3 followed by heating and cooling of the samples The absorbance of the samples and standards were measured at 7 nm using an HP 843 UV-vis spectrophotometer Results were calculated as parts per million of tannic acid per 1 g fresh leaves weight ppm TA ppm TA 7 ppm TA 1 ppm TA 1 ppm TA 1 ppm TA 17 ppm TA ppm TA TA extract oncentration of Tannin Extract = 11.6 ppm Linear Regresion of conc. of TA vs. at 767 nm.7 y =.7x R = oncentration of TA (ppm)

3 ondensed Tannin Determination with Vanillin-Hl Solutions of (+)-atechin standard were used for the vanillin assay 4% vanillin (w/v) in methanol and concentrated Hl were added to crude tannin extract dissolved in methanol and to the (+)-atechin solutions The absorbance of the sample and standard solutions were measured at nm with a UV- Vis spectrophotometer. The interference background of the crude extract was corrected by preparing the test without vanillin 9.9E-1 7.9E-1.9E-1 3.9E-1 1.9E-1 ondensed Tannin Determination Vanillin-Hl Assay -1.E Wavelength (nm ) ppm atechin ppm atechin 1 ppm atechin 1 ppm atechin ppm atechin TA Extract Tannin oncentration = 6.7 ppm ondensed Tannin determination - at nm y =.39x +.89 R = oncentration of atechin std. (ppm) HPL Analysis HPL of Standards Hewlett Packard Series 11 HPL System was used in the analysis of extracted polyphenolics. Mobile phases were solvent A: 1% HPLgrade methanol; solvent B: 1% HPL acetonitrile; and solvent :. M KH P 4 (ph 3.). oncentration of standards ((+)-catechin hydrate, ellagic acid, gallic acid, rutin hydrate) was 1 mg/ml. (+)-atechin hydrate Rutin hydrate Ellagic Acid Gallic Acid HPL of rude Tannin Extract HPL of Tannin Extract (Ammonia fraction from polyamide mini-column chromatography) = (+)-atechin 1 = Ellagic acid, Rutin 1 = (+)-atechin 13 = (+)-atechin, Ellagic acid, Rutin

4 F F Fluorescence Titration Analysis Fluorescence of Tannin Extract Instrument: Shimadzu RF-31 P Spectrofluorophotometer Parameters: Excitation Emission wavelength = 4 nm Excitation wavelength range = 8-4 nm Emission Excitation wavelength = 34 nm Emission wavelength range = 36-6 nm Slit width = Ex: 1; Em: 1; Sensitivity = High 3 ml of standards were pipetted into a quartz cuvette and titrated with.1 M of metal ions. The Stern-Volmer equation was used to calculate the binding capacity or quenching constant of the metals Excitation Emission Emission Spectra of Standards and Extract Wavelength Extract Gallic Acid atechin Rutin Stern-Volmer Equation Used to calculate binding/quenching constant for metals o φ f = 1+ K q [ Q] φ f K q = binding/quenching constant K q = m/b m is the slope of the graph of F vs. [ ] of metal b is the y-intercept of the graph Fluorescence of 1 mg/l (+)-atechin titration with.1 M Al µl. µl 1. µl 1. µl. µl. µl 3. µl 3. µl 4. µl 4. µl 1 1.E+ Kq = 6,93 y = 416x R =.961 Kq = 1,739 y = 677x R =.88.E- 4.E- 6.E- 8.E- 1.E-4 1.E-4 1.4E-4 1.6E-4 onc. after adding.1 M Al 3+ to atechin Fluorescence of 1 mg/l Gallic Acid titration with.1 M Al µl. µl 1. µl 1. µl. µl. µl 3. µl 3. µl 4. µl 4. µl K q =41,678 y = 76899x R =.996 E- 4E- 6E- 8E onc. after adding.1 M Al3+ (M) 4

5 F Fluorescence of 1 mg/l Rutin titration with.1 M Al µl. µl 1. µl 1. µl. µl. µl 3. µl 3. µl 4. µl 4. µl 6 4 y = x -.48 R =.9871 G-Derivitization Derivitization of tannin extract was performed using Tri Sil Z and Tri Sil TBT for analysis by gas chromatography. The standards (+)-catechin, ellagic acid, gallic acid, and rutin were also derivitized but did not yield results. 3 K eq =8,844 1.E+ 4.E- 8.E- 1.E-4 1.6E-4 G analysis was not successful for any of the samples. onc. after adding.1 M Al 3+ to rutin hydrate Discussion The excessive time needed to extract the acids from the soil and plant samples limited the amount of work that was done afterwards. Even after the long extraction process, the amount of extracts obtained were not enough for all the intended investigations. Not having enough samples also introduced the issue of concentration differences between each batch of extracts and the analysis they were used for. onclusion The standards of (+)-catechin hydrate, ellagic acid, gallic acid, and rutin were analyzed using fluorescence titration analysis during the tannins extraction process. The HPL analysis indicated that (+)-catechin, ellagic acid, and rutin were the possibly present in the extract. This was the first trial so further trials could yield better results. Further extractions and analysis must be performed to confirm the binding of tannin extract to metal ions. References Unpublished results. Miller, J. et. al. Isolation and Analysis of Humic and Fulvic from Savannah, GA Marsh Soils and Its Binding apacity for Aluminum. Department of hemistry and Physics, Armstrong Atlantic State University. Tannins: Properties. toxicagents/tannin/chem_anl.html (accessed Apr 14, 8). Siriwoharn, T.; Wrolstad, R. E. Polyphenolic omposition of Marion and Evergreen Blackberries. J. Food Sci. 4, 69, Acknowledgements Dr. Nivens, Department of hemistry & Physics, Armstrong Atlantic State University. Dr. Matt Gilligan, Marine Science Department, Savannah State University AASU Department of hemistry and Physics