1 Isolation and Analysis of Humic, Fulvic and Tannic Acids from Savannah, GA Marsh Soils and their Binding Capacity for Metal Ions. Eugenia S. Narh Advisor: Delana Nivens Department of Chemistry and Physics Armstrong Atlantic State University Savannah, GA 31419 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 2. • 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. Chemical 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: 2000 – 5000 – The phenolic groups consist of hexahydroxydiphenic acid, which spontaneously dehydrates to the lactone form, ellagic acid • Condensed Tannins – polymers of 2 – 50 flavonoid units Chemical Structures of Phenolic Acids/Tannins O O Flavone O O O H2C O O C O HO OH OH C O HO HO HO C O HO HO O C O OH OH HO O C O HO OH O C O OH OH O C O OH OH HO C O OH OH OH Tannic acid O O O HO HO O OH OH COOH HO OH OH Ellagic acid Gallic acid
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Isolation and Analysis of Humic, Fulvic and tannic acids · • Humic, fulvic, and tannic acids are complex organic molecules produced when plants, fats, excrement and organisms decompose
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1
Isolation and Analysis of Humic, Fulvic
and Tannic Acids from Savannah, GA
Marsh Soils and their Binding
Capacity for Metal Ions.
Eugenia S. NarhAdvisor: Delana Nivens
Department of Chemistry and PhysicsArmstrong Atlantic State University
Savannah, GA 31419
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 highermolecular weight but are soluble only above pH 2.
• 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 aquaticenvironments. Studying these acids from natural environments can provide valuable information about their interaction with biologically hazardous metals.
Chemical 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. HTsinclude 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: 2000 – 5000 – The phenolic groups consist of hexahydroxydiphenic acid, which spontaneously
dehydrates to the lactone form, ellagic acid
• Condensed Tannins – polymers of 2 – 50 flavonoid units
Chemical Structures of PhenolicAcids/Tannins
O
O
Flavone
OOO
H2C
O O
C O
HO
OH
OH
C
OHO
HO
HO
C
O
HO
HO
O
CO
OH
OH
HO
O
CO
HO
OH
O C
OOH
OH
O
C O
OHOH
HO
C
O
OH
OH
OH
Tannic acid
O
O
O
HO
HO
O
OH
OH COOH
HO
OH
OH
Ellagic acid Gallic acid
2
Experimental Details
• Collection and preparation of samples
– Five samples each of Spartina grass and marsh soil were obtained along the Savannah marsh
Collection and Preparation of Samples
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 70% acetone and 30% 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
• Condensed Tannin Determination with Vanillin-HCl
• HPLC analysis
• Fluorescence titration analysis
• GC-Derivitization
Total Phenolic 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 20% Na2CO3 followed by heating and cooling of the samples
• The absorbance of the samples and standards were measured at 755 nm using an HP 8453 UV-vis spectrophotometer
• Results were calculated as parts per million of tannic acid per 10 g fresh leaves weight.
Total Phenolics Determination
0
0.1
0.2
0.3
0.4
0.5
0.6
400 500 600 700 800 900 1000
Wavelength (nm)
Absorb
ance
25 ppm TA 50 ppm TA 75 ppm TA 100 ppm TA 125 ppm TA 150 ppm TA
175 ppm TA 200 ppm TA TA extractLinear Regresion of conc. of TA vs.
Absorbance at 767 nm
y = 0.0027x + 0.0125
R2 = 0.9584
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 50 100 150 200 250
Concentration of TA (ppm)
Ab
so
rban
ce
Concentration of Tannin Extract = 101.6 ppm
3
Condensed Tannin Determination with Vanillin-HCl
• Solutions of (+)-Catechin standard were used for the vanillin assay
• 4% vanillin (w/v) in methanol and concentrated HCl were added to crude tannin extract dissolved in methanol and to the (+)-Catechinsolutions
• The absorbance of the sample and standard solutions were measured at 500 nm with a UV-Vis spectrophotometer.– The interference background of the crude extract was
Fluorescence of 15 mg/L Rutintitration with 0.1 M Al3+
0
5
10
15
20
25
30
35
40
45
50
400 450 500 550 600
Wavelength (nm)
Inte
ns
ity
0 µL
0.5 µL
1.0 µL
1.5 µL
2.0 µL
2.5 µL
3.0 µL
3.5 µL
4.0 µL
4.5 µL
y = 333817x - 0.4082
R2 = 0.9871
0
10
20
30
40
50
60
0.0E+00 4.0E-05 8.0E-05 1.2E-04 1.6E-04
Conc. after adding 0.1 M Al3+ to rutin hydrate
F
Keq=82,844
GC-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 derivitizedbut did not yield results.
• GC analysis was not successful for any of the samples.
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.
Conclusion
• The standards of (+)-catechin hydrate, ellagic acid, gallicacid, and rutin were analyzed using fluorescence titration analysis during the tannins extraction process.
• The HPLC analysis indicated that (+)-catechin, ellagicacid, 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 Capacity for Aluminum. Department of
Chemistry and Physics, Armstrong Atlantic State University.