Introduction
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
• 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
•
•
Chemical Structures of Phenolic
Acids/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
OH
OH
HO
O
O C
OH
O
O C
O
O
C
HO
Tannins – core of D-glucose carbohydrate esterified with phenolic groups
HO
•
OH
OH
HO
Gallic acid
– Most famous source of gallotannins is tannic acid obtained from the twigs galls of
Rhus semialata Murray plant
HO
C O
O
OH
OH
O C
O
O
HO O
HO
C O
O C
O
H2 C
O
C O
OH
OH
OH
HO
HO
OH
OH
•
•
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
Tannic acid
O
O
HO
HO
COOH
OH
O
OH
HO
O
O
Ellagic acid
OH
OH
O
Gallic acid
Flavone
1
Collection and Preparation of
Samples
Experimental Details
• Collection and preparation of samples
– Five samples each of Spartina grass and marsh soil
were obtained along the Savannah marsh
Experimental Details
Experimental Details
• Total phenolics determination
• 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
• Condensed Tannin Determination with VanillinHCl
• HPLC analysis
• Fluorescence titration analysis
• Extraction of polyphenolics
• GC-Derivitization
– Polyamide mini-column chromatography was utilized
to separate flavanols and ellagic acid derivatives
Total Phenolic Determination
Total Phenolics Determination
0.6
0.5
0.4
Absorbance
0.3
0.2
0.1
0
400
500
600
700
800
900
1000
Wavelength (nm)
25 ppm TA
50 ppm TA
75 ppm TA
175 ppm TA
200 ppm TA
TA extract
100 ppm TA
125 ppm TA
150 ppm TA
Concentration of Tannin Extract = 101.6 ppm
Linear Regresion of conc. of TA vs.
Absorbance at 767 nm
Absorbance
• 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.
y = 0.0027x + 0.0125
2
R = 0.9584
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
50
100
150
200
250
Concentration of TA (ppm)
2
Condensed Tannin
Determination with Vanillin-HCl
Condensed Tannin Determination
– Vanillin-HCl Assay
9.90E-01
7.90E-01
5.90E-01
3.90E-01
1.90E-01
-1.00E-02
390
440
490
540
590
Wavelength (nm )
25 ppm Catechin
50 ppm Catechin
100 ppm Catechin
150 ppm Catechin
200 ppm Catechin
TA Extract
Condensed Tannin determination - Absorbance
at 500 nm
– The interference background of the crude extract was
corrected by preparing the test without vanillin
Absorbance
Absorbance
• 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 (+)-Catechin
solutions
• The absorbance of the sample and standard
solutions were measured at 500 nm with a UVVis spectrophotometer.
Tannin Concentration = 26.7 ppm
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
y = 0.0039x + 0.0289
R2 = 0.9922
0
50
100
150
200
250
Concentration of Catechin std. (ppm)
HPLC Analysis
HPLC of Standards
(+)-Catechin hydrate
Ellagic Acid
200
180
400
160
350
140
300
120
250
100
mAU
mAU
• Hewlett Packard Series 1100 HPLC System was
used in the analysis of extracted polyphenolics.
80
100
40
• Mobile phases were solvent A: 100% HPLCgrade methanol; solvent B: 100% HPLC
acetonitrile; and solvent C: 0.05 M KH2PO4 (pH
3.5).
200
150
60
50
20
0
0
0
10
20
30
40
50
0
60
10
20
30
40
50
60
Time (min)
Time (min)
Rutin hydrate
Gallic Acid
70
1000
900
60
800
50
700
600
40
mAU
mAU
• Concentration of standards ((+)-catechin
hydrate, ellagic acid, gallic acid, rutin hydrate)
was 1 mg/mL.
30
500
400
300
20
200
10
100
0
0
0
10
20
30
40
50
0
60
10
HPLC of Crude Tannin Extract
20
30
40
50
60
Time (min)
Time (min)
HPLC of Tannin Extract
(Ammonia fraction from polyamide mini-column chromatography)
50
120
45
1
40
100
35
30
mAU
mAU
80
3 = (+)-Catechin
10 = Ellagic acid, Rutin
12 = (+)-Catechin
13 = (+)-Catechin, Ellagic acid, Rutin
60
25
20
15
40
9
10
11
20
2
5
13
3
4
5
6 7
8
10
12
0
0
0
10
20
30
Tim e (m in)
40
50
60
0
10
20
30
40
50
60
Tim e (m in)
3
Fluorescence Titration Analysis
•
Instrument: Shimadzu RF-5301 PC Spectrofluorophotometer
•
Parameters:
–
Fluorescence of Tannin Extract
70
Excitation
Emission wavelength = 420 nm
Excitation wavelength range = 280-450 nm
60
50
–
Emission
Excitation wavelength = 340 nm
Emission wavelength range = 360-600 nm
Intensity
–
40
Excitation
Emission
30
Slit width = Ex: 10; Em: 10; Sensitivity = High
20
•
3 mL of standards were pipetted into a quartz cuvette and titrated with 0.1 M of metal
ions.
•
The Stern-Volmer equation was used to calculate the binding capacity or quenching
constant of the metals.
10
0
280
330
380
430
480
Wave length (nm )
Emission Spectra of Standards
and Extract
Stern-Volmer Equation
• Used to calculate binding/quenching constant for
metals
700
600
o
φf
= 1 + K q [Q ]
φf
Intensity
500
Extract
Gallic Acid
Catechin
Rutin
400
300
• Kq = binding/quenching constant
Kq = m/b
• m is the slope of the graph of F vs. [ ] of metal
• b is the y-intercept of the graph
200
100
0
380
400
420
440
460
480
500
520
540
Wavelength
Fluorescence of 15 mg/L Gallic
Acid titration with 0.1 M Al3+
Fluorescence of 15 mg/L
(+)-Catechin titration with 0.1 M Al3+
20
120
18
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
100
16
0 µL
Intensity
1.0 µL
1.5 µL
12
2.0 µL
40
3.0 µL
8
20
3.5 µL
4.0 µL
6
0
400
4.5 µL
25
420
440
460
480
500
520
540
560
580
600
Wavelength (nm)
4
Kq = 1,739
140
20
2
450
500
Wavelength (nm)
550
15
600
10
Kq = 62,593
y = 242106x + 3.8679
R2 = 0.9561
y = 706899x + 16.961
R2 = 0.9596
120
y = 26757x + 15.386
R2 = 0.8282
100
Kq=41,678
80
F
0
400
60
2.5 µL
10
F
Intensity
80
0.5 µL
14
60
40
5
20
0
0.0E+0 2.0E-05 4.0E-05 6.0E-05 8.0E-05 1.0E-04 1.2E-04 1.4E-04 1.6E-04
0
Conc. after adding 0.1 M Al 3+ to Catechin
0
0
2E-05 4E-05 6E-05 8E-05 0.0001 0.0001 0.0001 0.0002
Conc. after adding 0.1 M Al3+ (M)
4
Fluorescence of 15 mg/L Rutin
titration with 0.1 M Al3+
GC-Derivitization
• Derivitization of tannin extract was
performed using Tri Sil Z and Tri Sil TBT
for analysis by gas chromatography.
50
45
40
0 µL
0.5 µL
35
1.0 µL
1.5 µL
2.0 µL
25
2.5 µL
3.0 µL
20
3.5 µL
15
60
4.5 µL
5
0
400
• The standards (+)-catechin, ellagic acid,
gallic acid, and rutin were also derivitized
but did not yield results.
4.0 µL
10
50
450
500
550
y = 333817x - 0.4082
R2 = 0.9871
600
40
Wavelength (nm)
F
Intensity
30
30
20
• GC analysis was not successful for any of
the samples.
Keq=82,844
10
0
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
Discussion
Conclusion
• The excessive time needed to extract the acids from the
soil and plant samples limited the amount of work that
was done afterwards.
• The standards of (+)-catechin hydrate, ellagic acid, gallic
acid, and rutin were analyzed using fluorescence titration
analysis during the tannins extraction process.
• Even after the long extraction process, the amount of
extracts obtained were not enough for all the intended
investigations.
• The HPLC analysis indicated that (+)-catechin, ellagic
acid, and rutin were the possibly present in the extract.
• Not having enough samples also introduced the issue of
concentration differences between each batch of
extracts and the analysis they were used for.
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.
• Tannins: Properties. http://www.ansci.cornell.edu/plants/
toxicagents/tannin/chem_anl.html (accessed Apr 14, 2008).
• Siriwoharn, T.; Wrolstad, R. E. Polyphenolic Composition of
Marion and Evergreen Blackberries. J. Food Sci. 2004, 69,
233-240.
• 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.
Acknowledgements
• Dr. Nivens, Department of Chemistry &
Physics, Armstrong Atlantic State
University.
• Dr. Matt Gilligan, Marine Science
Department, Savannah State University
• AASU Department of Chemistry and
Physics
5