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

corrected by preparing the test without vanillin

Condensed Tannin Determination – Vanillin-HCl Assay

-1.00E-02

1.90E-01

3.90E-01

5.90E-01

7.90E-01

9.90E-01

390 440 490 540 590

Wavelength (nm )

Ab

so

rba

nc

e

25 ppm Catechin 50 ppm Catechin 100 ppm Catechin

150 ppm Catechin 200 ppm Catechin TA Extract

Condensed Tannin determination - Absorbance

at 500 nm

y = 0.0039x + 0.0289

R2 = 0.9922

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 50 100 150 200 250

Concentration of Catechin std. (ppm)

Ab

so

rban

ce

Tannin Concentration = 26.7 ppm

HPLC Analysis

• Hewlett Packard Series 1100 HPLC System was used in the analysis of extracted polyphenolics.

• Mobile phases were solvent A: 100% HPLC-grade methanol; solvent B: 100% HPLC acetonitrile; and solvent C: 0.05 M KH2PO4 (pH 3.5).

• Concentration of standards ((+)-catechinhydrate, ellagic acid, gallic acid, rutin hydrate) was 1 mg/mL.

HPLC of Standards(+)-Catechin hydrate

0

20

40

60

80

100

120

140

160

180

200

0 10 20 30 40 50 60

Time (min)

mA

U

Ellagic Acid

0

50

100

150

200

250

300

350

400

0 10 20 30 40 50 60

Time (min)

mA

U

Rutin hydrate

0

10

20

30

40

50

60

70

0 10 20 30 40 50 60

Time (min)

mA

U

Gallic Acid

0

100

200

300

400

500

600

700

800

900

1000

0 10 20 30 40 50 60

Time (min)

mA

U

HPLC of Crude Tannin Extract

0

20

40

60

80

100

120

0 10 20 30 40 50 60

Time (min)

mA

U

9

3 = (+)-Catechin

10 = Ellagic acid, Rutin

12 = (+)-Catechin

13 = (+)-Catechin, Ellagic acid, Rutin

1

34

2

56 7 8 10

11

12

13

HPLC of Tannin Extract(Ammonia fraction from polyamide mini-column chromatography)

0

5

10

15

20

25

30

35

40

45

50

0 10 20 30 40 50 60

Time (min)

mA

U

4

Fluorescence Titration Analysis

• Instrument: Shimadzu RF-5301 PC Spectrofluorophotometer

• Parameters:– Excitation

Emission wavelength = 420 nmExcitation wavelength range = 280-450 nm

– EmissionExcitation wavelength = 340 nmEmission wavelength range = 360-600 nm

– Slit width = Ex: 10; Em: 10; Sensitivity = High

• 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.

Fluorescence of Tannin Extract

0

10

20

30

40

50

60

70

280 330 380 430 480

Wavelength (nm)

Inte

nsit

y

Excitation

Emission

Emission Spectra of Standards and Extract

0

100

200

300

400

500

600

700

380 400 420 440 460 480 500 520 540

Wavelength

Inte

nsit

y ExtractGallic AcidCatechinRutin

Stern-Volmer Equation

• Used to calculate binding/quenching constant for metals

• 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

[ ]QK q

f

o

f+= 1

φ

φ

Fluorescence of 15 mg/L

(+)-Catechin titration with 0.1 M Al3+

0

2

4

6

8

10

12

14

16

18

20

400 450 500 550 600

Wavelength (nm)

Inte

nsit

y

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 = 26757x + 15.386

R2 = 0.8282

y = 242106x + 3.8679

R2 = 0.9561

0

5

10

15

20

25

0.0E+0

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

Conc. after adding 0.1 M Al3+

to Catechin

F

Kq = 62,593

Kq = 1,739

Fluorescence of 15 mg/L Gallic Acid titration with 0.1 M Al3+

0

20

40

60

80

100

120

400 420 440 460 480 500 520 540 560 580 600

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 = 706899x + 16.961

R2 = 0.9596

0

20

40

60

80

100

120

140

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)

F

Kq=41,678

5

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.

• 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.

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