isolation and analysis of humic and fulvic acid from...

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Isolation and Analysis of Humic and

Fulvic Acid from Savannah, GA Marsh

Soils and Its Binding Capacity for

Aluminum

Isolation and Analysis of Humic and

Fulvic Acid from Savannah, GA Marsh

Soils and Its Binding Capacity for

Aluminum

Miller, Jessica, Waller, Andy: Undergraduate Students

Lynch, Will; Nivens, Delana; Elkins, Kelly: Academic Advisors

Department of Chemistry and PhysicsArmstrong Atlantic State University

Savannah, GA 31419

AbstractAbstract

Fulvic acid was purified from marsh soils in the Savannah, GA using a purification method from the International Humic Substances Society. Humic and fulvic acids are complex organic molecules produced by the oxidative

decomposition of organic material including carbohydrates, proteins, lignins, lignans, and fats from ground litter, roots, dead organisms and the excrements of

living organisms by micro-organisms and other processes. Within the complex humic group, fulvic acids are the lowest molecular weight substances with the

highest oxygen content and solubility over the entire pH range. Humic acids are higher molecular weight substances that are soluble only above pH 2. These materials have been documented to affect the pH of natural waters, trace metal

aquatic chemistry, bioavailability, and the degradation and transport of hydrophobic organic molecules. Many metal ions, including Al(III), are becoming

increasingly prevalent in the ‘free’ (aqua) state in both fresh and sea waters, as a consequence of acid rain and other environmental processes. Studying these

acids from natural environments can provide valuable information on how they bind to biologically hazardous metals, such as copper, cadmium and lead, as well as organic pollutants such as the herbicides and pesticides 2,4-

dichlorophenoxyacetic acid, 2-(2,4-dichlorophenoxy-)propionic acid, triclosan, and permethrin. After drying the marsh soil, a lengthy purification procedure was

performed which involved the removal of large debris, acid equilibration, centrifugation, and column chromatography on a DAX-8 resin. The nature of the

purified materials and their chemical reactivity was probed using various analytical techniques.

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Savannah State University:

Sampling Site

Savannah State University:

Sampling Site

Dr. Matt Gilligan of SSU and Jessica Miller of AASU

BackgroundBackground

1. What is Humic Acid (HA)?

Humic acids are a complex mixture of partially "decomposed" and otherwise transformed

organic materials. The chemistry of their formation is quite complex and an official structure of the molecule is unknown. Saltwater humic acids can come from a variety of sources, most of which are on land/marsh. These substances wash into rivers, undergoing further

transformations along the way, and ultimately into the ocean. Most, but certainly not all, of the marine humic acids also ultimately have their origin on land. Almost all of the lignins found in

marine environments originate on land. There are several subclasses of humic acids: tannins, lignins, fulvic acids.1

2. What are lignans?

Antioxidants found in plants such as flax seed, broccoli, and some berries

3. What are tannins?

Polyphenols found in plants that bind and precipitate proteins, used in tea and wine for coloring and favoring.

4. What is Fulvic Acid (FA)?

Fulvic acids are 'naturally occurring' organic acids that are part of the structure of rich soil. It is

an acid created in extremely small amounts by the action of millions of beneficial microbes, working on a soil environment with adequate oxygen. It is of relatively low molecular weight

and is biologically very active.2 Not to mention it is the target Humic subclass purified from our marsh soil sample.

Columns: The resin used for the column in this experiment is a DAX-8 resin.

The column is prepared and stored in methanol. The purpose of the column is to concentrate the FA solution by first adhering the solution onto the column by

means of pH manipulation. By washing the column with NaOH (basic) the acidic solution is extracted off of the resin and retained in a beaker as a concentrated

FA solution.

Infrared spectra (IR): Infrared spectroscopy is used in this experiment to

determine structural components of the isolated compound. From the peaks of the spectra, correlations between the samples can be made as well as

components of the known compared to components of the unknown to determine if they are indeed the same.

Fluorescence: Fluorescence is used to determine how well the compound can bind various metals and the metals effect on the compound; aluminum in this

particular experiment.

InstrumentationBackground

InstrumentationBackground

Proposed Structures of

Fulvic Acid

Proposed Structures of

Fulvic Acid*

**

* www.enerex.ca (March 20, 2006), proposed Fulvic acid structures

** www.ar.wroc.pl (March 20, 2006), proposed Fulvic acid structures

Phenol

Carboxylic Acid

Alcohol

2

PhysicalPhysical

Properties of Fulvic Acid

Properties of Fulvic Acid

Concentrated after column

Procedure:Purification

Procedure:Purification

Core Sample

Suspension in Solution

Acidify Solution

Centrifuge Sample

Membrane Filtration

Instrumental Analysis



Procedure:DAX-8 Resin/Column Preparation

Procedure:DAX-8 Resin/Column Preparation

Reflux Glassware Column Packed with CleanResin in 0.1 M NaOH



ProcedureFluorescence Titration with Aluminum(III)

ProcedureFluorescence Titration with Aluminum(III)



Initial Fluorescence-1


0

20

40

60

80

100

120

240 290 340 390 440 490 540 590

wavelength (nm)

Excitation Excitation

Fulvic 1Fulvic 1

Emission Emission

Fulvic 1Fulvic 1

Maximum = 326.2 nmMaximum = 433.2 nm

Fulvic 1 corresponds to the FA purified from the first core sample

(0-3 cm depth)

0

20

40

60

80

100

120

140

160

180

200

240 290 340 390 440 490 540 590

wavelength (nm)



Excitation Excitation

Fulvic 8Fulvic 8

Emission Emission

Fulvic 8Fulvic 8

Maximum = 441.6 nmMaximum = 325.6 nm

Fulvic 8 corresponds to the FA purified from the second core sample (2-6 cm depth)

3

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

200 250 300 350 400 450 500 550 600

wavelength (nm)

Fulvic 1 Fulvic 4

“Shoulder” seen around 250 nm indicates an aromatic organic ring in the compound

UV-Vis forUV-Vis for

Samples 1 and 4Samples 1 and 4

• Fulvic 1 corresponds to the FA purified from the first core sample (0-3 cm depth)

• Fulvic 4 corresponds to the FA purified from the first core sample (3-6 cm depth)

Wavenumber (cm-1)

450950145019502450295034503950

Infrared Spectra for

Fulvic Acids 1,4, and 8

Infrared Spectra for

Fulvic Acids 1,4, and 8

11

44

88

Broadening/weakening of peaks can be attributed to the amount of salts in each

sample.

Carboxylic Acid -OH

OH

Terminal Alkyne

Carboxylate Salts Phenols

100

150

200

250

300

375 395 415 435 455 475 495

wavelength (nm)

Series1 Series2 Series3 Series4 Series5 Series6 Series7 Series8

Series9 Series10 Series11 Series12 Series13 Series14 Series15

FluorescenceFluorescence

Series 1 corresponds to 0 µµµµL of Al3+ added, and with

each series increase 5 µµµµL are added. Thus series 2

contains 5 µµµµL and series 3 contains 10 µµµµL, etc… The maximum amount of Al3+ held is seen at series 13 ≈

60 µµµµL.

Indicates approximate location of the λλλλmax which undergoes a hypsochromic or blue shift in response to the increasing

aluminum and thus increasing structural rigidity. A shift to

lower λλλλ (blue shift) indicates higher energy, which is needed to bind more Al3+ as the Fulvic Acid becomes more rigid with

bound metal.

y = -276.97x + 1.0136

R2 = 0.9499

y = -105.27x + 0.8693

R2 = 0.9651

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

0.0000 0.0005 0.0010 0.0015 0.0020 0.0025

[Al3+] in

ββββ1= 276.97ββββ1= 276.97

ββββ2= 105.27ββββ2= 105.27

The two distinct slopes seen from the graph indicate that the reaction that takes place for

aluminum to bind is two steps, or there are two sites that can be attached to. Each ββββ value

corresponds to a piece of the overall rate of formation or formation constant (Kf).

Fluorescence analysis

Fluorescence analysis

ββββ1* ββββ2 = Kf ββββ1* ββββ2 = Kf

2.916×104 = Kf 2.916×104 = Kf

M

1

ConclusionsConclusions

In conclusion, the compound isolated is Fulvic Acid based on the

Infrared: 3565 cm-1 indicating the -OH of the carboxylic acid, 3565-3159 cm-1 for -OH, 1607cm-1 for carboxylate salts, and 1196 cm-1 for

the phenols; and UV-Vis spectra supporting the theorized structure

of the compound.

Also, it was seen that aluminum(III) binds to the fulvic acid. This

binding enhances and shifts the fluorescence.

The stoichiometric ratio appears to be 2:1 aluminum to fulvic acid.

The next steps are to further purify fulvic acids and characterize their binding with other metals. Fluorescence, IR and NMR will be

used.

We would also like to purify humic acids from similar marsh soils

and characterize them in the same manner.

ReferencesReferences

1. www.thekrib.com/Chemistry/humic.html

2. www.active-liquid-minerals.com/fulvic.htm

Thurman, Earl M.; Malcom, Ronald L. Preparative Isolation of Aquatic Humic

Substances. Environmental Science & Technology 1981, 15, 463-465

3. Chefetz, Benny; Hatcher, Patrick G.; Hadar, Yitzhak; Chen, Yona Characterization of Dissolved Organic Matter Extracted from Composted Minicipal Solid Waste. Soil

Sci.Soc. Am. 1998, 62, 326-332

4. Turner, R. Eugene; Swenson, Erick M.; Milan, Charles S.; Lee, James M.; Oswald, Thomas A. Below-Ground biomass in healthy and impaired salt marshes. Ecological

Research 2004, 19, 29-35 (accessed March 15, 2006), humic acid.

5. Aiken, G. R., McKnight, D. M., Thorn, K. A., and Thurman, E. M. Isolation of Hydrophilig Organic Acids from Water Using Nonionic Macroporous Resins. Org.

Geochem 1992, 18, 567-573

5. www.ihss.gatech.edu/soilhafa.html

6. www.earth.google.com

7. www.olypusfluoview.com/theory

(March 15, 2006), humic acid

(March 15, 2006), fulvic acid

(January 9, 2006), isolation of humic and fulvic acid

(March 15, 2006), Savannah State Univ.

(March 30, 2006), fluorescence theory

4

AcknowledgementsAcknowledgements

Special thanks are given to:

• Dr. Matt Gilligan and the Savannah State University Marine Science Department

• Armstrong Atlantic State University Department of Chemistry and Physics

• Armstrong Atlantic State University Department of Biology

• My fellow colleagues: Cassandra Dyal and Justin Lybarger

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