Tannins and Terpenoids

Scientifi c Investigations Report 2004-5276

U.S. Department of the Interior

U.S. Geological Survey

as Major Precursors of Suwannee 

River Fulvic Acid
Tannins and Terpenoids as Major 

Precursors of Suwannee River Fulvic Acid

By Jerry A. Leenheer and Colleen Rostad

U.S. Department of the Interior

U.S. Geological Survey

Scientific Investigations Report 2004-5276
U.S. Department of the Interior

Gale A. Norton, Secretary

U.S. Geological Survey

Charles G. Groat, Director

U.S. Geological Survey, Reston, Virginia: 2004

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Suggested citation:

Leenheer, J.A., and Rostad, C.E., 2004, Tannins and terpenoids as major precursors of Suwannee River fulvic acid: 

U.S. Geological Survey Scientific Investigations Report 2004-5276, 16 p.
iii

Contents

Abstract ..................................................................................................................................................... 1

Introduction ................................................................................................................................................... 1

Materials and Methods ............................................................................................................................... 2

Sample collection .............................................................................................................................. 2

Suwannee River Fulvic Acid Fractionations ................................................................................. 2

Normal Phase Fractionation .................................................................................................... 2

Reverse-Phase Fractionation .................................................................................................. 3

Spectral Characterizations of SRFA Fractions and Subfractions .............................................. 4

Elemental Analyses ............................................................................................................................ 4

Tannins and Terpenoids as Major Precursors........................................................................................ 4

Two-Stage Suwannee River Fulvic Acid Fractionation Pattern .............................................. 5

Molecular Structures Detected by Infrared Spectra ................................................................. 5

Molecular Structures Detected by 13C-NMR Spectra .................................................................. 7

Implications for Molecular Models and Conclusions about Precursors ........................................ 13

Summary 13

References Cited ........................................................................................................................................ 15

Figures

Figure 1. Map showing location of the Suwannee River sample site (map modified from 

Pircke and others, 1977). ......................................................................................................... 2

Figure 2. Graphs showing fraction masses recovered from two-stage normal and reversephase fractionation of Suwannee River fulvic acid. ......................................................... 4

Figure 4. Graphs showing infrared spectra of subfractions recovered from reverse-phase 

fractionation of SFRA fraction Si-3. ...................................................................................... 5

Figure 3. Graphs showing infrared spectra of subfractions recovered from silca gel fractionation (A) and reverse-phase fractionation of fraction Si-2 (B) of Suwannee River 

fulvic acid. ................................................................................................................................. 5

Figure 5. Graphs showing infrared spectra of subfractions recovered from reverse-phase 

fractionation of SRFA fraction Si-4. ...................................................................................... 6

Figure 6. Graphs showing infrared spectra of subfractions recovered from reverse-phase 

fractionation of SRFA fraction Si-5. ...................................................................................... 6

Figure 7. Graphs showing infrared spectra of subfractions recovered from reverse-phase 

fractionation of SRFA fraction Si-6. ...................................................................................... 6

Tables

Table 1. Fraction designations, eluent composition, and eluent volumes. ...................................... 3

Table 2. Data used to determine structural characteristics of selected SRFA subfractions. .. 12
iv

Figure 8. Graphs showing 13C-NMR spectra of selected major subfractions recovered from 

reverse-phase fractionation of Suwannee River fulvic acid. .......................................... 6

Figure 9. Graphs showing 13C-NMR dipolar-dephased spectra of selected major subfractions 

recovered from reverse-phase fractionation of Suwannee River fulvic acid. ............. 7

Figure 10. Graphs showing 13C-NMR spectra of subfractions recovered from reverse-phase 

fractionation of SRFA fraction Si-2. ...................................................................................... 7

Figure 11. Graphs showing 13C-NMR spectra of subfractions recovered from reverse-phase 

fractionation of SRFA fraction Si-3. ...................................................................................... 8

Figure 12. Graphs showing 13C-NMR spectra of subfractions recovered from reverse-phase 

fractionation of SRFA fraction Si-4. ...................................................................................... 8

Figure 13. Graphs showing 13C-NMR spectra of subfractions recovered from reverse-phase 

fractionation of SRFA fraction Si-6. ...................................................................................... 8

Figure 14. Graphs showing 13C-NMR scpectra of subfractions recovered from reverse-phase 

fractionation of SFRA fraction Si-6. ...................................................................................... 8

Figure 15. Graphs showing 13C-NMR spectra of SRFA fraction SI-7. ............................................... 9

Figure 16. Graphs showing 13C-NMR dipolar-dephased spectra of subfractions recovered 

from reverse-phase fractionation of SRFA fraction Si-2. .................................................. 9

Figure 17. Graphs showing 13C-NMR dipolar-dephased spectra of subfractions recovered 

from reverse-phase fractionation of SRFA fraction Si-3. ................................................ 10

Figure 18. Graphs showing 13C-NMR dipolar-dephased spectra of subfractions recovered 

from reverse-phase fractionation of SRFA fraction Si-4. ................................................ 10

Figure 19. Graphs showing 13C-NMR dipolar-dephased spectra of subfractions recovered 

from reverse-phase fractionation of SRFA fraction Si-5. ................................................ 10

Figure 20. Graphs showing 13C-NMR dipolar-dephased spectra of subfractions recovered 

from reverse-phase fractionations of SRFA fraction Si-6. .............................................. 10

Figure 21. Graphs showing 13C-NMR spectra of unfractionated Suwannee River fulvic acid at 

1 ms and 5 ms contact times. ................................................................................................ 11

Figure 22. Diagram showing structural models of selected subfractions of Suwannee River 

fulvic acid. A and B represent aromatic rings designations in parent condensed 

tannin structures (MW = molecular weight in Daltons). ................................................ 11
Tannins and Terpenoids as Major Precursors

 of Suwannee River Fulvic Acid

Abstract

Suwannee River fulvic acid (SRFA) was fractionated 

into 7 fractions by normal-phase chromatography on silica 

gel followed by reverse-phase fractionation on XAD-8 resin 

that produced 18 subfractions. Selected major subfractions 

were characterized by 13C-nuclear magnetic resonance (NMR), 

infrared spectrometry, and elemental analyses. 13C-NMR spectra of the subfractions were more indicative of precursor structures than unfractionated SRFA, and gave spectral profiles that 

indicated SRFA mass was about equally split between tannin 

precursors and terpenoid precursors. Lignin precursors were 

minor components. Synthesis of 13C-NMR data with elemental 

data for subfractions derived from both tannin and terpenoid 

precursors revealed high ring contents and low numbers of 

carbon per rings which is indicative of fused ring structures 

that are extensively substituted with carboxyl and methyl 

groups. These results ruled out extended chain structures for 

SRFA. This information is useful for determining sources and 

properties of fulvic acid in drinking water supplies as tannins 

are more reactive with chlorine to produce undesirable disinfection by-products than are terpenoids.

Introduction

Suwannee River fulvic acid (SRFA) is likely the most 

studied aquatic humic substance since a large sample was 

isolated in 1982, (Malcolm and others, 1994) and was adopted, 

distributed, and sold by the International Humic Substances 

Society (IHSS) as a standard fulvic acid for various scientific 

studies. The IHSS and other SRFA samples have been used 

for basic structural studies (Averett and others, 1994), for 

non-polar organic contaminant binding interactions (Chiou 

and others, 1987), for metal-binding interactions (Brown and 

others, 1999), and for production of disinfection by-products 

(DBPs) (Croué and others, 2000). These and other studies too 

numerous to reference would benefit by a better understanding of organic precursor structures and diagenetic processes 

that result in aquatic fulvic acid so that reactivity and reaction 

mechanisms can be better elucidated. Tannins are more reactive with chlorine to produce undesirable disinfection byproducts than are terpenoids (Larson and Weber, 1994). This 

information can aid water managers in determining sources of 

aquatic fulvic acids in drinking water supplies and in design of 

drinking-water-treatment processes specific to waters containing aquatic fulvic acid.

The prevailing viewpoint of the origin of terrestrial 

aquatic humic substances is that they are derived from lignin. 

This viewpoint is derived from the application of various 

degradation procedures such as alkaline cupric oxide oxidation (Ertel and others, 1984), a comparative study of cupric 

oxide, chlorine, permangante oxidations and alkaline hydrolysis (Sonnenberg and others, 1989); and thermochemolysis 

with tetramethylammonium hydroxide (del Rio and Hatcher, 

1995) in which phenolic carboxylic acids oxidation products 

were derived from aquatic humic substances that were similar 

in structure to oxidation products derived from lignin. A 

recent non-degradative study of SRFA (Kujawinski and others, 

2002) that used high-resolution mass spectrometry seemed to 

confirm the lignin hypothesis origin, because many peaks in 

the SRFA sample occurred at exactly the same mass as peaks 

in an extract of a degraded wood lignin. 1

U.S. Geological Survey

By Jerry A. Leenheer1 and Colleen E. Rostad1
2 Tannins and Terpenoids as Major Precursors of Suwannee River Fulvic Acid

The lignin origin hypothesis, however, is still open to 

some questions about lignin 

comprising the bulk of aquatic 

fulvic acid derived from terrestrial sources. 1) Do the 

small percentages of identified fulvic acid fragments 

from degradative procedures 

represent the whole sample? 

2) Do tannins that are isomerically similar to lignins, give 

similar degradation products 

and similar mass ions in mass 

spectrometric studies? 3) Why 

should lignin degradation 

products predominate over 

tannin degradation products 

when parent lignins are insoluble and tannins are soluble in 

water? 4.) The recent finding that terpenoids are major 

precursors of certain aquatic 

fulvic acid also showed that 

pyrolysis and thermochemolysis degradative procedures 

failed to give diagnostic information regarding this class 

of compounds (Leenheer and 

others, 2003), which begs the 

question of missing precursor 

compounds.

Tannins derived from 

plants consist of two types, hydrolyzable and condensed tannins. Hydrolyzable tannins consists of gallic acid esterified to 

sugars such as glucose, and condensed tannins are three-ring 

flavonols units that are polymerized and conjugated to other 

plant components such as carbohydrates and terpenoids (Robinson, 1980). Lignin is the most abundant natural polyphenol 

polymer in wood, but tannins are more abundant in leaves and 

needles with contents as high as 20 percent (Hernes and others, 2001). Natural polyphenols are substituted at the 4; 3,4; 

and 3,4,5 positions on aromatic rings of lignin and also with 

the B ring (note structure in report title) of condensed tannnis, 

and at the 3,4,5 position of hydrolyzable tannins. The A ring 

(note structure in report title ) of condensed tannins is different, with aromatic phenol and phenolic ethers being substituted at the 5,7,9 positions, a meta substitution pattern. This 

difference can be exploited to differentiate tannins from lignin 

precursors.

 The objective of this report is to describe structural 

studies selected to identify specific structures indicative of 

organic precursors in the bulk SRFA sample. These techniques (infrared spectrometry, 13C-nuclear magnetic resonance 

(NMR) spectrometry, and elemental analyses) are selected 

to provide quantitative structural information. The complex 

mixture of SRFA components 

is simplified by first performing 

normal-phase chromatography 

using silica gel, followed by 

reverse-phase chromatography 

using XAD-8 resin. This twostage fractionation was done to 

better resolve spectral peaks and 

more specific elemental composition in the fractions that gave 

diagnostic information about 

organic precursors.

Materials and 

Methods

Sample collection 

The Suwannee River was 

sampled at its origin at the outlet 

of the Okefenokee Swamp near 

Fargo, Georgia, in November 

1983 (fig 1). Onsite, 8104 liters 

(L) of water were processed 

through a filtration and columnadsorption system that fractionated and isolated organic solutes 

into hydrophobic-neutral, strong 

hydrophobic fulvic acid, weak 

hydrophobic fulvic acid, hydrophilic acid, and hydrophilicneutral fractions (Leenheer and Noyes, 1984). The strong 

hydrophobic acid fraction, designated in this report as Suwannee River fulvic acid (SRFA), constituted 66 percent of the 

dissolved organic carbon concentration which was 38.4 milligrams per liter (mg/L) at the time of sampling.

Suwannee River Fulvic Acid Fractionations 

Normal Phase Fractionation

Silica gel, 40-150 mesh, obtained from Woelm Pharma, 

was packed dry in a 110 cubic centimeter (cc) bed volume 

glass chromatographic column 1.6 centimeter (cm) internal 

diameter (ID) by 55 cm long. The silica gel was deactivated 

by pumping 100 milliliters (mL) of 75 percent acetonitrile 

(ACN)/25 percent water (H2O) through the column. This 

was followed by 200 mL of methylene chloride. The SRFA 

sample (3.47 grams) was dissolved in 10 mL of 75 percent 

ACN/25 percent H2O and this solution was applied to the 

column. The seven fractions were eluted with solvents of 

Figure 1. Map showing location of the Suwannee River 

sample site (map modified from Pircke and others, 1977).
Reverse-Phase Fractionation 3

increasing polarity: Si-1, 1 L methylene chloride; Si-2, 1 L 

ethyl acetate; Si-3, 1 L ACN; Si-4, 1 L 95 percent ACN/5 

percent water (H2O), Si-5, 1 L 90 percent ACN/10m percent 

H2O; Si-6, 1 L of 75 percent ACN/25percent H2O, and Si-7, 

0.5 L 75 percent ACN/25 percent H2O containing 0.25 molar 

(M) oxalic acid. Fractions Si-1 to Si-6 were recovered by 

vacuum-rotary evaporation of the solvents. The fractions were 

dissolved in water, freeze-dried, and weighed. Fraction Si-7 

was subjected to an additional adsorption/desorption step on 

XAD-8 resin to remove the oxalic acid.

Reverse-Phase Fractionation 

Two glass chromatography columns filled with Amberlite 

XAD-8 resin, a macroporous methylmethacrylate polymer, 

were used to fractionate the fractions eluted from the silica 

gel column into subfractions: Column A (5 cm ID by 30 cm 

long) has a bed volume of 589 cc and was used to fractionate 

the larger mass fractions (Si-2 and Si-4). Column B (3 cm ID 

by 30 cm long) has a bed volume is 212 cc and was used to 

fractionate the smaller mass fractions (Si-3, Si-5, and Si-6). 

Fractions Si-1 and Si-7 were not fractionated because of their 

small mass. Fractions applied to column A were dissolved in 

500 mL H2O acidified to pH 2 with formic acid, and fractions 

applied to column B were dissolved in 250 mL H2O acidified to pH 2 with formic acid. These samples were pumped 

through the columns to adsorb the fractions on the XAD-8 

resin. The XAD-8 column eluent composition, eluent volumes, 

and subfraction designations are given in table 1. The fractions 

were concentrated by vacuum-rotary evaporation of the solvents followed by freeze-drying and weighing of the fractions.

Table 1. Fraction designations, eluent composition, and eluent volumes.

[F, fraction; 2/1, fraction number from silica gel, and subfraction number from XAD-8 resin; percent; L, liter]

Fraction from silica gel 

(normal-phase fractionation)

Subfraction from XAD-8 resin 

(reverse-phase fractionation)

Eluent Composition (Percent acetonitrile in water acidified to pH 2 

with formic acid)

Eluent Volume 

(Liters)

Si-2 F 2/1 10% 2 L

F 2/2 15% 2 L

F 2/3 20% 2 L

F 2/4 30% 2 L

F 2/5 75% 0.5 L

Si-3 F 3/1 0% 0.35 L

F 3/2 10% 0.4 L

F 3/3 15% 0.4 L

F 3/4 25% 0.4 L

F 3/5 75% 0.2 L

Si-4 F 4/1 0% 2 L

F 4/2 5% 2 L

F 4/3 10% 2 L

F 4/4 15% 2 L

F 4/5 20% 2 L

F 4/6 75% 0.5 L

Si-5 F 5/1 0% 0.5 L

F 5/2 15% 0.4 L

F 5/3 25% 0.4 L

F 5/4 75% 0.2 L

Si-6 F-6/1 0% 0.4 L

F 6/2 20% 0.4 L

F 6/3 75% 0.2 L
4 Tannins and Terpenoids as Major Precursors of Suwannee River Fulvic Acid

Spectral Characterizations of SRFA Fractions 

and Subfractions

Infrared spectra were collected by using 2 to 5 milligrams 

(mg) of SRFA fraction and subfractions isolates in KBr pellets. The Perkin Elmer System 2000 FT-IR used an infrared 

source with a pulsed laser carrier and a deuterated triglycine 

sulfate detector. The instrument was scanned from 4,000 to 

400 reciprocal centimeters (cm-1) averaging 10 scans at 1.0 

cm-1 intervals with a resolution of 4.0 cm-1. All spectra were 

normalized after acquisition to a maximum absorbance of 1.0 

for comparative purposes. Cross polarization magic angle 

spinning (CPMAS) 13C-NMR spectra were obtained on 20 to 

200 mg of SRFA fractions. Freeze-dried samples were packed 

in zirconia rotors. CPMAS 13C-NMR spectra were obtained on 

a 200-megahertz Chemagnetics CMX spectrometer with a 7.5-

millimeter-diameter probe. The spinning rate was 5,000 hertz. 

The acquisition parameters included a contact time of 5 milliseconds (ms), pulse delay of 1 second (s), and a pulse width of 

4.5 microseconds (μs) for the 90o pulse. The dipolar dephased 

spectra of SRFA fractions used a 1 ms contact time and a coupling time of 30 μs.

Elemental Analyses

Elemental analyses were conducted by Huffman Laboratories, (Golden, Colorado). Elemental analyses (C, H, O, 

N, and ash) of selected SRFA subfractions were conducted 

according to reported methods (Huffman and Stuber, 1985). 

Tannins and Terpenoids as Major 

Precursors

SRFA fractions from the normal-phase fractionation and 

subfractions from the reverse-phase fractionation were analyzed to determine major precursors. These analyses indicate 

whether tannins and terpenoids are more important precursors 

than lignin in SRFA.

Figure 2. Graphs showing fraction masses recovered from two-stage normal and reversephase fractionation of Suwannee River fulvic acid.
Molecular Structures Detected by Infrared Spectra 5

Two-Stage Suwannee River Fulvic Acid 

Fractionation Pattern 

A bar diagram of the two-stage SRFA fractionation is 

presented in figure 2. Fraction recovery was 91.5 percent from 

the normal phase fractionation on silica gel, and 95.0 percent 

from the subsequent reverse phase fractionation on XAD-8 

resin for an overall recovery of 86.9 percent. The Si-2 fraction 

was the largest, constituting 47 percent of the mass; only 1.5 

mg was recovered in the Si-1 fraction. 

Molecular Structures Detected by Infrared 

Spectra 

The infrared (IR) spectra of the silica-gel fractionation 

are presented in figure 3A. Fraction Si-1 (most nonpolar) 

is rich in aliphatic hydrocarbon structures (peaks at 2,963, 

2,934, 1,459, and 1,384 cm-1) and esters (peaks at 1,774 and 

1,725 cm-1). The 1,774 cm-1 ester peak likely indicates fivemembered ring lactones of natural origin, whereas the 1,725 

cm-1 ester peak is accompanied by peaks at 1,287, 1,125, and 

Figure 3. Graphs showing infrared spectra of subfractions recovered from silca gel fractionation (A) and reverse-phase fractionation of fraction Si-2 (B) of Suwannee River fulvic acid.

Figure 4. Graphs showing infrared spectra of subfractions 

recovered from reverse-phase fractionation of SFRA fraction 

Si-3.
6 Tannins and Terpenoids as Major Precursors of Suwannee River Fulvic Acid

Figure 5. Graphs showing infrared spectra of subfractions 

recovered from reverse-phase fractionation of SRFA fraction 

Si-4.

Figure 6. Graphs showing infrared spectra of subfractions 

recovered from reverse-phase fractionation of SRFA fraction 

Si-5.

Figure 7. Graphs showing infrared spectra of subfractions 

recovered from reverse-phase fractionation of SRFA fraction 

Si-6.

Figure 8. Graphs showing 13C-NMR spectra of selected major 

subfractions recovered from reverse-phase fractionation of 

Suwannee River fulvic acid.
Molecular Structures Detected by 13C-NMR Spectra 7

1,075 cm-1 that are indicative of phthalate ester contaminants 

likely leached from plastic components during sampling and 

processing. The Si-1 fraction contains most of the volatile 

“tea-like” fragrance of the SRFA.

Fractions Si-2 to Si-7 produced similar infrared spectra 

with increasing intensity of the broad peak at 1,620 cm-1 in 

progression from fraction Si-2 to Si-6. This peak has been 

assigned to aromatic double bonds and hydrogen-bonded 

conjugated ketones in humic substances (Stevenson, 1994). 

The strong carbonyl peak near 1,720 cm-1 accompanied by the 

broad shoulder near 2,500 cm-1 is characteristic of carboxylic 

acids. Subtraction of spectra (not shown) revealed an ester 

shoulder near 1,780 cm-1, characteristic of five-membered 

lactones that were the most intense in the Si-2 fraction and 

progressively decreased in the later fractions.

The infrared spectra of the reverse phase fractionation 

are presented in figure 3B and figures 4 to 7. Peaks indicative 

of aliphatic hydrocarbon (2,920 to 2,980 cm-1) and aromatic 

hydrocarbon (1,620 cm-1) structures increased as the fraction 

number increased. This progression was expected because in 

reverse-phase fractionations polar structures rich in carboxylic 

acids and alcohols elute before nonpolar structures rich in 

hydrocarbon structures. Fractions 5/1 (fig. 6) and 6/1 (fig. 7) 

also contained peaks indicative of amino acids and peptides, 

with the peak near 3,200 cm-1 assigned to the N-H stretch, and 

the peak near 1,600 cm-1 assigned to the carbonyl stretch of the 

carboxylate zwitterions. 

Molecular Structures Detected by 13C-NMR 

Spectra 

The 13C-NMR spectra of selected subfractions of SRFA 

are shown in figure 8, and the dipolar-dephased spectra of 

these subfractions are shown in figure 9. The subfractions 

were selected to represent the mass range of SRFA shown in 

figure 2. 

There are major compositional differences between 

the Si-2 subfraction isolates, which represent nearly half the 

mass of SRFA, and the higher-numbered silica-gel subfractions. Both the normal and dipolar-dephased 13C-NMR spectra 

for the Si-2 subfractions are typical for fulvic acid recently 

reported (Leenheer and others, 2003) to be derived from 

terpenoid precursors. The aliphatic hydrocarbon peak at 0-60 

parts per million (ppm) is resolved into two peaks with the 

peak near 20 ppm assigned to branched methyl groups and the 

peak near 45 ppm assigned to methine and quaternary carbons 

(shown in the dipolar-dephased spectra) that typify highly 

branched and ring structures of terpenoids. The aliphatic quaternary carbon peak at 45 ppm in the dipolar-dephased spectra 

(fig. 9) is unique to the Si-2 subfractions. The C-O region (60-

100 ppm) has a significant quaternary C-O peak near 85 ppm 

(fig. 9) that is attributed to lactone esters of tertiary alcohols 

in the terpenoid structure (Leenheer and others, 2003). The 

aromatic carbon peak (100-160 ppm, subfraction 2/4 of fig. 8) 

only has small subpeaks caused by phenolic structures (140-

Figure 9. Graphs showing 13C-NMR dipolar-dephased spectra 

of selected major subfractions recovered from reverse-phase 

fractionation of Suwannee River fulvic acid.

Figure 10. Graphs showing 13C-NMR spectra of subfractions 

recovered from reverse-phase fractionation of SRFA fraction Si-2.
8 Tannins and Terpenoids as Major Precursors of Suwannee River Fulvic Acid

Figure 12. Graphs showing 13C-NMR spectra of subfractions 

recovered from reverse-phase fractionation of SRFA fraction 

Si-4.

Figure 11. Graphs showing 13C-NMR spectra of subfractions 

recovered from reverse-phase fractionation of SRFA fraction 

Si-3.

Figure 13. Graphs showing 13C-NMR spectra of subfractions 

recovered from reverse-phase fractionation of SRFA fraction 

Si-6.

Figure 14. Graphs showing 13C-NMR spectra of subfractions 

recovered from reverse-phase fractionation of SRFA fraction 

Si-6.
Molecular Structures Detected by 13C-NMR Spectra 9

160, 105-120 ppm) that are prominent in lignins and tannins. 

Much of the aromatic ring is substituted with C-C linkages 

(peak near 130 ppm, subfraction 2/4 of fig. 9). The peak at 105 

ppm in figure 9 may be caused by quaternary anomeric carbon 

of ketals or lactols, or by a C-substituted aromatic structure 

between meta-substituted aromatic C-O linkages as found in 

substituted phloroglucinol rings (Wershaw and others, 2000). 

Naturally occurring phloroglucinol derivatives of terpenoids 

occur in a wide variety of plants in compounds such as 

humulone, which is one of the bitter substances found in hops 

(Robinson, 1980).

Subfractions derived from fractions Si-3 to Si-6 (fig. 8) 

have lesser amounts of the branched methyl peak at 20 ppm, 

and the aromatic carbon region has prominent subpeaks near 

153 and 105 ppm that are characteristic of plant condensed 

tannins (Benner and others, 1990; Preston and Sayer, 1992, 

Preston and others, 1997; Wilson and Hatcher, 1988). The 

most diagnostic peak for condensed tannins is the 105 ppm 

peak in the dipolar dephased spectra of figure 9, that is a quaternary C-C linkage between meta-substituted C-O linkages in 

the A-ring of condensed tannins. Lignins lack this substituted 

C-C linkage at 105 ppm although syringyl lignins have protonated C-2 and C-6 carbons near 105 ppm that are detected in 

normal CPMAS 13C-NMR spectra (Preston and others, 1997). 

The dipolar dephased spectra of subfractions derived from 

fractions Si-3 to Si-6 (fig. 9) all show the distinct 105-ppm 

peak characteristic of condensed tannins, and this peak cannot 

be attributed to ketal anomeric carbons of carbohydrates due 

to insufficient concentrations of alcohols from 60-90 ppm, as 

shown in the spectrum of fraction 6/3 (fig. 8). 

The normal 13C-NMR spectra of the reverse-phase subfractions of each silica fraction are presented in figures 10 to 

15, and the dipolar-dephased spectra are presented in figures 

16 to 20. Peaks indicative of aliphatic hydrocarbon (0-60 

ppm) and aromatic hydrocarbon (100-160 ppm) structures 

increased as the fraction number increased, which is expected 

in the polar-to-nonpolar progression of reverse phase fractions. 

The increase in aromatic carbon content was much greater 

than the increase in aliphatic carbon content for the fulvic acid 

fractions derived from condensed tannins. 

At this point in the discussion of 13C-NMR results, the 

question about why previous investigators (including the 

authors of this report) did not report terpenoid and tannin 

structures in SRFA should be addressed. The first answer 

to this question is the lack of fractionation studies of SRFA. 

Structural models of SRFA (Leenheer and others, 1994) were 

based on unfractionated 13C-NMR spectra as presented in 

figure 21. Peaks are broadened and obscured by the mixture 

characteristics so that indicator peaks, such as the branched 

methyl groups of terpenoids near 20 ppm and the phenol peak 

at 153 ppm, are difficult to differentiate in the unfractionated SRFA. In the aromatic carbon region (100-160 ppm), 

the combination of the aromatic carbon profile of terpenoid 

fractions (fig. 10) with the aromatic carbon profile of the tanFigure 15. Graph showing 13C-NMR spectra of SRFA fraction 

SI-7.

Figure 16. Graphs showing 13C-NMR dipolar-dephased spectra of subfractions recovered from reverse-phase fractionation 

of SRFA fraction Si-2.
10 Tannins and Terpenoids as Major Precursors of Suwannee River Fulvic Acid

Figure 17. Graphs showing 13C-NMR dipolar-dephased spectra 

of subfractions recovered from reverse-phase fractionation of 

SRFA fraction Si-3.

Figure 18. Graphs showing 13C-NMR dipolar-dephased spectra 

of subfractions recovered from reverse-phase fractionation of 

SRFA fraction Si-4.

Figure 19. Graphs showing 13C-NMR dipolar-dephased spectra of subfractions recovered from reverse-phase fractionation 

of SRFA fraction Si-5.

Figure 20. Graphs showing 13C-NMR dipolar-dephased spectra of subfractions recovered from reverse-phase fractionations of SRFA fraction Si-6.
Molecular Structures Detected by 13C-NMR Spectra 11

nin fractions (figs. 11-15) gives a combined aromatic carbon 

profile that is consistent with lignin structures. Liquid-state 

quantitative 13C-NMR spectra of SRFA (Thorn, 1994; Thorn 

and others, 1989) give better peak resolution, but the mixture 

characteristics of these spectra still limit the correct assignment of precursor structures.

The second reason why terpenoids and tannins have not 

been detected in SRFA is that quaternary carbon linkages in 

these structures have not been representatively detected in previous studies because of short contact times in CPMAS 13CNMR spectra, and because few dipolar-dephased studies have 

been done that selectively detect quaternary linkages. Figure 

21 shows the difference between 1 ms and 5 ms contact times. 

For the 1 ms contact time, aliphatic quaternary C-C at 45 ppm, 

C-O at 85 ppm, aromatic C-C at 105 ppm and C-O at 140-160 

ppm are under-represented because of low cross-polarization efficiency of these carbons. Therefore, the contact time 

was lengthened to 5 ms in accordance with the findings of 

Alemany and others (1983) who report more comparable 

responses at this longer contact time for molecules of low or 

remote protonation. Wershaw and others (2000) confirmed 

Figure 21. Graphs showing 13C-NMR spectra of unfractionated 

Suwannee River fulvic acid at 1 ms and 5 ms contact times.

Figure 22. Diagram showing structural models of selected subfractions of Suwannee River fulvic acid. A and B represent aromatic rings designations in parent 

condensed tannin structures (MW = molecular weight in Daltons).
12 Tannins and Terpenoids as Major Precursors of Suwannee River Fulvic Acid

Table 2. Data used to determine structural characteristics of selected SRFA subfractions.[F, fraction; 2/1, fraction number from silica gel, and subfraction number from XAD-8 resin; %, percent; #, number; NMR, nuclear magnetic resonance].

Determination Method Subfraction 2/1 Subfraction 2/4 Subfraction 3/2 Subfraction 4/6

Empirical Formula

(1,000) Dalton

Elemental Analyses C42.5H 44.7O 27.3N 0.4 C47.6H46.6O23.8N0.3 C43.2H38.6O27.4N0.6 C46.7H39.4O24.3N1.1

Aliphatic C-HPlus C-N Carbons

%C from 13C-NMR times

# C’s in Empirical Formula

18.0 16.4 11.1 12.6

Aliphatic C-OCarbons

%C from 13C-NMR times

# C’s in Empirical Formula

7.9 6.8 8.5 6.1

Aromatic plusOlefinic Carbons

%C from 13C-NMR times

# C’s in Empirical Formula

2.8 15.2 13.5 19.2

Carboxyl plusEster Carbons

%C from 13C-NMR times

# C’s in Empirical Formula

11.9 7.4 8.3 7.0

Keytone

Carbons

%C from 13C-NMR times

# C’s in Empirical Formula

1.9 1.8 1.8 1.8

Index of HydrogenDeficiency (Φ)

Φ = [(2C+2) – (H – N)]/2 21.4 25.5 25.5 28.5

# Rings (θ) θ = Φ – C = OC – 0.5

(Aromatic plus Olefinic C)

6.2 8.7 8.4 10.1

# C’s per Ring (Ω) Ω = Total C/θ 6.9 5.5 5.1 4.6
Summary 13

the findings of Alemany and others (1983) by reporting that 

CPMAS 13C-NMR spectra of several aquatic fulvic acids gave 

the best quantitation at 5 ms contact time when compared to 

quantitative liquid-state 13C-NMR spectra of these samples. 

Implications for Molecular Models and 

Conclusions about Precursors

Average molecular models were derived by combining 

elemental data: infrared, and 13C-NMR data with degradation 

of postulated tannin and terpenoid precursor structures. The 

data used in this modeling are presented in table 2, and the 

average molecular models are presented in figure 22. These 

models conform to the empirical formula, molecular weight 

distribution, functional group content, and ring content data. 

These models are much better approximations of actual 

structures than models presented previously for unfractionated 

SRFA (Leenheer and others, 1994), because the extensive fractionation of this study produced more homogeneous fractions.

The large number of rings and the low carbon number per 

ring in the data of table 2 indicate aryl/aliphatic rings, some of 

which are fused, that are extensively substituted with carboxyl 

and methyl end groups. These models of primary molecular structure indicate more spherical structures than chain 

structures that can fold or unfold depending on the solution 

chemistry. 

The structural models of fraction 2/1 and 2/4 (fig. 22) 

were derived from cyclic diterpenoids such as abietic acid. 

Digenetic pathways to fulvic acid from terpenoid precursors 

include double bond cleavage and oxidation, condensation of 

carboxyl groups with double bonds to produce cyclic tertiary 

lactones, and terminal methyl group oxidation to carboxyl 

groups (Leenheer and others, 2003). Fractions 3/2 and 4/6 

were derived from condensed tannins that are composed of 

flavonoid units. The carbons in the A ring of the parent flavonoid with its phloroglucinol substitution pattern are completely substituted as indicated by the 13C-NMR data; and the 

B ring, with its single phenol groups is absent in fraction 3/2, 

but present in fraction 4/6. Tannins extracted from undegraded 

leaves have unsubstituted carbons in the A ring as indicated by 

a 13C-NMR peak at 95 ppm (Preston and others, 1997), but this 

peak was absent in tannins found in mangrove leaves at various stages of decomposition (Benner and others, 1990) indicating that A-ring substitution occurs during leaf senescence. 

Ortho-phenol structures in the B-ring of certain condensed 

tannins are indicated by a 13C-NMR peak at 145 ppm (Benner 

and others, 1990), but this peak was found in minor amounts 

in only a few fractions of this study indicating selective 

decomposition of ortho-phenol structures as was found in the 

mangrove leaves study. The center pyran ring of the flavonoid 

has been opened, but other condensation reactions produce 

additional rings as indicated by studies of flavonoid reactivity 

(Haslam, 1998).

 Lignin-derived fulvic acid may be present as a minor 

component in SRFA, but fulvic acids more likely to be derived 

from lignin sources such as is found in the standard International Humic Substances Society soil and peat fulvic acids, 

give different 13C-NMR aromatic carbon profiles as compared 

to aquatic fulvic acids (Thorn and others, 1989). Lignins and 

tannins are isomers with respect to elemental composition, so 

a recent high-resolution mass spectrometric study (Stenson 

and others, 2003) of SRFA elemental composition of resolved 

ions is not definitive with respect to a lignin origin. The basis 

of the argument for tannins and terpenoids as major precursors of SRFA resides in the comprehensive fractionation and 

spectral characterization approach of this study.

Summary

SRFA is likely the most studied aquatic humic substance 

since a large sample was isolated in 1982, and was adopted, 

distributed, and sold by the IHSS as a standard fulvic acid for 

various scientific studies. The IHSS and other SRFA samples 

have been used for basic structural studies, for non-polar 

organic contaminant binding interactions, for metal-binding 

interactions, and for production of DBPs . These and other 

studies too numerous to reference would benefit by a better 

understanding of organic precursor structures and diagenetic 

processes that result in aquatic fulvic acid so that reactivity 

and reaction mechanisms can be better elucidated. Tannins are 

more reactive with chlorine to produce undesirable disinfection by-products than are terpenoids. This information can aid 

water managers in determining sources of aquatic fulvic acids 

in drinking water supplies and in design of drinking-watertreatment processes specific to waters containing aquatic 

fulvic acid.

The objective of this report is to describe structural 

studies selected to identify specific structures indicative of 

organic precursors in the bulk SRFA sample. These techniques (infrared spectrometry, 13C-nuclear magnetic resonance 

(NMR) spectrometry, and elemental analyses) are selected 

to provide quantitative structural information. The complex 

mixture of SRFA components is simplified by first performing 

normal-phase chromatography using silica gel, followed by 

reverse-phase chromatography using XAD-8 resin. This twostage fractionation was done to better resolve spectral peaks 

and more specific elemental composition in the fractions that 

gave diagnostic information about organic precursors. These 

analyses indicate whether tannins and terpenoids are more 

important precursors than lignin in SRFA.

There are major compositional differences between 

the Si-2 subfraction isolates, which represent nearly half the 

mass of SRFA, and the higher-numbered silica-gel subfractions. Both the normal and dipolar-dephased 13C-NMR spectra 

for the Si-2 subfractions are typical for fulvic acid derived 

from terpenoid precursors. The dipolar dephased spectra of 

subfractions derived from fractions Si-3 to Si-6 all show the 
14 Tannins and Terpenoids as Major Precursors of Suwannee River Fulvic Acid

distinct 105-ppm peak characteristic of condensed tannins, 

and this peak cannot be attributed to ketal anomeric carbons 

of carbohydrates due to insufficient concentrations of alcohols 

detected in the infrared and 13C-NMR spectra of these subfractions. The basis of the argument for tannins and terpenoids as 

major precursors of SRFA resides in the comprehensive fractionation and spectral characterization approach of this study.
References 15

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