Influence of humic substance structure and composition on interactions with hydrophobic organic compounds

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Influence of humic substance structure and composition on interactions with hydrophobic organic compounds
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Davis, William McCord, 1953-
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Thesis (Ph. D.)--University of Florida, 1993.
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Includes bibliographical references (leaves 206-215).
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by William McCord Davis.
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Vita.

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INFLUENCE OF HUMIC SUBSTANCE STRUCTURE AND COMPOSITION
ON INTERACTIONS WITH HYDROPHOBIC ORGANIC COMPOUNDS
















By

WILLIAM MCCORD DAVIS


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1993













ACKNOWLEDGMENTS


I wish to thank Dr. Joseph J. Delfino for his

encouragement and support throughout my association with the

Department of Environmental Engineering Sciences at the

University of Florida. He has been a true mentor.

The other members of my advisory committee were also

supportive of my research efforts during my graduate studies

here. They provided needed guidance, suggestions and

equipment to assist in the accomplishment of this research.

I would like to thank Drs. G. Bitton, P. Chadik, P.S.C. Rao

and R. Yost.

Many other members of the University of Florida

provided assistance and equipment that made my research

possible. I am reluctant to try to name them all because I

am sure to leave someone out. However, I would like to

thank them too, so I will try. Nancy Engle, Norm Cabrera

and Candace Biggerstaff assisted me in my day to day

laboratory work and kept the glassware clean. Chris

Erickson and Dr. Cliff Johnston assisted me in acquiring the

infrared spectra. Dr. Dave Powell and Mel Courtney provided

the elemental analyses. Linda Lee was very helpful in

allowing the use of Dr. Rao's liquid chromatograph for

polynuclear aromatic hydrocarbon analysis. Dr. Bill Cooper

ii








at Florida State University provided the 13C nuclear

magnetic resonance spectral data. I would also like to

thank Dr. Bitton for the use of the freeze-drier and Dr.

Chadik for the use of the total organic carbon analyzer and

sampling pump.

Shirley Jordan has provided valuable friendship as well

as an endless store of needed laboratory supplies during my

time at Black Hall. Mrs. Jo David deserves thanks for

guiding me through the graduate school bureaucracy.

Berdenia Monroe has always been more than helpful in the

acquisition of the needed tools to carry out research. I

would also like to acknowledge the support and friendship of

Sandra James and Eleanor Merritt.

I wish to acknowledge the long term encouragement of my

family. My mother and father always believed that there

children could do anything they were determined to do. My

brothers and sisters all supported me in my graduate

studies.

Finally, I thank my wife and daughter who encouraged

and assisted me every step of the way in this graduate

program. Without their help and love, I would not have been

able to accomplish this goal.










iii





______________ J











TABLE OF CONTENTS


page
ACKNOWLEDGMENTS......................................... ii

ABSTRACT......................... ............ .......... vi

CHAPTERS

1 INTRODUCTION ..... ...................... ... ...... 1

Interactions of Humic Substances with
Hydrophobic Organic Compounds.................. 2
Soil and Sediment Humic Substances.......... 2
Dissolved Humic Substances................... 4
The Role of Humic Substance Structure and
Composition in Interactions with
Hydrophobic Organic Compounds.................... 6

2 COLLECTION AND ISOLATION OF HUMIC SUBSTANCES...... 12

Sample Collection.................... ............. 12
Isolation of Humic Substances................... 16
Aqueous Humic Substances........................ 17
Materials and Methods........................ 18
Isolation................. ................. 22
Soil/Sediment Humic Substances................. 27
Discussion....... ................. ............... 29

3 CHARACTERIZATION OF HUMIC SUBSTANCE STRUCTURE
AND COMPOSITION................................ 35

Elemental Analysis................................ 38
Methods................... ...................... 39
Results......................... .. ......... 44
Ultraviolet/Visible Spectroscopy.................. 49
Methods............................. ........ .. 51
Results ........................... ............. 52
Total Acidity and Copper Binding Capacity
of Humic Substances............................. 62
Total Acidity.................................. 63
Methods......... ............. ...... ....... 64
Results......................... ............ 67
Copper Binding Capacity........................ 70
Methods ............... ...... ............. 71
Results..................................... 76








Infrared Spectroscopy of Humic Substances......... 84
Methods..................................... 86
Results..................................... 90
Discussion.................................. 115
13C Nuclear Magnetic Resonance Spectroscopy....... 124
Methods..................................... 125
Results ..................................... 131
Summary ........................................... 146
Conclusions ....................................... 149

4 INTERACTIONS OF HYDROPHOBIC ORGANIC COMPOUNDS
WITH DISSOLVED HUMIC SUBSTANCES ................ 151

Introduction...................................... 151
Methods........................................... 152
Results and Discussion........................... 162
Influence of HOC Hydrophobic Character on
Kd ...................................... 170
Thermodynamic Basis of Partition
Coefficients................................. 171
Similarity Between Octanol and
Dissolved Humic Substances as
Solvents in HOC Partitioning............ 175
Partitioning Description of Dissolved
Humic Substance/HOC Interactions............ 179
Variation in Partitioning of HOCs with
Different Dissolved Humic
Substances... .............................. 182
Influence of the Structure and Composition
of Humic Substances on Interactions
with HOCs... ....................... ...... 188
Summary ........................................... 197
Conclusions....................................... 198

5 SUMMARY AND CONCLUSIONS .......................... 201

LITERATURE CITED........................................ 206

APPENDICES

A ADDITIONAL DATA ON THE CHARACTERIZATION OF HUMIC
SUBSTANCE STRUCTURE AND COMPOSITION............ 216

B ADDITIONAL DATA ON THE INTERACTIONS OF
HYDROPHOBIC ORGANIC COMPOUNDS WITH
DISSOLVED HUMIC SUBSTANCES..................... 231

BIOGRAPHICAL SKETCH.................................... 244














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

INFLUENCE OF HUMIC SUBSTANCE STRUCTURE AND COMPOSITION
ON INTERACTIONS WITH HYDROPHOBIC ORGANIC COMPOUNDS

By

William McCord Davis

May 1993

Chairman: Joseph J. Delfino
Major Department: Environmental Engineering Sciences

The influence of the structure and composition of humic

substance on interactions with hydrophobic organic compounds

was investigated for thirteen different humic substances.

The structure and composition of the humic substances were

determined using a variety of analytical techniques

including elemental analysis, ultraviolet/visible

spectroscopy, copper binding capacity, total acidity

titration, infrared spectroscopy and "C nuclear magnetic

resonance spectroscopy. The results of these analyses

indicated that there were significant variations in the

structure and composition of the different humic substances.

Partition coefficients (Kda) were measured for each

humic substance with two different homologous series of

hydrophobic organic compounds, i.e. polychlorinated

biphenyls (PCBs) and polynuclear aromatic hydrocarbons








(PAHs). Linear correlations were observed between the

aqueous solubility of the hydrophobic organic compounds and

the partition coefficients for each humic substance. The

PCBs were observed to have higher activity coefficients for

each humic substance than did the PAHs. These observations

support a partition mechanism for these interactions.

The partition experiments revealed at least one order

of magnitude variation in the Kda values for a particular

hydrophobic organic compound and the different humic

substances. Correlations were found between the log(Kda)

values for both the PCBs and PAHs with a number of

structural and compositional characteristics of the humic

substances. The aromatic carbon and carboxylic acid content

of the humic substances appeared to influence interactions

with the hydrophobic organic compounds. The Kdo values

determined for the PCBs were more sensitive to oxygen-

containing functional groups than those for the PAHs.

Differences among the activities of the PCBs and PAHs toward

the humic substances were correlated with the atomic

oxygen/carbon ratio and total acidity values.

The results of this investigation support a partition

mechanism for the interactions between hydrophobic organic

compounds and dissolved humic substances. Both the aromatic

carbon and carboxylic acid content are important humic

substance structural characteristics in determining the

strength of their interactions with hydrophobic organic

compounds.


vii














CHAPTER 1
INTRODUCTION


Humic substances are a class of naturally occurring

organic compounds which are the products of the biological

and chemical degradation of biological substances. Although

they are of biological origin, humic substances are highly

resistant to biodegradation. The biological oxygen demand

(BOD) of water containing dissolved humics has been shown to

be essentially zero (Black and Christman, 1963; Malcolm,

1985).

Humic substances are ubiquitous in soil, sediment and

water. These complex macromolecules do not consist of a

single unique chemical structure and are diverse mixtures of

polyfuntional macromolecules which span a large molecular

weight range. Due to their complex and varied structure,

humic substances have been operationally defined by their

solubility in aqueous acid and base (Aiken et al., 1985).

These definitions are:

Fulvic Acid: that fraction of humic substances that is

soluble under all pH conditions

Humic Acid: that fraction of humic substances that is

not soluble at pH 2, but which is soluble

at higher pH values








2

Humin: that fraction of humic substances that is

not soluble in water at any pH.

Recently, a new operational definition for aquatic humic

substances has been proposed (Clesceri et al., 1989). This

definition is based on the interaction of the humic

substances with a hydrophobic adsorbent, XAD-8 resin. This

adsorbent has been extensively used to concentrate dissolved

aquatic humic substances from aqueous samples (Leenheer,

1981; Aiken, 1985).

Despite the lack of knowledge of the specific structure

of humic substances, these compounds have been shown to play

major roles in soil, sediment and water chemistry as

photochemical sensitizing agents, precursors to disinfection

by-product formation, ion exchangers, surfactants, chelating

agents and sorbents for hydrophobic organic compounds

(HOCs).


Interactions of Humic Substances with Hydrophobic Organic
Compounds


Soil and Sediment Humic Substances


The operational definition of humic substances has

obscured the basis for many of the observed interactions of

humic substances with other classes of chemicals found in

the environment. Humic substances are known to interact

with both heavy metals (Truitt and Weber, 1981b; McKnight et

al., 1983) and hydrophobic organic pollutants (Karichkoff,










1981; Thurman, 1985). The role of humic substances in the

binding and transport of pollutants has been extensively

studied in both the dissolved and condensed phases (Chiou et

al., 1979; Karichkoff, 1981; Carter and Suffet, 1982;

Stevenson, 1982; Landrum et al., 1984; Whithouse, 1985;

Chiou et al., 1986; Morehead et al., 1986; Gauthier et al.,

1987; McCarthy et al., 1989; Jota and Hassett; 1991).

However, due to the complex nature of humic substances, the

actual mechanisms for these interactions have yet to be

determined.

Early attempts to empirically describe the sorption of

hydrophobic organic compounds from water by soil and

sediment humic substances using classical solid/liquid

sorption isotherms were quite successful (Chiou et al.,

1979; Karichkoff, 1981). The organic carbon content of the

soil and the hydrophobicity of the organic compound were

found to be the major factors controlling the distribution

of the pollutant between the water and soil. Many

correlations have been found between the aqueous solubility

or octanol/water partition coefficient (Kw) of the

hdyfrophobic organic compound (HOC) and the soil/water

partition coefficient which has been normalized to the soil

organic carbon content (K.). Means et al. (1980) derived a

general expression for the sorption of 22 hydrophobic

compounds by a variety of soils and sediments:












logK, = logK, 0.317 r2 = 0.980 (1.1)


These results are in agreement with those reported by Chiou

et al. (1979) who observed a similar relationship for the

behavior of 15 hydrophobic compounds using different soils.

Although these two groups obtained similar results, their

interpretations of the basis for the correlations were very

different. Means et al. (1980) used a standard adsorption

model to explain the interaction, while Chiou et al. (1979)

proposed a partition model for the same interactions.

Adsorption is generally used to describe an interaction

between a solute and adsorbent which is based on physical or

chemical bonding forces. Partitioning refers to the

distribution of a solute between two immiscible or partially

miscible phases. Partition behavior is governed primarily by

van der Waals forces (Kile and Chiou, 1989).


Dissolved Humic Substances


The interaction of aqueous dissolved humic substances

with hydrophobic organic pollutants presents conceptual

problems for either a sorption or partition model. If the

humic material is truly dissolved, there is no second bulk

phase for sorption or partitioning (Carter and Suffet, 1982;

Chiou et al., 1986; Chiou et al., 1987). A large number of

investigators have found that the presence of dissolved








5
organic carbon (DOC) increases the apparent water solubility

of hydrophobic pollutants. Poirrier et al. (1972) reported

that the presence of DOC in natural stream water increased

the apparent solubility of 1,1-bis(4-chlorophenyl)-2,2,2-

trichloroethane (DDT) fourfold. The presence of dissolved

humic substances has been reported to decrease the

partitioning of hydrophobic pollutants into sediment,

suspended solids and biota (Means et al., 1980; Hassett and

Anderson, 1982; Oliver and Nilmi, 1983). The presence of

DOC in water has also been shown to decrease the toxicity of

pollutants to a variety of aquatic organisms (Eadie et al.,

1982; McCarthy, 1983; Landrum et al., 1985; Bitton et al.,

1986). Regardless of how these interactions are viewed,

information reported in the literature indicates that

dissolved humic substances can greatly effect the transport

and fate of hydrophobic organic pollutants.

Equilibrium constants for the DOC/hydrophobic organic

compound interactions (Kda) have been measured for a variety

of natural waters and humic substances extracted from soil

and sediments which have been dissolved in water. The

magnitude of the interactions for a given HOC with DOC from

different sources has been reported to vary considerably.

Chiou et al. (1987) reported that a soil- derived,

commercial humic acid had Kdo values for two PCBs which were

4 to 20 times the Kd. values measured for natural, aquatic

DOC. Other research has shown that the K, of the HOC










accounts for only 46-50% of the observed variation in the

Kdc values measured with different sources of dissolved

humic substances (Landrum et al., 1987; Evans, 1988).

Observations of this type have prompted many investigators

to postulate that the variations in Kd for a given HOC with

different sources of dissolved humic substances is due to

the chemical and structural characteristics of the humic

substances (Whitehouse, 1985; Chiou et al., 1986; Morehead

et al., 1986; Gauthier et al., 1987; McCarthy et al., 1989;

Jota and Hassett; 1991). Although many researchers have

come to this conclusion, few have attempted to correlate the

structure or chemical composition of humic substances with

the variation of the Kdo values measured for HOCs.


The Role of Humic Substance Structure and Composition in
Interactions with Hydrophobic Organic Compounds


The chemical composition of humic substances has been

extensively investigated by geochemists (Stevenson, 1982;

Ertel and Hedges, 1983; Steelink et al., 1983; Huffman and

Stuber; 1985; MacCarthy and Rice, 1985; Wilson, 1987; Rice

and MacCarthy, 1991). Humic materials from both terrestrial

and aquatic systems have been characterized by a variety of

physical and chemical methods. For example, infrared (IR)

and nuclear magnetic resonance (NMR) spectroscopic

investigations of the structure and functional group

composition of humic substances have revealed some general

differences between humic materials from different sources.










NMR spectroscopy has shown that the aromatic carbon content

of humic materials varies from 20 to 75% (Hatcher et al.,

1983; Wilson, 1987). Terrestrial humic materials have been

shown to be more aromatic in character than marine humic

substances by both NMR (Hatcher et al., 1983; Wilson, 1987)

and IR (Stevenson, 1982) spectroscopy. Both these

techniques indicate that fulvic acids are generally less

aromatic than humic acids, but fulvic acids contain more

carbohydrate character (Stevenson, 1982; Aiken et al.,1985).

Other techniques which have been used to investigate the

chemical composition of humic substances include elemental

analysis (Huffman and Stuber, 1985; Steelink, 1985), total

acidity titrations (Schnitzer and Khan, 1972; Stevenson,

1982; Perdue; 1985), metal binding capacity (Steveson et

al., 1982; McKnight et al., 1983; Thurman, 1985) and UV/VIS

spectroscopy (Stevenson, 1982; Ertel and Hedges, 1983;

Gauthier et al., 1987; Traina et al., 1990; Novak et al.,

1991).

Since humic substances are mixtures of extremely

complex macromolecules, any attempt to determine their

structure or chemical composition is difficult. The

application of any analytical method to determine the

functional group composition, molecular weight or tertiary

structure of humic substances is in reality the application

of the technique to a mixture. Therefore, the information










gathered in these experiments tends to be net or average

responses (MacCarthy and Rice, 1985).

The difficult nature of the investigation of the

relationship between the structure and composition of humic

substances and the variation in the Kdc has generally

prevented researchers from gathering the data needed for a

full understanding of the mechanism of these interactions.

Gauthier et al. (1987) has investigated their relationship

for pyrene and a number of dissolved humic substances from

both terrestrial and marine sources. The humic substances

used in this study were characterized by elemental analysis,

NMR and IR spectroscopy. They observed a linear correlation

(r2 = 0.48) between the Kd. for pyrene and the atomic H/C

ratio of the humic substances. The Kdg was also found to

correlate linearly (r2 = 0.94) with the aromatic carbon

content (measured by NMR) of four of the humic substances.

Since both aromatic and carboxylic acid functional groups

have the same atomic H/C ratio, the authors suggested that

the carboxylic acid functional groups adversely affected the

correlation between the atomic H/C ratio and the Kda. These

data support the postulates of many researchers who believe

that the variation in Kdc for a particular HOC with

different sources of humic substances is due to their

structure and composition.

Although little data are available on the role of

oxygen in the interactions of humic substances and










hydrophobic pollutants, its effect on the polarity of humic

compounds is strong (Kile and Chiou, 1989). Garbarini and

Lion (1986) have found a correlation between the Kdo for

toluene and trichloroethene and a mathematical function

which includes both the C and 0 content of the humic

substances. The reported decrease of Kd. for a particular

HOC with a single humic source as the pH is increased also

suggests the importance of oxygen-containing carboxylic acid

functional groups in these interactions (Kile et al., 1989;

Jota and Hassett, 1991).

The work of Gauthier et al. (1987) and others (Kile et

al., 1989; McCarthy et al., 1989; Jota and Hassett, 1991)

indicates that the structure and composition of dissolved

humic substances is an important factor in the strength of

their interactions with HOCs. However, the data needed to

fully understand the mechanism of these interactions are

currently not available in the literature (Gauthier et al.,

1987; Evans, 1988; Kile and Chiou, 1989; McCarthy 1989).

The research described in this dissertation was designed to

obtain data which will be used to evaluate the importance of

the structure and composition of humic substances in their

interactions with hydrophobic organic compounds. The data

needed to understand the interactions of humic substances

with HOCs can be divided into two areas: (i)

characterization of the structure and composition of the

humic substances and (ii) characterization of the HOC/humic










substance interactions (measurement of Kd.) for the humic

substances used in (i).

Humic substances with a wide range of structure and

composition were isolated from soils, sediments and water.

A variety of different humic sources were chosen to increase

the variation in interactions with HOCs and measured

compositional characteristics. The investigation of the

structure and functional group composition of humic

substances was carried out using the following analytical

techniques:

1. Elemental Analysis

2. UV/VIS Spectroscopy

3. Total Acidity Titration

4. Copper Binding Capacity

5. Infrared Spectroscopy

6. Nuclear Magnetic Resonance Spectroscopy

The information obtained from these structural and

compositional analyses were used to investigate the role of

humic substance structure and composition in deterniming the

mechanism and strength of their interactions with two

homologous series of HOCs.

Chapter 2 of this dissertation describes the isolation

of thirteen different humic substances from soil, sediment

and water. The structural and compositional

characterization of these humic substances by the methods

discussed above is covered in Chapter 3. The interactions








11
of the HOCs with the humic substances isolated in this study

are detailed in Chapter 4. This Chapter also includes a

discussion of the correlations obtained between the log(KFd)

values measured for these humic substances and their

structure and composition. The implications of the

composition and structure of these humic substances for

understanding their interactions with hydrophobic organic

compounds are summerized in Chapter 5.














CHAPTER 2
COLLECTION AND ISOLATION OF HUMIC SUBSTANCES


Sample Collection


Humic substances with a wide range of compositional and

structural variation were needed to perform this research.

The rationale for this was that a wide range of humic

substance sources would likely exhibit the variation in

elemental composition and functionality necessary to

investigate the effect of these parameters on the

interactions between hydrophobic organic compounds and humic

substances. Therefore, a variety of humic substances were

isolated from several aqueous, terrestrial and sedimentary

environments.

Aqueous samples were obtained from two blackwater

rivers in Northcentral and Northeast Florida. Additional

aqueous samples were obtained from an eutrophic lake and a

surficial groundwater source in a planted pine forest. Soil

samples were collected from a planted pine forest and a

mixed hardwood forest. Sediment samples were collected from

the bottom of a eutrophic lake. The sampling and source

information for the humic substances used in this

investigation is summarized in Table 2.1.















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15
Water samples containing dissolved organic matter (DOM)

were obtained at each site using a submersible centrifugal

pump (K-VA Analytical Systems, model XP-100 Minipump). The

pump was placed approximately 30 cm below the surface of the

water and the pump and associated sampling tubing were

purged with the source water at a flow rate of approximately

1 L/min for five minutes before the sample was collected.

The water was pumped into glass carboys (6 or 12 L) that

were transported to the laboratory. The carboys had been

previously cleaned in the laboratory with phosphate-free

detergent and tap water, and rinsed sequentially with

deionized water, dilute HCI and deionized water. The pump

and sampling tubing were cleaned after each sampling by

purging with deionized water for approximately 15 min. The

water samples were transported to the laboratory at ambient

temperature and processed as described later.

Soil samples were collected using a stainless steel

trowel to obtain approximately 500 g of material at a given

site. The soil was placed into a plastic bag and

transported to the laboratory on ice. Soil samples were

stored at 10 C until they were extracted. Sediment samples

were obtained using a Livingstone piston corer. The cores

were return to the laboratory, extruded and then sectioned

into 1 cm sections (Gdttgens, 1992). Three sections

covering the range from 16 to 24 cm were combined from each

sampling location to form a single sample for extraction of










humic substances. The soil and sediment samples were

extracted as described below.

Three additional humic substances were obtained in

extracted form from the International Humic Substances

Society (Golden, CO). These samples included two aqueous

humic substances and a soil humic substance. These samples

were isolated as described by Malcolm et al. (1989),

procedures which were essentially those used for the

isolation of similar samples in this research. Aldrich

humic acid (Cat. No. H1675-2, Lot No. 121137) was also

included as a humic substance; this was known to be derived

from a soil source. Aldrich humic acid was included because

it has been studied extensively and would allow for ready

comparison of literature results with those in this

research.


Isolation of Humic Substances


Isolation of aquatic and soil/sediment organic matter

from natural matrices was necessary for a number of reasons.

Since the objective of this research was to investigate the

interactions between hydrophobic organic compounds (HOC) and

dissolved humic substances, the extraction of soil/sediment

derived humic substances from the solid matrix and

subsequent dissolution for experiments with HOC was

necessary. The aqueous humic substances were extracted to

provide a concentrated source of this type of humic










substance. Once isolated, aqueous and soil/sediment humic

sources could be dissolved in water for investigations

carried out under conditions of controlled pH and ionic

strength. This was an important consideration because the

interactions of a given HOC with a particular humic

substance has been shown to vary with ionic strength and pH

(Carter and Suffet, 1982; Jota and Hassett, 1991). The

final extracts for both aqueous and soil/sediment humic

substances were freeze dried. Humic substances in this

form, when stored in the dark, are quite stable (Aiken,

1985). The procedures used to characterize the structure

and functional group composition of the humic substances

were performed collectively. Therefore, the ability to

store isolated humic substances in a stable form was

important.


Aquatic Humic Substances


Aquatic humic substances were isolated from water

samples using the procedure described in Standard Method

5510 C (Clesceri et al., 1989). The basic procedure

involves acidification to pH 2 of the 0.45 pm filtered water

sample, concentration on a macroporous resin, and elution of

the aquatic humic substances by backflushing the column with

dilute base. This method is an analytical isolation method

and was modified for preparative scale isolation by simply

scaling up the size of the resin column. The procedure is








18
similar to the isolation of the hydrophobic acid fraction of

dissolved organic carbon from natural waters reported by

Leenheer (1981). This method has been used to reproducibly

isolate aquatic humic substances from a variety of water

sources on a preparative scale.


Materials and methods


Ionic strength measurements were made using a digital

conductivity bridge (Fisher Scientific Co., Cat. no. 09-325-

360) which was calibrated by measuring a standard 0.001 M

KC1 solution (Fisher Scientific Co., Cat. no. P212-100).

The pH measurements were made using a Fisher Scientific

Model 900 pH meter equipped with a combination pH electrode

(Fisher Scientific Co., Cat. no. 13-602-108). The pH meter

was calibrated before each use with buffer solutions at pH

1, 4 and 7 (Fisher Scientific Co., Cat. nos. SB140-500,

SB101-500 and SB107-500, respectively).

Total organic carbon (TOC) measurements were carried

out using an lonics Model 555 Organic Carbon Analyzer. The

dissolved CO2 present in liquid samples to be analyzed for

TOC was removed by acidifying the sample with concentrated

nitric acid (Fisher Scientific Co., Cat. no. A467) and

stripping with a nitrogen purge. This instrument operates

by combusting liquid samples over a copper oxide catalyst at

900 C under nitrogen flow (Airco, UHP Grade, 99.9995%) to

produce CO2. The CO2 produced is detected by an Horiba








19
Model PIR-2000 Infrared Detector. Potassium acid phthalate

(Mallinckrodt, Inc., Cat. no. 6704) solutions were prepared

from solid dried primary standards and used to calibrate

this instrument in the range of 1.0 to 10.0 mg/L TOC.

Water samples were filtered under 40 psi nitrogen

pressure through a 10.1 cm diameter, 0.45 pm pore size nylon

filter (Fisher Scientific Co., Cat. no. N04SP14225)

positioned in a flow through filter holder (Fisher

Scientific Co., Cat. no. 09-753-17E). Feed water for

filtration was provided from a 5 gal stainless steel

reservoir. Filtered water was stored in glass carboys and

the pH adjusted to 2.0 with concentrated HC1 (Fisher

Scientific Co., ACS Certified, Cat. no. A144-212).

The Amberlite XAD-8 resin (Rohm and Haas,lot# CG-360,

16-50 mesh size) was obtained from Supelco, Inc. (part# 2-

0278). The resin was packed into a glass low pressure

liquid chromatography column (Spectrum Medical Industries,

Inc., part# 124026). The column was 45 cm long and had a

2.5 cm internal diameter (ID), yielding 4.91 mL/cm bed

volume. The column was fitted with an adjustable plunger

endcap (Spectrum Medical Industries, Inc., part# 124122).

The column was also fitted with a shut-off valve (Spectrum

Medical Industries, Inc., part# 124420) at the exit and a

combination shut-off valve/metering valve (Spectrum Medical

Industries, Inc., part# 124432) at the entrance. Liquid was










run through the XAD-8 column by gravity feed and the flow

rate controlled using the metering valve.

The XAD-8 resin is a macroporous methylmethacrylate co-

polymer. The resin is contaminated with leachable organic

carbon when it is obtained from the manufacturer. Therefore

it must be repeatedly extracted to remove this organic

carbon before it is used to concentrate aquatic humic

substances (Leenheer, 1981; Clesceri et al., 1989). The

resin was initially wetted with 0.1 M NaOH (Fisher

Scientific Co., Reagent Grade, Cat. no. S320-500) and the

fine particle contaminants were decanted. The resin was

then stored in 0.1 M NaOH for at least 24 hours. This

solution was decanted and the resin was repeatedly washed

with deionized (DI) water followed by a final rinse with

methanol (Fisher Scientific Co., Optima Grade, Cat. no.

A454-4). The resin was sequentially Soxhlet extracted with

methanol, hexane (Fisher Scientific Co., Optima Grade, Cat.

no. H303-4) and methanol to remove organic contaminants.

The glass chromatography column was packed with a

methanol/resin slurry and 8 L of DI water pumped through

the column to remove the methanol. The column was further

rinsed with 8 L of 0.1 M NaOH followed by 8 L of 0.1 M HC1.

The column was considered ready for isolation of humic

substances from natural waters when the TOC of the 0.1 M HC1

eluent was less than 1 mg/L.










The Spectra/Gel Type 50X8 cation exchange resin

(Spectrum Medical Industries, Inc., lot# 16599) was obtained

from Fisher Scientific, Inc. (Cat. no. 11 187-130). The

resin was reported by the manufacturer to have an exchange

capacity of 1.7 milliequivalents/mL (meq/mL). The cation

exchange resin was purified by Soxhlet extraction with

methanol to remove leachable organic carbon (Leenheer,

1981). The resin was slurry packed into a 35 cm long by 2.5

cm ID glass chromatography column (Fisher Scientific Co.,

Cat no. K420280-0242) fitted with a 300 mL reservoir. The

end of the chromatography column was fitted with a scintered

glass frit and a stopcock. Flow through the column was

under gravity feed and was controlled at approximately 20

mL/min by the stopcock. The column was packed with a

methanol/resin slurry to just below the reservoir and a

glass wool plug was used to top the resin. This yielded a

bed volume of approximately 150 mL. The column was rinsed

with 5 L DI water, then 1 L of 2.0 M HC1 to convert the

resin to the hydrogen ion form. The final preparation of

this column was repeated rinsing with DI water (6-8 L) to

remove the HC1. The column was considered ready for use

when the conductivity of the column effluent was less than

10 pmhos/cm (Leenheer, 1981).

The calculated cation exchange capacity of the 150 mL

bed volume, based on the manufacturer's reported capacity

for this resin, was 0.255 meq. The capacity of the bed was








22
determined experimentally by measuring the volume of 0.1 M

NaOH required to achieve column breakthrough. The

calculated volume of this solution required to exceed the

column exchange capacity was 2.55 L. The results obtained

in this experiment are illustrated in Figure 2.1. The

effluent pH was monitored to determine when NaOH was eluting

from the column. The pH of the effluent remained at 5 until

2.60 L of 0.1 M NaOH had passed through the column. The pH

of the effluent was 8.0 at 2.65 L and 11 at 2.7 L. This

confirmed that the exchange capacity reported by the

manufacturer was accurate.

Once the cation exchange column had been used, it was

regenerated by eluting 4-5 L DI water to remove excess DOC.

The Na ions were displaced from the column by eluting 1 L

of 2 M HC1. The excess HCl was eluted with DI water

(approximately 2-3 L) until the conductivity of the column

effluent was less than 10 pmhos/cm.


Isolation


Once the XAD-8 column had been prepared and conditioned

as described, isolation of aquatic humic substances was

initiated. The water samples which had been filtered and

adjusted to pH 2.0 were placed approximately 1 m above the

XAD-8 column inlet. The flow through the column was set to

12-16 mL/min using the column inlet metering valve. This

flow rate was 6 to 8 bed volumes/hr, which is significantly





















.4.


4




+


+


.9


0.23


0.24


Figure 2.1
determination.


0.25 0.26 0.27 0.28 0.2
Column Capacity (meq Na)


Cation exchange column capacity


9


16


14-


12-


-10-




6-
6]










less than the suggested maximum rate of 30 bed volumes/hr

(Leenheer, 1981).

The column effluent total organic carbon (TOC) was used

to monitor the breakthrough of both humic and non-humic

dissolved organic carbon. The influent TOC was used as the

measure of total dissolve organic carbon (DOC) in the

sample. The nonhumic DOC fraction was represented by the

TOC measured immediately after the XAD-8 column dead volume

had been exceeded (75 mL) The humic fraction was considered

to be the difference between the total DOC and the non-humic

DOC. Based on the TOC measured after the dead volume was

eluted, the non-humic DOC fraction generally was about 50%

of the total DOC in the water samples investigated. This is

in the range reported for this fraction for natural waters

by Thurman (1985). The TOC concentration was monitored

every 2 L to determine the point at which the capacity of

the XAD-8 resin for retaining humic substances had been

exceeded. The application of the water sample was stopped

once approximately 30% of the humic fraction broke through

the column. The application of water was stopped at this

point to maximize the total amount of humic substance which

could be isolated from a given water source.

The breakthrough of both non-humic and humic DOC is

illustrated for the Orange Heights DOC sample in Figure 2.2.

In this graph, it can been seen that the non-humic DOC

begins eluting from the column almost immediately. The














18

16-

14-

12-

10-



6-1

4-

2
0 10 20 30 40 50 60 70 80
Effluent Volume (L)


Non-Hum. TOC + Non-Hum.+Hum. TOC ------ Influent TOC

Figure 2.2. XAD-8 column carbon breakthrough
determination for Orange Height DOC sample. Note the
non-humic TOC breakthrough at 4 L of effluent volume
and the non-humic plus humic TOC breakthrough at
approximately 26 L. Influent TOC was 17 mg/L and the
column void volumn was 75 mL.










non-humic DOC in the column effluent breakthrough occurred

within 4 L of the initial elution and maintains a relatively

constant level of 7.9 mg/L TOC. The humic DOC began to

breakthrough between 24 and 26 L and increased until

application of the sample was halted at 36 L (effluent TOC

at 10.5 mg/L). Based on the results of this breakthrough

curve, the non-humic fraction of the DOC in this sample was

7.9 mg/L (46% of the total DOC concentration of 17 mg/L) and

the humic DOC fraction was 9.1 mg/L. The elution was

stopped when 2.6 mg/L of humic DOC was breaking through the

column (28.5% of the humic DOC). Depending on the total DOC

concentration of the water and the fraction of the total DOC

which was humic DOC, the effluent volume required to reach

the 30% humic DOC breakthrough point was between 24 and 40 L

for the water samples investigated.

Aquatic humic substances were eluted from the XAD-8

column by backflushing the column with 0.1 M NaOH. The

column was inverted and the 0.1 M NaOH flow rate was set to

1-2 mL/min using the column metering valve. Elution of the

humic substances was considered complete when no color was

visible in the column effluent. A volume of 125 to 300 mL

of NaOH was required to elute the aquatic humic substances

from the column. This concentrate was stored in the dark in

a 250 mL glass erlenmeyer until it was eluted through the

cation exchange column.








27

The 0.1 M NaOH solutions of concentrated aquatic humic

substances were eluted through the cation exchange column

which had been prepared as described earlier to convert the

humic substances to the H* form. The solutions were eluted

at a rate of approximately 1-2 mL/min. This eluate was

frozen at -40C and freeze dried to provide the aquatic

humic substances in dried form. The dried humic substances

were stored in 40 mL glass vials with Teflon lined caps

(Fisher Scientific Co., Cat. no. 03-339-18A). These samples

were stored at room temperature in a desiccator in the dark

until needed.


Soil/Sediment Humic Substances


Soil and sediment humic substances were extracted from

the solid matrices using 0.5 M NaOH. Aqueous solutions of

NaOH have been the solvents most often used for extraction

of soil humic substances for approximately two centuries

(Hayes, 1985). Although some alteration of the humic

substances extracted with dilute NaOH does occur (Aiken,

1988) NaOH is still the most widely used solvent for

extraction of humic substances from soils (Schnitzer and

Khan, 1978). The predominant reaction occurring in basic

solution has been reported to be ester hydrolysis (Gregor

and Powell, 1987; Aiken, 1988). The minor alteration of

extracted humic substances is unavoidable and not of great

concern in this investigation because all sources of humic








28
substances were characterized for composition and structure

after extraction and isolation procedures were complete.

The humic substances were extracted from soil or

sediment by placing the soil/sediment in 0.5 M NaOH at a 1/3

ratio (weight g/volume mL). The mixture was stirred

thoroughly and allowed to stand overnight. The supernatant

was decanted into 250 mL polycarbonate centrifuge tubes

(Fisher Scientific Co., Cat. no. 05-579) and centrifuged at

7500 rpm for 1.5 hrs. After centrifugation, the supernatant

was removed from the pellet by pipet and filtered through a

47 mm diameter, 0.45 pm pore size nylon filter (Fisher

Scientific Co., Cat. no. N04SP04700). Soil/sediment humic

substance extracts were stored in glass, in the dark, until

they were processed through the cation exchange column.

The soil/sediment extracts were converted to the H*

form by eluting the NaOH extracts through the cation

exchange column in the same manner as that described for the

aquatic humic substance concentrates. It should be noted

that the XAD-8 column eluate concentrates of aquatic humic

substance contained only the Na ions present in the 0.1 M

NaOH solution. However, the soil/sediment extracts could

also contain co-extracted metal ions. The capacity of the

cation exchange resin was chosen to insure that it exceeded

the capacity needed to remove the Na4 ions present in the

0.5 M NaOH extracts by a factor of at least four (this

assumes 125 mL of 0.5 M NaOH). This leaves sufficient








29
cation exchange capacity to remove divalent cations from the

extract solution up to a concentration equivalent to 200

mg/kg in the original soil.

The soil/sediment humic substance extracts obtained

from the cation exchange procedure were frozen at -400C and

freeze dried to obtain the humic substances in dried form.

These humic substances were stored in 40 mL glass vials with

Teflon lined caps and the vials were stored in a desiccator,

in the dark, until analyzed further.


Discussion

Humic substances are operationally defined by the

procedures used to extract them from their natural

environment (Aiken, 1988). This has been the case since the

initial work was performed on the colored extracts of soils.

Even the "dissolved" designation of aqueous humic substances

is operationally defined as that portion of aqueous humic

substances that is not retained by a 0.45 pm filter

(Clesceri et al., 1989). Many different methods have been

used to isolate humic substances for water (Aiken, 1985).

This has lead to difficulty in directly comparing results

from studies using different isolation techniques. However,

the recently proposed method for isolation and quantitation

of aquatic humic substances in Standard Methods (Clesceri et

al., 1989) appears to be a move toward a more uniform

operational definition of these compounds. This method is










the result of extensive research over the past 20 years on

the use of macroporous resins for the isolation of humic

substances.

The isolation and concentration of the low levels of

naturally occurring dissolved humic substances is generally

necessary in any study which includes chemical and/or

structural investigation of aquatic humic substances (Aiken

et al., 1979; Thurman and Malcolm, 1981). A number of

different adsorbents have been used to isolate humic

substances from natural waters. These include a number of

different synthetic styrene divinylbenzene and acrylic ester

polymer resins as well as activated carbon (Aiken et al.,

1979; Aiken, 1985; Aiken, 1988) and diethylaminoethyl

cellulose (Clesceri et al., 1989; Miles et al., 1983).

The use of activated carbon for the isolation of humic

substances from water is simple and inexpensive. However,

many authors have noted irreversible sorption of some

organic compounds onto the activated carbon (Aiken, 1985).

Slow sorption kinetics have also been noted which often

limit flow rate. Also, chemical alteration of the isolated

organic matter has been reported (Aiken, 1985).

These problems have led many investigators to seek

other sorbents for the isolation of aquatic humic

substances. Macroporous non-ionic polymer resins have a

number of advantages over activated carbon for the

isolation of aquatic humic substances. These resins are










also simple, but they have higher capacities for humic

substances than activated carbon. Even though these resins

are more expensive than activated carbon, they can be easily

regenerated and reused. The sorbed humic substances are

easily eluted once concentration is complete. The main

disadvantage of these macroporous resins is the bleed of

organic material from the column material. This problem can

be minimized by thorough preparation of the column material

before isolation is initiated (Leenheer, 1981).

A number of different macroporous resins have been used

to isolate aquatic humic substances. These include both

styrene divinylbenzene polymers (XAD-1, XAD-2 and XAD-4) and

acrylic ester polymers (XAD-7 and XAD-8). The acrylic ester

resins have been shown to have faster sorption kinetics,

higher capacities and more efficient elution of sorbed

aquatic humic substances than the styrene divinylbenzene

resins (Aiken et al., 1979). These advantages have resulted

in the adoption of this resin as the sorbent in Standard

Method 5510 C (Clesceri et al., 1989). for the isolation and

quantitation of aquatic humic substances. The XAD-8 column

adsorption procedure has also been selected by the

International Humic Substances Society as the isolation

method used for the their standard and reference aquatic

humic substances.

The procedures used for the isolation and concentration

of aquatic humic substances in this research were chosen to










allow the most direct comparison with results available in

the literature. Although some alteration of the aquatic

humic substances, such as ester hydrolysis (Aiken, 1988),

has been shown to occur in the presence of NaOH (used to

elute the AHS from the column). This problem was minimized

by running the basic XAD-8 column eluates through the cation

exchange column and freeze drying these concentrates (Aiken,

1985).

Once the extracts have been freeze dried and placed in

the dark, they can be stored until needed without chemical

degradation (Aiken, 1985). This is important for the

research conducted in this investigation. Since the main

objective was to compare the humic substances obtained for

different sources in a variety of analytical procedures, it

is imperative that the humic substance isolates be stable.

Isolation of humic substances from soil and sediment

has received much attention (Stevenson, 1972; Schnitzer and

Khan, 1978; Hayes, 1985). Many organic and inorganic

solvents systems have been used to extract organic matter

from soils. Hayes (1985) compared the humic substances

extracted from soils using thirteen different solvent

systems. These included dipolar aprotic organic solvents as

well as aqueous buffer solutions and NaOH. Hayes (1985)

noted slightly higher extraction efficiencies for 2.5 M

ethylenediamine (EDA) than for 0.5 M NaOH. However,

elemental analysis revealed an increased nitrogen content of








33
the EDA extracts, indicating incorporation of the amine into

the humic substances. A similar result was obtained with

dimethylformamide (DMF). Dimethylsulfoxide (DMSO) was found

to extract similar levels of organic matter from soils

compared to 0.1 and 0.5 M NaOH. The problem with this

extraction procedure was that the recovery of the "classic"

humic fraction from the DMSO extract required the addition

of NaOH, resulting in the same degradation reactions that

the organic solvent was designed to prevent. The solvent of

choice for the extraction of humic substances from soils and

sediments still appears to be aqueous NaOH.

The extracts obtained from the soil and sediment

samples in this investigation were passed through a cation

exchange column to remove the Na* ions and produce the H*

form of the humic substances. This procedure also removed

the OH" ion by converting it to H20. Thus, once the

soil/sediment extracts had been passed through the cation

exchange resin, the hydrolysis of esters was prevented. As

noted for the aquatic humic substances, once the soil humic

substances were frozen and freeze dried they were not

subject to further degradation (Aiken, 1985).

The aquatic, soil and sediment humic substances were

stored in the dark in sealed glass vials in a desiccator

until needed for the different characterization experiments

described in Chapter 3. The isolation of different types of

humic substances to yield solid substances which could be








34
dissolved in aqueous solution under controlled pH and ionic

strength conditions was important for direct comparison of

experimental results among these humic sources. The control

of these two parameters is significant, considering the

complex nature of the humic substances themselves.














CHAPTER 3

CHARACTERIZATION OF HUMIC SUBSTANCE STRUCTURE AND
COMPOSITION

The current understanding of the factors that control

the association of hydrophobic organic compounds (HOC) with

natural dissolved organic carbon (DOC) is inadequate. The

association of different HOC with a particular DHS increases

as the water solubility of the organic compound decreases

(Carter and Suffet, 1983; Chiou et al., 1987; Lara and

Ernst, 1989). However, the amount of a given HOC that

associates with different DOCs has been shown to vary

significantly among different sources of DOC (Chiou et al.,

1987; Gauthier et al., 1987; Evans, 1988; McCarthy et al.,

1989). Sorption of HOC to soils and sediments has been

shown to be controlled by the sorbent organic carbon content

(Chiou et al., 1979 ; Karickhoff et al., 1979) Since less

than 50% of the variation in the sorption of HOC by DOC has

been attributed to the DOC organic carbon content it has

been postulated that structure and composition of DHS is the

cause for the observed variation in HOC sorption (Chiou et

al., 1987; Gauthier et al., 1987).

The fact that humic substances are complex mixtures of

macromolecules has impeded studies designed to investigate










the mechanisms of these interactions. To elucidate the

attributes of DOC that affect their sorption of HOC, humic

substances obtained from different sources (Table 2.1) were

characterized by a variety of physical and chemical

techniques. The elemental composition data reported by a

number of investigators indicate that humic substances from

different sources vary in composition (Huffman and Stuber,

1985; Malcolm and MacCarthy, 1986; Rice and MacCarthy,

1991).

The elemental composition of the humic substances was

determined to investigate whether the elemental composition

of humic substances could be used to predict their

association with HOC. Oxygen-containing functional groups

are abundant structural components of humic substances

(Schnitzer and Khan, 1972; Stevenson, 1982). The

definitions of humic and fulvic acids are based on aqueous

solubility that is related to their oxygen-containing

functional groups (Thurman, 1985). Therefore, oxygen was

expected to play an important role in the interactions

between DHS and HOC. The total acid functional group

content and copper ion binding capacities were measured as

overall estimates of humic substance oxygen-containing

functional groups.

Ultraviolet/visible (UV/Vis) spectroscopy has been used

extensively by soil scientistS for the investigation of

humic substances (Thurman, 1985). The absorbance at 272 nm








37
for DHS has been correlated with the aromatic carbon content

(Traina et al., 1990). The ratio of absorbance at

465nm/665nm (E4/E6) has been correlated with the aromatic

carbon content and molecular weight of DHS (Chen et al.,

1977). The UV/Vis spectra of the humic substances were

investigated because of these relationships and because

UV/Vis spectrosocpy is a readily available analytical tool.

Fourier transform infrared (FTIR) and "C nuclear

magnetic resonance (NMR) spectroscopic techniques were

employed to determine the functional group composition of

the different humic substances. FTIR and "C NMR yield

similar information but are also somewhat complementary

(Inbar et al., 1989). The powerful combination of FTIR and

13C NMR for the investigation of the structure as well as

the composition of these complex systems has been reported

(Lobartini and Tan, 1988; Inbar et al., 1989; Lobartini et

al., 1991).

A description of the methods used to characterize the

composition and structure of the humic substances is

provided in this chapter. The results of each

characterization are included as is a discussion of the

results relative to the structure and composition of the

humic substances investigated.










Elemental Analysis


The elemental composition of a chemical substance can

be used to derive the empirical formula and assist in

determining a molecular structure. However, with non-

stoichiometic materials like humic substances, the

usefulness of empirical formulas is limited (Rice and

MacCarthy, 1991). Nevertheless, elemental analysis is

probably the most widely used method to characterize humic

substances (Huffman and Stuber, 1985). Recent reviews of

the literature indicate that, even though the data obtained

for elemental analysis of humic substances represent

averages, there are significant differences among the

elemental composition of humic substances from different

sources (Steelink, 1985; Rice and MacCarthy, 1991).

The most commonly used method for the determination of

C,H, and N is the catalytic combustion of the sample in an

oxidizing atmosphere followed by separation and

quantification of the resulting C02, H20 and N2. The oxygen

content is most often determined by difference from 100 %

(after correction for moisture and ash content), based on

the measured quantities of the other three elements (Rice

and MacCarthy, 1991). The calculation of oxygen content

based on the C,H and N composition assumes that there are no

other elements present in humic substances. Although this

assumption can present difficulties, it is reasonable

(Schnitzer and Khan, 1972; Thurman, 1985) and will be










discussed later in detail. The above method was used to

determine the elemental analysis of humic substances

investigated in this study.

A potential problem encountered in the determination of

oxygen, as well as hydrogen, is moisture. The presence of

moisture in samples leads to incorrectly high hydrogen and

oxygen values. The most reliable method to avert this

problem is to determine the moisture content of the samples

used for elemental analysis and to correct the resulting

data for the contributions of moisture (Huffman and Stuber,

1985). Another important consideration in the accurate

determination of the elemental composition of humic

substances is ash content. The ash content of an isolated

humic substance is considered a measure of the total

inorganic content of the sample. The procedure for

measuring ash content is straightforward. The humic

substance is placed in a crucible and the organic fraction

is oxidized at high temperature (750 C), leaving an ash

which represents the inorganic residue.


Methods


Elemental analyses were carried out using a Carlo Erba

Model 1106 CHN analyzer. Freeze dried humic substance

samples (approximately 2 mg each) were weighed into tin

boats and combusted in the presence of oxygen gas over

silver cobaltus cobaltus oxide at 18000 to 1850 C. The










resulting combustion gases were separated using a 2 m long,

0.25 inch internal diameter column containing 80 mesh

PoraPak QS with helium carrier gas. The analyte gases were

detected and quantified employing a thermal conductivity

detector that had been previously calibrated with organic

compounds of known C, H, and N composition. In this

procedure, every fifth sample was a quality control check

sample. The elemental composition of all samples of humic

substances were analyzed in duplicate. The results of these

analyses are presented in Table 3.1.

The moisture content of the humic substances

investigated was determined by measuring weight loss on

drying, a common method used by researchers in the field

(Schnitzer and Khan, 1972; Huffman and Stuber, 1985). Humic

substances (approximately 10 mg) were weighed into 4 mL

borosilicate glass vials (Fisher Scientific Co., Cat. no.

03-339-25B) and placed in a desiccator. Samples were dried

in a vacuum oven (Thelco Model 19, Precision Sci. Co.) at

60 C that was evacuated to 11 mm Hg. The vacuum was

released through a CaS04 drying tube after 24 hr. Samples

were transferred to a desiccator, allowed to cool to room

temperature and weighed. Weight measurements were carried

out using a Mettler Model HK60 balance. The drying

procedure was repeated and the samples were reweighed. No

difference was noted between the first and second drying.

Therefore, a single 24 hour drying under these conditions










.5























Om
-1I





























II
.9


























III$r-


'0 0i 0
o o o






-i r-4 -4




* ,Ln 0
`4 c14 o0
H- H- 0


8 O iON 0TA Ho '6m H 6znk mor
CO CN Cq CM H (N0 O N m( 0H O
Ln tzp- C- 4~ -' Ln %r -i'-


gO OO' ;:z H 00 H0 rN'
i 6 H T 0 r-O NO ON (1 (NH-1 in AH rNo




NN LALA LAL HH 00 o'o 'DD Or>


(n N H Mn lzr LAt) m v m HN

CON A' L- LA' 9'- '0( on c c -
m- n LO n)- .T *',r- 'tr- *ir Qi.


o o o o in


0 0 0 0 0 LA
S 0 (0 N N
H H




(N H (N H


0 LA






O H4


H '0 a)
`l r-4 r-I



.U .U .-I M




".41
zi d


ko *4 in It Ln
o o 0 0 0




r, c0 rc ai k0
N- W- N rQ5 l-
Hl H i~- 1-1 H n




'O r-. -i n

o 0 o H H4 H















r- '.

o 0












o -0
0"l ,-1


-4X ml- 0cr-


JN 09 0 %-
I0


00 00 00



(14(14 00 0 0
99 O1 -! 00 1



r-'y ;F m ;Z m
-N (N (N
r-4 q 0v 0 V
i L i





rn La (N

0 (N %0


0 0

t-m L





0 0 O L
o 0














O o


A -

00 0



a'c. .





^Ln tN .

Ln- Ln -


0 IN
I I


'0 *


0 0-




m 0
'. t-
I I
0N 0


r- Ln
un w0

in In
m -V


CN 0 C

0 N N
0 0













6 2 M


m
4J











8

0>





I) -
4








S*Q
0' ) -
0 q'S

.4*d )
g T




1..i
r-CM4J


U


Ai








43
was considered sufficient for moisture determination. The

conditions employed for moisture determination were those

suggested by Huffman and Stuber (1985) who have conducted an

extensive investigation of the factors effecting moisture

determinations of humic substances. They found that weight

loss on drying at 60" C corresponded closely with the

moisture determined by Karl Fischer titration.

The ash content of the humic substances investigated

was determined by measuring the weight lost after ignition

at 750 C for two hours. The Mettler Model HK60 balance was

also used for the ash determination weighing. Before

samples were ashed, the 4 mL covered ceramic crucibles

(Coors, Fisher Scientific Co., Cat. nos. 07-965B, porcelain

crucible; 07-970C, cover) used in this procedure were

checked for constant tare weight by repeated heating to

750 C and cooling in a desiccator. This procedure

confirmed that the weights of the crucibles were constant

and unaffected by repeated heating to 750* C.

The humic substances were weighed (approximately 10 mg)

into tared crucibles. The crucibles were placed in a cool

muffle furnace which was then heated to 7500 C and the

samples were ignited for 2 hours. The furnace was then

cooled to near 1000 C and the crucibles were removed and

placed in a desiccator. This insured that the ash did not

become saturated with water from the air. Once the samples

reached room temperature, they were weighed. The process








44
was then repeated. The second heating showed no additional

weight loss, indicating that the first heating was

sufficient for ash determination.

The moisture and ash content of each humic substance

were determined in duplicate. The averages for each of

these determinations are included in Table 3.1. The

elemental compositions in Table 3.1 have been corrected for

moisture and ash content. The oxygen content was determined

by difference (from 100%) based on the C,H and N content,

after these values had been corrected for moisture and ash.


Results


The most reliable information obtained from the

elemental analysis of humic substances is the carbon and

hydrogen content (Huffman and Stuber, 1985). The low levels

of nitrogen generally found in humic substances lead to

lower precision in its determination. The calculation of

oxygen content based on the results of C,H and N measurement

has a number of associated problems. First, this procedure

magnifies the errors in the measurement of the other three

elements. Also, the calculation assumes that no elements

other than C,H,N and 0 are present in humic substances.

This assumption can be considered a good approximation if

the data available on sulfur and phosphorous are considered.

The sulfur content of humic substances has been

reported by Schnitzer and Kahn (1972) to be generally near








45
zero. The average S content of aquatic humic substances has

been reported to be between 0.96 and 0.40 percent (Thurman,

1985). Thurman (1985) points out that a S content of 0.6

percent for a humic substance with an average molecular

weight of 1,500 would yield one sulfur atom per every four

molecules of humic substances. Similarly, phosphorous has

average values in aquatic humic substances of 0.1 to 0.4

percent (Thurman, 1985). Considering the low levels

reported for these two elements, it is doubtful that the

error of including them in the calculated oxygen content

will create a significant error for each of the elemental

analyses. It is also improbable that elements occurring at

such low levels would have a major effect on the structure

or behavior of the humic substances.

The data presented in Table 3.1 show a variation of

elemental compositions for the humic substances

investigated. The elemental compositions are within the

ranges for individual elements reported in the literature.

Trends in the elemental composition of humic substances from

different sources have been reported (Stevenson, 1982:

Steelink, 1985; Rice and MacCarthy, 1991). Humic acids

generally have higher carbon and lower oxygen content than

fulvic acids (Stevenson,1982; Rice and MacCarthy,1991).

However, there is a great deal of overlap between the range

of elemental compositions reported for these two groups.








46
Rice and MacCarthy (1991) have recently carried out an

extensive review of the available elemental data reported

for humic substances. The ranges reported for humic and

fulvic acid percent carbon were 37.2-75.8 and 35.1-75.4,

respectively. These ranges were obtained from 410 different

humic acids sources and 214 fulvic acid sources. This

compilation of available elemental composition data for

humic substances is by far the most extensive review of its

type ever undertaken. It should be noted that Rice and

MacCarthy (1991) found sulfur data reported for only 160 of

the 410 humic acids and 71 of the 214 fulvic acids.

Although the ranges of carbon content reported for this

large data base were wide, the observed standard deviations

for the data sets were unexpectedly small. The mean values

and 95 percent confidence intervals (mean 1.97 standard

deviations) for elemental compositions of the humic

substances reviewed by Rice and MacCarthy (1991) appear in

Table 3.1. The values for the humic substances investigated

in this dissertation fall within the 95 percent confidence

interval for all samples except for the low percent carbon

and high percent oxygen values for Newnans Lake 16 and 18

sediments. The hydrogen and nitrogen composition of the

Newnans Lake Sediment samples are among the highest values

obtained in this study, perhaps reflecting the high

eutrophic nature of this lake. Similar elemental

compositions have been reported for sedimentary humic








47
substances (Steelink, 1985). The higher nitrogen content of

sedimentary humic substances may be a result of the relative

decrease of lignin and an increase of amino acids as humic

substance precursors in these systems (Steekink, 1985).

Ratios of elemental composition are often used to

compare different humic substances (Stevenson, 1982; Ertel

and Hedges, 1983; Steelink,1985; Rice and MacCarthy, 1991).

Therefore, the atomic ratios have been included with the

elemental composition data presented in Table 3.1. Again,

the means and 95 percent confidence intervals for humic and

fulvic acids reported by Rice and MacCarthy (1991) are

included for comparison. The most common atomic ratios

reported for humic substances are H/C and O/C. The H/C

ratio has often been used to indicate the degree of

unsaturation or aromaticity of humic substances(Rice and

MacCarthy, 1991; Thurman, 1985). A small H/C ratio may

indicate that the humic substance contains a large number of

carbon to carbon double bonds. This reasoning has been

questioned recently because it does not consider the

contribution of carbon to oxygen double bonds to the H/C

ratio (Rice and MacCarthy, 1991; Gauthier et al., 1987;

Perdue, 1984).

The ratios reported in Table 3.1 for the humic

substances investigated in this dissertation fall within the

95 percent confidence intervals reported by Rice and

MacCarthy (1991) except for the O/C ratios of Newnans Lake










16 and 18 sediment samples. As noted earlier, sedimentary

humic substances have been reported to have higher H/C and

O/C ratios than soil humic acids (Steelink, 1985;

Ishiwatari, 1985). This has been postulated to be due to

the difference in source material for lake sedimentary humic

substances. The more aromatic lignin available in

terrestrial environments is generally present in lakes

through runoff and detritus (Stevenson, 1982). However, an

eutrophic lake such as Newnans Lake (G6ttgens, 1992) would

be expected to contain significant aliphatic sources of

humic precursors from the water column phytoplankton. The

relative increase in the carbohydrate and amino acid content

of the humic substance source materials would also account

for the higher O/C ratio and higher nitrogen content of the

Newnans Lake sedimentary humic substances. Lake sediment

humic substances tend have aromatic ratios more similar to

marine humic substances than to terrestrial humic substances

(Ishiwatari, 1985; Rice and MacCarthy, 1991). The higher

the amount of autochthonous material that contributes to the

formation of lake sediment humic substances, the more

aliphatic are the humic substances formed (Ishiwatari,

1985).

The elemental ratios obtained in this study for Aldrich

humic acid compare well with those reported by Steelnik

(1985) for an Aldrich humic acid. However, Steekink (1985)

did not include the lot number for the Aldrich humic acid








49
used, therefore it was not possible to determine if it was

the same lot that was used in this dissertation research.

The H/C atomic ratio reported by Steelink (1985) was 0.80

compared with 0.84 measured in this research. The N/C

atomic ratio of 0.01 reported by Steelink (1985) is the same

value found for this ratio in the current study. However,

the O/C atomic ratio of 0.66 measured in this study is

different from the 0.46 value reported by Steelink (1985)

and may indicates that the Aldrich humic acids are from

different sources or atleast different lot numbers.

Based on comparison with the data available in the

literature, the elemental compositions of the humic

substances investigated for this dissertation (Table 3.1)

are typical of humic substances from different sources.


Ultraviolet/Visible Spectroscopy


The relative simplicity, wide availability and small

amount of sample required for this analysis have led to the

wide application of ultraviolet/visible (UV/Vis)

spectroscopy to investigations of humic substances

(Schnitzer and Khan, 1972). The UV/Vis spectra of humic

substances from both soil and aquatic environments have been

found to be relatively featureless (Schnitzer, 1978;

Stevenson, 1982) with absorptivity increasing as the

wavelength of radiation deceases (Ertel and Hedges, 1983).

This limits the application of UV/Vis spectra for the direct








50

determination of humic substances' structure (MacCarthy and

Rice, 1985). However, the ratio of absorbances of a given

humic substance at different wavelengths has been correlated

with a variety of structural properties for different humic

substances (Chen et al., 1977).

Absorbance measurements were recorded at 254, 272, 465

and 665 nm for all humic substances investigated. The 254

and 272 nm spectral regions include n n* electronic

transitions for phenolic arenes, benzoic acids, polyenes,

and polynuclear aromatic hydrocarbons (Silverstein and

Bassler, 1967). All these organic structures are known

components of most humic substances (Novak et al., 1992).

The ratios of absorbances at 465 and 665 nm have been

labeled the E4/E6 ratio by soil scientists and have been

investigated extensively for humic substances (Chen et al.,

1977; Schnitzer, 1978).

UV/Vis spectra of an individual humic substances have

been found to follow the Beer-Lambert Law for the

relationship between the concentration of the humic

substance and the absorption at a particular wavelength of

radiation (Black and Christman, 1963; Stevenson, 1982). The

relationship is given by:


A = abc


(3.1)










where A is the absorbance at a particular wavelength, a is

the absorptivity, b is the path length of light through the

sample, and c is the concentration of the chromophore in the

sample. The absorptivity of humic substances is generally

reported in units of L/(g humic carbon-cm) (Stevenson, 1982;

Gauthier et al., 1987; Traina et al., 1990; Novak et al.,

1992). The absorptivity of humic substances at a particular

wavelength has been reported to vary with pH (Chen et al.,

1977; MacCarthy and Rice, 1985; Thurman, 1985). A maximum

in the E4/E6 ratio has been seen for a variety of humic

substances between pH 7 and 8 (Chen et al., 1977). A review

of the literature reveals that UV/Vis spectra of humic

substances are often recorded from solutions at or near pH 7

in either 0.05 M NaCl (Traina et al., 1990; Novak et al.,

1992) or 0.05 M NaHCO3 (Chen et al., 1977; Ertel and

Hedges, 1983). Since the desired use of the UV/Vis

absorbance data was to compare results among the different

humic substances in this investigation and with work

reported elsewhere, all measurements were made at pH 7.


Methods


The UV/Vis absorbance values were recorded using a

Perkin Elmer Model 552 double beam grating

spectrophotometer. Solutions were prepared for UV/Vis

spectroscopy by dissolving approximately 10 mg of each humic

substance in 10 mL of 0.01 M NaCIl (Fisher Scientific Co.,










ACS Certified, Cat. no. S271-500). The pH of the solution

was adjusted to 7 using 0.01 M HCl (Fisher Scientific Co.,

ACS Reagent, Cat. no. A144-212) or 0.01 M NaOH (Fisher

Scientific Co., ACS Reagent, Cat. no. S320-500) (Traina et

al., 1990). Measurements were performed using 1.0 cm cells

(220 nm cut-off, Fisher Scientific Co., Cat. no. 14-385-

910C) with 0.01 M NaCI, pH 7 as a reference. Absorbance

values were recorded for each humic substance at 254, 272,

465 and 665 nm against the 0.01 M NaCl reference. The

absorptivities for each humic substance at the four

wavelengths were calculated using equation 3.1. The E4/E6

ratio was calculated by dividing the absorptivity at 465 nm

by the absorptivity at 665 nm (Schnitzer and Khan, 1972;

Stevenson, 1982).


Results


The Beer-Lambert Law has been used for quantitative

analysis for individual humic substances (Schnitzer and

Kahn, 1972; Stevenson, 1982). The absorptivity at a

particular wavelength for humic substances has been found to

vary for different sources (Thurman, 1985; Gauthier et al.,

1987; Traina et al., 1990; Novak et al., 1992). This

variation been attributed to differences in the structure

and functional group composition of the humic substances

(Stevenson, 1982; MacCarthy and Rice, 1985; Traina et al.,

1990).








53

The relatively featureless nature of UV/Vis spectra of

humic substances has been attributed to the fact that they

are complex mixtures of molecules which contain a number of

chromophores per molecule (MacCarthy and Rice, 1985). A

linear relationship (r2 = 0.88) between the absorptivity at

272 nm and the percent aromatic carbon (determined by 11C

NMR) has been reported for 12 soil humic substances (Traina

et al., 1990). A more recent report comparing the same two

parameters for eighteen terrestrial and aquatic humic

substances found a weaker correlation (r2 = 0.40) (Novak et

al., 1992). When the soil humic substances were considered

alone, the correlation increased (r2 = 0.72). There are a

large number of functional groups in addition to aromatic

moieties that are known to absorb light at or near 272 nm

(Gauthier et al., 1987). Since the absorptivity measured

for a particular humic substance at 272 nm represents an

average of all chromophores present, it is not surprising

that the correlation with percent aromatic carbon reported

by Novak et al., (1992) is weak.

The decrease in the correlations discussed above with

an expanded data set underscores an important consideration

in humic substance research. When drawing general

conclusions about humic substances from small data sets, one

should be very cautious. The strength of large data sets,

such as the one discussed earlier for elemental analysis








54
(Rice and MacCarthy, 1991), is that they allow correlations

to be identified that are more statistically significant.

Traina et al. (1990) also reported elemental ratios for

the humic substances investigated by UV spectroscopy and 13C

NMR. Combining their data with the data reported by

Gauthier et al. (1987) these authors reported a weak

correlation (r = 0.42) between the percent aromatic carbon

and the atomic H/C ratio. Since both studies indicated a

correlation between absorptivity of humic substances at 272

nm and percent aromatic carbon, Traina et al. (1990)

concluded that perhaps a relationship exists between the

absorptivity at 272 nm and the atomic H/C. Therefore, the

data for the humic substances investigated in dissertation

research (Tables 3.1 and 3.2) were combined with the data of

Traina et al. (1990) and Gauthier et al. (1987) to form a

data set which contained 35 different humic substance

sources. These sources included humic substances from

soils, marine and freshwater sediments as well as aquatic

humic substances.

Data from three independent sources indicate that

absorptivities increase as the humic substance H/C atomic

ratios decrease (Figure 3.1). The correlation (r = 0.62, p

< 0.01) was stronger than that reported by Novak et al.

(1992) for the correlation (r = 0.40) of percent aromatic

carbon with absorptivity at 272 nm for a diverse group of

humic substances. This finding suggests that the
















Table 3.2. Ultra-violet and visible spectral analysis results of humic
substances.



HUMIC SOURCE ABSORPTIVITY (LqCC'*cml) E4/E6

254 nm 272 nm 465 nm 665 nm




Aldrich Humic Acid 74.72 67.76 2.81 1.26 2.23

IHSS Humic Acid 76.42 71.58 3.66 0.99 3.69

Pine Mt. Soil 22.69 19.46 2.41 0.30 8.14

Orange Hts. Soil 63.29 55.59 2.05 0.48 4.28

Newnans Lake Sediment 11 36.03 31.97 1.41 0.40 3.53

Newnans Lake Sediment 16 33.50 30.16 1.13 0.31 3.65

Newnans Lake Sediment 18 35.57 31.79 1.76 0.43 4.11

Santa Fe River DOC 46.53 39.90 2.88 0.40 7.21

St. Marys River DOC 53.12 46.48 2.72 0.56 4.85

Orange Hts. DOC 47.49 40.80 2.42 0.47 5.15

Newnans Lake DOC 46.21 39.95 1.84 0.36 5.06

Suwannee River Fulvic 39.86 33.80 4.71 0.66 7.14

Suwannee River Humic 60.65 53.55 2.54 0.62 4.07















1.8

1.6-

o 1.4-
-,-
(: 1.2-
o
6 1-

0.8-
-1-

m 0.6-

0.4-

0.2


0 10 20 30 40 50 60 7C
Absorptivity @ 272 nm


80 90


100


This Study + Gauthier Traina

Figure 3.1. Absorptivity (L-gC-1-cm-) at 272
nm was correlated with the H/C atomic ratio (r2 =
0.64, p < 0.01) for the combined data of this study,
Gauthier et al. (1987), and Traina et al. (1990).


v


+


i










absorptivity at 272 runm includes information about the

concentration of other functional groups in addition to

aromatic carbon.

Since both -HC=CH- and -COOH have H/C atomic ratios of

one, the presence of carboxylic acid functional groups would

adversely affect the use of the H/C atomic ratio as a

measure of aromatic carbon content (Gauthier et al., 1987).

However, both these functional groups absorb at or near 272

nm. The data presented in Figure 3.1 support the conclusion

that the atomic H/C ratio is not a good measure of the

aromatic carbon content of humic substances. Further, the

correlation between the absorptivity at 272nm and the H/C

atomic ratio is likely the result of the combined affects of

both aromatic and carboxylic acid functional groups.

The E4/E6 ratio has been used to characterize humic

substances extracted from soil (Schnitzer and Khan, 1972;

Chen et al., 1977; Schnitzer, 1978; Stevenson, 1982) and

isolated from water (Thurman, 1985). The E4/E6 ratio for a

variety of humic substances has been shown to remain

constant over a wide range of concentrations (Chen et al.,

1977). The ratio varies for humic substances extracted from

different sources (Schnitzer and Khan, 1972). The ratio has

been reported to be in the range of 2 to 5 for soil humic

acids, 6 to 8.5 for soil fulvic acids (Schnitzer and Khan,

1972) and 5 to 22 for aquatic fulvic acids (Thurman, 1985).










The E4/E6 ratio has been correlated with a variety of

compositional and structural characteristics of humic

substances. It has been widely reported to be inversely

correlated with the degree of condensation or aromatic

carbon content of humic substances (Schnitzer and Khan,

1972; Schnitzer, 1978; Stevenson, 1982): lower ratios

reflect higher amounts of aromatic carbon. The extensive

study by Chen et al. (1977) of 15 different soil humic

substances however, found no evidence to support this

hypothesis. For the humic substances investigated by Chen

et al. (1977), the strongest correlation (r2 = 0.90, p <

0.01) was found to be with the particle size and molecular

weight of the humic substances. Weaker correlations were

found between the E4/E6 ratio and the percent carbon (r2 =

-0.53, p < 0.05), percent oxygen (r2 = 0.67, p < 0.01) and

total carboxylic acid functional groups (r2 = 0.40, p <

0.05).

The E4/E6 values of the soil and sediment humic

substances measured in this study (Table 3.2) fall within

the range for humic acids (Stevenson, 1982; Thurman, 1985)

with the exception of the Pine Mt. Soil extract. This peat

soil has a relatively high E4/E6 ratio. High E4/E6 ratios

have been reported for other peat humic acids (Ertel and

Hedges, 1983). The E4/E6 ratios for the aquatic humic

substances fall in the range reported for fulvic acids

(Schnitzer, 1978; Thurman, 1985) except for the St. Marys










River DOC sample. The value for this sample falls very

near the lower limit generally attributed to aquatic fulvic

acids (Thurman, 1985). The St. Marys River DOC sample was

obtained at a time when the river was at flood stage. This

may account for its E4/E6 ratio being between humic and

fulvic acid values. The E4/E6 ratios measured for the humic

substances in the current study indicate that the soil and

sediment humic substances are predominately humic acids and

the aquatic humic substances are predominately fulvic acids.

This is consistent with reported compositions of humic

substances from these sources. Extractable humic substances

from soil are predominately humic acids (Stevenson, 1982)

and aquatic humic substances are generally 85 to 90% fulvic

acids in lakes and greater than 80% fulvic acids in streams

and rivers (Thurman, 1985).

The relationship between the E4/E6 and humic substance

structure and composition is unclear. Ertel and Hedges

(1983) reported a linear correlation (r2 = 0.85) between the

E4/E6 ratio and the H/C atomic ratio for seventeen

terrestrial and synthetic humic acids. The E4/E6 ratios for

the humic substances investigated in this dissertation

(Table 3.2) are also correlated with the H/C atomic ratio.

This relationship is further illustrated by combining data

of Ertel and Hedges (1983) and from Nissenbaum and Kaplan

(1972) with the results from this dissertation reserach

(Figure 3.2). The correlation between the E4/E6 ratio and















1.6
+

1.4-
+ +
0- +
1.2- + +

.a m = m _



0.8-


0.6-


0 .4 -11 1
1 2 3 4 5 6 7 8 9 10 11
E4/E6



Terr.& Aquatic + Marine & Sed.


Figure 3.2. The E4/E6 ratio was correlated with
the H/C atomic ratio (r2 = 0.50, p < 0.01) for the
combined data from this study, Nissenbaum and Kaplan
(1972), and Ertel and Hedges (1983). Marine and
sedimentary humic substances have been reported to
have high H/C atomic ratios compared with their E4/E6
ratios (Ertel and Hedges, 1983).










the H/C atomic ratio (Figure 3.2; r2 = 0.49, p < 0.01) was

attributed by Ertel and Hedges (1983) to be the result of

decreasing aromatic carbon content of humic substances as

the H/C atomic ratio increased. However, data presented

earlier for the relationships between the H/C atomic ratio

and absorptivity at 270 nm (Figure 3.1) indicate that the

H/C atomic ratio is a composite value resulting from a

number of functional groups, including aromatic carbon and

carboxylic acids. The weak relationship between H/C atomic

ratio and the aromatic carbon content of humic substances

reported by Traina et al. (1990) further illustrates the

composite nature of the H/C atomic ratio. This leads to the

conclusion that the E4/E6 ratio is likely the result of

multiple functional groups.

The marine sediment humic acids and the Newnans Lake

sediment humic substances appear above the other humic

substances in Figure 3.2. Ertel and Hedges (1983) found

that marine humic acids had low E4/E6 ratios compared to

their H/C atomic ratios. This was attributed to reported

low E4/E6 ratios for marine humic substances due to pigment

absorption near 665 nm (Nissenbaum and Kaplan, 1970).

Marine humic substances have higher H/C atomic ratios than

their terrestrial counterparts (Rice and MacCarthy, 1991)

Also, as noted earlier, lake sedimentary humic substances

are similar to marine humic substances in their H/C atomic

ratios (Ishiwatari, 1985). The higher H/C atomic ratios for








62
lake sedimentary humic substances and lower E4/E, ratios for

marine sedimentary humic substances may explain low E4/E6

ratios for the Newnans Lake Sediment samples compared to

their H/C atomic ratios.

Although the UV/Vis spectra of humic substances do not

yield direct information about their structure, the data

presented here and in the literature indicate that there are

relationships between the composition of humic substances

and their UV/Vis spectra. Both the absorptivity at 272 nm

and the E4/E6 ratios appear to be the result of multiple

functional groups, including aromatic and carboxylic acid

carbon. Further, the E4/E6 ratio for marine and sedimentary

humic substances, when compared with their elemental

composition, suggest that these two types of humic

substances may be similar in their structure as well as

their composition.


Total Acidity and Copper Binding Capacity of Humic
Substances


The ability of humic substances in both the condensed

and dissolved phases to complex metal ions has received a

great deal of attention in recent years. Humic substances

have been reported to complex both divalent and trivalent

metal ions (Stevenson, 1982). The metal ions most strongly

bound by humic substances are reported to be Hg* and Cu"+

(Mantoura et al., 1978; Schnitzer, 1978; Stevenson, 1982;

Thurman, 1985). Humic substances have been shown to control










the bio-availability of metals in soils and aqueous

environments (Stevenson, 1982; McKnight et al., 1983).

Metal-humic substance interactions have also been reported

to decrease the toxicity of heavy metals in solution

(Tuschall and Brezonik, 1983).

The oxygen containing functional groups are believed to

be the functional groups primarily responsible for metal ion

binding by humic substances (Schnitzer and Khan, 1972;

McBride, 1978; Stevenson, 1982) Both carboxylic acids and

phenolic hydroxyl groups have been reported as ligands

involved in these completing reactions (Vinkler et al.,

1976; Lakatos et al., 1977; Piccolo and Stevenson, 1982;

Stevenson, 1985). The acidic character of humic substances

has also been attributed to these functional groups (Perdue,

1985; Thurman, 1985). Therefore, the total acidity and the

copper binding capacity of the humic substances were

investigated as measures of their oxygen containing

functional groups.


Total Acidity


A large number of methods have been used for the

analysis of acidic functional groups in humic substances

(Schnitzer and Khan, 1972; Davis, 1982; Stevenson, 1982;

Perdue, 1985). These include direct titrations,

discontinuous titrations, indirect titrations, direct

titrations coupled with distillation or ultrafiltration,










nonaqueous titration, irreversible reactions of acidic

hydrogens with various reagents and other methods

(Stevenson, 1982). The barium hydroxide (Ba(OH)2) total

acidity titration, which is an indirect titration method

(Stevenson, 1982), was originally developed for brown coal

and was adapted for humic substance research by Schnitzer

and co-workers (Schnitzer and Gupta, 1965; Schnitzer and

Khan, 1972). The Ba(OH)2 titration method is by far the

most often applied method for total acidity measurement of

humic substances (Perdue, 1985) and was chosen as the

method for this dissertation research.

The Ba(OH)2 titration is based on the extreme

insolubility of barium salts. In this method, the humic

substance is allowed to equilibrate with a solution

containing Ba(OH)2. Humic substances containing acidic

functional groups react with the Ba" ions and precipitate.

The samples are filtered to remove the precipitate and the

resulting filtrate is titrated with HC1 to a pH of 8.4. The

total acidity of the humic substance is calculated by

difference based on the initial Ba(OH)2 concentration and

the equilvalents of Ba(OH)2 remaining in the filtrate.


Methods


In my research, 50-100 mg of each humic substance was

allowed to equilibrate for 24 hours with 20 mL of 0.1 M

Ba(OH)2 (Fisher Scientific Co., Cat. no. B46-250) under a










nitrogen atmosphere. During this period, humic substances

containing acidic functional groups reacted with the Ba*

ions and precipitated. A blank sample which consisted of 20

mL of Ba(OH)2 was also allowed to equilibrate under the same

conditions for the same period of time. The samples were

then filtered (Whatman no. 41, ashless filter paper) to

remove the precipitated Ba-humic acid. The filter was then

thoroughly rinsed with CO2 free DI water. The resulting

filtrate and rinse water were combined and titrated with

0.050 M HCI to a pH of 8.4. The difference between the

volume of titrant required by the sample and the blank

titration was used to calculate the amount of Ba(OH)2

remaining in solution.

The total acidity of the humic substance investigated

was calculated using the following equation:



meq H*/g humic sub. = (Vb V,) X M HCl/g humic (3.2)



where meq H* is the total acidity per gram of a specific

humic substance, Vb and V, are the volumes of titrant

required to titrate the blank and sample, respectively. The

total acidity values measured for the humic substances

(Table 3.3) were normalized for the weight of humic

substance and the weight of carbon for each source. Each

humic substance was titrated in duplicate and the difference

between replicate titrations was generally less than 5%















Table 3.3. Total acidity of humic substances.



Humic Source meqH*gH"' meqH'-gC'




Aldrich Humic Acid 7.37 14.50

IHSS Humic Acid 6.23 10.85

Pine Mt. Soil 4.59 7.80

Orange Hts. Soil 8.90 18.66

Newnans Lake Sediment 11 9.91 21.53

Newnans Lake Sediment 16 9.03 22.28

Newnans Lake Sediment 18 8.42 19.88

Santa Fe River DOC 8.40 17.00

St. Marys River DOC 9.24 18.06

Orange Hts. DOC 7.83 15.50

Newnans Lake DOC 7.61 15.15

Suwannee River Fulvic Acid 8.20 15.25

Suwannee River Humic Acid 6.89 12.72










(average, 3.7%; maximum, 9.9%, minimum, 0.1%). The mean

variation for four replicate blank titrations was 1.0%.


Results


There has been some debate about the validity of the

Ba(OH)2 method for determining the total acidity of humic

substances (Davis, 1982; Stevenson, 1982; Perdue, 1985).

The presence of CO2 will cause the measured total acidities

to be high due to the precipitation of BaCO3 during the

equilibration period. However, this can be avoided by

working under a nitrogen environment. In addition, there is

some debate over whether or not the less acidic functional

groups (i.e. phenols) react with the Ba" (Stevenson and

Gupta, 1965; Davis, 1982; Stevenson, 1982). The pKa values

for substituted phenols have been reported to be as high as

13, but most are near 10 (Perdue, 1985; Thurman, 1985). The

Ba(OH)2 solution has a pH greater than 13, which should

assure the reaction of the phenolic ions. Recent reports of

the direct potentiometric titration of aquatic humic

substances have shown that less than 17% of their total

acidity occurs at pKa values greater than 10.3. Therefore,

the values obtained from the Ba(OH)2 method for total

acidity of humic substances should yield reasonable

estimates (Perdue, 1985).

The total acidity values measured in this research

(Table 3.3) are within the ranges reported for humic










substances (Schnitzer and Khan, 1972; Stevenson, 1982;

Perdue, 1985). The value of 8.2 meq H/gH obtained for the

Suwannee River Fulvic Acid sample is very close to the

reported value of 8.3 meq HW/g measured by potentiometric

titration (Bowles et al., 1989). Fulvic acids and aquatic

humic substances generally contain more acidic functional

groups than soil derived humic substances (Schnitzer and

Khan, 1972; Thurman, 1985), although the ranges reported for

different types of humic substances overlap (Stevenson,

1982). The total acidity values of the aquatic humic

substances measured were higher than the values for the soil

derived humic substances, with the exception of the Orange

Heights Soil sample. The sediment derived humic substances

all had high total acidity values. The higher total acidity

values measured for the aquatic humic substances in this

study, compared with the soil humic substances, are

consistent with the conclusion based on the E4/E6 ratios

that the aquatic humic substances are predominately fulvic

acids and the soil humic substances are predominately humic

acids.

The total acidities of all humic substances

investigated varied linearly (Figure 3.3; r2 = 0.83, p <

0.01) with the O/C atomic ratio. The acidity of humic

substances has been attributed to the oxygen containing

functional groups (Schnitzer, 1972; Thurman, 1985), the

majority of which are reported to be carboxylic acid and









69





1 -1

1-

0.9-

0.8-

E 0.7-
0.5-
I 06-8

0 .4 ...."..







0.4
0 .3 ..,,-


6 8 10 12 14 16 18 20 22 24 26
Total Acidity



Figure 3.3. The O/C atomic ratio was linearly
correlated (r = 0.83, p < 0.01) with the total
acidity (meq H+*g Carbon--) measured for the humic
substances in the current study.










phenolic hydroxyl groups (Stevenson, 1982; Perdue, 1985).

Non-acidic oxygen containing functional groups reported to

be present in humic substances include ketone and aldyhr

carbonyls, alcohol hydroxyls, and ether groups (Steelink,

1985). The relationship between the total acidity and O/C

atomic ratio suggests that acidic functional groups are more

predoment than other oxygen containing functional groups in

the humic substances included in this dissertation research.


Copper Binding Capacity


A number of methods have been used to measure the metal

binding capacities of humic substances. These include ion

selective electrode potentiometry, anodic stripping

voltammetry, fluorescence quenching spectrophotometry,

ultrafiltration, liquid chromatography and equilibrium

dialysis (Thurman, 1985). Each of these methods has

advantages and disadvantages which have been discussed

extensively (Sarr and Weber, 1982). The method employed in

this dissertation research was the equilibrium dialysis

titration method of Truitt and Weber (1981a).

Equilibrium dialysis titration was used to determine

the copper binding capacity of humic substances. The method

involves equilibrating an aqueous solution of copper ions

and a particular humic substance with DI water held in a

dialysis bag (Guy and Chakrabarti, 1976; Truitt and Weber,

1981a). The dialysis membrane allows the freely dissolved,








71

hydrated metal ions to pass between the two solutions, while

the metal ions completed to humic substances are retained

outside the membrane. After reaching equilibrium, the

concentration of metal in each solution is measured by

atomic absorption spectrophotometry. The metal ion

concentration inside the dialysis bag represents the freely

dissolved metal concentration and the metal ion

concentration outside the membrane is the total metal ion

concentration (freely dissolved plus metal ion/humic

complexes). The titration involves incremental addition of

metal ions, equilibration and AAS measurement of the metal

ion concentration in the two solutions. The addition of

copper is continued until the completing capacity of the

humic substance is past saturation (Truitt and Weber,

1981a). A linear regression calculation of the free metal

ion concentration versus the total metal ion concentration

yields an intercept that represents the total completing

capacity of the humic substance.


Methods


The dialysis titration experiments were performed

according to the method of Truitt and Weber (1981a). The

Spectra/Por 6 dialysis tubing (molecular weight cut off of

1,000, Fisher Scientific Co., Cat. no. 08-670-12B), required

cleaning before use to remove preservatives. The tubing was

rinsed with DI water and then soaked in 0.1% Na2S (Fisher










Scientific Co., Cat. no. S426-212) at 60 C for 15 minutes.

The tubing was then rinsed in warm DI water, soaked in 3%

H2SO4 (Fisher Scientific Co., ACS Reagent, Cat. no.

A300-212) at 60 C for 5 minutes, rinsed with DI water and

stored in DI water until needed.

The dialysis titration experiments were performed in 1

L Nalgene bottles which had been rinsed with 50% nitric acid

(Fisher Scientific Co., Cat. no. A202-212) to remove any

metal ions. Solutions of humic substances (approximately 20

mg/L) were prepared in 0.001 M NaCl at pH 7. The pH of the

solution was adjusted as needed using 0.1 M HC1 or NaOH.

Initially, 0.50 mL of 1000 mg/L Cu' standard solution

(Fisher Scienctific Co., Cat. no. SC194-100) was added to

each humic substance solution to yield a solution

concentration of 0.5 ppm Cu. The humic substance/copper

ion solution was placed in the 1 L bottle and a dialysis bag

containing 40 mL of 0.001 M NaCl at pH 7 was added and the

bottle sealed. The bottle was placed on a shaker table

overnight and then allowed to equilibrate for an additional

day. Solutions were equilibrated for 48 hours between metal

additions. This was probably more than sufficient time to

achieve equilibration between the two solutions since Guy

and Chakrabarti (1976) reported no change between the two

solutions for a number of humic substances after about 20

hours. The equilibrated solutions were subsampled by

removing 1 to 2 mL of the solutions from both inside and










outside the membrane. The copper concentration of each

subsample solution was then determined by AAS. After

samples were obtained, another aliquot of Cu standard

solution was added to each humic substance solution and the

equilibration procedure repeated. As mentioned earlier, the

titration of Cu++ was complete when the humic substances

were all past saturation (slope of titration curve is one)

(Truitt and Weber, 1981a).

Each batch of dialysis titration experiments included a

blank dialysis sample which consisted of a 0.001 M NaCl/Cu

solution outside the dialysis bag and 0.001 M NaCl inside

the bag. The blank samples were run for the duration of the

humic titration experiments to confirm that freely dissolved

Cu ions passed through the dialysis membrane and did not

significantly adsorb to the membrane. In all cases, the

Cu* concentrations inside the dialysis tubing were found to

be the same as solution concentrations outside the tubing.

Also, no adsorption of metal to the dialysis membrane was

observed since the concentrations of Cu measured in these

blank samples was the same as predicted based on the amount

of Cu" added to the solutions.

Another potential problem with the dialysis titration

technique is the leakage of humic substances across the

dialysis membrane. Any leakage of humic substances across

the membrane would lead to an underestimation of the Cu

binding capacity. Truitt and Weber (1981a) noted that at pH










5 approximately 30% of humic substances investigated were

able to pass through the dialysis membrane, while at pH 7

less than 5% passed through the membrane. These values were

based on the percentage of the retentate color (UV

absorbance at 260 nm) that diffused across the membrane.

The lack of color of the retentate solutions after

equilibrium in this research indicates that there was no

leakage of humic substances across the dialysis membrane.

This was in agreement with the reported low level of humic

substances breakthrough at pH 7 (Truitt and Weber, 1981a).

The AAS analysis was performed using a Perkin Elmer

Model 5000 AAS with an air/acetylene flame. The hollow

cathode lamp (Perkin Elmer, Inc., Cat. no. 303-6024) was

energized at 15 mA and the 325 nm copper emission was

monitored in the absorbance mode using a 0.7 nm slit width.

Each time samples were analyzed, a standard curve for Cu was

constructed using the following concentrations of standards

(in ppm Cu): 0.5, 2, 4, 6, 10. Typical calibration curves

were linear (r2 k 0.99) and indicated very little or no bias

(i.e. intercepts at or near 0 ppm Cu and 0 absorbance

units).

The Cu" binding capacity was obtained by plotting the

freely dissolved Cu" concentration vs. the total Cu"

concentration (Guy and Chakrabarti, 1976; Truitt and Weber,

1981a). A typical dialysis titration plot is presented in

Figure 3.4 for Newnans Lake DOC. It should be noted that















4

3.5
?3-5



2 .52.






S0.5- .



0 '
0 0.5 1 1.5 2 2.5 3 3.5

Total Copper (mg*L^-1)


Figure 3.4. Copper dialysis titration for
Newnans Lake DOC sample (20 mg humic*L-1) at pH 7 and
0.001 M NaC1.










the first data point of the titration curve was below the

Cu*" saturation point and that the linear portion of the

curve above the Cu* saturation point was extrapolated to

the abscissa to obtain the Cu binding capacity (Guy and

Chakrabarti, 1976).


Results


The Cu" binding capacities of the humic substances

measured in this study (Table 3.4) are in the range for Cu"

binding data previously reported for humic substances.

McKnight et al.(1983) reported the Cu" binding capacities

for 18 humic substances isolated from surface waters using

XAD-8 resin. They found copper binding capacities ranging

from 0.54 to 2.55 pM Cu/mg C. Copper binding capacities

found during this study cover a similar range and fall

within the range of Cu binding capacities reported by

McKnight, et al (1983).

The Cu binding capacity of the International Humic

Substances Society's Suwannee River Fulvic Acid Reference

(SRFAR) material has been reported to be 1.2 to 1.4 pM

Cu/mgC in 0.001 M KNO3 (McKnight et al., 1983; McKnight and

Wershaw, 1989). Ventry et al. (1991) reported the Cut"

binding capacity for this humic material to be between 4.6

and 6.3 pM Cu/mgC when measured in 0.1 M NaClO4. The value

measured for SRFAR measured in my research (0.70 pM

Cu"/mgC) is lower than either of these values. However,
















Table 3.4. Copper binding capacity of humic substances.



HUMIC SOURCE PM Cu,'*mg C' r2



Aldrich Humic Acid 2.25 0.971

IHSS Humic Acid 0.7 0.997

Pine Mt. Soil 2.11 0.999

Orange Hts. Soil 1.24 0.999

Newnans Lake Sediment 11 0.91 0.997

Newnans Lake Sediment 16 1.04 0.999

Newnans Lake Sediment 18 1.2 0.997

Santa Fe River DOC 1.74 0.999

St. Marys River DOC 2.06 0.998

Orange Hts. DOC 1.64 0.999

Newnans Lake DOC 0.8 0.998

Suwannee River Fulvic Acid 0.7 0.999

Suwannee River Humic Acid 0.71 0.999










the value reported in Table 3.4 is closer to that reported

by McKnight et al. (1983) who indicated that the value was

probably good to within a factor of two. The values

reported by Ventry et al. (1991) are high and are

significantly beyond the ranges reported by either McKnight

et al. (1983) or this study. Also, the conditional

stability constants for Cu complexation by SRFAR reported

by Ventry et al. (1991) were an order of magnitude lower

than those previously reported for this humic substance

(McKnight et al., 1983; McKnight and Wershaw, 1989).

The reason for this discrepancy between reported copper

binding capacities may be related to the ionic strength of

the solutions used to make the measurements. The strength

of the interactions between metal ions and humic substances

(as measured by the conditional stability constant)

decreases as the ionic strength is increased (Schnitzer and

Khan, 1972). The measured Cu" binding capacity of a humic

substance has been reported to be dependent on the ionic

strength of the solution (McKnight, 1983). The Cu binding

capacity of SRFAR was observed to decrease from 1.4 pM

Cu/mgC at 0.00001 M Ca(N03)2 to 0.73 piM Cu/mgC at 0.01 M

Ca(N03)2 (McKnight and Wershaw, 1989). Also, the Cu**

binding capacities of six natural waters containing DOC

showed strong negative correlations with alkalinity (r2 =

-0.91) and conductance (r2 = -0.89) (Truitt and Weber,

1981b). Thus, the Cu" binding capacity of SRFAR would be








79
expected to be lower in 0.1 M NaCl04 than in either 0.001 M

NaCl or 0.001 M KN03. The ionic strength dependence of Cu*

binding capacity may also explain the difference between the

SRFAR values measured by McKnight et al. (1983; 1989) and

the value I obtained during this research. The pH of the

solutions used in the dialysis titrations were adjusted with

0.1 M HC1 and 0.1 M NaOH. The ionic strength of the

solution could have been greater than 0.001 M and been

closer to 0.01 M. The Cu binding capacity for SRFAR in

0.01 M Ca(N03)2 has been reported to be 0.73 pM Cu++/mgC

(McKnight et al., 1989) which is close to the value of 0.70

pM Cu+/mgC measured in the this study.

Copper is one of the most strongly bound metals when

chelated in organic complexes with oxygen containing ligands

(Schnitzer, 1978; Stevenson, 1982; Thurman, 1985). Humic

substances have been reported to bind Cu++ more strongly

than any other divalent cation with the exception of Pb"+

(Guy and Chakrabarti, 1976). Mantoura et al. (1978) found

that 99.9% of the copper present in lake and river waters

was completed to dissolved humic substances. Copper has

been reported to displace more weakly bound cations when it

complexes with humic substances (Truitt and Weber, 1981b;

Stevenson, 1982; Albert et al., 1989). The stronger

retention of Cu" compared with other metals (Mn, Mg, and

Cr+) has been investigated using electron spin resonance

spectrometry (Lakatos et al., 1977; McBride, 1978). The










results indicate that oxygen containing functional groups

are able to form inner sphere complexes with Cut, but not

with the other metal ions. This means that oxygens in humic

substances displace the water of hydration when coordination

complexes are formed with Cu**.

The metal completing behavior of humic substances has

generally been attributed to oxygen containing functional

groups. Carboxylic acids and phenolic hydroxyl groups are

the most frequently mentioned functional groups responsible

for these interactions (Schnitzer and Khan, 1972; Stevenson

1982; Thurman, 1985; Gamble et al., 1970; McKnight et al.,

1983; McBride, 1978; Lakatos et al., 1977; Vinkler et al.,

1976; Piccolo and Stevenson, 1982). Nitrogen containing

functional groups are also capable of completing metal ions

and no doubt are responsible for a portion of the measured

metal binding capacities of humic substances (Stevenson,

1982; Thurman, 1985). However, since the number of nitrogen

containing functional groups in humic substances is small

compared to the number of oxygen containing functional

groups, the latter would be expected to play the dominant

role in interactions with metal ions. The exception would

be marine humic substances which often contain significant

amounts of nitrogen (Harvey and Boran, 1985; Rice and

McCarthy, 1991).

The mechanism of binding between humic substances and

metal ions is not clear. Two main types of chelation










reactions between metals and humic substances have been

proposed. These include the "salicylate-like" site which

contains a carboxylic acid adjacent to a phenolic hydroxyl

group and the "phthalate-like" site which contain ortho-

carboxylic acid functions (Piccolo and Stevenson, 1982;

Gamble, et al. 1970). Inter-molecular bidentate complexes

between different humic molecules and a single metal ion

also are likely to occur (Schnitzer, 1978). Vinkler et al.

(1976) reported that metal ions interact predominately with

carboxylate functional groups based on the IR spectra of

humic substances and their metal complexes. However,

another investigation indicated that humic substance metal

binding reactions involved both carboxylate and hydroxyl

functional groups (Gamble et al., 1970). This conclusion

was based on the fact that the greatest reduction in Cu**

binding capacity for a series of humic substances was

observed when both carboxylate and phenolic hydroxyl groups

were chemically blocked. Blocking either one of these

functional groups alone reduced the Cu" binding capacities

of the humic substances investigated, but to a lesser extent

than when both groups were blocked. A similar study using

functional group blocking concluded that the major reaction

of metals with humic substances involves a phenolic hydroxyl

and a carboxyl group (Schnitzer and Kahn, 1972). Another

minor reaction involves two carboxylic acid functional










groups. Aliphatic hydroxyl groups were not found to be

important in metal binding interactions.

The exact nature of metal/humic substances interactions

is difficult to discern due to the complex nature of humic

substances themselves. There is little disagreement that

oxygen containing functional groups are important in these

interactions (Stevenson, 1982; Thurman, 1985). This

conclusion is supported by the data obtained in this

research. The Cu binding data from Table 3.4 is plotted

in Figure 3.5 against the H/O atomic ratio. There is a

strong negative correlation (r2 = 0.88, p < 0.01) between

the Cu*" binding capacity and the H/O atomic ratio for the

humic substances investigated in this study, with the

exception of the Pine Mt. Soil humic substance. As noted in

the earlier discussion of elemental composition in this

Chapter, the Pine Mt. Soil sample appears to be an outlier

with respect to its H/O atomic ratio. It is interesting to

note that although the Cu" binding capacities correlated

well with the H/O ratio for the humic substances

investigated, they did not correlate with the total acidity

(r = 0.16, p > 0.5). Stevenson and Goh (1974) have

reported free, uncomplexed carboxylic acid functional groups

in the infrared spectra of humic substances which have been

saturated with Cu". The data presented for the humic

substances investigated in this dissertation research

indicate that there is no simple relationship between the








83





2.4-

2.2-

* 2-

S 1.8 ... ...

1.4- .....

S 1.4- "".....***... ... .


Copper Binding


I Pine Mt. Soil


Figure 3.5. Copper binding capacity (gMole
Cu++*mg Carbon-1) was linearly correlated (r = 0.88,
p < 0.01) with the H/O atomic ratio for the humic
substances in the current study. Pine Mt. Soil was
excluded from the correlation calculation.










acidic functional group content and the Cu** binding

capacity. The lack of correlation between these two

seemingly related parameters may be due to the fact that

some acidic functional groups are inaccessible to Cu.


Infrared Spectroscopy of Humic Substances


Infrared (IR) spectroscopy has been widely used for the

investigation of humic substances (MacCarthy and Rice, 1985;

Lobartini and Tan, 1988) and has provided a great deal of

information on the nature, reactivity and structural

arrangement of oxygen containing functional groups in humic

substances (Stevenson, 1982). Infrared spectra of humic

substances are the result of the absorption of IR radiation

by a complex mixture of molecules which are themselves

multi-functional. The resulting IR spectra contain a

variety of bands which are indicative of the different

functional groups present in these complex mixtures

(Stevenson, 1982). IR spectra have been used to investigate

the interactions of humic substances with metals (Vinkler

et al., 1976; Piccolo and Stevenson, 1982; MacCarthy and

Rice, 1985) and pesticides (Kahn, 1974; Kahn, 1978).

IR spectroscopy, like UV/Vis spectroscopy, is a

quantitative technique that follows the Beer-Lambert Law

(Schnitzer and Khan, 1972; Stevenson, 1982). The sample

size required to obtain an IR spectrum is 1 to 10 mg

(MacCarthy and Rice, 1985). This small sample size is an










advantage when IR spectroscopy is compared with 1C NMR

spectroscopy, which also provides information on functional

groups, but requires sample sizes a 100 mg (Wershaw, 1985).

Fourier transform IR spectroscopy (FTIR) has a number of

advantages compared to more traditional dispersive IR

techniques. These advantages include better resolution,

higher signal to noise ratios (due to spectra averaging) and

higher energy throughput (MacCarthy and Rice, 1985). The

increased resolution is not a great advantage in the current

study due to the broad bands produced by humic substances

(Schnitzer and Khan, 1972). However, the increased energy

throughput and higher signal to noise ratio lead to

increased sensitivity. Although FTIR instruments have been

available for sometime, relatively little work with these

instruments has been reported for humic substances.

The ease of obtaining an IR spectrum, coupled with the

large amount of published information on the IR spectra of

humic substances, make this an attractive technique for the

investigation of the functional group composition of humic

substances. The fact that IR analysis is potentially

quantitative is also attractive, although, the quantitative

application of IR spectroscopy to humic substances is rarely

reported (Schnitzer and Khan, 1972; Stevenson, 1982).

The desired result of the application of IR

spectroscopy in my study was to obtain quantitative

measurements of specific functional group compositions among








86
the different humic substances investigated. Therefore, an

internal standard FTIR method was developed for the

investigation of humic substances. The technique involves

use of an internal standard to compensate for the variation

in sample presentation and instrumental variation between

different samples. The internal standard approach allowed

the direct comparison of the intensities of specific IR

peaks among different spectra and therefore between

different humic substances. The IR data obtained using this

method allowed the absorption bands assigned to specific

functional groups to be quantitatively compared.


Methods


Sample presentation is an important consideration in IR

spectroscopy. The most common method of sample presentation

for obtaining IR spectra of humic substances is the KBr

pellet (Stevenson, 1982, MacCarthy and Rice, 1985). In this

technique, pellets are made by mixing 1 to 2 mg of dried

humic substance and 100 to 200 mg of Kbr (Stevenson, 1982;

Schnitzer, 1978). The pellet is pressed into a disk. The

disk is then illuminated with IR radiation and the spectrum

recorded. These types of samples are used for qualitative

comparisons among the functional groups of different humic

substances. A disadvantage of this technique is that, under

certain conditions, KBr has been reported to catalyze the

formation of cyclic anhydrides from carboxylic acids, thus








87
altering the humic substances under investigation (Stevenson

and Goh, 1974; Wagner and Stevenson, 1965).

Other techniques for sample presentation in IR

spectroscopy include the preparation of mulls of samples

with a hydrocarbon oil (Bellamy, 1975; MacCarthy and Rice,

1985) and cast films placed on IR transparent plates

(MacCarthy and Rice, 1985). The mull technique has the

disadvantage that the hydrocarbon oil used to prepare the

mull absorbs strongly in the aliphatic carbon region of the

spectrum, a spectral region of interest in this research.

The cast film technique involves applying a solution of the

humic sample to an IR transparent plate followed by

evaporation of the solvent to dryness (MacCarthy and Rice,

1985). The IR spectrum is recorded from the dried deposit.

Cast film deposits are less susceptible to adsorption of

moisture than the more hydroscopic KBr pellets. The

disadvantage of this method is that a satisfactory film is

not formed for all materials. The cast film sample

technique was chosen for this research because of the

inherent homogeneity of liquid solutions. Homogeneity of

the sample/internal standard solution is a basic requirement

for the success of any internal standard analysis.

The optimum compound for an internal standard for IR

analysis of humic substances would be one that has a single,

strong absorption band in a spectral region where humic

substances do not absorb. The compound must also be soluble










in the solvent used to make the film deposits and be

unreactive with the humic substances. The spectra of humic

substances are void of absorption bands between 2400 cm-'

and 1800 cm-1 (Stevenson, 1982). Potassium thiocyanate

(KSCN) was chosen as an internal standard because it

contained a strong absorption band at 2050 cm-1 (Sadtler

Infrared Index, 1973) and was freely soluble in water and

50% dimethylsulfoxide (DMSO), the two solvent systems used

to make film deposits.

Two solvent systems were used because cast films of all

humic substances could not be obtained from a single

solvent. It was found that the humic substances

investigated all formed satisfactory films from aqueous

solution at pH 11. However, at this elevated pH, all

ionizable protons are dissociated, causing the carboxylic

acid absorption band at approximately 1700 cm-1 to shift to

lower frequencies indicative of the carboxylate anion (near

1600 cm-1 and 1400 cm-1) (Piccolo and Stevenson, 1982). The

1600 cm-1 region of the spectrum is also where aromatic

carbon absorption occurs (Eltantawy and Baverez, 1978)

Therefore, IR spectra were recorded for all humic substances

investigated on films deposited from aqueous solution at pH

11 and from 50% aqueous DMSO at pH 4. The latter solvent

system was employed to obtain spectra of humic substances

with carboxylic acids in the un-ionized form. Aqueous DMSO

has been reported to extract humic substances from soils










which are identical in composition to the humic substances

extracted with aqueous NaOH (Hayes, 1985).

Solutions for FTIR analysis were prepared by dissolving

humic substances (approximately 10mg/mL) into a solution

containing a fixed amount of KSCN (1.97 mg/mL) (Fisher

Scientific Co., Cat. no. P317-100) in either DI water or 50%

DI water/ 50 % DMSO (Fisher Scientific Co., Spectranalyzed

Cat. no. D136-1). The pH of the solutions were adjusted

with 0.001 M NaOH or HC1. Cast films were prepared by

depositing 50 pL of the humic substance/internal standard

solution onto a 3 mm thick ZnSe plate (Harrick Scientific

Corp., Cat. no. WMD-U37) and placing the plate in a drying

oven at approximately 70 C for 15 to 20 minutes. The dried

deposit was then placed in a sample holder with an

adjustable aperture set to a diameter of 5.5 mm. The

deposit was then placed into the sample chamber of the FTIR

spectrometer and the chamber evacuated to a pressure of

approximately 1-3 millitorr before the IR spectrum was

recorded. The spectra were recorded using a Bomem DA3.10

FTIR spectrometer scanning over the frequency range from

4000 cm-1 to 400 cm-1 at a resolution of 2 cm-1. The

spectrometer was equipped with a Globar source, a Ge/KBr

beam spliter and a liquid nitrogen cooled Hg/Cd/Te detector.

To insure a high signal to noise ratio, the spectrum for

each sample was calculated from the average of 100

repetitive scans.








90
Spectra were obtained from at least duplicate deposits

for each humic substance at pH 4 and 11. The peak heights

for each peak of interest were measured and normalized to

the peak height of the internal standard peak at 2050 cm-'.

The data are reported as peak height per gram of humic

substance normalized to the peak height at 2050 cm-1 per

gram of KSCN (units for peak height ratio: g KSCN/g humic

substance).

A number of conditions are required for the internal

standard method to be successful. First, the internal

standard and the humic substances must be uniformly mixed in

the solution. Second, the internal standard/humic substance

solution must form a uniform film when it is dried on the

ZnSe plate. Finally, the internal standard must behave in a

similar manner as the humic substances. In order to

investigate whether the KSCN was suitable for use as an

internal standard, experiments were performed which used

potassium acid phthalate (KHP) as a model for humic

substances. Solutions with varying KHP concentration (Table

3.5) and constant KSCN concentration were prepared and

analyzed using the cast film method described above.


Results


The IR spectra obtained for the KHP were those expected

from the literature (Sadtler Infrared Index, 1976). The

peak heights and areas for the two major KHP peaks (1560 and













Table 3.5. Potassium hydrogen phthalate internal standard calibration.


PEAK HEIGHT PEAK AREA 2

KHP KHP KSCN KHP/KSCN KHP KSCN KHP/KSCN

CONC. 1560 1410 2050 1560 1410 1410 2050 1410
(mg*mL') (cm') (cm"') (cm*) (cm") (cm")




3.86 155.8 156.4 117.9 1.166 1.228 31.20 3.90 8.000

3.86 211.6 237.2 207.3 1.021 1.144 29.50 3.76 7.846

3.86 212.4 236.3 208.5 1.019 1.133 22.10 2.94 7.517

1.93 239.0 206.0 393.0 0.608 0.524 27.05 8.84 3.060

1.93 177.0 189.0 334.0 0.530 0.566 23.92 8.09 2.957

1.93 205.0 191.0 413.0 0.496 0.462 15.27 5.24 2.914

0.97 88.1 56.1 260.0 0.339 0.216 6.90 5.59 1.234

0.97 76.5 75.3 298.0 0.257 0.253 8.98 7.30 1.230

0.97 48.6 71.6 201.0 0.242 0.356 7.49 3.55 2.110

0.48 52.9 54.0 277.0 0.191 0.195 7.11 7.11 1.000

0.48 44.3 56.5 299.0 0.148 0.189 6.71 6.32 1.062

0.48 45.3 57.5 280.6 0.161 0.205 5.58 5.44 1.026


' Absorbance (x 103)
2 Arbitrary units
3 KSCN concentration was constant at 1.97 mg.mL1










1410 cm-1, COO-) were measured and normalized to the peak

height and area for the KSCN peak at 2050 cm-1 (Table 3.5).

A strong linear correlation was found between the normalized

peak area ratios and peak height ratios for the KHP peak at

1410 cm-1 (Figure 3.6, r2 = 0.98, p < 0.001). A similar

relationship was obtained for the KHP peak at 1560 cm-" (r2 =

0.96, p < 0.001). These results demonstrate that either the

peak area or height ratio can be measured to yield the same

result. The relative complexity of the IR spectra of humic

substances, compared to that of KHP, led to the decision to

measure peak height rather than peak area in their IR

spectra. The normalized peak heights for the KHP peak at

1560 cm-1 were found to vary linearly with the KHP

concentration (Figure 3.7, r2 = 0.97, p < 0.001). It should

be noted that these data (Table 3.5) demonstrate that the

peak height for a given concentration of KHP or KSCN can

vary by greater than 200%, but the variation in the KHP/KSCN

ratio is less than 30% in all cases and generally below 15%.

These data support the conclusion that KSCN is a suitable

internal standard for the quantitative application of the IR

cast film method used in the current research.

The final questions remaining for the application of

the KSCN internal standard cast film method for the semi-

quantitative IR investigation of humic substances are:

whether or not the KSCN act in the same manner when mixed

with humic substances as it did with KHP and whether









93





10



8..
0 0.2 70.4 0.6 0.8 1.2 1.4 1.6



















area ratios for the KHP peak at 1410 cmc and the
< 6
5d


Z 4-












8 to 3.6 mg while thei KSN concentration
area ratios for the KHP peak at 1410 cm-1 and the
KSCN peak at 2050 cm-1 were linearly related (r2 =
0.98, p <0.01). The KHP concentration varied from
0.48 to 3.86 mg'mL-1 while the KSCN concentration
remained constant at 1.97 mg'mL-~.




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