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Comprehensive Isolation of Natural Organic Matter from Water for Spectral


Characterizations and Reactivity Testing

Article  in  ACS Symposium Series · August 2000


DOI: 10.1021/bk-2000-0761.ch005

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ISOLATION, FRACTIONATION AND CHARACTERIZATION OF

NATURAL ORGANIC MATTER IN DRINKING WATER


ii
ISOLATION, FRACTIONATION AND CHARACTERIZATION OF NATURAL
ORGANIC MATTER IN DRINKING WATER

Prepared by:

Jean-Phillipe Croue(1), Gregory V. Korshin (2), Jerry Leenheer(3)


and Mark Benjamin(2)

(1)
Laboratoire de Chimie de l’Eau et de l’Environnement, UPRESA CNRS 6008

Ecole Supérieure d’Ingénieurs de Poitiers, Université de Poitiers

86022 Poitiers Cedex, France


(2)
Department of Civil Engineering, Box 352700, University of Washington
Seattle, WA 98195-2700
(3)
U.S. Geological Survey
Denver, Colo.

Jointly Sponsored by:


AWWA Research Foundation
6666 Quincy Avenue
Denver, CO 80235-3098

and

HDR Engineering, Inc.,(4) SAUR,(5) and SAUR Services(6)


Seattle Wash.(4); Maurepas, France(5); West Sussex, England(6)

Published by:
AWWA Research Foundation and
American Water Works Association
DISCLAIMER

This study is funded by the AWWA research Foundation (AWWARF). AWWARF


assumes no responsibility for the content of the research study reported in this
publication or for the opinions or statements of fact expressed in the report. The mention
of trade names for commercial products does not represent or imply the approval or
endorsement by AWWARF. This report is presented solely for informational purposes.

iv
Copyright ©1998

by

AWWA Research Foundation

and

American Water Works Association

Printed in the U.S.A.

ISBN0-00000-000-0

v
CONTENTS

TABLES .......................................................................................................................... xiii

FIGURES....................................................................................................................... xviii

Foreward ........................................................................................................................ xxvi

acknowledgments.......................................................................................................... xxvii

EXECUTIVE SUMMARY ......................................................................................... xxviii

ABBREVIATIONS ..................................................................................................... xxxix

CHAPTER 1 Introduction..................................................................................................1

NOM Fractions and Water Quality Issues ...............................................................5

CHAPTER 2 Literature Review ........................................................................................4

Overview..................................................................................................................4

Preliminary characterization ....................................................................................5

Techniques for Concentration, Isolation and Fractionation of NOM......................7

Techniques Intended Primarily for Concentration of NOM ........................7

Evaporation ......................................................................................7

Freeze concentration and freeze-drying...........................................8

Membrane technologies ...................................................................8

Sorption Techniques for Concentrating, Isolating, and Fractionating NOM11

Quantitative Analysis of the Use of Adsorptive Columns for NOM


Isolation and Fractionation ................................................12

vi
Sorption onto Al and Fe oxides and ion exchange resins ..............16

Sorption onto hydrophobic sorbents (XAD and similar resins) ....18

Desalting ....................................................................................................21

Ion exchange ..................................................................................22

Precipitation and Co-precipitation .................................................22

Desalting with XAD resins ............................................................23

Losses of NOM During Processing ...........................................................25

NOM Characterization...........................................................................................26

13C and H-NMR spectroscopy..................................................................27

FTIR spectroscopy .....................................................................................30

Pyrolysis-GC-MS.......................................................................................32

Elemental Analysis ....................................................................................35

UV / Visible and Fluorescence Spectrometry............................................37

Nature of Light Absorption by NOMError! Bookmark not defined.

The Three-Band Theory of UV Spectra of NOMError! Bookmark not defined.

Nature of Light Emission by NOMError! Bookmark not defined.

Compound Class Identification .................................................................41

CHAPTER 3 Materials and Methods...............................................................................46

Sample Collection..................................................................................................46

NOM isolation protocols .......................................................................................49


vii
Membrane-Based NOM Isolation Protocols..............................................50

European Water Samples...............................................................50

Pacific Northwest Water Samples .................................................53

NOM Isolation Using Adsorption and Elution Processes .........................55

European Water Samples...............................................................55

Pacific Northwest Water Samples .................................................56

NOM desalting...........................................................................................59

Experimental Procedures for Characterizing NOM reactivity ..............................60

Chlorination Studies ..................................................................................61

Coagulation - Flocculation Study ..............................................................61

Analytical Methods................................................................................................62

Inorganic Species .......................................................................................62

Inorganics in Source Waters ..........................................................62

Elemental Analysis of NOM Isolates ............................................64

Total Organic Halides (TOX) ........................................................65

Trihalomethanes.............................................................................66

Haloacetic Acids ............................................................................66

Total Dissolved Amino Acids....................................................................67

Total Dissolved Carbohydrates..................................................................69

Pyrolysis-GC-MS.......................................................................................70

viii
NMR and FTIR Spectra .............................................................................71

UV Absorbance and Fluorescence Emission Spectra................................72

CHAPTER 4 NOM Concentration, Isolation and Fractionation .....................................74

Overview................................................................................................................74

Case Studies with the Goal of Maximal Recovery and Fractionation...................76

Suwannee River .........................................................................................76

South Platte River ......................................................................................82

Comparison of NOM Recovery from the Suwannee and S. Platte Rivers90

Case Studies Focusing on Maximal NOM Recovery without Fractionation.........92

Membrane Processes for NOM Recovery from European Waters............92

Salt Concentration and Recovery ..............................................................94

Use of XAD-8 /XAD-4 for NOM Recovery from European Waters ........98

Combination of RO with XAD Resin for NOM Recovery from European


Waters ..........................................................................................100

NOM Concentration and Isolation of Judy Reservoir and Tolt River Water
By Reverse Osmosis ....................................................................103

Comparison of NOM Concentration and Isolation Using IOCS, IOCO, and


XAD Resins for Judy Reservoir and Tolt River Water ...............105

Competition Between Sulfate and NOM for Oxide Adsorbents .............112

Recovery of Various NOM Fractions with IOCO ...................................113

ix
Summary and Comparison of NOM Concentration-Isolation-Fractionation
Techniques ...................................................................................114

CHAPTER 5 NOM Characterization: Suwannee and South Platte Rivers ...................122

Introduction..........................................................................................................122

Bulk Fractionation and Elemental analysis .........................................................123

FTIR Characterization .........................................................................................129

13
C-NMR Characterization ..................................................................................134

1
H-NMR Characterization ...................................................................................138

Dissolved Amino Acids and Carbohydrates ........................................................138

Pyrolysis - gas chromatography - mass spectrometry .........................................145

UV Spectra...........................................................................................................150

Relationship between various structural and spectral properties of NOM fractions160

Comparison of NOM Characteristics between the Suwannee and South Platte


Rivers .......................................................................................................169

Reactivity of NOM ..............................................................................................171

Coagulation-Flocculation.........................................................................171

Removal of DOC and A254 .........................................................171

Chlorination of NOM: Reactions of Different Fractions and


Changes in NOM Characteristics ....................................176

DBP formation potentials ....................................................................176

Relationships between DBPFP and Spectral Properties......................181

x
Summary and Comparison of Suwannee River and South Platte River NOM
Characterizations .....................................................................................186

CHAPTER 6 Characteristics of NOM Captured by Different Techniques ...................191

General Characterization of NOM in the Blavet River .......................................191

Elemental Analysis and SUVA of the NOM isolates from the Blavet River ......195

Total dissolved Amino acids and Carbohydrates ................................................198

13
C-NMR and FTIR Data for the Blavet River NOM Isolates.............................201

Pyrolysis - Gas Chromatography - Mass Spectrometry for the Blavet River NOM
Isolates .....................................................................................................204

Chlorination of the Blavet River NOM Isolates ..................................................207

UV and fluorescence spectra of the Blavet NOM concentrates ..........................210

Correlations between spectral and structural characteristics of NOM in Blavet


River fractions..........................................................................................213

Conclusions..........................................................................................................222

CHAPTER 7 Correlations between Data From Structure-Sensitive Methods and UV and


Fluorescence Spectroscopy..............................................................................................225

Introduction..........................................................................................................225

The Major Parameters of UV and Fluorescence Spectra of NOM ......................227

Correlations between 13C-NMR spectroscopy and the major parameters of UV


and fluorescence emission spectra ...........................................................227

Correlations between Pyr-GC-MS spectroscopy and the major parameters of UV


and fluorescence emission spectra...........................................................232

xi
Conclusions..........................................................................................................234

CHAPTER 8 Summary, Conclusions and Recommendations.......................................236

Comparison of NOM concentration methods ......................................................236

Comparison of Chemical Composition of NOM Isolates....................................240

Desalting of NOM Isolates ..................................................................................242

Variability of NOM .............................................................................................244

Seasonal Changes of NOM..................................................................................248

Effects of NOM Isolation and Concentration on DBP Precursors ......................248

Comparison of Ex Situ and In Situ Methods for NOM Characterization............249

13
C CPMAS NMR....................................................................................249

Fourier Transform IR Spectroscopy (FTIR) ............................................250

Pyrolysis GC-MS .....................................................................................251

Total Dissolved Amino Acids and Carbohydrates ..................................252

Elemental Analysis ..................................................................................253

UV Spectroscopy .....................................................................................253

Fluorescence ............................................................................................255

Relationship Between Data From in Situ and ex Situ Analyses..............256

Appendix..........................................................................................................................257

references .........................................................................................................................258

xii
TABLES

Table 1.1 Major chemical classes of compounds included into NOM and associated water
quality problems ..........................................................................................................7

Table 1.2 Association between specific types of disinfection by-products and major
chemical classes of NOM ............................................................................................1

Table 2.1 Some literature values for DOC rejection and recovery efficiencies by RO.......9

Table 2.2 Structural assignments for 13C-NMR spectra ....................................................28

Table 2.3 Structural assignments for 1H-NMR spectra .....................................................29

Table 2.4 Infrared frequency bands for biomolecular structures in NOM isolates ...........31

Table 2.5 Characteristic infrared spectral peaks of inorganic solutes (in KBr pellets) .....32

Table 2.6 Origin of biopolymers and their respective specific pyrolysis fragments .........33

Table 2.7 Elemental analysis of hydrophobic acids and transphilic acids isolated from
surface waters ............................................................................................................36

Table 2.8 Average elemental analysis of humic substances (with or without fractionation
to humic and fulvic acids) and transphilic acids isolated from surface waters..........37

Table 2.9 Concentrations of amino acids and total dissolved sugars in various surface
waters .........................................................................................................................43

Table 2.10 Amino acid and sugar concentrations in various NOM fractions....................44

Table 3.1 Water quality characteristics of untreated water samples..................................49

Table 3.2 Characteristics of the membranes used to process European samples ..............51

xiii
Table 3.3 Characteristics of the composite iron oxide-based media used for NOM
adsorption...................................................................................................................57

Table 3.4 Operation parameters for concentration of NOM by adsorption for Pacific
Northwest waters .......................................................................................................58

Table 3.5 Acceptable concentration ranges for elemental analysis ...................................64

Table 3.6 TDAA content of the IHSS standard Suwannee River fulvic acid....................68

Table 3.7 TDCA content of the IHSS standard Suwannee River fulvic acids ..................70

Table 4.1 Fraction name conventions and their correlation with the previously used
terminology ................................................................................................................75

Table 4.2 Yields and recovery of dissolved natural organic matter (NOM) fractions from
the Suwannee River ...................................................................................................81

Table 4.4 Yields and recovery of NOM fractions from the second South Platte River
sample ........................................................................................................................89

Table 4.5 Volume and DOC content of the solutions collected from reverse osmosis and
nanofiltration processing of five surface waters ........................................................92

Table 4.6 Isolation of NOM: Efficiency of RO and NF membranes.................................93

Table 4.7 Concentration of anions in permeate and concentrate produced by reverse


osmosis and nanofiltration .........................................................................................95

Table 4.8 Anion recoveries for reverse osmosis and nanofiltration ..................................96

Table 4.9 Recovery of anions in the concentrated water produced by RO treatment of a


synthetic solution .......................................................................................................98

Table 4.10 DOC content of the XAD-8 and XAD-4 permeates of the three water sources99

Table 4.11 XAD-8/XAD-4 DOC distribution and DOC recoveries..................................99


xiv
Table 4.12 Purification of RO-concentrated water: RO and XAD resin desalting in series102

Table 4.13 Results for the fractionation of IOCO effluent for treatment of Judy Reservoir
water.........................................................................................................................114

Table 5.1 Elemental analysis of the Suwannee River NOM isolates ..............................126

Table 5.2 Elemental analysis of the South Platte River NOM isolates ...........................126

Table 5.3 C/O, C/H and C/N ratios of the Suwannee River NOM isolates.....................128

Table 5.4 C/O, C/H and C/N ratios of the South Platte River NOM isolates..................128

Table 5.5 Integrated areas of 13C-NMR Spectra of Suwannee River NOM fractions.....135

Table 5.6 Integrated areas of 13C-NMR spectra of South Platte River NOM fractions from
first ...........................................................................................................................136

13
Table 5.7 Integrated areas of C-NMR Spectra of South Platte River NOM Fractions
from second sampling ..............................................................................................136

Table 5.8 TDCA and TDAA in the Suwannee River NOM isolatesError! Bookmark not defined.

Table 5.9 TDCA and TDAA in the South Platte River and its isolates fractionError! Bookmark not defined

Table 5.10 Major compounds identified by Pyr-GC-MS analysis of NOM fractions.....146

Table 5.11 Relative proportions of biopolymers in South Platte River NOM isolates ...149

Table 5.12 Relative proportions of biopolymers in Suwannee River NOM isolates ......149

Table 5.13 UV and fluorescence parameters for Suwannee River NOM fractions.........152

Table 5.14 UV and fluorescence parameters for South Platte River NOM fractions......158

Table 5.15 Aromatic carbon content and SUVA of the Suwannee River NOM isolatesError! Bookmark not

xv
Table 5.16 Aromatic carbon content and SUVA254 of the South Platte River NOM
isolates .......................................................................Error! Bookmark not defined.

Table 5.17 DOC and A254 removals of Suwannee River NOM isolates during
coagulation-flocculation with aluminum at pH 6.5. ................................................172

Table 5.18 DOC and A254 removals of South Platte River NOM isolates during
coagulation-flocculation with aluminum at pH 6.5. ................................................172

Table 5.19 Chlorine demand and disinfection by-products formation potentials of the
Suwannee River NOM isolates................................................................................177

Table 5.20 Chlorine demand and DBP formation potentials of the South Platte River and
its isolated NOM fractions.......................................................................................178

Table 5.21 DBP formation potentials of the Suwannee River NOM isolates .................179

Table 5.22 DBP formation potentials of the South Platte River and its NOM isolated
fractions ...................................................................................................................180

Table 6.1 TDCA, TDAA, and BDOC in the Blavet River ..............................................194

Table 6.2 Apparent molecular weight distribution of the NOM of the Blavet River ......195

Table 6.3 Elemental analysis of the Blavet River NOM isolates ....................................196

Table 6.4 C/O, C/N and C/H ratios with SUVA of the Blavet River NOM....................198

Table 6.5 TDAA content of the Blavet River NOM isolates (winter sample) ................199

Table 6.6 Integrated areas of 13C-NMR spectra of the Blavet River NOM isolates .......203

Table 6.7 Relative proportions of biopolymers in the Blavet River NOM isolates ........204

Table 6.8 Some chlorinated DBPs formed by chlorination of the Blavet River and its
NOM isolates ...........................................................................................................208

xvi
Table 6.9 UV and fluorescence parameters for NOM samples concentrated from the
Blavet River .............................................................................................................211

Table 7.1 Summary of results from 13C-NMR, UV absorbance and fluorescence emission
analysis for 27 samples of concentrated or fractionated NOM from the Blavet, South
Platte, Suwannee and Tolt Rivers. ...........................................................................228

Table 7.3 Ranges of major parameters of Pyr-GC-MS, UV absorbance and fluorescence


emission analyses of 13 samples from three sources (Blavet, South Platte and
Suwannee Rivers) ....................................................................................................232

Table 7.4 External and internal correlations of the UV and fluorescence spectral
parameters with Pyr-GC-MS results for 13 samples from the Blavet, South Platte
and Suwannee Rivers...............................................................................................233

Table 8.1 Comparison of NOM concentration methods..................................................237

Table 8.2 Comparison of distribution of biopolymers in RO and XAD-8/XAD-4 samples,


based on UV and Pyr-GC-MS .................................................................................241

xvii
FIGURES

Figure 2.1 Conceptual representation of electronic transitions caused by the absorbance


of light for benzene and NOM ...................................................................................39

Figure 2.2 Summation of three composite absorption bands and formation of


unconvoluted UV absorbance spectrum of NOM......................................................40

Figure 3.1 Schematic for NOM concentration using reverse osmosis and adsorption......54

Figure 4.1 Flow chart for fractionation and isolation of hydrophobic and transphilic
NOM fractions from the Suwannee River .................................................................77

Figure 4.2 Flow chart for fractionation and isolation of hydrophilic NOM (k' = 5-100)
from the Suwannee River...........................................................................................79

Figure 4.3 Flow chart for fractionation and isolation of ultra-hydrophilic NOM fractions
(k' < 5) from the Suwannee River..............................................................................80

Figure 4.4 Flow chart for fractionation and isolation of ultra-hydrophilic NOM fractions
from the South Platte River .......................................................................................85

Figure 4.7 Breakthrough curves for treatment of concentrated NOM solutions with
XAD-4 resin.............................................................................................................101

Figure 4.8 DOC breakthrough curves for treatment of water from Judy Reservoir using
IOCO, IOCS and XAD-8.........................................................................................105

Figure 4.9 A254 breakthrough curves for treatment of water from Judy Reservoir using
IOCO, IOCS and XAD-8.........................................................................................106

Figure 4.10 Cumulative DOC retention efficiency for treatment of Judy Reservoir water
using IOCO, IOCS and XAD-8 ...............................................................................107

xviii
Figure 4.11 Cumulative A254 retention efficiency for treatment of Judy Reservoir water
using IOCO, IOCS and XAD-8 ...............................................................................108

Figure 4.12 DOC breakthrough curves for processing of Tolt River water using IOCO,
IOCS and XAD-8 adsorbents...................................................................................109

Figure 4.13 A254 breakthrough curves for processing of Tolt River water using IOCO,
IOCS and XAD-8 adsorbents...................................................................................110

Figure 4.14 Cumulative DOC retention efficiency by IOCO, IOCS and XAD-8
processing Tolt River water.....................................................................................111

Figure 4.15 Cumulative A254 retention efficiency by IOCO, IOCS and XAD-8
processing Tolt River water.....................................................................................112

Figure 4.16 Sulfate breakthrough curves for IOCS mediumError! Bookmark not defined.

Figure 4.17 Relation between initial SUVA and XAD-8 resin adsorbability for several
surface waters ..........................................................................................................116

Figure 4.18 Relative amounts of NOM sorbed onto XAD-8 and XAD-4 resins in series117

Figure 4.19 Relationship between the adsorption of DOC and SUVA using XAD-8 and
XAD-4 resins in series versus XAD-8 alone ...........................................................118

Figure 4.20 Relationship between DOC concentration efficiency using reverse osmosis
and SUVA for surface waters ....................................Error! Bookmark not defined.

Figure 5.1 DOC distribution of the Suwannee River.......................................................124

Figure 5.2 DOC distribution of the South Platte River....................................................125

Figure 5.3 FTIR spectra of hydrophobic and transphilic Suwannee River NOM fractions130

Figure 5.4 FTIR spectra of hydrophilic and ultrahydrophilic NOM fractions from the
Suwannee River .......................................................................................................130

xix
Figure 5.5 FTIR spectra of hydrophobic and transphilic NOM fractions from the first
sampling of the South Platte River ..........................................................................132

Figure 5.6 FTIR spectra of hydrophilic and ultrahydrophilic NOM fractions from the first
sampling of the South Platte River ..........................................................................133

Figure 5.7 FTIR spectra of neutrals NOM fractions isolated from the second South Plate
River sampling .........................................................................................................134

13
Figure 5.8 C-NMR spectra of the hydrophobic and transphilic fractions of Suwannee
River NOM ................................................................Error! Bookmark not defined.

13
Figure 5.9 C-NMR spectra of the hydrophilic and ultrahydrophilic fractions of
Suwannee River NOM...............................................Error! Bookmark not defined.

Figure 5.10 13C-NMR spectra of the hydrophobic and transphilic fractions of South Platte
River NOM from the first sampling event.................Error! Bookmark not defined.

Figure 5.11 13C-NMR spectra of the hydrophilic and ultrahydrophilic fractions of South
Platte River NOM from the first sampling event.......Error! Bookmark not defined.

13
Figure 5.12 C-NMR spectra of the NOM fractions of South Platte River from the
second sampling event ...............................................Error! Bookmark not defined.

Figure 5.13 1H-NMR spectra of NOM fractions from the Suwannee RiverError! Bookmark not defined.

Figure 5.14 TDCA and TDAA contents of the Suwannee River NOM isolates .............139

Figure 5.15 TDCA and TDAA contents of the South Platte River and its NOM isolates140

Figure 5.16 TDCA distribution of the South Platte River and its NOM isolates ............142

Figure 5.17 TDCA distribution of the Suwannee River and its NOM isolates ...............143

Figure 5.18 TDAA distribution of the South Platte River and its NOM isolates ............144

Figure 5.19 TDAA distribution of the Suwannee River NOM isolates...........................144


xx
Figure 5.20 Pyrolysis GC/MS chromatograms of hydrophilic acid (HPIA) and
hydrophilic neutral (HPIN) fractions of NOM from Suwannee RiverError! Bookmark not defined.

Figure 5.21 Pyrolysis GC-MS chromatogram of hydrophilic base (HPIB) fraction of


NOM from Suwannee River ......................................Error! Bookmark not defined.

Figure 5.22 Pyrolysis GC-MS chromatograms of hydrophobic acid (HPOA) and


hydrophobic neutral (HPON) fractions of NOM from South Platte RiverError! Bookmark not defined

Figure 5.23 Pyrolysis GC-MS chromatograms of transphilic acid (TPHA) and transphilic
neutral (TPHN) fractions of NOM from South Platte RiverError! Bookmark not defined.

Figure 5.24 Specific UV absorbance (SUVA) spectra of Suwannee River NOM fractions.151

Figure 5.25 Correlation between SUVA254 and half-width of the ET band for Suwannee
River NOM concentrates .........................................................................................153

Figure 5.26 Set of selected fluorescence emission spectra of Suwannee River NOM
fractions .....................................................................Error! Bookmark not defined.

Figure 5.27 Normalized fluorescence emission spectra of selected Suwannee River NOM
fractions ...................................................................................................................154

Figure 5.28 Comparison of the fluorescence yield of the Suwannee River NOM fractions155

Figure 5.29 Relationship between the half-width of the ET band in the UV absorbance
spectra of the Suwannee River NOM fractions and the position of the maximum in
the fluorescence emission spectra............................................................................156

Figure 5.30 Specific UV absorbance spectra of South Platte River NOM fractions.......157

Figure 5.31 Normalized fluorescence emission spectra of South Platte River NOM
fractions ...................................................................................................................159

Figure 5.32 Tentative correlation between nitrogen content and TDAA content for NOM
fractions from the Suwannee and South Platte Rivers.Error! Bookmark not defined.
xxi
Figure 5.33 Relation between the relative proportion of proteins and TDAA content in
NOM fractions from the Suwannee and South Platte Rivers. .................................161

Figure 5.34 Relationship between the relative proportion of polysaccharides and the
anomeric carbon content in NOM fractions from the Suwannee and South Platte
Rivers. ......................................................................................................................162

Figure 5.35 Correlation between aromatic C and SUVA in NOM fractions from the
Suwannee and South Platte Rivers. .........................................................................163

Figure 5.36 Correlation between the relative proportion of PHA and SUVA254 in NOM
fractions from the Suwannee and South Platte Rivers.............................................164

Figure 5.37 Correlation between the values of SUVA254 and λmax and the percentage of
nitrogen in the South Platte River fractions estimated using elemental analysis.Error! Bookmark not d

Figure 5.38 Correlation between the aromaticity of the Suwannee River NOM fractions
and ∆ET. ..................................................................................................................165

Figure 5.39 Dependence of the fluorescence yield of the South Platte River NOM
fractions vs. the concentration of tyrosine and phenylalanine.Error! Bookmark not defined.

Figure 5.40 Correlation between the aromaticity of the Suwannee River NOM fractions
and the position of the maximum in the fluorescence emission spectra..................166

Figure 5.41 Correlation between the position of the emission maxima in the fluorescence
spectra of the Suwannee River NOM fractions and the aromatic carbon content of
the sample, based on Pyr-GC-MS data. ...................................................................167

Figure 5.42 Correlation between the position of maximum in the fluorescence emission
spectra and the content of proteinaceous and polyhydroxyaromatic carbon (based on
Pyr-GC-MS analysis) in the South Platte River NOM fractions .............................168

Figure 5.43 Comparison of FTIR spectra of transphilic neutral fractions between


Suwannee River and South Platte River. .................................................................170

xxii
Figure 5.44 Relationship between DOC or A254 removals and SUVA254....................175

Figure 5.45 Relationships between DBPFPs and SUVA254 for NOM fractions from the
Suwannee and South Platte Rivers ..........................................................................184

Figure 5.46 Relationship between TOXFP and THMFP in Suwannee and South Platte
River NOM fractions ...............................................................................................186

Figure 5.47 Relationship between TCAAFP and DCAAFP for the Suwannee River and
South Platte River NOM............................................Error! Bookmark not defined.

Figure 5.48 TOXFP versus destruction of A254 by chlorination for Suwannee and South
Platte NOM fractions .................................................Error! Bookmark not defined.

Figure 6.1 Bromide concentrations in the Blavet River during a one-year sampling period192

Figure 6.2 DOC and A254 in the Blavet River during a one-year sampling period .......193

Figure 6.3 SUVA254 in the Blavet River during a ne-year sampling period..................193

Figure 6.4 TDAA distribution of the Blavet River (winter sample)................................199

Figure 6.5 TDCA distribution of the Blavet River (winter sample)................................200

Figure 6.6 TDAA distribution of the Blavet River (summer sample) .............................200

Figure 6.7 TDCA distribution of the Blavet River (summer sample) .............................201

Figure 6.8 FTIR spectra of NOM fractions isolated from Blavet River (winter sample)
using XAD resins.......................................................Error! Bookmark not defined.

Figure 6.9 FTIR spectra of NOM fractions isolated from Blavet River (summer sample)
using XAD resins.......................................................Error! Bookmark not defined.

13
Figure 6.10 C-NMR spectra of NOM fractions isolated from Blavet River (winter
sample) using XAD resins .........................................Error! Bookmark not defined.

xxiii
13
Figure 6.11 C-NMR spectra of NOM fractions isolated from Blavet River (winter
sample) using membranes with and without further desalting proceduresError! Bookmark not defined

13
Figure 6.12 C-NMR spectra of NOM fractions isolated from Blavet River (summer
sample) using XAD resins .........................................Error! Bookmark not defined.

13
Figur 6.13 C-NMR spectra of NOM fractions isolated from Blavet River (summer
sample) using membranes with and without further desalting proceduresError! Bookmark not defined

Figure 6.14 Pyrolysis GC-MS chromatogram of XAD-8/XAD-4 mixture NOM isolate


from Blavet River (winter sample) ............................Error! Bookmark not defined.

Figure 6.15 Pyrolysis GC-MS chromatograms of RO and NF NOM isolates (without


desalting) from Blavet River (winter sample) ...........Error! Bookmark not defined.

Figure 6.16 Pyrolysis GC-MS chromatograms of RO and NF NOM isolates with further
desalting from Blavet River (winter sample).............Error! Bookmark not defined.

Figure 6.17 Pyrolysis GC-MS chromatogram XAD-8/XAD-4 mixture and RO NOM


fractions isolated from Blavet River (summer sample)Error! Bookmark not defined.

Figure 6.18 Pyrolysis GC-MS chromatogram of XAD-8/XAD-4 mixture and RO


(without desalting) NOM isolates from Blavet River (summer sample)Error! Bookmark not defined.

Figure 6.19 Set of UV spectra of NOM concentrates from the Blavet River winter
sampling period........................................................................................................210

Figure 6.20 Correlation between SUVA254 and ∆ΕΤ for NOM concentrates from the
Blavet River .............................................................................................................214

Figure 6.21 Selected fluorescence emission spectra of NOM concentrates from the Blavet
River.........................................................................................................................215

Figure 6.22 Normalized fluorescence emission spectra of selected NOM concentrates


from the Blavet River winter sample .......................................................................216

xxiv
Figure 6.23 Correlation of the ET band half-width and the wavelength of maximum
fluorescence emission intensity ...............................................................................217

Figure 6.24 Correlation between the percentage of carbon in low-ash NOM concentrates
13
and percentages of aromatic carbon as determined through C-NMR and
Pyr-GC-MS data ......................................................................................................218

Figure 6.25 Correlation between the values of SUVA254 and the percentage of
polyhydroxyaromatic and total aromatic carbon .....................................................220

Figure 6.26 Correlation between ∆ET and the percentage of polyhydroxyaromatic and
total aromatic carbon in the Blavet NOM fractions based on Pyr-GC-MS data .....221

Figure 6.27 Correlation between the position of maximum in the fluorescence emission
spectra and the percentage of aromatic carbon based on 13C-NMR data ................222

13
Figure 7.1 Correlation between the aromaticity of NOM estimated using C-NMR
spectroscopy and corresponding values of SUVA254 and ∆ET .............................229

13
Figure 7.2 Correlation between the aromaticity of NOM estimated using C-NMR
spectroscopy and corresponding λmax values ...........................................................230

xxv
FOREWARD

xxvi
ACKNOWLEDGMENTS

The authors are grateful to the following water utilities and individuals for their
support, cooperation and participation in this project:

Skagit County Public Utility District, Mt. Vernon, Wash., Greg Peterka and Greg
Hamilton

Seattle Public Utilities, Seattle, Wash., David Hilmoe and Bryan Hoyt

A great deal of the experimental work was conducted by Dr. Laurence Labouyrie-
Rouillier and David Violleau (MS student) at the Université de Poitiers (France) and Dr.
Chi-wang Li at the University of Washington. Dr. George Aiken of the USGS (Denver,
Colo.) carried out the XAD-8 and XAD-4 isolations of the water from the South Platte
River. The experimental effort contributed by these three individuals, as well as their
input to help interpret the experimental results, are greatly appreciated. The advice of the
Project Advisory Committee (PAC) – including Erika Hargesheimer, City of Calgary,
Calgary, Alb.; Peter Huck, University of Waterloo, Waterloo, Ont.; Gayle Newcombe,
Australian Water Quality Centre, Adelaide, South Australia; Douglas Owen, Malcolm-
Pirnie, Inc., Carlsbad, Calif.; and Earl Peterkin, Philadelphia Water Dept., Philadelphia,
Pa. – and of AWWARF project officer Jeff Oxenford, are also appreciated.

xxvii
EXECUTIVE SUMMARY

BACKGROUND

The material referred to as natural organic matter (NOM) is an extremely


complex mixture of organic compounds found in all potable water sources. NOM is
typically dominated by humic substances generated by biological activity both in the
watershed surrounding a water source (allochthonous NOM) and within the water source
itself (autochthonous NOM). In addition to humic substances, proteins, polysaccharides,
and other classes of biopolymers also contribute to NOM. Monomeric species such as
simple sugars and amino acids are also present in water sources, but they are less
abundant because they are subject to relatively rapid biodegradation. Although the name
implies that NOM is of natural origin, as a practical matter the molecules collected as
NOM from any water source include many organic compounds contributed by human
activities.

NOM is important in many different reactions and processes that affect water
quality. For instance, it provides precursor material for most halogenated and oxygenated
disinfection by-products, substrate for biogrowth in water treatment and distribution
systems, and complexation sites for binding of heavy metals, and it affects the behavior
of colloidal matter by binding to the colloids’ surfaces. To understand these processes
better and to control their effects on drinking water quality, it is necessary to understand
the chemistry of NOM. However, due to the diversity of the molecules that constitute
NOM and the relatively low concentration of NOM in potable water sources (typically, 2
to 10 mg/L when quantified as dissolved organic carbon (DOC)), methods are needed
that can either characterize NOM in dilute solutions containing a variety of other
chemicals (i.e., in situ) or that can isolate NOM without altering its properties. The
current research project addressed these issues.

xxviii
APPROACH

A three-fold research approach was used. First, the performance of several NOM
isolation methods was compared. The techniques investigated included evaporation,
reverse osmosis (RO), nanofiltration (NF), and adsorption onto several adsorbents,
including XAD-4 and XAD-8 resins and iron-oxide-coated sand and olivine (IOCS and
IOCO). These techniques were compared with respect to the efficiency with which the
NOM was collected and separated from other constituents of solution and the type of
NOM that was lost (i.e., that could not be collected efficiently) during the process.

Next, NOM from two water sources was fractionated based on the hydrophobicity
and acidity of the molecules, and the various fractions were compared using several state-
13
of-the-art analytical methods. These methods included C-NMR spectroscopy;
pyrolysis-gas chromatography-mass (Pyr-GC-MS) spectrometry; Fourier transform
infrared (FTIR) spectroscopy; analyses of elemental composition and total dissolved
carbohydrates and amino acids (TDCA and TDAA); and ultraviolet (UV) and
fluorescence spectroscopy. This part of research shed light on the variability of molecules
contributing to NOM, its site-specificity, and, to a limited extent, the effects of seasonal
processes on NOM composition. It also highlighted the potential value and limitations of
the analytical methods employed in NOM research.

Finally, the effects of NOM isolation procedures on the chemical composition and
reactivity of the isolates was investigated. This part of the study used several water
sources from Europe and the Pacific Northwest of the United States, but the report
focuses on the results from the source that was studied most intensively: the Blavet River
in France.

In the report, the feasibility and adequacy of in situ and ex situ techniques for
studying NOM are addressed. Overall, this project represents a concerted and consistent
effort to gain more insight into the intrinsic properties of NOM and to develop practical
methods to isolate and study it.

xxix
RESULTS AND CONCLUSIONS

NOM Concentration Methods. Isolation of 100% of the NOM in a water sample


is an admirable, but unattainable goal. No technique currently available is capable of
efficiently isolating low molecular weight, low aromaticity organic compounds.

Evaporation is a very efficient technique for concentrating the non-volatile NOM


in a sample. However, this technique requires a great deal of time and effort, and it does
not isolate the NOM from the inorganic matrix. This concentration approach might be
adequate for subsequent analysis of the NOM by Pyr-GC-MS, but not for analysis by
13
C-NMR or FTIR spectroscopy, or for determination of elemental composition.

RO and NF afford relatively convenient ways to concentrate NOM from large


volumes of water. RO allows isolation and recovery of 90 to 95% of the DOC and
virtually 100% of the UV absorbance in a sample. NF membranes are usually 5 to 15%
less efficient than RO with respect to recovery of both DOC and UV absorbance. Due to
the high surface area of RO and NF membranes, it is not uncommon for up to 10% of the
NOM to be trapped within the cartridge. At least part of this NOM can be eluted by
rinsing the membranes with deionized water or a sodium hydroxide solution, but the
efficiency of such a step and the compatibility of the NaOH with the membrane depend
on the particular water and membrane under study. Like evaporation, RO and NF
concentrate the inorganic salts in the sample along with the NOM. In all these cases, the
inorganic (ash) content of the material isolated by RO or NF depends on the inorganic
content of source water.

The XAD series of macroreticular resins has been used extensively in the past to
concentrate and isolate certain fractions of NOM. Passage of a sample through XAD-8
and XAD-4 resins in series collects 60 to 80% of the NOM and yields NOM concentrates
of high purity (ash <5%). This NOM concentration method is slower than RO and NF
and requires extensive preparation of the media.

Iron-oxide-coated sand (IOCS) and olivine (IOCO) have recently been shown to
be good adsorbents for NOM under certain conditions. The efficiency of NOM collection
xxx
using IOCS is typically 60 to 80% when processing a few hundred bed volumes of water,
and higher if fewer bed volumes are processed. Co-adsorption of sulfate with NOM
causes sulfate to accumulate in the NOM isolates and may compromise the effectiveness
of IOCS in high-sulfate waters. As opposed to adsorption onto XAD resins or IOCS,
adsorption onto IOCO retains a substantial amount of NOM at pH 7. Therefore, use of
IOCO is attractive if one wishes to collect NOM with minimal or no sample pre-
treatment. However, the capacity of IOCO for NOM at neutral pH is lower than that of
IOCS under acid conditions by a factor of 2 to 3. The capacity of XAD-8 at pH 2 in some
cases (e.g., Judy Reservoir) exceeded that of IOCO by ca. 40%, while in others (e.g., Tolt
River) it was lower by ca. 25%. Therefore, either larger amounts of media are needed to
process a given volume of sample or the NOM must be eluted more frequently when
IOCO is used to collect the NOM.

Chemical Composition of NOM Isolates. RO and NF concentrates are richer in


amino sugars and proteins than XAD concentrates, while the latter are enriched in
polysaccharides. The XAD-8 NOM isolates are aromatic, hydrophobic, and rich in
carbon but depleted in nitrogen and oxygen. The XAD-4 samples are less aromatic but
contain notable amounts**JP1 of carboxylic acids, aliphatic alcohols, ethers and esters.
RO and NF retained 80 to 90% of the total dissolved amino acids in the samples studied,
compared with 70 to 80% retention using the XAD-8/XAD-4 tandem. Thus, the
membrane isolation procedure appears to be more useful for evaluating NOM
composition with respect to specific biopolymers, while XAD isolates are more useful for
examination of the aromatic and hydrophobic humic species in NOM. The analysis of the
IOCO and IOCS isolates for aminoacids and carbohydrates was hindered by the presence
of sulfate retained by these media, and the relevant data were not available. Based on the
affinity of the surface of iron oxides to carboxylic groups, it is concluded that non-humic
and non-acidic fractions of NOM are less likely to be retained by IOCO/IOCS, while the
humic fraction of NOM, including the hydrophilic acidic fraction are expected to be
efficiently retained by these media.

The various isolates were also compared with respect to their tendency to form
chlorinated DBPs. Upon chlorination, membrane isolates formed less DBPs than did the
xxxi
raw water, but this result could be an artifact related to the difficulties of re-dissolving
lyophilized (freeze-dried) membrane isolates. XAD and IOCS isolates behaved similarly
to the raw water.

Desalting of NOM Isolates. Non-XAD NOM isolation methods retain inorganic


species present in the solution. These inorganic species may interfere with analysis of the
elemental composition and TDAA and TDCA content of NOM, as well as
13
characterization of NOM by C-NMR, FTIR, and in some cases UV and Pyr-GC-MS.
Desalting of NOM isolates using adsorption on XAD resins followed by elution with an
acetonitrile/ water mixture and zeotrophic distillation with acetic acid is an efficient but
very laborious technique that requires substantial skill. Desalting may be also achieved
using the XAD-8/XAD-4 tandem without subsequent zeotrophic distillation, but this
modification decreases the final recovery of NOM by JP2** to **%. Possible NOM
alteration caused by desalting procedures remains an unresolved issue that needs to be
carefully examined in the future.

Intrinsic NOM Chemistry. The intrinsic variability of NOM and its site-
specificity was addressed by comparing NOM from the Blavet, Suwannee and South
Platte Rivers. NOM from each source was fractionated into hydrophobic (HPO),
transphilic (TPH), hydrophilic (HPI) and ultrahydrophilic (uHPI) fractions, each of which
was further divided into acid, neutral and basic sub-fractions, using a modification of the
technique first proposed by Leenheer (1981). Suwannee NOM is primarily
allochthonous, while South Platte NOM is derived from both allochthonous and
autochthonous inputs. Suwannee NOM is hydrophobic and highly aromatic, whereas
South Platte NOM is dominated by hydrophilic and transphilic molecules.

Comparison of NOM characteristics in various fractions or between the two


sources led to the following observations. The C/O, C/N, and C/H ratios decrease with
increasing hydrophilic character for both acid and neutral fractions in all waters. The acid
fractions are characterized by lower C/O ratios than the basic and neutral fractions,
suggesting that most of the acidic groups are carboxylic. The C/N and C/H ratios of the
neutral fractions are lower than those of the acid fractions. The base fractions have the

xxxii
highest nitrogen content, consistent with the expectation that the basicity is related to
amino and amide functionalities. TDAA accounts for the majority of nitrogen in the
hydrophobic fractions of both waters, but accounts for only 5 to 20% of the nitrogen in
the other fractions. TDCA and TDAA concentrations are greater in the South Platte River
than in the Suwannee River, but they account for only a small concentration of the DOC
in both waters. Glucose is the dominant sugar in most NOM fractions. A high ornithine
concentration distinguishes Suwannee River NOM from South Platte River NOM.

Specific UV absorbance (SUVA) varies substantially in Suwannee River NOM


fractions, in the order HA > HPO > TPI > HPI, where HA denotes the fraction of humic
acids that has a mixed colloidal/polymeric nature. A similar trend applies in South Platte
River NOM fractions, but the SUVA values are much lower in the South Platte than for
the corresponding fractions from the Suwannee River. For the Suwannee River NOM
fractions, fluorescence emission yields increase and the peak in the emission spectrum
shifts to lower wavelengths (it is ‘blue-shifted’) as the hydrophilic character of the NOM
increases. This trend probably reflects a decrease in average molecular weight as
hydrophilic character increases. Fluorescence data for South Platte River NOM fractions
suggest that the average molecular weight in this NOM is less than for the corresponding
Suwannee River NOM fractions, but that molecular weight tends to increase as the
hydrophilic character of the fraction increases. The protein-rich fractions derived from
both NOM sources exhibit strong fluorescence that is blue-shifted compared with that of
highly aromatic fractions.

Moderate correlations were found between SUVA and coagulability of the


fraction. Acid fractions were more efficiently coagulated with alum than were neutral or
base fractions. The coagulability of the Suwannee NOM fractions was higher than that of
the South Platte NOM fractions.

Seasonal changes of NOM were studied using NOM extracted from the Blavet
River during summer and winter sampling periods. The hydrophobic and transphilic
NOM comprised 57 and 21%, respectively, of the NOM in the summer sample, and 79
and 11% in the winter sample. The most dramatic differences between the summer and

xxxiii
winter samples were the higher concentrations of TCAA and TDAA in the summer
sample. The properties of the humic components of the NOM were not significantly
different in the two seasons.

Ex Situ and In Situ NOM Characterization. A combination of methods is


required to explore the chemistry of NOM and to probe its reactions in potable water
systems. Among the structure-sensitive methods investigated, 13C-NMR is notable for its
ability to quantify the abundance of organic carbon types. This analysis requires dry,
desalted NOM samples containing at least 50 mg of organic carbon. The precision of
13
C-NMR in estimating the fraction of the carbon that is associated with aromatic and
13
carbonyl or carboxyl functional groups in NOM is limited, so C-NMR data should be
considered as semi-quantitative in this regard.

Pyr-GC-MS requires a few milligrams of dry sample, but unlike the case for
13
C-NMR analysis, the sample need not be desalted. Pyr-GC-MS is a semi-quantitative
analytical tool that yields information about the distribution of molecules belonging to
various biopolymer classes. The interpretation of NOM pyrochromatograms is more
subjective than that of NMR spectra, since only a fraction of the pyrolysis fragments is
used for the interpretation. The interpretation may be further complicated by the presence
of secondary reactions of pyrolysis fragments. Pyr-GC-MS is one of the newest analytical
techniques available for NOM characterization, and more effort is needed to establish
and verify approaches for interpreting the output. However, it is clear that this technique
provides a unique and distinct NOM fingerprint that can be a valuable adjunct to other
information for characterizing NOM.

FTIR analysis also requires only a few milligrams of dry sample and is useful as a
monitoring tool for ascertaining the inorganic composition of NOM isolates, since it can
detect the presence of ammonium, bicarbonate, carbonate, nitrate, silicate, and sulfate
(but not that of inorganic halides). It was used for that purpose in the current study. FTIR
is a qualitative spectrometric tool that is most valuable as a source of supplementary
structural information about inorganic and organic components of the sample, in
13
conjunction with more quantitative methods such as C-NMR and elemental analyses.

xxxiv
For example, the carbonyl group of acids, amides, and esters is not resolved in 13C-NMR
spectra, but there is sufficient resolution of these groups in FTIR spectra to indicate
whether an NOM isolate is predominately an acid (as in humic substances), an ester (as
in certain tannins), or an amide (as in proteins and amino sugars). Complementary
information on aliphatic hydrocarbon and aliphatic alcohol composition of NOM isolates
is also provided by FTIR spectrometry. The sensitivity of FTIR spectrometry to inorganic
constituents is both a useful feature and a problem: it is useful because the FTIR signal
can indicate whether or not the sample has been desalted successfully, but it is
problematic because, if the sample has not be efficiently desalted, the signal from the
organic constituents cannot be discerned.

Analyses for elemental composition and specific classes of chemical species in


NOM can complement structure-sensitive analytical techniques. Elemental analysis can
be reliably conducted only on dry, low-ash NOM isolates. Such analyses provide the
most direct measure of the efficiency of protocols for both concentrating and desalting
NOM. Although elemental analysis does not appear to be a very specific characterization
tool, it does provide important information, such as elemental ratios that allow various
NOM fractions isolated from the same source to be compared. For instance, the C/O ratio
is an indicator of the oxygenated functional group content in NOM, the C/N ratio
indicates the content of nitrogenous functional groups, and the C/H ratio indicates the
degree of unsaturation of the NOM. The C/N ratio might also be a good indicator of the
origin (autochthonous versus allochthonous) of the NOM.

Analysis for total dissolved amino acids and carbohydrates can be conducted on
natural or treated waters or on NOM isolates. Typically, amino acids and carbohydrates
comprise a few percent of the DOC of surface waters. The distribution of monomeric
species in TDAA and TDCA is not very site-specific or indicative of the NOM
generation processes. The only exception is ornithine, which might be a good indicator of
microbial (algal, rather than microbial**? JP) activity in natural waters. Given the effort
required to carry out these analyses, the evaluation of TDAA and TDCA content does not
merit high priority for structural characterization of NOM. On the other hand, these

xxxv
analyses are certainly useful in NOM biodegradability studies, since the analytes
represent a significant part of the biodegradable DOC (BDOC) of the NOM.

Techniques that can be used on unaltered samples typically require much less
time and effort per analysis than do the techniques described above. The techniques
meeting this criterion that were utilized in the current study were UV and fluorescence
spectroscopy. Only carbon that is in aromatic moieties has been shown unambiguously to
affect the UV absorbance spectrum of NOM. The SUVA value at 254 nm (SUVA254) can
be used as a reasonably good indicator of the aromaticity of NOM (as quantified by
13
either C-NMR or Pyr-GC-MS). Determination of SUVA254 is much easier and less
expensive than analysis of NMR or Pyr-GC-MS spectra, but even SUVA254 requires
some off-line analysis (for DOC). An alternative parameter that seems to provide
comparable information is the width of the so-called electron-transfer band (∆ET) in the
UV absorbance spectrum, which can be calculated using the ratio of absorbances at 350
and 280 nm (A350/A280) without any off-line analysis. ∆ET is correlated to both NOM
aromaticity and, presumably, its molecular weight. The dependence of ∆ET on NOM
properties should be investigated in more detail.

Fluorescence spectroscopy is an extremely sensitive method that permits NOM to


be studied in solution at DOC concentrations <1 mg/L. The fluorescence emission of
NOM is governed primarily by the identity and concentration of aromatic functional
groups in NOM molecules, but nitrogenous groups might make a significant contribution
as well. In the latter case, the emission is blue shifted, and the fluorescence may be useful
as a probe for investigating the relative amounts of aromatic and nitrogenous species in
NOM. The emission of aromatic units in NOM is substantially affected by the molecular
weight distribution and conformation of the molecules (Frimmel and Kumke 1998,
Kumke, Abbt-Braun and Frimmel. 1998, Ewald, Belin et al. 1992, Donard, Belin and
Ewald 1987, Stewart and Wetzel 1981). This fact may allow fluorescence to be used to
track reactions of NOM in water treatment processes or, alternatively, to probe the origin
of NOM and mixing processes in water distribution systems, if sources with dissimilar
NOM are blended.

xxxvi
13
Data from C-NMR, Pyr-GC-MS, UV absorbance and fluorescence emission
analyses (parameterized by SUVA254, ∆ET, and λmax) are correlated, since they all are
sensitive to aromatic structures in NOM. Some correlations between UV spectral
parameters and the 13C-NMR aromaticity and/or the polyhydroxyaromatic (PHA) content
of NOM (indicated by Pyr-GC-MS) were identified in this research, but the correlations
are not strong enough to allow use of UV spectroscopy as a substitute for the other, more
complex analyses. Given the wide range of the properties of the NOM samples and the
semi-quantitative nature of the aromaticity or PHA estimates, the scatter observed in
these correlations is not surprising. Thus, while it appears that SUVA254, ∆ET, λmax can
potentially be used to monitor the concentration and transformations of aromatic moieties
of NOM, additional research is needed to clarify the relationships among these
characteristics, the output from other analytical probes, and NOM structure.

Research Needs. This research has identified several issues relevant to


exploration of NOM concentration and isolation that deserve further exploration. To
improve NOM concentration and isolation, new membranes and perhaps new designs of
membrane processing systems are needed that minimize NOM binding to the membrane
and occlusion in the system. Development of methods to prevent the adsorption of sulfate
onto iron-oxide based media is also a priority. More work is warranted with XAD or
similar resins to explore their use in combination with RO and NF, in order to improve
isolation recoveries and yield high-purity samples. For evaporative concentration,
development of approaches for circumventing the effects of silica on concentrated NOM,
and for removal of sulfates and hardness cations would be valuable.

With respect to the development of NOM characterization methods,


improvements in analytical methods that reduce the mass of NOM necessary to obtain
13
high-quality signals (e.g., from C-NMR spectrometry) would represent a significant
advance, as well as approaches for improving the precision of data from NMR analysis so
that the content of aromatic and carbonyl carbon in a sample can be quantified more
reliably. Continued development of the data base for NOM pyrochromatograms is also a
very high priority.

xxxvii
With respect to improvements in analytical techniques that can be applied in situ,
the effects of the molecular weight distribution on UV spectra, and in particular on the
characteristics of the electron transfer band in those spectra, need clarification. The
possible impact of furan structures and nitrogen bases also needs to be elucidated. The
correlation between the activation of aromatic moieties in NOM and the parameters of
the corresponding UV spectra should also be quantified in more detail. A high priority is
research leading to the development of a consistent, unified mathematical theory of NOM
fluorescence. Such a model should address the effects of the molecular weight
distribution, protonation of acidic sites and molecular conformation on the emission of
NOM. Further development of numerical methods to process UV and fluorescence
spectra of NOM and improvements in the precision and interpretability of 13C-NMR and
Pyr-GC-MS spectra will enhance the versatility and predictive capacity of both in situ
and ex situ analytical methods.

xxxviii
ABBREVIATIONS

Ai light absorbance at a wavelength of i nm

ala alanine

amu atomic mass units (Daltons)

Ao,i maximum light absorbance associated with absorbance band i

arg arginine

AS amino sugars

asp aspartic acid

BDOC biodegradable dissolved organic carbon

BVbreakthrough number of bed volumes treated at breakthrough

BV empty bed volume

Bz benzenoid band in UV absorbance spectrum

°C degrees Celsius

C 'ads mass of organic carbon adsorbed per liter of packed bed

C/H mass ratio of carbon to hydrogen

C/N mass ratio of carbon to nitrogen

C/O mass ratio of carbon to oxygen

Cads concentration of adsorbed material in a packed bed (mg adsorbed/L of


bed)

CF concentration factor

Cinfluent influent concentration

cm centimeter

xxxix
COD chemical oxygen demand

CPMAS cross polarization magic angle spinning

DBP disinfection by-product

DBPFP disinfection by-product formation potential

DCAA dichloroacetic acid

DCAAFP dichloroacetic acid formation potential

DOC dissolved organic carbon

DPD N,N-diethyl-p-phenylenediamine

∆ET width of the ET band in the absorbance spectrum (eV)

∆i width of band i in the absorbance spectrum (eV)

ECD electron capture detector

EPA Environmental Protection Agency

eV electron volts

ε void fraction of a packed column

FP formation potential

FTIR Fourier transform infrared

g grams

GC gas chromatography

glu glucose

gly glycine

gpm gallons per minute

xl
h hours

HA humic acid

HAA haloacetic acid

HAAFP haloacetic acid formation potential

his histidine

HPI hydrophilic

HPIA hydrophilic acid

HPIA-2 hydrophilic acids collected on second pass through column

HPIB hydrophilic base

HPIN hydrophilic neutral

HPLC high pressure liquid chromatography

HPO hydrophobic

HPOA hydrophobic acid

HPON hydrophobic neutral

η removal efficiency (%)

ICP-AE inductively coupled plasma - atomic emission

ID inside diameter

ile isoleucine

IOCO iron oxide coated olivine

IOCS iron oxide coated sand

IX ion exchange

k proportionality constant

xli
k' column capacity factor

km kilometer

L liter

LE local excitation band in UV absorbance spectrum

leu leucine

lys lysine

λmax wavelength of maximum fluorescence emission (nm)

meq milliequivalent

met methionine

min minute

mm millimeter

mol mole

mS milliSiemens

ms millisecond

MS mass spectrometry

MTBE methyl tert-butyl ether

MW molecular weight

MX 3-chloro-4(dichloromethyl)-5- hydroxy-2(5H)-furanone

µg microgram

µL microliter

µm micrometer

µmol micromole

xlii
N normality (eq/L)

NF nanofiltration

nm nanometer

nM nanolmoles/L

nmol nanomole

NOM natural organic matter

NTU nephelometric turbidity units

orn ornithine

PHA polyhydroxyaromatic

phe phenylalanine

ppm parts per million

PR proteins

PS polysaccharides

psi pounds per square inch

Pyr pyrolysis

PZC point of zero charge

R2 regression coefficient

RF radio frequency

RO reverse osmosis

Rsplit splitting ratio

SDS simulated distribution system

xliii
ser serine

SUVA specific ultraviolet absorbance (L/mg-m)

t time

TCAAFP trichloroacetic acid formation potential

TDAA total dissolved amino acid

TDCA total dissolved carbohydrate

THM trihalomethane

THMFP trihalomethane formation potential

thr threonine

TOC total organic carbon

TOXFP total organic halogen formation potential

TPH transphilic

TPHA transphilic acid

TPHN transphilic neutral

tyr tyrosine

UA unsubstituted aromatic

UFC uniform formation conditions

uHA ultrahydrophilic humic acid

uHPI ultrahydrophilic

uHPIAMe methylated ultrahydrophilic acid

uHPIN ultrahydrophilic neutral

USGS United States Geological Survey

UV ultraviolet

xliv
Vwater/Vbed ratio of water volume treated to bed volume

xlv
CHAPTER 1 INTRODUCTION

Diverse organic compounds generated by biological processes both in a water


body (autochthonous material) and in the surrounding watershed (allochthonous material)
are found in all surface and many ground waters. Collectively, these compounds, along
with organic compounds that enter the water as a result of human activities, are referred
to as natural organic matter (NOM). The concentrations of NOM and of the sub-groups
of molecules that contribute to it are usually quantified in terms of the amount of carbon
in the molecules. Typically, values are reported as the concentration of dissolved organic
carbon (DOC) in a sample.

Because NOM includes literally thousands of distinct chemical species,


evaluation of its properties based on a comprehensive compilation of the individual
compounds is not realistic. Rather, researchers have attempted to characterize NOM by
grouping the NOM molecules into a limited set of categories (fractions). As a practical
matter, the assignment of molecules to particular categories is always operational,
although the categorization is often described in terms of fundamental or conceptual
characteristics. An implicit expectation of NOM fractionation is that fractions isolated
from independent water sources by the same set of procedures will have similar
composition and properties, though the concentration of a given NOM fraction in
different sources may differ.

NOM characterization has been and remains a priority for the water treatment
industry, in part because such characterization holds the key to understanding, predicting
and perhaps controlling NOM reactivity under water treatment conditions. NOM
characterization techniques can be classified broadly as probing the properties of NOM
molecules (e.g., hydrophobic or hydrophilic), their structure (e.g., functional group
content), or their reactions with other molecules (e.g., DBP formation). Differences
between the properties of NOM and inorganic molecules are exploited to isolate and
concentrate NOM, and differences in the properties of different NOM molecules are

1
exploited to fractionate NOM; by the same token, the fractionation results can be used to
draw inferences about the properties of the molecules.

Like snowflakes, NOM molecules are all unique while also sharing many
common properties. Fractionation of NOM selects a sub-group molecules from the
mixture that share a narrower range of properties than does the entire aggregate. Ideally,
fractionation collects 100% of the molecules that share these properties and 0% of those
that do not. In truth, of course, fractionation selects imperfectly for molecules that fit the
criteria, always leading to both positive and negative errors: some molecules are
collected that do not fall in the target group, and some that fall into the target group are
not detected and/or collected. Procedures intended to concentrate NOM non-selectively
invariably also fractionate the sample to some extent. Many techniques (e.g., adsorption
on resins) can be used to concentrate small amounts of NOM almost quantitatively, but
when applied to larger amounts they collect only a (selective) portion, thereby by default
becoming fractionation techniques.

The need for concentration and/or isolation of NOM is largely driven by the
sensitivity of the characterization methods: if it were possible to use the characterization
methods effectively on unconcentrated and unfractionated samples, many problems
associated with the analysis (e.g., reactions among molecules in concentrates that are
different from those in dilute samples; losses upon concentration and/or isolation) could
be avoided. Unfortunately, at present, most NOM characterization methods are
insufficiently sensitive to be applied to unmodified raw water samples.

The effort and cost associated with NOM collection and characterization is
related to the recovery efficiency and information content. With extensive effort and
utilization of several techniques, it is now possible to recover nearly 100% of the organic
carbon in low-inorganic isolates. However, substantial recovery (e.g., 65 to 85% of the
NOM) can be achieved with much less effort, and for some applications these lower
amounts of effort and recovery are acceptable.

2
One common approach for characterizing NOM divides the mixture into
hydrophilic and hydrophobic fractions. The hydrophilic fraction is expected to include
carboxylic acids, carbohydrates, amino acids and amino sugars, and proteins, while the
hydrophobic fraction includes so-called humic species. All these groups of compounds
are likely to be present in all natural waters, though their absolute and relative
concentrations are expected to vary from site to site. Despite the site specificity of NOM
and some variability of its properties over time (often related to seasonal cycles of
biological activity), humic species typically dominate the NOM on a mass basis,
contributing from ~50 to >90% of the DOC in most natural waters (Thurman 1985).

Most dissolved humic substances are thought to have molecular weights of a few
hundred to a few thousand atomic mass units (amu) (McIntyre et al. 1997, Remmler et al.
1995, Wershaw and Aiken 1985). Humic molecules contain aromatic, carbonyl, carboxyl,
methoxyl, and aliphatic units (Stevenson 1982, Christman et al. 1989, Perdue 1985,
Gjessing 1976), with the phenolic and carboxylic functional groups providing most of the
protonation and metal complexation sites. As opposed to synthetic polymers and many
biological polymers (e.g., proteins), humic molecules are not comprised of unique, highly
reproducible monomeric building blocks (Christman et al. 1989, Hess and Chin 1996,
Pompe et al. 1992). Rather, a group of similar building blocks is probably present in
many humic molecules, but the sequence and frequency of occurrence of the building
blocks, and the exact structure of the regions between adjacent building blocks, is
probably different in every humic molecule. For this and other reasons, the chemistry of
humic substances is distinct in many ways from that of conventional polymers.

Previous investigations of NOM from a wide variety of sources has led to some
generalizations about the characteristics of NOM molecules in different environments.
For instance, environments in which water is exposed to mineral surfaces that complex
and adsorb NOM contain low concentrations of dissolved NOM, especially humic
substances. NOM in lakes and reservoirs of moderate to high trophic status is often
dominated by material generated in the water body (autochthonous material), whereas
low-order rivers and streams usually carry more NOM that is generated exterior to the
water body (allochthonous NOM). Allochthonous NOM has large C/N ratios (near
3
100:1), is highly colored, and has significant aromatic carbon content, whereas
autochthonous NOM has lower C/N ratios (near 10:1), is almost colorless, and has low
aromatic carbon content (Aiken, McKnight et al. 1991).

No attempt to fractionate NOM can be completely successful, for both practical


and theoretical reasons. For instance, separation of the humic fraction from the non-
humic fraction is impeded by the fact that molecules from the two groups can form
complexes or otherwise associate with one another in solution (Lytle and Perdue 1981,
Leenheer et al. 1989, Boerschke et al. 1996, Cook and Langford 1998, Volk et al. 1997,
Jahnel and Frimmel 1996). Nevertheless, conventional operational approaches have been
developed for carrying out these separations.

The most common approach for distinguishing between hydrophobic and


hydrophilic dissolved NOM is to define them as the organic matter that is adsorbable and
non-adsorbable, respectively, on XAD-8 resins. Hydrophobic NOM is often further
separated into humic acids (defined as the fraction that coagulates at pH<1 or 2,
depending on the researcher) and fulvic acids (the non-coagulable fraction). A more
elaborate fractionation scheme in which NOM is separated into four fractions
(hydrophobic, transphilic, hydrophilic and ultrahydrophilic) is described in this report.

A significant portion of the hydrophilic NOM can be associated with identifiable


compounds, or at least combinations of such compounds that have formed condensation
products or other composites. However, such is not the case for the hydrophobic fraction
of NOM. Therefore, in order to characterize this fraction in a consistent manner,
approaches have been proposed to fractionate it into groups that exhibit gradually varying
chemical properties. These fractionation schemes may be based on various parameters
such as molecular weight and size, density of electric charge, surface activity toward
standard surfaces, hydration energy, or affinity for protons or metal ions (Leenheer 1981,
Leenheer 1984, Hongve et al. 1989, Dycus et al. 1995, Ciavatta et al. 1995). These
fractionation parameters can be viewed as complementary: if one of them is used in the
separation, the NOM fractions so obtained may be further processed based on the other
parameters. Thus, any discussion of NOM properties must begin with a recognition that

4
NOM is a multi-dimensional chemical entity, and that no single characterization tool can
adequately describe it.

Many analytical (characterization) techniques have been applied both to highly


and slightly fractionated NOM samples. These analyses can be used conjunctively to
understand NOM. For example, the hydrophobic fraction of the NOM can be isolated,
spectral techniques can probe the structure of this fraction before and after reaction with
an oxidant, and the stoichiometry and kinetics of DBP formation when the fraction is
chlorinated can be followed. Collectively, the results can be used to infer linkages among
molecular structure, properties and reactivity.

Interpretation of the results of such analyses is straightforward and quantitative in


some cases (elemental composition), but is complex and qualitative in others. Few
studies have attempted to integrate the information from numerous characterization
approaches. The objectives of the fractionation studies in the current project were to
optimize and expand the preparative DOC fractionation approach that was originally
proposed by Leenheer and Huffman in 1976 and that has evolved extensively since that
time. One specific goal was to modify the procedures to maximize recovery of NOM,
with special attention focused on development and testing of new approaches for
recovery and characterization of hydrophilic NOM fractions.

NOM FRACTIONS AND WATER QUALITY ISSUES

For the potable water industry, the major goal of NOM characterization is to
understand and predict the reactivity of NOM or its fractions in specific treatment
processes. Any water treatment plant is likely to have specific compliance issues and/or
operational problems that require attention and for which certain types of NOM
characterization are useful. For example, if biological activity in the distribution system
is a major issue, then attention should focus on chemical classes contributing to the
biodegradable compounds in solution (e.g., proteins, carbohydrates, amino acids) and
less attention can be paid to humic species (unless they have been altered by ozonation).

5
Alternatively, if the concentration of disinfection by-products is the major concern, the
humic part of NOM should receive special attention, and losses of carbohydrates and
proteins during the NOM collection process will not affect the results dramatically.
However, for some halogenation products (e.g., haloacetonitriles, trihalonitromethanes,
and cyanogen halides), minor chemical classes of NOM may be disproportionately
important, and NOM concentration techniques that fail to retain these classes will yield
concentrates whose properties do not represent those of the original NOM. Since no
concentration technique is able to retain all NOM, the selection of appropriate
concentration and characterization technique is a matter of judgment.

Table 1.1 provides a general evaluation of water quality issues associated with
NOM, and the fractions of the NOM that are most likely to be relevant for each issue.
Table 1.2 provides general information relating different NOM fractions to the formation
of some important disinfection by-products. When NOM concentration is necessary in
order to explore a particular issue, knowledge regarding which NOM classes are
concentrated efficiently and which are lost becomes very important.

6
Table 1.1
Major chemical classes of compounds included into NOM and associated water quality problems
Associated compliance problems
Chemical class Disinfection Disinfection Biological Color Transport of Transport of Taste and odor
of compounds by-products, by-products, activity pesticides heavy
chlorination ozonation metals
Humic species Major role Major role Little Major Major role Major role Secondary
impact * role importance
Carbohydrates Not known, Probably not Major role None Significant? Insignificant Insignificant
probably significant
insignificant
Amino acids Important May be Major role Major Significant? Secondary Insignificant
significant † importance
Proteins Important Important † Major role Major Significant? May be Insignificant
significant
Carboxylic Important Generated Secondary None None Insignificant Insignificant
acids by ozonation importance
Other Primarily geosmin
and 2-methyl-
isoborneol (2-MIB)
* ozonated NOM and ozonation by-products form a major group of BDOC
† see LeLacheur and Glaze (1996), Hureiki et al. (1993), Hureiki (1993).

7
Table 1.2
Association between specific types of disinfection by-products and major chemical
classes of NOM

Class of chemical compounds


Class of disinfection Humic species Carbohydrates Amino acids Proteins Carboxylic acids
by-products
Trihalomethanes Primary Not known, Minor * Important * Secondary
(THM) source probably minor source†
Haloacetic acids Primary Not known, Not known, Not known, Secondary
(HAA) source probably minor probably may be source
insignificant significant
Chlorophenols Primary Insignificant Insignificant Insignificant Insignificant
source
MX Primary Not known, Not known, Not known, Insignificant
source‡ probably probably probably
insignificant insignificant insignificant
Haloketones Primary Not known, Not known, Not known, Insignificant
source may be probably may be
significant insignificant significant
Chloral hydrate Primary Not known, Not known, Not known, Insignificant
source probably probably probably
insignificant insignificant insignificant
Haloacetonitriles Important Not known, Important Important Insignificant
probably
insignificant
Trihalonitromethane Important Not known, Important Important Insignificant
probably
insignificant
Cyanogen halide Important Not known, Important Important Insignificant
probably
insignificant
Aldehydes Primary Not known, Not known, Important Not known,
source § may be probably probably minor
significant insignificant
Contribution to the Predominant Little known, Important Important Probably 5-10%
pool of DBP (80-90%) probably <5% (5-10%) (5-10%)
precursors
* some amino acids produce THMs (Larson and Weber 1994) but their concentration is much smaller than
that of humic species
† β-hydroxy and β-keto acids have been shown to produce THMs upon chlorination (Larson and Weber
1994)
‡ Xu et al. 1997, DeMarini et al. 1995, Smeds et al. 1997, Conrad and Huck 1996
§ Paode et al. 1997

1
The research project described in this report represents an attempt to compare
approaches for concentrating, isolating, and fractionating NOM from natural waters. The
overall goals of the project were: (1) to obtain a better understanding of what can be
learned about NOM from each of several analytical techniques; (2) to develop techniques
to better integrate the available characterization methods with one another and with
fractionation techniques; (3) to draw (preliminary) generalizable conclusions about
similarities and differences among NOM fractions and NOM sources; and (4) to forge
more links between structural information and reactivity.

The work was carried out by three research groups on two continents. Water
samples that were tested came from rivers and reservoirs in the Pacific Northwest, the
Southwest and the Southeast of the United States, and from several locations in France
and one in England. Each water supply was subjected to processing and testing at each of
the participating laboratories, but the intensity with which a given water sample was
studied varied from laboratory to laboratory and from one sample to the next.

In the course of the research, an unexpected and remarkably strong relationship


between the formation of chlorinated disinfection by-products and the change in UV
absorbance of a sample was discovered. That relationship was studied intensively, and
the results of that effort represent a significant outcome of the research project. However,
that study was sufficiently distinct from the rest of the project that, given the amount of
data collected in the characterization, isolation, and fractionation studies, a decision was
made to exclude the results from this report. Some of the results relating to the DBP-UV
relationship have been published in journal articles (Korshin et al. 1996, 1997a; Li et al.
1998), and other publications are forthcoming.

The remainder of this report is divided into seven chapters. Chapter 2 provides a
literature review and context for the current research. Chapter 3 describes the sampling,
processing, and analytical approaches that were used in the research. Chapter 4 focuses
on NOM concentration and isolation methods, i.e., methods intended to collect NOM as
efficiently as possible from a water source and to isolate it from solution and from
inorganic salts. Chapter 5 provides a detailed description of NOM characterization

2
techniques, using the Suwannee River and South Platte River NOM as case studies.
NOM from these two rivers was subjected to the most intensive characterization, and the
similarities and differences between the NOM from the two samples are instructive for
understanding both the universal and the site-specific aspects of NOM composition and
behavior. In Chapter 6, the results from analysis of fractionated NOM samples from a
range of water sources are presented. The thrust of the chapter is both to describe those
fractions in a way that provides information about the water source and to show whether
and how different NOM processing approaches might lead to slightly different inferences
about the NOM characteristics. Chapter 7 describes some of the correlations that were
obtained among different types of analyses, suggesting ways that information about
NOM might be obtainable from some in situ analytical techniques that have not been
utilized to their full potential in the past. Chapter 8 summarizes the key findings of the
research and offers suggestions for future research.

3
CHAPTER 2 LITERATURE REVIEW

OVERVIEW

This chapter provides background information on techniques that have been


utilized to concentrate, isolate, fractionate, and characterize NOM. The variety of
approaches that have been used to accomplish these goals is enormous, and a
comprehensive review is beyond the scope of the current effort. Rather, an attempt has
been made to summarize the historical development of the approaches that are in
common use and to describe the key advantages and disadvantages of the techniques used
in the current project.

The first section of the chapter is not formally a literature review, but rather
provides background information regarding the preliminary steps that usually are (or, in
the authors’ opinions, should be) carried out before NOM can be analyzed and
characterized confidently. The remainder of the chapter is organized into two broad
sections: one on concentration, isolation, and fractionation techniques, and one on NOM
analysis.

As noted in Chapter 1, while it is relatively easy to draw conceptual distinctions


among various concentration, isolation, and fractionation procedures, no NOM
processing approach accomplishes any of these three goals completely, and many
processing steps accomplish more than one of the goals simultaneously (albeit
incompletely). For instance, adsorption of NOM onto resins and its subsequent elution is
used to collect specific sub-groups (fractions) of the NOM in the sample, to separate
those molecules from water and dissolved salts, and to concentrate the NOM into a small
volume of eluent.

On the other hand, some NOM processing techniques (e.g., reverse osmosis,
evaporation, and freezing) accomplish concentration with minimal isolation and
fractionation. In waters with little salt, such as some soft, black-water rivers, these
4
techniques by themselves might be adequate for collecting NOM samples to characterize
the organic matrix of the water. These techniques are described next, followed by those
techniques that accomplish substantial isolation and/or fractionation. In the latter portion
of the chapter, techniques for characterizing NOM are reviewed.

PRELIMINARY CHARACTERIZATION

Preliminary analyses should be conducted on water samples to determine


combinations of concentration, fractionation, and isolation techniques that are applicable.
The most important of these measurements are determinations of total and/or dissolved
organic carbon (TOC or DOC) concentration, turbidity and/or suspended solids
concentration, and specific conductance, as a surrogate for the salt concentration. All of
these parameters can be analyzed by standard techniques (Standard Methods 1995).

These analyses provide information about the composition of the water sample
and the amount of NOM that can be isolated from it, and guidance regarding the
combination of techniques that can be used to collect the NOM. For instance, because
NOM is approximately 50% by mass, the mass of NOM that can potentially be isolated
from a given water sample can be estimated as the product of the water volume and twice
the TOC or DOC concentration.

Because ion exchange resins are used in many NOM fractionation and isolation
techniques (Leenheer 1981, 1984; Leenheer and Noyes 1984), knowledge of the ionic
salt concentration of samples is essential to avoid exceeding resin exchange capacities.
As a first approximation, the salt concentration of many natural water samples can be
estimated by Equation 2.1 (Fireman and Reeve 1948):

⎛ meq ⎞
Salt concentration ⎜ ⎟ = 12.5 x Specific Conductance at 25 C (mS)
o
(Equation 2.1)
⎝ L ⎠

5
Note, however, that certain dissolved inorganic solutes (e.g., silicic and boric acids) are
nonionic at neutral and acidic pH values and are not detected by specific conductance
measurements.

Finally, organic contaminant discharges into the water sources should be noted;
especially as water reuse increases, organic matter composition and concentration in
some water bodies may become dominated by societal inputs.

Most efforts to characterize NOM in drinking water sources have focused on the
solution phase, which typically contains >90% of the NOM in the sample. In such cases,
particulate and colloidal organic carbon is separated from the sample prior to its
characterization, generally by some combination of centrifugation, filtration and/or
ultrafiltration methods (Aiken and Leenheer 1993, Rees et al. 1991). Particulate (supra-
colloidal) carbon is sometimes defined as organic carbon in particles greater than 1 µm in
size (**MB—change ref to list authors, Characterization of Natural Organic Matter
1993), but as a practical matter, it is most often separated from the aqueous phase by
filtration through filters with 0.45-µm pores.

There is no generally accepted definition of the size cut-off between colloidal and
particulate matter. Although many membrane filters have well-defined pore sizes, the
effective particle-size cutoff using such membranes is indefinite because of the
progressive plugging of membrane pores (Horowitz 1996). Glass fiber cartridge filters
are less susceptible to plugging than membrane filters (Leenheer and Noyes 1984), but
are not immune to it. The least ambiguous way to separate particles of various sizes from
a natural water sample (and from one another) appears to be continuous-flow
centrifugation followed by tangential-flow (sometimes called crossflow) ultrafiltration.
This sequence minimizes the possibility of filter plugging and allows large volumes of
water with high suspended sediment concentrations to be processed (Leenheer et al.
1989). The choice of which separation process to use in a particular situation depends on
the objectives of the study, the equipment available, and the suspended sediment
concentration of the water.

6
TECHNIQUES FOR CONCENTRATION, ISOLATION AND FRACTIONATION
OF NOM

Techniques Intended Primarily for Concentration of NOM

Evaporation

Vacuum evaporation at smaller scales has often been used as a preliminary step to
concentrate NOM and thereby make subsequent resin sorption procedures more efficient
(Aiken and Leenheer 1993), and it is extensively used to remove water or organic
solvents prior to freeze-drying (lyophilization) of an NOM isolate. It is also used to distill
water from a solution containing a miscible organic solvent such as acetic acid in a
procedure called zeotrophic distillation. This procedure can be used to partially desalt
certain NOM fractions (Aiken and Leenheer 1993). However, much larger evaporators
than are commonly used in the laboratory are required to concentrate the NOM from
large quantities of water, and such units have not been widely employed for this purpose.
One such unit, employed by the USGS research group in the current research, processes
water at a rate of 2 to 4 L/h.

Water samples should not generally be taken to dryness with vacuum evaporation
because the combination of heat from the water bath and high salt concentrations can
cause the NOM to hydrolyze, dehydrate, and polymerize with itself and inorganic
constituents such as silica.

The water processing rate typically achievable by vacuum evaporation is slow


relative to other, more widely used NOM concentration techniques (membrane- and
sorption-based procedures), although it is rapid relative to freeze-drying and freeze
concentration techniques. Some volatile NOM solutes are lost with evaporation, but such
species usually account for a very small percentage of the DOC (Thurman 1985). On the
other hand, small non-volatile hydrophilic solutes are recovered by vacuum evaporation

7
but are frequently lost when membrane- or sorption-based concentration techniques are
used.

Freeze concentration and freeze-drying

Freeze concentration of water is based upon the principle that ice excludes solutes
during the freezing process. It is a very gentle concentration technique and is especially
good for the recovery of semi-volatile solutes that are lost with evaporative techniques.
However, the technique is slow and its use is not widespread for concentrating water
samples. The apparatus most often used for freeze concentration was designed by Shapiro
(1961). A review of applications of freeze concentration to water samples is given by
Leenheer (1984).

Freeze-drying (lyophilization) is generally accepted as the best method for


producing dry NOM isolates that have been minimally altered from their dissolved
composition and that have physical properties that are reasonably convenient for
subsequent sample handling (Malcolm 1968). Only a few liters of water per day can be
lyophilized using a typical freeze-dryer. Care must be taken to avoid losses of fluffy low
density isolates (usually hydrophobic NOM fractions) that can become entrained in the
flow of water vapor exiting the sample. Vacuum evaporation of the sample prior to
freeze-drying produces higher density isolates that are less prone to escape. Alternatively,
very porous filters can be used in the necks of freeze-drying flasks to trap some of the
fluffy NOM isolates without restricting the vapor flow. If salts or inorganic acids are
present in solution, the freezing point of the solution decreases during the freeze-drying
process. In such cases, the ice might melt, and the isolate can become a sticky mixture of
NOM and salt that is difficult to remove from the flask.

Membrane technologies

The efficiency with which NOM is retained by membranes depends strongly on


the membrane composition and pore size, the volume concentration factor (CF), and the
8
ionic strength of the solution. Relatively few studies have investigated the use of
membranes specifically to isolate aquatic NOM. These studies were reviewed by Aiken
and Leenheer (1993). However, a large number of research projects have evaluated the
efficiency of membrane processes for the production of drinking water, and these studies
provide some data on the behavior of NOM in such systems. Both nanofiltration (NF)
and reverse osmosis (RO) can be highly efficient processes for removing natural organic
matter from water. The results from a few studies in which RO was used are summarized
in Table 2.1.

Table 2.1
Some literature values for DOC rejection and recovery efficiencies by RO

Source DOC rejection (%) Reference


Moose Pit Brook (USA) 98.2 Clair et al. (1991)
Moose Pit Bog (USA) 98.5 Clair et al. (1991)
Tupper Lake (USA) 95.9 Clair et al. (1991)

DOC recovery (%)


Ogeechee River 83 to 94 Sun et al. (1995)
Watersheds (Coweeta) 74 to 92 Sun et al. (1995)
Clinch River (TN) 88 Sun et al. (1995)
Groundwater (Hanford-WA)) 89 to 96 Sun et al. (1995)
Suwannee River 85 Sun et al. (1995)
Suwannee River (4/1987) 89.6 Serkiz and Perdue (1990)
Suwannee River (10/1988) 90.1 Serkiz and Perdue (1990)

Serkiz and Perdue (1990) suggested that aromatic polyamide membranes would
be superior to cellulose acetate membranes for recovery of NOM. DiGiano et al. (1993)
used a thin surface film composite of polysulfone (a negatively charged membrane) to
concentrate NOM. They cited articles that indicated that NOM charge and other physical/
chemical interactions between the membrane and NOM can significantly influence the
rejection efficiency.

9
The use of RO or NF to isolate NOM offers three significant advantages over
adsorbent-based NOM isolation techniques (discussed below). First, a large volume of
water can be processed in a short period of time; second, the NOM is never subjected to
extreme pH values that could alter its structural properties; and third, according to at least
one report (Serkiz and Perdue 1990), membranes seem to be more efficient than XAD-8
resin at collecting polysaccharides and polypeptides.

The major drawback of membrane isolation procedures is that they usually


concentrate inorganic salts along with the NOM, albeit not necessarily with the same
efficiency. Although some waters contain a sufficiently low concentration of inorganic
salts to allow NOM concentration by RO without pre-treatment, the need to pre-treat
most natural waters to reduce the risk that inorganic species such as CaCO3 and CaSO4
will precipitate on the surface of the membrane is well accepted. Even if salts do not
interfere with operation of the RO unit, subsequent analyses of the organic matter
collected by RO may require separation of salts that are concentrated with the NOM.

Sample pre-treatment to remove the salts of concern is often accomplished by


passing the water through a Na+-saturated cation exchange resin. Na+ is preferred as the
exchangeable ion over H+ to avoid lowering the pH of the water, because acidification of
the water could protonate low molecular weight acids and cause them to pass through the
membrane (Chian et al. 1976, Clair et al. 1991, Sun et al. 1995). Acidification could also
cause humic substances to precipitate in the membrane or at the membrane surface (Clair
et al. 1991, Clark and Jucker 1993, Sun et al. 1995). However, if the solution is subjected
to cation exchange to prevent precipitation of calcium, the basic fractions of NOM might
be retained by the resin. Thus, pre-treatment using ion exchange has drawbacks as well as
benefits, and the importance of removing hardness cations from a given sample prior to
NOM concentration needs to be carefully evaluated before deciding whether and how to
pre-treat the sample. At present, no membrane can separate NOM efficiently from
inorganic salts while maintaining a high NOM recovery efficiency.

Sulfate and silica are particularly problematic inorganic species that get
concentrated by RO membranes (Serkiz and Perdue 1990, Sun et al. 1995). Silica can

10
cause siliceous precipitates to form inside the membrane system and clog it. Sulfate is
problematic because it forms sulfuric acid if H+-cation exchange is used as a desalting
step downstream of the RO process and prior to lyophilisation. The sulfuric acid might
then react with the NOM to form sulfonated compounds, or to modify it in other ways
during the subsequent processing steps.

Another potential problem with using membranes to concentrate NOM is that the
NOM may foul the membrane surface or its pores. A number of researchers (e.g.,
Wiesner and Chellam 1992, DiGiano et al. 1993) have reported that NOM can adsorb
onto membranes and that high molecular weight organics (i.e., humic substances) adsorb
preferentially. If the NOM concentration near the membrane exceeds some critical value,
it can form a gel-like layer that increases the resistance to water flux and is sometimes
very hard to remove (Chang and Benjamin 1996, Chang et al. 1998).

While the drawbacks noted above can decrease the attractiveness of membrane
techniques for concentrating NOM, it should be noted that RO is one of the most efficient
NOM concentration techniques that has been applied to natural water samples. For
instance, Serkiz and Perdue (1990) used RO to process 150-180 L/h of water from the
Suwannee River using a field-portable unit mounted on a medium-sized hand truck. The
DOC recovery efficiency was 90%. That work probably presents a best-case application
of RO because of the low salt content of the sample that was processed. The overall loss
of 10% of the NOM resulted from passage of about 5 to 7% of the DOC through the RO
membrane and, presumably, sorption of a few percent of the DOC onto either the
membrane or the ion exchange media. The sorbed compounds were thought to be high
molecular weight dissolved or colloidal material.

Sorption Techniques for Concentrating, Isolating, and Fractionating NOM

Sorption-based techniques can simultaneously concentrate, isolate, and


fractionate NOM. In this section, the general operation of column adsorption systems is

11
discussed, and then the results of efforts to collect NOM with specific adsorbents are
reviewed.

Quantitative Analysis of the Use of Adsorptive Columns for NOM Isolation and
Fractionation

Sorption is used both in small-scale systems to process NOM for subsequent


analysis (in which case the process is often referred to as chromatography) and in large-
scale processes for drinking water treatment. As water containing NOM passes through a
column packed with adsorbent particles, some of the NOM molecules sorb to the packed
media. The sorption efficiency depends on the characteristics of the NOM molecules, the
media, and other aspects of the solution composition (especially pH, but also the
concentration of competing adsorbates and other molecules that interact with the NOM).
Eventually, the capacity of the media to sorb the molecules under the given conditions is
reached, and the molecules appear in the effluent in substantial amounts (i.e., they ‘break
through’ the column).

The ratio of the volume of water treated to the volume of the packed media
(Vwater/Vbed) is referred to as the number of empty bed volumes (or simply the number of
bed volumes, BVs) of water treated. The number of empty bed volumes treated can be
converted to the number of void volumes treated by dividing the former by the porosity
of the bed.

In the water treatment literature, it is common to characterize the amount of water


processed before breakthrough in terms of the number of bed volumes treated. In the
chromatography literature, this same concept is commonly quantified in terms of the
adsorptive affinity of a molecule that would be present in the column effluent at a
concentration equal to 50% of its influent concentration, i.e., that would be at 50%
breakthrough, when a given volume of water had been processed. In other words, the
water treatment literature typically describes the water volume processed prior to
breakthrough for molecules with a particular affinity for the surface, whereas the

12
chromatography literature typically describes the affinity for the adsorbent of molecules
that break through after a particular volume of water has been processed. It is important
to recognize that these are simply two different ways to express the same information.

When discussing the efficiency with which NOM molecules is retained by a


column, it is important to distinguish between the instantaneous NOM concentration in
the effluent and the integrated NOM concentration in the effluent from the beginning of
the test. If the breakthrough curve is sharp and can be reasonably approximated as an
instantaneous increase in the effluent concentration from zero to complete breakthrough
(a square wave), one can treat the system as though all of the molecules entering before
the time of breakthrough are retained, and all of those entering thereafter are not retained.
In such a case, the volume of water processed at 50% cumulative breakthrough equals
twice the volume corresponding to 50% instantaneous breakthrough (Figure ** MB
prepare figure). This latter approximation can also be made if the breakthrough curve is
approximately symmetric even if it is not sharp, but in this case the approximation that all
of the material entering the column prior to 50% breakthrough is adsorbed does not
apply.

Because NOM is a collection of diverse molecules, different fractions of the


NOM break through the column at different times, corresponding to processing of
different numbers of bed volumes of influent. The higher the affinity of a group of NOM
molecules for the media surface, the larger is the number of bed volumes of water that
can be processed before the molecules break through.

For any adsorption column, the overall, cumulative NOM retention efficiency (η)
can be computed as follows:

∫ ( DOC influent − DOC effluent )dV


η=
0
V (Equation 2.2)
∫ DOC
0
influent dV

13
where V is the volume of liquid that has been processed. If the flow rate through the
column is constant, the volume integrals on the right-hand side of Equation 2.2 can be
replaced by time integrals:

∫ ( DOC influent − DOCeffluent )dt


η= 0
t (Equation 2.3)
∫ DOC
0
influent dt

If the breakthrough curve can be approximated as a square wave, then DOCeffluent


is zero before breakthrough and DOCinfluent after breakthrough. In that case, η can be
computed as follows:

η = 100% at t < t breakthrough ⎫



t breakthrough ⎬ (Equation 2.4)
η= at t > t breakthrough ⎪
t ⎭

Consider a group of NOM molecules that have strong and approximately equal
affinities for the adsorbent, so that they break through the column as a square wave. After
breakthrough, the concentration of these NOM molecules in solution throughout the
column is Cinfluent. The concentration of NOM adsorbed to the media after breakthrough is
also constant throughout the column and can be represented as the product of a constant
and Cinfluent:

C ads = k C influent (Equation 2.5)

where Cads is the concentration of adsorbed NOM in mg of organic carbon per liter of
adsorbent media, and k is the conditional adsorption equilibrium constant (applicable for
the given influent composition), sometimes called a distribution coefficient. The
adsorbed concentration can also be expressed as the mass of organic carbon adsorbed per

liter of packed bed ( C 'ads ) by multiplying both sides of Equation 2.5 by the bed porosity

ε. Defining k' as k ε, Equation 2.5 becomes:

14
C 'ads = C ads ε = k εC influent = k' C influent (Equation 2.6)

k' is called the column capacity factor. It is the parameter used in the
chromatography literature to characterize the affinity of adsorbents for dissolved
molecules. At the time of breakthrough, the total mass of the molecules of interest that
are in the column includes εVbedCinfluent in solution plus Vbed k' Cinfluent bound to the
media, for a total of (ε + k')VbedCinfluent. The total mass of such molecules applied to the
column up to the time of breakthrough is VinfluentCinfluent. Assuming 100% retention up to
that time, these latter two terms can be equated to yield:

(ε + k')VbedCinfluent = VinfluentCinfluent (Equation 2.7)

Vinfluent
ε + k' = = BVbreakthrough (Equation 2.8)
Vbed

where BVbreakthrough is the number of bed volumes treated before breakthrough. Typically,
k′>>ε, so Equation 2.8 can be approximated as:

Vinfluent
k' = = BVbreakthrough (Equation 2.9)
Vbed

Equation 2.9 makes the point mathematically that is described in text above, that the
column capacity factor (which is used to describe sorption in chromatography columns)
is directly related to the number of bed volumes of water that can be processed prior to
breakthrough (which is used to describe sorption in columns used for water treatment).

In the literature, adsorptive columns are sometimes described as being operated


‘at a column capacity factor of x’. What this really means is that the amount of sample
being applied to the column corresponds to the amount that would cause molecules with
a column capacity factor (k') equal to x to just barely break through the column. Put
another way, if a column is operated at a column capacity factor of x, the number of bed
volumes of water being processed equals x. Under these conditions, molecules with k'
equal to or greater than x would be almost completely sorbed (assuming a square wave

15
breakthrough pattern), and molecules with k' values less than x would break through the
column before the processing is complete and would be incompletely captured. The
lower the value of k' for a group of molecules relative to x, the less efficiently the
molecules will be captured by the adsorbent.

Sorption onto Fe oxides and ion exchange resins

Both iron and aluminum oxides are used to collect NOM and aid in its removal
from drinking water in coagulation processes. The mechanisms by which the coagulants
react with NOM are essentially identical. In this section, the focus is on NOM
interactions with iron oxides, since iron oxides were used as NOM adsorbents in the
research.

The ability of iron oxides to adsorb many inorganic ions and NOM molecules is
well established (Dzombak and Morel 1990; Levashkevich 1966; Parfitt et al. 1977;
Parfitt and Russell 1977; Tipping 1981; Tipping and Cooke 1982; Loder and Liss 1982;
Gu et al. 1994, 1995; Korshin et al. 1996). NOM binds to iron oxides via specific
chemical interactions, as indicated by the fact that the oxide surface acquires a negative
charge when sufficient NOM sorbs to it (Tipping and Cooke 1982, Loder and Liss 1982).
Many of the specific chemical interactions are believed to involve replacement of
surface-coordinated H2O or OH− groups by carboxyl functional groups of the NOM
molecules (Parfitt and Russell 1977; Gu et al. 1994, 1995) in reactions that can be
represented generically as follows:

R-COO− + ≡FeOH ↔ ≡FeOOC-R + OH− (Reaction 2.1)

Consistent with Reaction 2.1, adsorption of NOM on hydrous iron oxides


generally decreases with increasing pH, with sorption typically becoming negligible at
pH > 8 (Tipping and Cooke 1982, Loder and Liss 1982). On the other hand, pH values
considerably higher than 8 are sometimes required to desorb NOM from oxides once it
has adsorbed at a lower pH. The above reaction also makes evident the importance of

16
acidic groups in NOM molecules for the performance of iron oxide-based adsorbent
media and explains why acidic fractions of NOM adsorb preferentially to such media.

Solution pH also affects NOM adsorption by altering the degree of ionization of


carboxyl groups in the NOM. At low pH’s, the degree of ionization of acidic groups in
NOM molecules decreases, and the overall charge on the molecules becomes less
negative or neutral. As a result, electrostatic repulsion between adsorbed NOM molecules
decreases and their maximum surface density (the apparent adsorption capacity of the
oxide surface) increases. Simultaneously, the charge reduction neutralizes the
electrostatic repulsion between ionized functional groups within individual molecules,
causing them to form more compact and more hydrophobic structures. This
conformational change can decrease the affinity of the molecules for cationic surface
sites on hydrophilic surfaces (e.g., for Fe sites on iron oxides), while enhancing NOM
adsorption onto hydrophobic surfaces (such as XAD-8 and XAD-4 resins). The net result
of the competing processes described above is that the adsorption of NOM onto oxide
surfaces increases as pH decreases from around 8 to 4 or 5, and then either remains
approximately constant or decreases as the pH is lowered further.

Although NOM removal efficiency can be controlled to a substantial extent by


adjusting solution pH and the concentration of adsorbent, a portion of the DOC cannot be
adsorbed even at very high oxide concentrations. The residual (non-adsorbable) NOM
typically includes lower-MW, less polar, and less acidic NOM molecules (Sinsabaugh et
al. 1986a, Semmens and Staples 1986, Korshin et al. 1997b).

The ability of iron oxide-based adsorbent media to retain NOM fractions may be
substantially compromised by competition from naturally occurring inorganic anions.
The major ion of concern in this respect is sulfate, which can be present in natural waters
at concentrations exceeding 100 mg/L. Sulfate interference with NOM adsorption is
expected to be most severe in high-sulfate, low-NOM waters, but even in low-sulfate
waters, sulfate ions may be problematic if they adsorb and subsequently elute with the
NOM. In such cases, additional desalting steps may be necessary before certain analyses

17
can be carried out on the NOM (e.g., analyses for elemental composition and IR
absorption characteristics).

Interactions of ion exchange media with NOM molecules bear some similarities
to those of iron oxides, although the different adsorbents might target somewhat different
groups of molecules, and the sorption of NOM onto iron oxides generally involves more
specific interactions. Ion exchange has been used by a number of researchers to collect
NOM (along with accompanying salts) and fractionate it into basic and acidic fractions.
Basic NOM can be efficiently adsorbed on hydrogen-form, strong acid cation exchange
resins and then can be eluted with ammonium hydroxide. However, reactions of ammonia
with ketone, ester, and quinone groups might alter the NOM in the process (Thorn et al.
1992). Acidic NOM is difficult to elute from strong-base anion exchange resins, but high
recoveries are obtainable using weak-base anion exchange resins to collect the NOM,
using sodium hydroxide as an eluent. As with iron oxide adsorbents, inorganic anions are
often co-eluted with the NOM from ion exchange media and may present a problem for
subsequent processing or analysis.

Sorption onto hydrophobic sorbents (XAD and similar resins)

Leenheer (1984) provided an extensive review of the use of hydrophobic sorbents


to isolate and fractionate NOM as of the mid-1980’s. Although activated carbon has high
sorption efficiencies for NOM and was used in a number of early studies as an NOM
sorbent, only half to two-thirds of the NOM could be eluted, and there was some
evidence that eluted NOM was chemically altered. Various polyamide sorbents have
been used as NOM adsorbents, but problems with NOM desorption have also limited
their use.

Presently, the procedure used most extensively for isolating aquatic NOM is
sorption onto XAD-type resins. These and similar non-ionic, macroporous resins
combine relatively high adsorption affinities for NOM with high elution efficiencies and
very low affinities for inorganic salts. The resins adsorb NOM through a combination of

18
non-polar (hydrophobic) and polar (hydrogen bonding and aromatic π-electron)
interactions. They have better sorption efficiencies and better stability when exposed to
acidic and basic eluents than the C-18 silica based sorbents that are popular for isolating
various contaminants from water, and they can be used with higher processing rates.
Although the hydrophobicity of the NOM molecules is the major determinant of their
tendency to sorb to XAD resins, the molecules’ acid-base characteristics and size play an
important role as well, since these characteristics have a strong effect on the
hydrophobic-hydrophilic nature of the molecules. (Charged molecules, whether acidic or
basic, are much more hydrophilic than their uncharged conjugate species.) Molecular size
also helps determine the extent to which NOM molecules bind to the resins, since much
of the binding capacity is in internal pores that might not be accessible to large
molecules.

Over two decades ago, Leenheer and Huffman (1976) proposed using XAD and
ion exchange resins in a hierarchical fractionation procedure that characterizes NOM
molecules based on their hydrophobic-hydrophilic and acid-base properties. This
approach was developed further in subsequent years, and a version of the protocol, first
proposed by researchers at the USGS in 1981 (Leenheer 1981, Thurman and Malcolm
1981) has become virtually a reference method for the isolation of humic and fulvic
acids. The approach has been further expanded and modified since that time (Aiken and
Leenheer 1993, Leenheer 1981, Leenheer and Noyes 1984, Leenheer 1997). It still
provides the basic framework for most fractionation studies and is the procedure against
which alternative fractionation approaches are measured.

In any version of the basic procedure, NOM is separated into two fractions, which
are referred to as humic and non-humic by some researchers and as hydrophobic and
hydrophilic, respectively, by others. The humic fraction comprises the NOM molecules
that adsorb at acidic pH onto an XAD-8 resin column using a column capacity factor (k')
on the order of 50 to 100 (different protocols use slightly different k' cut-offs), while the
non-humic fraction passes through the column. Each fraction is often further fractionated
into acidic, basic, and neutral fractions by selective elution and/or subsequent sorption

19
and elution procedures. Generally, more than 90% of the material adsorbed on XAD-8
can be eluted with base and is therefore identified as hydrophobic acids.

Typically, the DOC in surface waters is approximately evenly split between the
XAD-8 adsorbable (humic or hydrophobic) and non-sorbable (non-humic or hydrophilic)
fractions (Leenheer and Huffman 1976, Leenheer 1981, Thurman and Malcolm 1981,
Martin-Mousset et al. 1997). Martin-Mousset et al. (1997) reported that the hydrophobic
fraction is generally slightly more abundant in reservoir water (51 to 62% for four water
sources) than in river water (41 to 50% for four water sources), perhaps due to the
adsorption of hydrophobic NOM onto river-borne sediments. Similar results were
obtained by Semmens and Staples (1986) and Collins, Amy and Steelink (1986).

Leenheer (1981) proposed a protocol for isolating and fractionating the NOM that
is not sorbed by XAD-8, but this protocol has not been used as extensively as the method
for isolating the adsorbed (hydrophobic) fraction. Recently, Aiken et al. (1992), Malcolm
and MacCarthy (1992), Croue et al. (1993a), and Andrews and Huck (1994) have used
XAD-4 resins at acidic pH to collect a portion of the NOM that does not sorb to XAD-8
under the conditions used to collect the hydrophobic NOM. These authors reported that
25 to 30% of the DOC of the raw water, corresponding to 50 to 60% of the NOM passing
through the column packed with XAD-8, adsorbs onto the XAD-4. About 75 to 80% of
the NOM that sorbs onto the XAD-4 resin can be eluted with base. Although previous
authors have referred to the NOM that sorbs to XAD-4 and is released by elution with
base as the ‘hydrophilic acid’ fraction or the ‘XAD-4 acids’, this NOM is designated as
‘transphilic’ in the current report, to distinguish it from more hydrophilic NOM that does
not sorb to either XAD-8 or XAD-4 under the specified conditions.

Processing samples as described above typically collects about 50% of the NOM
if only an XAD-8 column is used, and about 75% of the NOM if both XAD-8 and
XAD-4 columns are used. In more sophisticated isolation and fractionation procedures,
additional steps can be employed to collect more of the NOM. As noted in Chapter 1,
these steps typically offer diminishing returns: more and more effort is required to collect
progressively smaller incremental amounts of NOM. Nevertheless, in cases where the

20
goals of the study justify these efforts, procedures have been proposed to capture more of
the NOM. For instance, Aiken et al. (1992) used XAD-8 and XAD-4 resins in series to
isolate essentially all hydrophobic and transphilic NOM molecules with column capacity
factors k' > 100, respectively. Recoveries from the XAD-4 resin can be improved by
using vacuum evaporation to concentrate the effluent from the XAD-4 resin to the point
of salt saturation and then passing the concentrated solution through another column
packed with XAD-4 to isolate the NOM molecules with k' between 5 and 100. Some
molecules sorb in the second exposure to XAD-4 but not the first exposure because fewer
bed volumes of sample are passed through the column in the second exposure
(corresponding to the lower k' value, per Equation 2.9) (Aiken and Leenheer 1993). That
is, in the first exposure step, enough sample is applied to the column to allow weakly
binding molecules to break through, whereas in the second exposure, the processing is
stopped when these same molecules have not yet broken through the column. In this
report, the NOM that adsorbs in the second exposure to XAD-4 is referred to as
‘hydrophilic’, and the NOM that does not adsorb on the XAD-4 resin at this point as
‘ultra-hydrophilic’.

An even more comprehensive NOM fractionation and isolation scheme using


XAD-8 resin to fractionate hydrophobic NOM with selective elution into hydrophobic
acid, base, and neutral fractions, and usng ion exchange resins to fractionate hydrophilic
NOM into acid, base, and neutral fractions has been incorporated by the USGS into a
mobile field laboratory. This system has been described by Leenheer and Noyes (1984).

Desalting

Inorganic salts interfere with many analytical procedures for characterizing NOM.
The need to remove these salts and the best approach for doing so depend on the nature
of the salt species and NOM. This section describes various approaches that have been
used to remove salts from raw water or, more often, from water that has been processed
to concentrate NOM and in which the salts have been concentrated as well.

21
Ion exchange

Anion exchange resins have been used by a number of researchers to concentrate,


fractionate, and isolate NOM acids (Leenheer 1984). The two major problems associated
with the use of anion exchange resins are: (1) NOM is difficult to elute from most anion
exchange resins, and (2) inorganic anions are also concentrated on the resins and are
often eluted with the NOM acids. The elution problem can be overcome by using a weak-
base anion exchange resin with a phenol-formaldehyde matrix that reverses its charge
from positive at low pH to negative at high pH (Leenheer 1981). One such resin was used
to concentrate organic hydrophilic NOM acids in this project.

Cation-exchange resins in the hydrogen-form and chelating resins in the cupric


ion form have been used to isolate amino acids and peptides from fresh and salt waters
(Degens and Reuter 1964, Siegel and Degens 1966). Amino acids and peptides can be
eluted from these resins with ammonium hydroxide, which does not displace inorganic
cations. Amino acids and peptides usually comprise only a very small percentage of the
NOM in water samples.

Precipitation and Co-precipitation

Leenheer (1984) has reviewed several methods that have been developed for
co-precipitating NOM with metal hydroxides (Al3+, Cu2+, Fe3+, Mg2+, Mn2+, and Pb2+).
These methods are selective for the acid fraction of the NOM and work especially well
on the acids that form complexes with metals. Concentrations of metal ions from about
30 to 50 mg/L have been found to remove about half of the DOC from fresh and sea
water samples. None of the early studies (1960’s and 70’s) using co-precipitation
attempted to recover and purify NOM from the precipitate. However, Aiken and
Leenheer (1993) recently described a process in which cupric hydroxide was used to
co-precipitate NOM, the precipitate was redissolved in acetic acid, co-precipitated sulfate
was removed by precipitation with barium chloride, and barium and copper were

22
removed by ion exchange, so that NOM could be isolated as a residue after vacuum
evaporation and freeze-drying of the ultimate solution.

Desalting with XAD resins

Hydrophobic NOM can be separated from salts dissolved in the sample by


sorption onto and subsequent elution from XAD-8 resins, and transphilic NOM can
desalted in an analogous way with XAD-4 resins. These processes can be applied either
to the raw water or to water in which the NOM has been pre-concentrated, e.g., by
reverse osmosis, vacuum evaporation, or ion exchange. Pre-concentration of NOM
facilitates its recovery, especially for transphilic NOM, because a significant amount of
NOM can be collected while processing a smaller volume of sample, thereby facilitating
collection of NOM molecules with lower column capacity factors (k') (Aiken and
Leenheer 1993). The practical minimum value for k' is 5 for desalting on XAD-4 resin
because of band spreading of the salt peak.

NOM that is not adsorbed by XAD-8 and XAD-4 resins (i.e., the ultra-
hydrophilic fraction) is much more difficult to desalt. Although substantial desalting of
this fraction is possible, it often requires several steps, each of which targets a few
specific salt ions. For instance, zeotrophic distillation (fractional distillation of two
miscible solvents that do not form an azeotrope) of a solution containing water and acetic
acid can lead to precipitation of sodium chloride and calcium sulfate as acetic acid is
enriched during distillation. Therefore, this process can efficiently separate these salts
from ultra-hydrophilic NOM (which remains soluble) during the distillation. However,
calcium and magnesium chloride, nitrate salts, phosphate salts, silicic acid, and boric acid
are soluble in acetic acid and therefore are not separated from the NOM by this process
(Audrieth and Kleinberg 1953). Boric acid can be removed as volatile trimethyl borate by
dissolving the isolate in methanol and heating to dryness three times (Aiken and
Leenheer 1993).

23
Calcium and magnesium can be removed by cation exchange (exchanging these
ions for H+), and HCl can then be evaporated from the NOM by azeotrophic evaporation
with acetonitrile (Aiken and Leenheer 1993). Alternatively, calcium and magnesium
hydroxide can be precipitated with sodium hydroxide at pH 12. Some of the NOM
co-precipitates with magnesium and calcium hydroxides in this procedure. However, the
NOM can be redissolved and separated from the calcium and magnesium by suspending
the solids in a solution of sulfuric and acetic acids and then applying zeotrophic
distillation. During this step, calcium and magnesium sulfate precipitate and are
separated from the NOM, which remains in solution.

Phosphates can be precipitated as magnesium ammonium phosphate (Aiken and


Leenheer 1993) or as tri-lithium phosphate (Leenheer 1997). The majority of the nitrate
in the sample can be removed by adding a 10-fold molar excess (relative to nitrate
concentration) of barium chloride to the ultra-hydrophilic NOM concentrate and
removing barium nitrate by zeotrophic distillation in acetic acid. Traces of nitrate can be
removed by evaporation with anhydrous formic acid that reduces nitric acid to gaseous
nitrogen dioxide, but it is not advisable to use this procedure with large amounts of nitric
acid because of the potential for oxidation and nitration of NOM by nitric acid.

Separation of silica from ultra-hydrophilic NOM is particularly problematic,


because silicic acid polymerizes with itself and with the NOM to form a silica-NOM
ester gel when taken to dryness. This problem can be overcome by hydrolyzing the ester
by gentle heating with 0.01 N HCl, a process that dissolves the NOM but not the silica
gel, so the latter can be removed by centrifugation.

Thus, if one knows which salts must be separated from the NOM, methods are
probably available to carry out the separation. However, each procedure is often
complicated and applicable to only one or a limited suite of salts. Analysis of inorganic
constituents is therefore a key prerequisite for designing a sensible and effective
concentration, fractionation, and isolation scheme for ultra-hydrophilic NOM
constituents in a particular sample.

24
Losses of NOM During Processing

NOM recoveries can be estimated at various stages of an isolation-concentration-


fractionation procedure, or for the overall procedure, by a mass balance on DOC, as long
as organic reagents are not used in the procedure. The goal of comprehensive NOM
isolation is, of course, 100 percent recovery of desalted NOM fractions. However,
practical considerations limit the recovery currently achievable to 70 to 90% for fresh-
water samples and less than 50% for seawater samples. The major limitation is imposed
by the presence of salts and is quantitatively related to the salt:DOC concentration ratio.
For a soft freshwater sample such as the Suwannee River, this ratio is about 0.2 mg/mg,
while for the Mississippi River at New Orleans, it is about 50 mg/mg (Hem 1971**JL
(year given as 1970 in refs), Leenheer et al. 1995), and for seawater it is 18,000 mg/mg
(Hem 1971, Benner et al. 1992). If all of the salts remain in solution when the
hydrophobic NOM is extracted from the sample, the salt:DOC ratio in the remaining
(transphilic and hydrophilic) fractions typically increases by a factor of two or more.
Therefore, efficient desalting techniques are particularly important if one is to avoid
losses of hydrophilic NOM during the processing steps.

Removal of salt by zeotrophic distillation, ion exchange, or selective precipitation


techniques invariably results in both non-selective and selective losses of NOM. Non-
selective losses (i.e., those that decrease the mass of NOM recovered but do not alter its
composition) are primarily because of mechanical operations. For instance, as discussed
previously, some NOM might be lost by sticking to the walls of freeze-drying vessels.
Also, losses of NOM by accidents, such as spills and breakage of containers, occur with
increasing frequency as the complexity and difficulty of the isolation procedure
increases.

Selective losses of certain classes of NOM can occur during various steps that are
intended to target other molecules. For instance, some NOM might sorb onto ion
exchange resins intended to remove salts from solution, some might volatilize or be lost
as foam during vacuum evaporation steps, and some might form precipitates at various

25
stages in the concentration procedures. In addition, organic colloids might be lost at
various points in a procedure.

NOM CHARACTERIZATION

Due to the complex, ‘multi-dimensional’ nature of NOM described earlier, a


variety of experimental methods has been used to characterize it. For purposes of
discussion, these methods can be separated into four tiers. The first tier addresses the
chemical identities of individual species included in the NOM pool, such as amino acids
and carbohydrates. The second tier addresses the nature and abundance of structural units
in the NOM molecules. The relevant methods include elemental composition analysis,
13
C- and 1H- nuclear magnetic resonance (NMR) spectroscopy, Fourier Transform
Infrared (FTIR) spectroscopy, and pyrolysis - gas chromatography - mass spectrometry
(Pyr-GC-MS). The techniques associated with the first two tiers provide chemically
specific information but require substantial sample preparation and cannot be used in
situ. The third tier of methods addresses issues related to the chemical behavior of NOM,
often focusing on the polymeric nature of NOM molecules. Techniques to probe the
molecular size distribution, acid-base and hydrophobic-hydrophilic properties, and
reactivity of NOM are included in this group. This tier of the experimental methods
generally requires less sample preparation than the first two. The fourth group of methods
comprises those that do not explicitly probe the chemical identities of functional groups
or molecules, but measure a spectral signature of NOM in toto, such as UV absorbance
and fluorescence spectroscopy. Due to the exceptional sensitivity and experimental
simplicity of these methods, they can be used to probe NOM in situ, without pre-
concentration and with minimal or no pre-treatment. However, interpretation of the data
generated by these techniques is usually less direct than for techniques in the other three
analytical tiers.

26
13
C and H-NMR spectroscopy

The theory and application of solution and solid-state NMR spectroscopy to


obtain quantitative and qualitative information on organic carbon structural distributions
has been reviewed by Nanny et al. (1997), Wershaw and Mikita (1987), and Wilson
(1987). Solution-state 1H-NMR provides information on non-exchangeable structural
proton distributions in NOM isolates. The acquisition and interpretation of 1H-NMR
spectra is discussed in a recent report by Leenheer et al. (1987).

Carbon (13C), hydrogen (1H), nitrogen (15N), and phosphorus (31P) nuclei in NOM
13
can be probed by NMR spectroscopy; however, only C and 1H have sufficient natural
abundance and relative receptivities (sensitivity) to allow routine determinations of NMR
spectra. NMR signals are generated by the absorption and emission of radio frequency
(RF) signals of spinning nuclei that are precessing about an axis in a magnetic field.
When the RF frequency matches the precession frequency during an RF pulse, RF energy
is absorbed and the nuclear spin is shifted to a different energy level. When the nuclei
relax to the ground state energy level between RF pulses, RF energy is emitted, and it is
this energy that is detected as the NMR signal.

The electron field around a nucleus is determined by its chemical structure and
affects the NMR signal to various degrees. The magnitude of this effect is defined as the
“chemical shift”. Chemical shifts are measured as parts per million frequency differences
relative to a standard compound. NMR signals generated in the time domain are
converted by Fourier Transform mathematics to frequency domain information that
graphs the intensity of an NMR signal (ordinate) to the chemical shift (abscissa). The
orientation of NMR nuclei relative to the applied magnetic field must be random to
achieve useful NMR signals. This randomization is accomplished either by dissolving the
sample (which randomizes the nuclei through molecular Brownian motion) or by
spinning a solid sample at a “Magic Angle” relative to the applied magnetic field. The
latter approach is referred to as cross-polarization magic angle spinning (CPMAS).

27
Both solution-state and solid-state capabilities are desirable for characterizing
NOM structure. Solid-state NMR is generally not as quantitative as solution-state NMR
(Thorn et al. 1989), assuming complete solution of NOM fractions at high concentrations.
The cost of NMR spectrometers is high ($300,000-$500,000), so NOM isolates are
frequently sent to institutions that run the samples. Approximately 50 mg of desalted
sample is required to run the analysis.

13
Possible structural assignments for C-NMR and 1H-NMR spectra of NOM
isolates are given in Tables 2.2 and 2.3, respectively. Both tables indicate considerable
overlap in the chemical shifts for various types of structures. In addition, in 1H-NMR
spectra, there is an intense and sometimes broad peak near 4.6 ppm from exchangeable
hydroxyl hydrogen that obscures some of the structural hydrogen groups near this region.
For these reasons, quantitative determinations of various structures in NOM is a
somewhat subjective exercise that depends on the judgment of the analyst.

Table 2.2
Structural assignments for 13C-NMR spectra
Chemical linkage Compound type Chemical shift
range (ppm)
C-H Hydrocarbon 0-55
C-N Amines, amides, proteins 40-55
O-CH3 Methoxy groups in tannins and lignins 55-60
C-O Aliphatic alcohols, ethers, and esters 60-90
O-C-O Anomeric carbon in carbohydrates, lactols 90-110
φ Aromatic carbon 95-165
φ-O Aromatic esters, ethers, and phenols 135-165
O=C-O,O=C-N Carboxylic acids, esters ,amides 160-190
O=C-C=C Flavones, quinones 170-200
O=C-C Aliphatic and aromatic ketones 190-220

28
Table 2.3
Structural assignments for 1H-NMR spectra
Chemical Linkage Compound Type Chemical Shift
Range (ppm)
R-CH3 Aliphatic Hydrocarbons 0.6-0.9
-CH2- Aliphatic Hydrocarbon Chains 0.9-1.4
O=C-C-CH3 α-Methyl ketones, carboxylic acids 0.9-1.2
O-C-C-H α-Oxy alcohols, ethers, esters 1.4-1.8
Aliphatic, alicyclic hydrocarbon 1.4-1.8

O=C-CH3 Methyl ketones, carboxylic acids 1.9-2.1


O=C-C-H α-Methylene and methine ketones, acids 2.1-
3.2
φ-C-H Aliphatic CH on aromatic rings 2.1-3.2
O-C-H Alcohols (carbohydrates), ethers, esters 3.2-5.2
O=C-CH-C=O β-Keto carboxylic acids, ketones
O=C-CH-φ β-Aryl carboxylic acids, ketones 3.5-5.5
C=C-H Olefinic hydrogen 5.0-6.4
O H-ortho phenols, phenol ethers, phenol esters 6.4-7.0
H

H Aromatic protons in general 6.4-8.5

O H-ortho carboxylic acids, ketones 7.6-9.5

13
C-NMR with CPMAS may have significant limitations that affect its precision
in estimating the contribution of aromatic and carbonyl or carboxyl carbon in NOM and
overemphasize the contribution of other types of carbon in the sample. Variable contact
13
time studies by Alamany et al. (1983) indicate that in the C-NMR cross-polarization
experiments, an optimal signal-to-noise ratio can be achieved at a 1 ms contact time.

29
However, these experimental conditions compromise the precision of the method in
estimating the contributions from different structural groups. Comparison of CPMAS
13
C-NMR spectra acquired with 1 ms contact times with highly-precision quantitative
13
liquid-state C-NMR of aquatic NOM isolates indicate that aromatic carbon is
underestimated by 20 to 40% (phenolic by 50%) and carbonyl (carboxyl, ester, amide)
carbon is underestimated by 30 to 50%, and that the aliphatic carbon is overestimated by
corresponding amounts. This problem exists because the aromatic rings in humic
substances have low and remote protonation.

Using a 5 ms contact time gives results much closer to quantitative liquid-state


spectra, but a 1 ms contact time has become the standard because the lower field
instruments for which the original methods were developed needed the sensitivity that
1 ms contact time provided. Due to this limitation, inferences about the structural
13
distribution of carbon derived from C-NMR data should be considered as semi-
quantitative, similar to those from Pyr-GC-MS data (discussed below). In particular,
estimates of NOM aromaticity obtained using 1 ms contact times should be considered as
minimum estimates.

FTIR spectroscopy

FTIR spectrometry detects various molecular vibrations (rotations and stretches).


When the frequency of the infrared radiation entering a solution or crystal of an organic
compound corresponds to the frequency of a molecular motion in the organic compound,
radiation is absorbed. A plot of percent transmission versus frequency indicates the
relative amounts of molecular stretching and bending vibrations of various atoms in the
molecule. Fourier transform mathematics converts the data into a spectrum showing
transmission or absorbance of infrared radiation on the ordinate versus the infrared
frequency on the abscissa. Infrared spectrometry is especially useful for qualitative
identification of oxygen and nitrogen functional groups in NOM.

30
Comprehensive interpretation of FTIR spectra of pure compounds is complex
because so many absorption bands are generated. Paradoxically, the complexity of
fractionated NOM simplifies interpretation of the spectra because only the strongest
bands can be identified and associated with the predominant structures. For interpretation
of the spectra of pure compounds, the reader is referred to Pouchert (1985), and for
analysis of complex biomolecular structures and humic substances, to Bellamy
(1975**JL (year given as 1960 in refs) and Stevenson (1982), respectively. Table 2.4
lists characteristic IR frequency bands for some complex biomolecules typically found in
NOM isolates.

Table 2.4
Infrared frequency bands for biomolecular structures in NOM isolates
Biomolecule Frequencies (cm−1) and Structure
Carbohydrates 3400-3300 (O-H); 1100-1000 (C-O)
Fulvic Acid 3400-3300 (O-H); 2700-2500 (COOH); 1760 (COOR); 1720 (COOH);
1660-1630 (φ-C=O); 1280-1150 (φ-O; COOH)
Hydrocarbons 2960 (CH3); 2940 (CH2); 1460 (CH2); 1380 (CH3)
Proteins 1660 (Amide-1 band; N-C=O); 1550 (Amide-2 band; N=C-O)

FTIR characterization of isolates can be particularly useful for identifying the


13
proteinaceous component of NOM, which is difficult to identify using C-NMR. In
addition, the relative abundances of hydrocarbons (hydrophobic) and carbohydrate
(hydrophilic) moieties indicated by the IR spectrum can be used as an indicator of the
hydrophobic-hydrophilic nature of NOM isolates.

FTIR spectrometry can also serve as an assay of the purity of NOM fractions
because it allows bicarbonate, carbonate, nitrate, phosphate, silicate, and sulfate salts in
the sample to be readily detected. Table 2.5 gives characteristic peaks that can be used to
identify inorganic contaminants in NOM fractions. By the same token, inorganic salts
(with the exception of chloride salts) can be a major interference, so purification

31
requirements are significant for FTIR spectrometry to be a useful tool for NOM
characterization.

Table 2.5
Characteristic infrared spectral peaks of inorganic solutes (in KBr pellets)
Inorganic solute Characteristic IR peaks (cm−1)
Boric acid 3212, 2260, 1450, 1194, 548
Sodium bicarbonate 2541, 1920, 1695, 1618, 1307, 1000, 837, 696
Sodium carbonate 1440, 880
Sodium nitrate 1385, 838
Phosphoric acid 1007, 490
Disodium hydrogen phosphate 1159, 1074, 950, 860, 544, 521
Silicic acid 1093, 964, 798, 468
Sulfuric acid 1288, 1176, 1071, 1012, 889, 852, 617, 577, 455
Sodium hydrogen sulfate 1251,1182, 1046, 865, 607,577, 481
Sodium sulfate 1122, 640, 608

Pyrolysis-GC-MS

If NOM is degraded thermally, macromolecules that were originally synthesized


from natural biopolymers (e.g., polysaccharides, proteins, amino sugars or polyhydroxy-
aromatics) tend to produce fairly specific by-products. Pyrolysis followed by gas
chromatography and mass spectrometry (Pyr-GC-MS) can separate and identify those
by-products.

While Pyr-GC-MS cannot be used as a strictly quantitative analytical technique, it


can provide a fingerprint of the NOM that is quite specific. For instance, it has been used
to distinguish among humic substances isolated from various natural waters (Peschel and
Wildt 1988 **JP – spelling is Pischel in ref list) and between fulvic and humic acids
isolated from the same water source (Bruchet et al. 1986). It is also useful for following
the evolution of NOM as a function of season or during drinking water treatment
processes (Bruchet et al. 1990, and Bornick. 1996). As pointed out by Saiz-
32
Jimenez (1994), one major advantage of the technique is that it does not require prior
hydrolysis, purification or fractionation of the organics.

Table 2.6 presents information about the presumed origin of some natural
biopolymers and their pyrolysis by-products. Based on the types and diversity of
fragments produced by Pyr-GC-MS, humic acids are reported to be structurally more
heterogeneous than fulvic acids and to contain carbohydrates as the most prevalent class
of constituents (Bruchet et al. 1986). Humic acids also appear to have a larger phenolic
and unsubstituted aromatic content than fulvic acids based on this technique (Gadel et al.
1992). Pyr-GC-MS analysis of NOM suggests that, despite their high specific UV
absorbance (SUVA) values, humic acids are highly aliphatic (Gadel and Bruchet 1987,
Bruchet et al. 1986), and that proteins and carbohydrates are much more substantial
components of NOM than is suggested by other types of analyses.

Table 2.6**MB landscape


Origin of biopolymers and their respective specific pyrolysis fragments
Type Origin Pyrolysis by-products
Polysaccharides Aquagenic (algae and Hexoses and pentoses: furan,
(stored, e.g., starch, and bacteria) and pedogenic furfural, levoglucosenone
structural, e.g., cellulose) (plant residuals)
Proteins Aquagenic (algae and Pyridines, pyrroles, indoles,
phytoplankton) nitriles; phenol, paracresol
(equal quantities, from
tyrosine); toluene, styrene,
phenylacetonitrile (from
phenylalanine); indole from
tryptophane
Amino sugars Cell walls of bacteria and Amides (acetamide,
fungi ethanamide)
Polyhydroxy-aromatics Plants (lignin) and aquatic Phenolic compounds (e.g.,
(PHA) (algae, diatoms, animals) methoxyphenols)
Source: Adapted from Bruchet et al. 1986; Gadel et Bruchet 1987; Bruchet et al. 1990; Biber et al. 1996.

33
The amounts of saturated and aromatic hydrocarbons generated by Pyr-GC-MS of
NOM might be useful as indicators of terrestrial organics in the NOM. For instance,
Schulten and Plage (1991) reported that benzene and allylbenzene are the major thermal
degradation by-products of humic acids isolated from soils. However, this category of
by-products can also be generated by pyrolysis of fatty acids, aromatic acids, aryl
aliphatics and alcohols (Saiz-Jimenez 1994, Göbbels and Püttmann 1997).

Croue et al. (1993b) published Pyr-GC-MS chromatograms of fulvic and


transphilic acids isolated from a French reservoir. The chromatogram of fulvic acids gave
clear evidence of aromatic structures (large peaks of phenol and cresol). These fragments
were present at lower concentrations in the chromatogram of the transphilic acids, which
is consistent with the observation that the fulvic acids had a higher SUVA than the
transphilic acids. By contrast, transphilic acids contained a higher proportion of sugars
and amino sugars (based on large peaks of furfural, methyl furfural, levoglucosenone and
acetamide), a fact that is undoubtedly related to their higher hydrophilic character.

Harrington et al. (1996) reported that, although phenol is generally the major peak
in the pyrochromatogram of hydrophobic NOM (XAD-8 isolates), the relative
proportions of the four biopolymer classes identified in Table 2.6 depend on the origin of
the humic materials. For the five isolates studied, they found a strong correlation between
the phenolic or polyhydroxy aromatic content (based on Pyr-GC-MS) and aromatic
13
carbon content (based on C-NMR spectra). Somewhat surprisingly, the correlation
between amino sugars and aliphatic carbon content was also strong. Using the same
approach, Martin (1995) established relationships between polyhydroxy aromatics and
the aromatic carbon content and between TDAA content (based on HPLC analysis) and
proteins. Further development and calibration of the Pyr-GC-MS technique might allow
it to become an important tool for NOM characterization.

34
Elemental Analysis

Elemental analysis is generally among the first approaches that researchers use to
the characterize NOM and its isolates. The elements analyzed commonly include carbon,
hydrogen, oxygen, nitrogen, and sulfur; the non-oxidizable element content is also
usually characterized and reported as ‘ash’. Phosphorus and halogens are analyzed in
some cases, but more rarely than the elements listed above. Results are typically given in
percent by weight, and some specific ratios (e.g., C/H, C/O and C/N ratios) are reported
and used as indicators of particular characteristics of NOM.

The elemental analysis data base available in the literature mainly includes
hydrophobic acid fractions (with or without fractionation into humic and fulvic acids),
along with some results on transphilic acid fractions. Table 2.7 provides a comparison of
elemental analyses of humic, fulvic and transphilic acids isolated from three different
water sources. In this table, the hydrophobic acid fraction isolated by Aiken et al. (1992)
from the Yakima river can be considered comparable to the fulvic acid fraction reported
for the other waters, since fulvic acids generally comprise the dominant portion of the
hydrophobic acid fraction.

35
Table 2.7
Elemental analysis of hydrophobic acids and transphilic acids isolated from surface
waters
Source Fraction C H N O S Ash
Contribution to Fraction Mass (%)
Yakima river* Hydrophobic acids § 56.1 4.95 2.2 35.5 0.97 1.1
Transphilic acids 50.5 4.4 3.0. 40.6 1.2 3.9
Lake Skjervatjern † Humic acids 55.8 3.58 0.96 36.9 0.32 1.18
Fulvic acids 54.2 3.96 0.56 39.3 0.24 0.33
Transphilic acids 50.2 4.0 0.97 43.8 0.51 0.85
Apremont reservoir ‡ Humic acids 48.1 4.9 3.04 36.1 2.58 4.7
Fulvic acids 49.7 4.9 2.14 39.5 1.88 1.5
Transphilic acids 41.1 4.4 3.1 41.1 1.6 nd**
*Aiken et al. 1992
† Malcolm et al. 1993**not in refs
‡ Martin 1995
§ Fulvic acids generally account for 90% of the hydrophobic acids;
**MB nd : not determined

For the three water sources described in Table 2.7, the transphilic acids contained
less carbon and more oxygen than did the humic and/or fulvic acids from the same origin.
These results indicate that oxygenated functional groups are more abundant in the
transphilic acids than the hydrophobic acids, as expected (since oxygen-containing
functional groups cause NOM molecules to be more hydrophilic). For all sources, fulvic
acids had the lowest proportion of nitrogen, while the nitrogen content of transphilic and
humic acids was similar.

Table 2.8 gives typical elemental analyses for various NOM fractions based on
literature data.

36
Table 2.8**MBlandscape
Average elemental analysis of humic substances (with or without fractionation to humic
and fulvic acids) and transphilic acids isolated from surface waters
Fraction C H O N S C/O C/N C/H n
Contribution to Mass (%)
Humic acids 53.1 4.5 37.4 2.1 1.5 1.4 28.3 12.1 12
±2.9 ±0.6 ±2.1 ±0.7 ±0.9 ±0.1 ±11.1 ±2.3
Fulvic acids
53.2 4.8 38.3 1.4 0.8 1.4 43.7 11.2 24
and HPOA *
±2.5 ±0.7 ±2.0 ±0.6 ±0.4 ±0.1 ±18.5 ±1.6
Transphilic 45.8 4.4 43.9 2.5 1.0 1.0 24.1 10.6 10
acids ±3.6 ±0.5 ±2.1 ±1.1 ±0.5 ±0.1 ±13.1 ±2.0

Source: Adapted from Reckhow et al. 1990, Aiken et al. 1992, Martin 1995.
*HPOA = hydrophobic acids

In a literature review prepared in 1985, Thurman suggested that significant


differences could be observed between humic substances isolated from ground waters,
surface waters and soils, and that these differences could be related to the physical and
chemical characteristics of the media. Nevertheless, only minor differences were
observed between humic substances that have a similar origin, indicating that elemental
analysis was not specific enough to distinguish among NOM samples isolated from
similar types of sources. Table 2.7, which presents more recent data, supports this
conclusion. However, the range of values for each element (minimum and maximum
values) does indicate that there can be significant differences in the elemental
composition of NOM from different sources. **JP add a sentence and reference Perdue’s
work.

UV / Visible and Fluorescence Spectrometry

The absorption of both visible and ultraviolet (UV) light by surface waters is
widely attributed to the aromatic chromophores (light-absorbing sub-units) present in
dissolved NOM, primarily in humic molecules. Humic molecules are also thought to be

37
largely responsible for the fluorescence of natural waters. As a result, the energy (related
to the wavelength, λ) and intensity of light absorption and/or emission can be used to
infer structural information about the NOM molecules. UV absorbance is attractive
analytically because it is simple to carry out, the required instrumentation is relatively
inexpensive, and minimal sample preparation is required.

Absorbance of ultraviolet light by NOM in the wavelength range from 200 to


400 nm is easier to assess than absorbance of visible light because few inorganic species
present in natural fresh waters absorb substantial amounts of light at λ>200 nm (in some
waters, bromide and nitrate absorb enough light to be problematic at wavelengths up to
230 nm (Ogura and Hanya 1966, 1968; Mrkva 1969).

13
The aromatic content of NOM, as found by C-NMR, has been reported to
correlate well with UV absorbance at 272 nm (A272), with regression coefficients in the
range from 0.70 to 0.94 (Traina et al. 1990, Novak et al. 1992). Resorcinol, catechol, and
benzoic, hydroxybenzoic and vanillic acids have all been suggested as model aromatic
chromophores that are likely to be incorporated into the structure of NOM (Christman et
al. 1989); other aromatic units undoubtedly contribute as well.

Because humic species are likely to be the predominant organic reactants in


reactions with disinfectants and coagulants, studies of the humic part of NOM can
provide important insights into these processes (Korshin et al. 1996, 1997c). In these
cases, UV spectrophotometry, which is inexpensive and virtually universally available,
can contribute substantially to our ability to predict and monitor the reactions of interest.

One drawback of using UV spectroscopy for studying NOM is that the spectra are
typically broad and nearly featureless (Ghosh and Schnitzer 1979, Wang et al. 1990).
Only minor peaks have occasionally been reported (see, for example, Baes and Bloom
1990), the number of possible types of chromophores is high, and none of the
chromophores possesses an easily distinguishable spectrum.

Although the UV absorbance spectrum of NOM from any given source could, in
theory, be deconvoluted into separate spectra contributed by distinct chromophores, such
38
an approach is not a practical possibility. As a result, the potential value of UV
spectroscopy in the study of NOM has remained unrealized. Most researchers have
limited their data collection to monitoring the absorbance at 254 nm, using these values
as a rough indicator of the overall NOM concentration. The value of SUVA at 254 nm
(SUVA254) is also often calculated and used as an indicator or the aromatic, hydrophobic
character of the NOM (Traina et al. 1990, Novak et al. 1992).

Korshin et al. (1996, 1997c) recently proposed modeling the UV absorbance


spectrum of NOM as a composite of three absorption bands, each of which in turn
represents a composite of the absorbance from certain electronic transitions in aromatic
chromophores in NOM molecules. They suggested that the transitions were similar to
those identified as the local excitation (LE), benzenoid (Bz), and electron transfer (ET)
bands in simple aromatic compounds (Figure 2.1).

A B
εmax>45,000 LE band
εmax=7,400 Bz band
εmax=204
ET band

Local excitation Benzenoid (Bz) Electron transfer


(LE) band, band, (ET) band,
max. @ 180 nm max. @ 203 nm max. @ 253 nm
(6.88 eV) (6.11 eV) (4.90 eV)

Ground electronic state Ground electronic state

Figure 2.1. (A) Conceptual representation of electronic transitions caused by the


absorbance of light for benzene (B) Conceptual representation of composite light-
absorption bands for NOM. Source: Adapted from Jaffee and Orchin (1962) and Scott
(1964).

They represented the absorbance intensity of each model spectrum as a Gaussian


function of the corresponding absorption energy. Typically, the three bands have peaks
near and 180, 203, and 253 nm, respectively. When the three model spectra are super-
imposed and summed, the resultant spectrum closely matches the experimental one
(Figure 2.2).

39
0.9
unconvoluted spectrum
Absorbance tailing part of LE band
0.6
Bz band
ET band
0.3

0.0
190 210 230 250 270 290 310 330
Wavelength, nm

Figure 2.2. Summation of three composite absorption bands and formation of


unconvoluted UV absorbance spectrum of NOM

Korshin et al. (1996, 1997c) proposed that the absolute and relative intensities of
the three model bands, their peak wavelengths (λi,max), and their widths (∆i) provide
useful structural information about the NOM molecules, and that alterations in these
parameters when the NOM is subjected to various physico-chemical processes could
provide information about the NOM reactions in those systems.

Fluorescence occurs when optically-excited molecules emit light whose


wavelength is longer (‘red-shifted’) than that of the excitation energy. In organic
molecules, the emission occurs in functional groups called fluorophores. Like the
chromophores that absorb light, fluorophores in NOM are thought to be associated with
phenolic groups in the humic portion of NOM (Ghosh and Schnitzer 1980, 1981;
Lochmueller and Saavedra 1986; Vinodgopal and Kamat 1992; Goldberg and Negomir
1989). Aromatic amino acids (e.g., tyrosine, tryptophan and phenylalanine) also fluoresce
intensely (Coble 1996) and thus may contribute to the emission (Laane and Koole 1982).
It is estimated that <1% of the aromatic fluorophores in NOM actually emit light, while
the others release the excitation energy via radiationless transitions (Lapen and Seitz
1982, Lochmueller and Saavedra 1986, Seitz 1981). Nevertheless, fluorescence
spectroscopy is at least an order of magnitude more sensitive to the presence of NOM
than UV absorbance (Pennanen and Mannio 1987, Brun and Milburn 1977).

40
Fluorescence spectra are usually obtained either by analyzing the intensity of
emitted light as a function of its wavelength, in which case they are called emission
spectra, or by analyzing the intensity of light emitted at a fixed wavelength while
scanning the wavelength of excitation, in which case they are called excitation spectra.
When both the excitation and emission wavelength are scanned but the difference
between them is kept constant, the resulting spectrum is referred to as a synchronous
spectrum.

NOM fluorescence is strongly affected by the molecular weight of the molecules,


their conformation, and the extent of their complexation with metallic ions and with other
organic molecules (Kalbitz et al. 1997, Kuckuk et al. 1997, Von Wandruszka et al. 1997,
Puchalsky and Morra 1992, Gauthier et al. 1986, Roemelt and Seitz 1982). In fact,
relationships between the fluorescence and the average molecular weight (MW) of NOM
are among the strongest and most consistent correlations obtained for all spectral
properties of NOM. NOM fractions with low average MW have high excitation and
emission intensity, and the position of the maximum in the emission spectrum (λmax)
shifts to lower wavelengths as the average MW decreases (Levesque 1972; Hall and Lee
1974; Smart et al. 1976; Stewart and Wetzel 1980, 1981; Hayase and Tsubota 1985;
Green et al. 1992). This effect may be attributed to the increased probability of
radiationless transitions and quenching of fluorophores in the larger molecules.

In this report, the UV absorbance and fluorescence of NOM are compared based
on several spectral parameters. These include SUVA254, the ratio of absorbances at 350
and 280 nm (A350/A280), the width of the electron-transfer band in the UV spectra (∆ET),
and the wavelength of maximum fluorescence (λmax). The value of ∆ET is calculated using
the A350/A280 ratio, based on the assumptions that the shape of the ET band (when plotted
as absorbance vs. energy) at λ > 250 nm is Gaussian and that the maximum absorbance of
the band is at ~4.90 eV (252 to 254 nm). Using those assumptions, ∆ET can be computed
from Equation 2.10 (Korshin et al. 1996, 1997c).

41
1

⎛ ⎛ A ⎞⎞ 2
∆ ET . ⋅ ⎜ ln⎜ 280 ⎟ ⎟
= 218 (Equation 2.10)
⎝ ⎝ A350 ⎠ ⎠

Compound Class Identification

Amino acids and sugars are present in both free and combined form in natural
waters. The combined forms, which dominate over the free forms in surface waters
(Thurman 1985), include associations with polypeptides, proteins, and polysaccharides or
with humic substances.

Ittekkot et al. (1982) demonstrated the importance of amino acids and sugars as
possible tracers of the different types of NOM transported by water bodies. For instance,
an increase in the concentration of arabinose correlates with increasing concentrations of
β-alanine and γ-amino-butyric acid and is an indicator of NOM of bacterial origin.

Total dissolved amino acids (TDAA) and total dissolved sugars (referred to in this
report as total dissolved carbohydrates, TDCA) are typically present in surface waters at
mean concentrations of 300 µg/L and 500 µg/L, respectively. These constituents
contribute about 2 to 5% and 5 to 10% of the chemical oxygen demand (COD) of such
waters. **JP provide a reference? Glutamic acid, glycine, serine and aspartic acid are the
major amino acids found in surface waters (Thurman 1985), and glucose is the most
abundant sugar. Concentrations of TDAA and TDCA in some surface waters are listed in
Table 2.9, and corresponding data for some fractionated NOM samples are presented in
Table 2.10.

42
Table 2.9**JP: see note 1

Source Types Analyzed Concentration Contribution to Reference


(µg/L) COD (%)

Mackenzie River 114-566


Indus River Total 347-1213 Ittekkot et al. (1982)
Orinoco River 65-284
Total dissolved 300 2-3 Thurman (1985)
Oise River Total dissolved 462 4.5
Marne River Total dissolved 372 5 Dossier-Berne (1994)
Seine River Total dissolved 378 5
Zelivka River 270
Vlata River Combined 450 Chudoba et al. (1986)
Berounka River 337
Sugars
Mackenzie River Total 520-1540 13-40
Indus River Total 267-1141 0.7-11 Ittekkot et al. (1982)
Orinoco River Total 103-970 1.7-9
Total dissolved 500 5-10 Thurman (1985)
Ado River Dissolved neutral 18 2 Ochiai et Nakajima
(1998)
Mano River Dissolved neutral 26 2.3

43
Table 2.10**JP see Note 1
Amino acid and sugar concentrations in various NOM fractions

Amino Acids
Source Fraction Concentration AA C/N Reference
(nmol/mg C) (% N)
Suwannee River Humic acids 110 - - Thurman and Malcolm
(1989)
Ohio River 307.5 - - Malcolm (1990**JP provide
full ref for ref list)
Apremont 324 17 15
Reservoir
260 14.5 16 Martin (1995)
Mayenne River 314 22 18
Shawsheen River Fulvic acids 127 - 35 McKnight et al. (1985)
Thoreau’s bog 78.5 - 71
Suwannee River 34 - - Thurman and Malcolm
(1989)
Ohio River 63.5 - - Malcolm (1990)
Lake Fryxell and 71-98 30.5- 17- McKnight et al. (1991)
Lake Hoare 4.9 22
Apremont 133 10 20
Reservoir
120 9 22 Martin (1995)
Mayenne River 205 18 24
Apremont Hydrophilic 198 90.5 12
Reservoir acids
217 12 14 Martin (1995)
Mayenne River 231 13 13

44
Fractions
Source Fraction sugars (µmol/mg C) Reference
Ohio River Humic acids 3.9 Malcolm (1990)
Ohio River Fulvic acids 6.8

McKnight et al. (1985) found that arabinose and mannose account for 74% and
15% of the carbohydrate in hydrolyzed hydrophilic (73.4%) and fulvic (74.6%) acids,
respectively. Thurman and Malcolm (1989) demonstrated that, in comparison with fulvic
acids, humic acids are enriched in basic, hydroxy-, sulfur-containing, and aromatic amino
acids. The major amino acids in fulvic acids are glycine and aspartic acid, and these acids
along with hydroxyproline are the dominant ones in humic acids.

While the detailed nature of the linkages between humic molecules and amino
acids are not well understood, three types of linkages might be important: hydrogen
bonds, bonds with metal ions, and covalent bonds. Understanding the nature of the bonds
is important both for assessing the biodegradability of these species and for assuring that
the analytical procedure used to hydrolyze the combined forms of the compounds is
appropriate when the total dissolved concentration is analyzed.

45
CHAPTER 3 MATERIALS AND METHODS

This chapter contains information about the water sources, materials and methods
used in the research. Because a major focus of the research conducted was the
development of methods for concentration and isolation of NOM (as opposed to the use
and testing of various methods), it is difficult to segregate the experimental methods used
in much of the research from the results. This is especially true of the work conducted at
the USGS, but is also true to a lesser extent of the work conducted at the other
participating laboratories. Therefore, in general, presentation of the methods that were
developed as part of the research is combined with the presentation of results in
Chapter 4. The material presented here is limited to information about more routine
aspects of sample collection, preliminary characterization, and analytical methods.

SAMPLE COLLECTION

Three rivers and one water supply reservoir in the U.S. and five rivers in Europe
were studied in this project. Two of the U.S. rivers with very different water quality and
NOM characteristics were selected for fractionation case studies: the Suwannee River in
southeastern Georgia and the South Platte River in Colorado. The Suwannee is a very
soft, “black water” river with low salt content. It contains a high concentration of NOM
that is derived principally from terrestrial plants and that has been minimally fractionated
by sorption onto soil mineral constituents. This NOM has been extensively characterized
(Averett et al. 1995) because of its use as a standard NOM by the International Humic
Substances Society. The river was sampled at its origin, at the outlet of the Okeefenokee
Swamp, on October 18, 1995. The entire sample (453 L) was filtered in the field through
two Balston glass-fiber cartridge filters in series (25-µm and 0.3-µm porosity) and was
then shipped in 40-L stainless steel milk cans to Denver. The sample was held in
refrigerated storage during processing.

46
The South Platte River, which serves as a major source of drinking water for
Denver, Colorado, was sampled in Waterton Canyon below Strontia Springs Reservoir on
February 7, 1996, when 408 L was collected, and March 27, 1996, when 440 L was
collected. The NOM in each sample was fractionated, but the corresponding fractions
from the two sampling events were combined prior to analysis. The river was almost
completely covered with ice on February 7, and on March 27, the ice cover was mostly
gone but the spring runoff had not yet begun. For reasons described below, the river was
re-sampled on November 21, 1996. The sampling point was on the South Fork of the
river about 15 miles upstream of the previous sampling point, because a forest fire on the
North Fork had caused massive quantities of ash to enter Strontia Springs Reservoir.

In contrast to the NOM in the Suwannee, a substantial portion of the NOM in the
South Platte is generated in the water itself, i.e., it is autochthonous NOM. The water in
the South Platte is moderately hard (**JL- typical value?) with moderate salt content
(**JL- typical value?), and the NOM content is low. In addition, there are extensive
mineral sediments and soils that act as solubility controls on the NOM content.

NOM was also obtained and concentrated from the Tolt River and Judy Reservoir
in Washington State, in the U.S. Pacific Northwest. The Tolt River, whose basin is in the
Cascade mountains, is a major potable water source for the city of Seattle, WA. Judy
Reservoir is the main water supply source for the city of Mt. Vernon, WA. Neither water
is subject to significant impacts from industry or agriculture. For both water sources, the
total dissolved solids are very low. More information on these waters is provided in the
Results section.

Five samples were collected from surface waters in Europe (the Thames River in
England, and the Vienne, Gartempe, and Blavet Rivers in France). The Blavet was
sampled on two occasions. Approximately 1,000 L of each water was sampled and was
filtered through a 0.45-µm porosity membrane on the same day. It was then stored at 4°C
in a refrigerated tank.

47
The Vienne and Gartempe Rivers are both in the primarily agricultural Vienne
region of France. The Gartempe River is a tributary of the Vienne. Only small industries
discharge into these rivers, except for a pulp and paper mill located ~50 km upstream of
the sampling point on the Vienne.

The Blavet River was sampled 500 m downstream of the Kerne Uhel Reservoir,
near the water treatment plant of Lanrivin (Côte D’Armor, Brittany region). This
reservoir is located in a rural area approximately 20 km from the Atlantic ocean. The
reservoir is surrounded by pine trees that were planted several years ago to define the
protected zone. The Blavet River was sampled in winter (December 1995) and in summer
(July 1996).

The Thames River was sampled at Bray, about 40 km west of London. At this
location, discharges of wastewater effluents may have already significantly impacted the
quality of the river.

Some general water quality characteristics of the untreated waters are summarized
in Table 3.1.

48
Table 3.1
Water quality characteristics of untreated water samples
Vienne Blavet Blavet Thames Gartempe Suwannee South Tolt Judy
River River River river river River Platte river Reser-
River voir

Sampling period 10/12/95 12/6/95 7/18/96 1/21/97 2/17/97 10/18/95 11/21/96 9/96, 6/96,
10/96 7/96
Location Belle- Kerne Kerne Bray Saulge Okeefen- *Waterton Mt. Mt.Verno
fonds Uhel Uhel okee Canyon Vernon, n not
Swamp Wash. Carna-
tion,
Wash.
DOC (mg/L) 4.9 12.0 6.6 3.9 6.4 46.8 3.0 1-2 3-4.5
SUVA254 3.6 5.1 4.8 3.2 4.4 4.6 2.4 2.9 3.3
(L/mg-m)
pH 7.6 7.0 7.8 7.4 7.9 nd** nd 6.5-7.1 6.6-7.4
Conductivity 125 133 145 740 90 30-60 400 25 50
(µS/cm)
Alkalinity 48.2 35.0 37.0 191 39.0 nd nd 4-6 6-10
(mg/L CaCO3)
Chloride (mg/L) 20.2 22.0 19.7 77 12.0 nd nd 0.7-1.0 1.0-3.0
Bromide (µg/L) 60 80 80 nd nd nd nd < 0.02 < 0.02
Nitrate (mg/L) 9.7 12.0 14.1 36.3 8.2 nd nd 0.1-0.5 0.5-1.5
Sulfate (mg/L) 6.3 8.6 7.7 nd 7.7 nd nd 1.0-4.0 3.0-6.0
Calcium (mg/L) 20.8 12.5 12.0 123 11.6 nd nd 5-8 4.0-6.0
Magnesium 3.4 4.3 4.9 nd 1.2 nd nd 0.4-0.6 1.40-1.70
(mg/L)
Sodium (mg/L) 10.5 15.0 14.5 nd nd nd nd nd nd
Potassium (mg/L) 2.3 nd 2.5 nd nd nd nd nd nd
TDAA (µg/L C) 222 342 664 nd nd nd nd nd nd
(µg/L N)
79 133 277
TDCA (µg/L C) 224 194 338 nd nd nd nd nd nd

First sample at Waterton Canyon; second sample 15 miles upstream of Waterton Canyon

NOM ISOLATION PROTOCOLS

49
NOM was concentrated and isolated at each of the participating laboratories. Both
the European water samples and those from the Pacific Northwest were subjected to two
types of isolation protocols: one based on membrane-based processes and one based on
adsorption/ elution processes. The membrane processes were often used in conjunction
with various approaches for desalting the solution. Adsorption processes that were
investigated used XAD-8 and XAD-4 resins in series for the European waters, and either
XAD-8 or an oxide-based adsorbent for the Pacific Northwest waters. These procedures
are described next.

Membrane-Based NOM Isolation Protocols

European Water Samples

NOM from European waters was concentrated using one RO and one NF
membranes. Throughout this report, these membranes are referred to by their brand
names, viz., TW30 and NF70, respectively.1 Both membranes are thin-film composites
made of a polyamide. Some characteristics of these membranes are provided in Table 3.2.

1
All manufactured by Film Tek Corp., Minneapolis, MN 55439.

50
Table 3.2
Characteristics of the membranes used to process European samples

Property Value
Membrane Identifier CTAB-2- TW30 (RO) NF70 (NF)
10HF (RO)
Max. Operating Pressure (psi) 125 300 250
Max. Feed Flow Rate (gpm) 0.021 17 16
pH range, Continuous 3 to 8 2 to 11 3 to 9
pH range, Cleaning (30 min) 1 to 12 1 to 11
Max. Operating Temp. (°C) 35 45 35
Max. Feed Turbidity (NTU) 5 1 1
Max. Feed Silt Density Index 5 5
Free Chlorine Tolerance <2 <0.1 <0.1
(mg/L)

Both a laboratory-scale and a pilot-scale membrane unit were used. The


laboratory-scale unit included a 10-L feed tank and could process 20 L/h using a ‘size
2514’ (2.5 inch diameter; 14 inch length) membrane. The pilot scale unit had a 200-L
feed tank and could process 300 L/h using two ‘size 4040’ (4.0 inch diameter; 40 inch
length) membranes in series. Both units were operated at a working pressure between 14
and 16 bars (**MB psi) with a concentrate-to-permeate flow ratio of 60%:40%.

NOM was concentrated using the pilot unit, sometimes followed by the lab unit.
Before being processed in the membrane units, the water was pre-filtered through 10-µm
and 0.45-µm porosity membranes (Millipore CR10 (polypropylene) and CWSC01
(cellulose acetate), respectively) in series and was then passed through a cation exchange
resin in the Na+ form. Between 300 and 400 L of water was processed at a time. During
the processing step, the concentrate was recycled into the feed tank until a final volume
of only 25 to 30 L of concentrate remained, corresponding to a concentration factor of
approximately 10. Part of the concentrate was used for analyses requiring dissolved
samples, and most of the rest was lyophilized, sometimes after further desalting. In some

51
cases, the concentrate was further concentrated using the lab-scale unit equipped with the
same type of membrane.

After a few runs, it became apparent that some dissolved constituents were being
retained by the membrane units (probably by adsorption or precipitation). Therefore, the
membranes were cleaned by circulating 40 L of 0.05 M NaOH through the system,
recycling both permeate and concentrate to the filling tank. The NaOH solution was
collected and analyzed for organics, as described below.

After the cleaning step using NaOH, the membrane units were cleaned again
using, successively, a solution containing 0.05 M NaOH + **MB 1 g/L EDTA4−, and
then a solution of deionized water acidified to pH 1.7 with HCl. The membranes were
then stored in contact with a solution containing **MB 40 mg/L of NaHSO3. Before
subsequent use, the membranes were rinsed with MilliRO® water (pilot scale unit) or
MilliQ® water (lab-scale unit) until both permeate and concentrate reached a dissolved
organic carbon (DOC) concentration equivalent to that of the MilliRO® or MilliQ® water.

Three procedures were tested in attempts to partially or completely desalt the


membrane concentrates. The first and simplest approach was the removal of carbonate
species by lowering the solution pH to 4 with HCl and stripping the CO2 under nitrogen
flow. This method was used in order to minimize interference from bicarbonate in the
13
C-NMR spectra. Samples treated using this procedure are identified below as ‘RO +
HCl’ or ‘NF + HCl’.

In a second approach, the membrane concentrate was passed through a column


packed with cation exchange resin (**JP brand name?) in the H+ form prior to
lyophilization, in order to remove sodium. Samples treated using this procedure are
referred to as ‘RO + Cat’ or ‘NF + Cat’.

The third desalting approach used XAD-8 or XAD-4 resins. In this procedure, the
membrane concentrate was passed through a column packed with one of the resins, using
a column capacity factor (k') of approximately 5. The resin column was then rinsed with
MilliQ® water acidified to pH 2 with formic acid, using a volume of rinse water equal to

52
four times the void volume of the column, after which the adsorbed organics were eluted
from the resin with a 75%:25% acetonitrile/ water mixture. This eluent was rotary
evaporated to remove acetonitrile and formic acid and was lyophilized. Samples treated
using this procedure are referred to as ‘RO + XAD’ or ‘NF + XAD’.

Pacific Northwest Water Samples

RO membranes used to concentrate NOM in the Pacific Northwest water samples


were cleaned by running MilliQ water through them for several hours. This step was
repeated each time the RO system was used with a new water source. The membranes
were operated at 6.8 bar (100 psi), at a production rate of 2.5 L/h. Prior to RO
concentration, water was passed through 5.0-, 1.0- and 0.45-µm filters and a Na+-
saturated cation exchange cartridge (Barnstead-Thermolyne D8904, Dubuque, Iowa) to
remove particulate matter and hardness cations (Figure 3.1). Due to the relatively low
flow rate, the samples were collected over a period of one to two weeks. The concentrate
was then shipped to the laboratory, and additional RO concentration was carried out until
the final volume of concentrate was approximately 4 L, corresponding to a concentration
factor of 100. The final RO concentrate was filtered through a 0.45-µm filter and then
acidified with HCl to pH 3.5 and purged with nitrogen to strip carbon dioxide. The pH
was then re-adjusted to 7.0. A part of this solution was lyophilized for subsequent NMR
and other analyses, and the remaining part was stored at 4ºC. When necessary, inorganic
salts were removed from the concentrate using the procedure described in the following
sections.

53
high-pressure reverse osmosis
5.0, 1.0 and 0.45 pump cartridge
µm filters and
cation exchange
cartridge
RO
retentate

permeate port for


peristaltic (to waste) injection
pump Adsorbing medium of eluent

Elution
intake
pump
intermediate
tank NOM
water intake concentrate
autosampler

Figure 3.1. Schematic for NOM concentration using reverse osmosis and adsorption

Typically, RO membranes used in this study2 did not release any detectable
organic carbon prior to the start of operations. However, a substantial amount of NOM
was always trapped in the membrane after the concentration step. The trapped organic
compounds were removed by pumping low-organic water into the cartridge to rinse its
surface. In most of these cases, >90% of the organic carbon retained by the membrane
was eluted using ~2 L of water. The resulting solution was combined with the rest of the
RO concentrate. No other attempts were made to collect NOM retained by the RO
membranes.

2
CTAB-2-10HF, AMETEK, Sheboygan, WI.

54
NOM Isolation Using Adsorption and Elution Processes

European Water Samples

NOM from European waters was also isolated using XAD-8 and XAD-4 resins in
series, following the approach of Croue et al. (1993a). For these experiments, the
filtration unit consisted of two 10-L glass columns, allowing up to 300 L of water to be
processed per run using a k' value of 50. All connections and tubing were made of teflon,
except that a small section of tygon tubing was used in the peristaltic pump. Smaller
columns packed with these resins (from 250 mL to 2 L) were used to further concentrate
the NOM in the eluents from the larger columns. These small columns were also made of
glass with connections and tubing of teflon, and they were used with a teflon membrane
pump.

Before their first use, the XAD-8 and XAD-4 resins were cleaned by sequential
Soxhlet extractions with methanol, methylene chloride, and acetonitrile. The extraction
was repeated several times with each solvent. After each NOM isolation procedure, both
resins were cleaned with methanol. Prior to processing a water sample, the resins were
rinsed with MilliQ® water and then successively with 0.1 N NaOH and 0.1 N HCl
prepared with MilliQ® water. After the final cleaning step, the DOC concentrations in the
rinse water from the XAD-8 and XAD-4 resins were generally ~0.1 and 0.2 to 0.3 mg/L,
respectively.

Before application to the XAD resins, water samples were pre-filtered (0.45-µm
pore size) and acidified to pH 2 with HCl. They were then pumped through the XAD-8
resin using a peristaltic pump at a flow rate of 10 L/h. The XAD-8 permeate was pumped
through the XAD-4 resin at the same flow rate using a second peristaltic pump. Permeate
from both columns was collected during the processing steps and was later analyzed for
DOC and UV absorbance at 254 nm (A254). When the adsorption step was completed,
both resin columns were rinsed with MilliQ water and were back-eluted with 0.1 N
NaOH. The eluents were then passed through a cation exchange resin in the H+ form.

55
The two desalted isolates were lyophilized separately or after mixing. The mixed
solution was prepared based on the distribution of the DOC in the two fractions. The
organics that adsorbed onto the XAD-8 resin are referred to below as the XAD-8 isolate
and correspond to the hydrophobic acid fraction of the NOM. Those that adsorbed onto
the XAD-4 resin are referred to as the XAD-4 isolate and correspond to the transphilic
acid fraction of the NOM. The combination of the two NOM fractions is referred to as
the XAD-8/XAD-4 mixture.

Pacific Northwest Water Samples

Pacific Northwest water samples were isolated using three different adsorbents:
iron oxide coated sand (IOCS), iron oxide coated olivine (IOCO), and XAD-8 resin. The
preparation of IOCO and IOCS included cleaning of the core material, preparation of an
iron hydroxide sludge by addition of sodium hydroxide to a solution of nitrate or chloride
salts of Fe(III), mixing the sludge with the core material, drying at a pre-set temperature,
and washing with deionized water. Details have been provided by Benjamin and co-
workers (1993).

Differences in the properties of the core material affected the performance of


these composite media. For instance, the points of zero charge (PZC) of the IOCS and
IOCO are substantially different (Table 3.3). IOCO can adsorb substantial amounts of
NOM at pH 7 and it is not stable at pH < 5 due to slow dissolution of the core. Therefore,
IOCO was tested as a medium for concentrating NOM directly from natural waters
without pH adjustment. On the other hand, IOCS does not sorb NOM at neutral pH, but it
is stable in mild acid and its ability to sorb NOM increases with decreasing pH.
Therefore, its performance with respect to NOM sorption was tested in acidic solutions.

56
Table 3.3
Characteristics of the composite iron oxide-based media used for NOM adsorption
Parameter IOCS IOCO

BET surface area (m2/g) 2.7 7.2

Weight percent iron on surface 2.5 5.0

Surface site density (mol sites/mol Fe) 0.095 0.036


pH of PZC 10.3 8.3

XAD-8 resin was purchased and was cleaned by repetitive Soxhlet extraction
with methanol and acetonitrile followed by rinsing with low-organic water until no traces
of solvent were detected.

Relevant operational parameters for adsorption-based NOM concentration


techniques used on Pacific Northwest waters are shown in Table 3.4. In all cases, the raw
water was filtered using 5.0, 1.0 and 0.45 µm filters in sequence and was passed through
a Na+-saturated cation-exchange column to remove hardness cations. This procedure
might remove some basic NOM molecules, but previous research had shown that these
waters contain very low concentrations of both hydrophilic and hydrophobic bases
(Korshin et al. 1997b).

57
Table 3.4
Operation parameters for concentration of NOM by adsorption for Pacific
Northwest waters
Parameter XAD-8 IOCO IOCS
Prefiltration (sequence of 5.0-, Yes Yes Yes
1.0- and 0.45-µm filters)

Removal of hardness cations Yes Yes Yes

pH of influent 2.0 No adjustment 3.8

Bed volume (L) 0.28 0.5 0.5

Flow rate (L/h) 1.12 2.0 2.0

Maximum number of bed volumes 700 700 700


passed before regeneration

Elution technique 0.1 M NaOH* 0.1 M NaOH 0.1 M NaOH

Removal of excess of Na+ H+ cation H+ cation H+ cation


exchange exchange exchange

*followed by methanol

The IOCS, IOCO and XAD-8 adsorbents were eluted with sodium hydroxide. The
eluents were neutralized immediately by cation exchange (H+-saturated Biorad
AG-MP-50 resin). If sulfate was present in the eluent, removal of Na+ from the eluent by
ion exchange was problematic because exchange of the Na+ for H+ yielded a strongly
acidic solution. In these cases, the alkaline regenerant was neutralized by adding
hydrochloric acid. The samples were then desalted by zeotrophic distillation, if
necessary. The NOM samples were lyophilized.

58
NOM desalting

The NOM concentrated by RO and, in some cases, IOCO and IOCS contained
concentrations of inorganic salts that were too high for the intended analyses. In such
cases, the samples were desalted and split into three groups that are referred to as humic
acids, fulvic acids and hydrophilic NOM. The fractionation scheme was developed by
Leenheer as part of the current research project, and the fractions are expected to
correspond qualitatively (but not exactly) to those isolated using the more elaborate
characterization and fractionation schemes presented in Chapter 4.

Humic acid was isolated by dissolving dried, concentrated NOM in 100 mL of


water adjusted to pH 1.0 with HCl. This solution was stirred for one hour to dissolve as
much NOM and salt as possible, after which it was centrifuged. The solids were collected
and the supernatant was saved for further processing. The solids were then washed with
50 mL 0.1 N HCl and again centrifuged. The resulting secondary supernatant was
combined with the initial supernatant. The washed solids were exposed to 50 mL of a
NaOH solution at pH 12. The suspension was briefly shaken, centrifuged, and non-
dissolved solids (presumably containing silica and insoluble organic colloids) were
discarded. The alkaline extract (containing the humic acid fraction of the NOM) was
acidified to pH 1 with HCl and was placed in a refrigerator overnight to allow the humic
acid to precipitate. This solution was centrifuged, and the resulting supernatant was
combined with the supernatants from previous processing. Humic acid so obtained was
resuspended in a minimum volume of distilled water and freeze-dried.

To desalt the fulvic acid fraction of the NOM, the supernatant was passed through
a column containing 100 mL of XAD-8 resin, followed by 200 mL of 0.1 N HCl. The
adsorbed fulvic acid was eluted with 100 mL of a 75% acetonitrile, 25% water mixture,
followed by 100 mL of H2O. The eluent containing fulvic acid was vacuum-evaporated
to a volume of 5 to 10 mL, to which 50 mL of 100% acetonitrile was added. This solution
was vacuum-evaporated to dryness, and 25 mL of 100% acetonitrile was added
immediately, after which the evaporation step was repeated. These cycles of evaporation
were performed to remove traces of HCl from the fulvic acid. The solids obtained were
59
dissolved in 2 mL of the 75% acetonitrile, 25% water mixture and transferred to a
freeze-drying flask. The flask of the rotary evaporator was rinsed with 20 mL of H2O,
which was transferred to the same freeze-dry flask. The solution was freeze-dried to
obtain the fulvic acid fraction of NOM.

To desalt the hydrophilic fraction of NOM, the supernatant remaining after the
separation of humic and fulvic acids was vacuum-evaporated to 20 mL, and 50 mL of
100% acetic acid was added. The resulting solution was vacuum-evaporated until a salt
slurry was formed in the rotovap flask. This slurry was never allowed to evaporate to
dryness. The slurry was vacuum-filtered through a 1.0-µm porous glass fiber filter placed
on a fritted glass disk. The filter was washed with 50 mL of 100% acetic acid. Then,
20 mL of a 0.5 M BaCl2 solution was added to precipitate sulfate from the solution. The
zeotrophic distillation with acetic acid was then repeated to remove barium sulfate,
barium chloride, and other salts. The resulting solution of the hydrophilic fraction of
NOM filtrate in acetic acid was passed through a column packed with 100 mL of a strong
acid ion exchange resin in the H+ form and was rinsed with 150 mL of distilled water.
The effluent was vacuum-evaporated to 10 mL, and 50 mL of 100% acetonitrile was
added. The solution so obtained was again vacuum-evaporated to 2 mL, and 50 mL of
acetonitrile was added. The solution was then vacuum evaporated to dryness. Twenty-
five mL of acetonitrile was added immediately, and the solution was once again
evaporated to dryness. The organic matter from the residues was extracted with 10 mL of
water. The desalted water solution containing the hydrophilic fraction of NOM was then
freeze-dried.

EXPERIMENTAL PROCEDURES FOR CHARACTERIZING NOM REACTIVITY

In some cases, NOM was subjected to various types of chemical processing as a


way to characterize its reactivity or to investigate its structure. The procedures used in
these tests are described next.

60
Chlorination Studies

Chlorination experiments on raw or reconstituted waters were conducted using


uniform formation conditions (Summers et al. 1994), and those on the NOM fractions
were conducted using formation potential (FP) conditions. Formation potential conditions
were preferred over simulated distribution system (SDS) conditions for two reasons.
First, the small amounts of some fractions that were available for testing limited our
ability to identify the appropriate chlorine dose to obtain the target residual for the SDS
test of 1 mg/L ± 0.3 after 24 hours; and second, FP conditions are more generally used as
a characterization tool, whereas SDS conditions are mainly applied to simulate a final
disinfection step. Residual chlorine was analyzed using the spectrophotometric method
proposed by Jadas-Hecart et al. (1992).

In the chlorination tests using NOM isolates, the DOC concentration was 4 to
7 mg/L, the (Cl2)dose/DOC ratio was 4 mg/mg, pH was maintained at 8.0 using borate
buffer, and samples were incubated for 72 hours in the dark at 20°C. At the end of the
test, residual Cl2 was quenched with sodium meta-arsenite. The only exception to these
conditions was that the DOC concentration was only 1 mg/L in the test with the
Suwannee River hydrophilic base fraction, due to a shortage of material.

NOM fractions were chlorinated both in the presence and absence of bromide. In
cases where bromide was added, it was injected into the samples as µL quantities of a
concentrated KBr solution to obtain the same Br−/DOC mass ratio as in the raw water
from which the NOM had been isolated.

Coagulation - Flocculation Study

The ability of aluminum (added as Al2(SO4)3) to coagulate the NOM was


sometimes used as a characterization tool. Experiments were conducted on 100 mL of
solution containing 10 mg DOC/L in 200-mL beakers mixed with a magnetic stir bar.
NOM solutions were prepared with non-buffered, high quality water. The aluminum dose

61
in these tests was 1 mg Al/mg DOC. The Al was added from a fresh stock solution using
a micro-pipette over the course of the first minute of the 15-min rapid mix step (300
rpm). The pH was continuously adjusted by dropwise addition of NaOH or HCl during
this step. This step was followed by 30 min of flocculation (100 rpm) and 15 min of
settling. Samples were then filtered using 0.45-µm porosity cellulose acetate membranes
washed with high purity water.3 The experiment was conducted at ambient temperature.

ANALYTICAL METHODS

Inorganic Species

Inorganics in Source Waters

Bicarbonates and carbonates in the European waters were analyzed by a


colorimetric standard method using methyl orange as the indicator (Standard AFNOR
method NFT 90-036). The precision of this analysis ranges from 2 to 10%, depending on
the analyte concentration. A carbon analyzer4 was used for these analyses in the Pacific
Northwest waters. Prior to the analysis, samples were typically filtered through a pre-
washed polycarbonate 0.4-µm filter. Ten-mL glass tubes baked at 400ºC were used for
DOC analyses. The carbon analyzer was calibrated using solutions of sodium biphthalate
and ultra-pure water boiled with an excess of sodium persulfate as described in the
instrument’s manual and in Standard Methods (1995). The calibration was checked daily.
Additionally, standards were run every ten samples.

3
MilliQ, Corp., St. Quentin en Yvelines, France
4
OI Model 700, OI Corporation, College Station TX

62
Other anions in the European waters were analyzed by ion chromatography after
separation on a 250 x 4.6 mm column packed with anion exchange resin,5 using a flow
rate of 1.5 mL/min. The eluent was a solution containing 0.49 g/L phthalic acid adjusted
to pH 4.9 with Na2BO4 and filtered through 0.45-µm membranes before use. The
detection limit for most of the major anions (chloride, nitrate, sulfate) was near 1 mg/L.
Sodium and potassium were analyzed by atomic absorption spectrophotometry.

For minor anions such as bromide, the analysis was conducted on an ion
chromatograph6 equipped with an ion auto-suppresser. Ions were separated on a
chromatography column7 using a carbonate-bicarbonate eluent. The detection limit for
bromide was 35 µg/L, although for low conductivity waters concentrations near 20 µg/L
could be analyzed reliably.

For the Pacific Northwest waters, most anions were analyzed using an ion
chromatograph8. The concentration of total dissolved organic and inorganic carbon was
measured using a carbon analyzer9.

Metals in Pacific Northwest waters were analyzed using a Jobin-Yvon inductively


coupled plasma atomic emission (ICP-AE) spectrometer10. Meinhard or cyclonic spray
nebulizers were employed for sample atomization. Prior to analysis, all metal-containing
samples were filtered if necessary and acidified with concentrated nitric acid. ICP-grade
standards were used for calibration.

5
VYDAC 302 IC4.6, Hesperia, CA, USA
6
DIONEX DX 300 equipped with conductimetric detector (PED-2), DIONEX Corp.,
Sunnyvale, CA, USA
7
AS 12 A (DIONEX Column), DIONEX Corp., Sunnyvale, CA, USA AS 12A
8
Dionex DX-500, with AS-11 column and CD-20 conductivity detector
9
OI Model 700, OI Corporation, College Station TX
10
Model Ultratrace JY-138, Jobin-Yvon S.A., Longjumeaux, France

63
Elemental Analysis of NOM Isolates

Elemental analysis of NOM isolates was conducted at the Service Central


d’Analyse du C.N.R.S. at Solaize, France. Analyses were conducted using microanalysis
protocols for organic materials that allow the determination of elemental composition
using only a few milligrams of homogeneous sample. Table 3.5 shows the acceptable
concentration ranges for analyses using these protocols.

Table 3.5
Acceptable concentration ranges for elemental analysis
Element Detectable range by weight
Carbon 0.3 to 100 %
Hydrogen 0.3 to 16 %
Nitrogen 0.3 to 70 %
Sulfur 0.3 to 100 %
Oxygen* 0.3 to 88 %
Ash 0.3 to 100 %
*The presence of ash may interfere with determination of the oxygen content, so an ash content <1% is
recommended.

The protocols used for the different elements are as follows:

Carbon and hydrogen: Total combustion of the NOM sample at 1050°C under
oxygen flow. The production of CO2 and H2O is quantified using infrared
detection.

Nitrogen: Total combustion of the NOM sample at 1050°C under mixed helium-
oxygen flow. Nitrogen oxides are reduced to N2 before quantification
using thermal conductimetric detection.

Sulfur: Total combustion of the NOM sample at 1320°C under oxygen flow.
Sulfur oxides are quantified using an acidimetric conductimetric method.

64
Oxygen: Total pyrolysis of NOM sample at 1080°C under nitrogen flow,
followed by production of CO from the oxygenated pyrolysis by-products
during filtration through activated carbon at 1120°C. CO is quantified by
infrared detection.

Ash: Total combustion of the NOM sample at 900°C under air flow. Ash content
is based on weight.

Results from tests using these protocols are generally accepted to have an error of
±0.3%.

Total Organic Halides (TOX)

TOX was analyzed by the adsorption-pyrolysis-titrimetric standard method


(Standard Methods 1995) using a TOX analyzer11. A 25-mL or 50-mL sample was
acidified with one drop of concentrated nitric acid to pH near 1.5 and was passed through
a series of two glass columns packed with activated carbon to adsorb halogenated organic
species. Inorganic halides retained by the activated carbon were removed by washing the
column with potassium nitrate solution. The activated carbon was pyrolyzed in a quartz
furnace in a stream of oxygen and carbon dioxide. The halogenated organics were
thereby transformed to corresponding volatile inorganic halides and carried by the gas
stream into an electrochemical titration cell. The cell used a silver working electrode and
a silver-sensitive reference electrode to quantify the amount of halides by coulometric
titration. The recovery of TOX was 100 ± 5%, and the sensitivity of the instrument was
10 ± 5 ng. When necessary, the samples were run in duplicates.

11
Mitsubishi TOX-10Σ

65
Trihalomethanes

THMs were analyzed in duplicate using a head space autosampler12 coupled with
a gas chromatograph13 equipped with a 63Ni electron capture detector and a split injector.
Ten mL of sample was injected into a 20-mL flask sealed with a teflon cap at 40°C. The
injection loop volume was 100 µL. THMs were separated on a wide bore (0.53 mm
internal diameter) column14 that was 30 m long and had a 3.0-µm film, using a
temperature program that increased temperature from 80°C to 120°C at 5°C/min. The
temperatures of the injector and the detector were 200 and 300°C, respectively.
Chromatograms were recorded, and peak areas were measured by an integrator.15

Haloacetic Acids

Haloacetic acids (HAAs) were analyzed in duplicate using a gas chromatograph16


equipped with an autosampler,17 an on-column injector, and a 63
Ni electron capture
detector. After liquid-liquid extraction with methyl tert-butyl ether (MTBE) at pH 1
(50 mL of sample per 5 mL of solvent), the sample was derivatized with concentrated
diazomethane (200 µL per 3 mL of solvent). The derivatization reaction was stopped
after a few minutes by adding two crystals of silica gel. HAAs were separated by
injection of 1 µL of sample onto a capillary column18 (0.32 mm ID, 30 m long, 0.3-µm

12
DANI HSS 3950, Monza, Italy
13
Varian 3300, Sunnyvale, CA, USA
14
DB-624, J&W Scientific, Folsom, CA, USA
15
Merck D-2500 chromate integrator, Darmstadt, Germany
16
Fisons 8000, FISONS Instruments SpA, Milano, Italy
17
Fisons AS 800, FISONS Instruments SpA, Milano, ItalyDohrmann DX-20A
18
DB-1701, J&W Scientific, Folsom CA, USA

66
film thickness) in the presence of dibromopropane (as an internal standard) using the
following temperature program: 35°C (20 min) to 150°C (1 min) at 4°C/min, to 200°C
(5°min) at 10°C/min. Chromatograms were recorded, and peak areas were measured on
an integrator.19

Total Dissolved Amino Acids

The total dissolved amino acid (TDAA) content of NOM isolates was determined
by analyzing a solution containing 5 mg/L DOC, according to the method of Dossier-
Berne et al. (1994). After acidic hydrolysis in 6 N HCl and heating to dryness at 120°C (3
hours), amino acids were derivatized with a mixture of orthophthaldialdehyde-
mercaptoethanol-borate buffer. They were then analyzed using a high performance liquid
chromatograph (HPLC). After derivatization, amino acids were separated on a C-18
column20 and were detected using a fluorescence detector21 operating at an excitation
wavelength of 335 nm and an emission wavelength of 425 nm. A methanol-water
gradient was used to enhance separation of the peaks. Data acquisition was facilitated by
software22 from the manufacturer.

The reproducibility of the analytical method was determined based on 10


replicate analyses of IHSS Standard Suwannee River Fulvic Acids. Each analysis used a
solution containing 5 mg/L DOC in MilliQ water. Table 3.6 gives details of the results
obtained on the 10 replicates for each amino acid. Based on the results in Table 3.6, the
relative standard deviation ranges from 20 to 25% for the majority of the amino acids
identified.

19
Merck D-2500 chromato integrator, Darmstadt, Germany
20
Waters Delta Pack (C18, 100A, 5 µm, 3.0x150 mm) Waters, Milford, MA, USA
21
Merck F-1050, Darmstadt, Germany
22
Millenium, version 2.00, Waters, Milford, MA, USA

67
Table 3.6
TDAA content of the IHSS standard Suwannee River fulvic acids (5 mg/L DOC solution)
Average value µg/L Standard deviation n*
Aspartic acid 8.7 2.4 8
Glutamic acid 9.7 2.6 8
Asparagine <dl † - -
Serine 12 3.1 9
Histidine 7.6 2.1 7
Arginine 13.6 2.16 8
Glycine 24.1 3.8 7
Threonine 27.4 3.3 9
Alanine 5.3 1.3 8
Tyrosine 2.53 0.5 8
Methionine <dl - -
Valine <dl - -
Phenylalanine 1.04 0.35 8
Isoleucine 6.95 1.56 9
Leucine 8.26 1.56 9
Ornithine 43.34 8.3 8
Lysine 9.94 3.8 9
* n: number of replicates used for the calculation of the standard deviation
† dl: detection limit

Following this preliminary study, the TDAA content of NOM isolates was
analyzed using 100-µL samples containing 5 mg/L of DOC. After acid hydrolysis (3
hours at 120°C) in the presence of 0.2 mL of 6 N HCl, the sample was dried and then
resolubilized in 100 µL of MilliQ water. Ten µL of this solution was injected into the
HPLC. For all isolates, the analysis was conducted on five replicates. For natural waters,
the same protocol was applied on 100 µL of sample filtered through 0.45-µm porosity
membranes.

68
Total Dissolved Carbohydrates

Analyses for total dissolved carbohydrates (TDCA) were conducted using an


HPLC with pulsed amperometric detecttion23 following the method developed by Panais
(1994). After acid hydrolysis with 1 M sulfuric acid at 100°C for 4.5 hours, samples were
filtered through a barium cartridge to remove sulfate before being injected onto an anion
exchange24 column fitted with a guard column.25 Sugars were separated with an alkaline
eluent (18 mM NaOH). The injection volume was 200 µL, and the flow rate was
1 mL/min. 2-Desoxy D-galactose at a concentration of 200 nM was used as the internal
standard. Data acquisition used software from the manufacturer.26

The reproducibility of the method was evaluated using the IHSS Standard
Suwannee River Fulvic Acids. The protocol was applied to five replicates of fulvic acid
solution containing 5 mg/L DOC prepared in MilliQ water. Table 3.7 gives the detailed
results.

23
Dionex DX 500 (Gradient pump, GP 40) with ED40 (Electrochemical Detector),
DIONEX Corp., Sunnyvale, CA, USA
24
DIONEX CarbopacTM PA1 (4x250 mm) DIONEX Corp., Sunnyvale, CA, USA
25
DIONEX CarbopacTM PA1 Guard (10-32), DIONEX Corp., Sunnyvale, CA, USA
26
PEAKNET software from Dionex, Sunnyvale, CA, USA

69
Table 3.7
TDCA content of the IHSS standard Suwannee River fulvic acids
Carbohydrate Average value Standard n*
(µg/L) deviation (µg/L)
Rhamnose 7.04 0.62 5
Arabinose 4.55 0.28 4
Glucosamine <dl - -
Galactose 6.5 0.46 5
Glucose 49.4 1.11 4
Mannose 71.4 17.85 5
Fructose 157.4 31.3 5

* n: number of replicates used for the calculation of the standard deviation

The relative standard deviations ranged from 20% to 25% for fructose and
mannose, respectively, and from 2 to 10% for the rest of detectable carbohydrates.

The TDCA content of the NOM isolates was analyzed using the same protocol on
10 mL of a 5 mg/L DOC solution prepared with MilliQ water. Acid hydrolysis was
carried out at 100°C for 4.5 hours after addition of 1 mL of 2 N H2SO4. The analysis was
then conducted on 200 µL of sample.

Pyrolysis-GC-MS

Lyophilized NOM samples were submitted to flash pyrolysis using a filament


pyrolyzer27. Approximately 500 µg of sample was placed in a 100-µL quartz tube. Quartz
wool was inserted at both ends of the tube to avoid loss of sample during its introduction
into the pyrolysis interface. The tube was placed into the platinum filament of the
pyrolysis probe, which was inserted into the pyrolysis oven. The oven was connected

27
Pyroprobe 2000, Chemical Data Systems, Oxford, PA

70
with the split/splitless injector of a gas chromatograph28 interfaced with a quadrupole
mass spectrometer29.

The pyrolysis oven was preheated to 200°C. Flash pyrolysis was performed by
programming the platinum filament to heat to 625°C at a rate of 20°C/ms, with a final
hold time of 20 min. The pyrolysis fragments were separated on a 30-m DB WAX fused
silica capillary column programmed to heat from 30°C to 220°C at a rate of 3°C/min. The
fragments were then identified by the mass spectrometer operating at 70 eV and scanning
from 20 to 450 amu at one scan/s.

NMR and FTIR Spectra

Solid-state, cross-polarization, magic-angle-spinning (CPMAS) 13C-NMR spectra


were obtained using a 200- megahertz (MHz) Chemagnetics CMX spectrometer with a
7.5-mm-diameter probe. The spinning rate was 5000 Hz. The acquisition parameters for
the freeze-dried samples included a contact time of 1 ms, pulse delay of 1 s, and a pulse
width of 4.5 µs for the 90o pulse. Variable contact time studies by Malcolm (1992)
indicate these are the optimum parameters for estimating different structural group
contributions to the spectra.

1
H-NMR spectra of NOM isolates dissolved in D2O at pH 7 were obtained on a
spectrometer30. On the 300 megahertz spectrometer, acquisition parameters used to
obtain quantitative spectra were: spectral window = 8,000 hertz; tip angle = 25o;
acquisition time = 1.0 second; and pulse delay = 5 seconds. These conditions were judged
to give quantitative spectra because proton spin-lattice relaxation times for both humic

28
Fisons GC 8086
29
Fisons MD 800
30
Varian XL-300

71
and fulvic acids from the Suwannee River were < 0.4 s by the progressive saturation
method (Thorn et al. 1989).

Infrared spectra were collected using 2 to 5 mg of NOM fraction isolates and


standard compounds in KBr pellets. The spectrometer31 used a pulsed laser source and a
deuterated triglycerine sulfate detector. All spectra were normalized after acquisition to a
maximum absorbance of 1.0 for comparative purposes.

UV Absorbance and Fluorescence Emission Spectra

UV spectra of raw and treated waters analyzed at the University of Washington


were recorded using a high precision, high dynamic range (up to 5.5 absorbance units)
dual-beam spectrophotometer32 with two coupled 5-cm quartz cells. For highly absorbing
samples, 1-cm coupled quartz cells were used. UV absorbance spectra were recorded in
the wavelength range 190 to 800 nm. Fluorescence emission spectra were recorded using
a fluorescence spectrometer33. A 1-cm rectangular quartz cell was always used for this
purpose. A pulsed xenon lamp was used for excitation. Light emission was recorded at a
90º angle relative to the excitation beam. The slit widths were 5 nm. The fluorescence of
NOM was excited at 320 nm, and the emission spectra were typically recorded from 360
to 600 nm. The performance of the fluorescence spectrometer was tested using a solution

31
Perkin Elmer System 2000 Fourier Transorm Infrared
32
Perkin-Elmer Lambda-18
33
Perkin-Elmer LS-50B

72
of quinine sulfate in 0.1 M sulfuric acid as recommended in (Velapoldi and Mielenz
1980). The intensity of the standard solution was stable within ±5%. High purity
deionized low-organic water was used as a blank.

73
CHAPTER 4 NOM CONCENTRATION, ISOLATION AND FRACTIONATION

OVERVIEW

This chapter deals with the concentration, isolation and fractionation of NOM.
The performance of various concentration techniques (evaporation, reverse osmosis,
XAD-8 and XAD-4 resins, iron-oxide-coated media) is compared. The chapter includes a
description of a sophisticated fractionation scheme developed by one of project
researchers (Jerry Leenheer) that, in conjunction with the associated desalting methods,
allows collection of both major and minor NOM fractions. Detailed examination of these
fractions, as described in the subsequent chapters, offers insight into both the diversity
and the unity of NOM from various sites, and demonstrates the type of information that
can be obtained using various analytical tools.

To discuss the characteristics of NOM fractions meaningfully, a convention is


needed for naming the fractions collected by specific techniques. The convention adopted
in this report defines four tiers of fractions with steadily decreasing hydrophobicity: the
hydrophobic, transphilic, hydrophilic and ultrahydrophilic fractions, respectively. The
name conventions used in this report are compared with some other commonly used
conventions in Table 4.1. More detailed descriptions of the separation techniques used to
collect each fraction are provided later in this chapter.

74
Table 4.1
Fraction name conventions and their correlation with the previously used terminology

Fraction name and Approach for collecting Other descriptors


acronym used in this fraction
report
Hydrophobic (HPO) Adsorption on XAD-8 Humic material; fulvic and
humic acid

Transphilic (TPH) Adsorption on XAD-4 of Acidic part of this fraction has


material that does not sometimes been referred to as
adsorb to XAD-8 hydrophilic acid

Hydrophilic (HPI) Adsorption on XAD-4 of Previously referred to as part of


material that does not sorb the non-adsorbable part of nom
in first pass through media

Ultrahydrophilic Zeotrophic distillation with Previously referred to as part of


(uHPI) acetic acid the non-adsorbable part of nom

Often, the fractions identified in Table 4.1 were further fractionated into acid,
basic, and neutral groups. The acronyms used for these sub-fractions are the same as
those shown in the table, with an A, B, or N appended (e.g., HPOA, HPIN, etc.).
Furthermore, in some cases, the HPIA fraction was thought to be incompletely collected
the first time that the sample was processed, and so it was processed a second time. In
those cases, the HPIA fraction collected during the second processing is identified by the
acronym HPIA-2. As part of the isolation procedure, the uHPIA fraction was methylated.
Therefore, when discussing the characteristics or behavior of this fraction in the raw
water, it is referred to as the uHPIA fraction, but when referring to the molecules after
fractionation, they are identified as part of the uHPIA-Me fraction. Finally, the fraction of
the NOM that did not sorb to XAD resins and precipitated at pH 1 was defined as
ultrahydrophilic humic acid and is represented by the acronym uHA.

75
CASE STUDIES WITH THE GOAL OF MAXIMAL RECOVERY AND
FRACTIONATION

Suwannee River

A flow chart showing the procedure that was used to isolate the hydrophobic and
transphilic NOM fractions from unconcentrated Suwannee River water is given in
Figure 4.1.

76
453 L of water sampled October 18, 1995

Field filtration through 25 µm and 0.3 µm Balston glass fiber cartridge filters

Adjust filtrate pH to 2.0 with HCl

1. Desorb with 0.1 N NaOH


XAD-8 resin,
2. Rinse with 0.01 N HCl, k' = 100
desorb with 75% aceto-
nitrile/ 25% water

3. Desorb with 0.1 N NaOH


XAD-4 resin,
4. Rinse with 0.01 N HCl, desorb
k' = 100 with 75% acetonitrile/25% water

Adjust to pH 4.0 with


NaOH; Save for MSC-1H
5. Desorb with 0.5 N subsequent isolation of cation-
MSC-1H
NaOH, acidify with hydrophilic NOM exchange
cation-
HCl to pH 2 resin
exchange
resin
6. Desorb with 75%
acetonitrile/ 25% water, Freeze-dry to isolate
Freeze-dry to isolate evaporate and freeze-dry transphilic acids
hydrophobic acids to isolate hydrophobic
b

Discard salts in rinse

7. Vacuum evaporate acetonitrile from step 2, freeze-dry to isolate hydrophobic neutrals


8. Vacuum evaporate acetonitrile from step 4, freeze-dry to isolate transphilic neutrals

Figure 4.1. Flow chart for fractionation and isolation of hydrophobic and transphilic
NOM fractions from the Suwannee River **JL what type of resin is in the final column?

77
The hydrophobic and transphilic NOM were collected from the sample by
sorption in columns packed with XAD-8 and XAD-4 resins, respectively. The volume of
the effluent from the XAD-4 column was then reduced by evaporative concentration, and
the hydrophilic NOM was isolated from this solution by adsorption on XAD-4 resin
(Figure 4.2). Thus, the hydrophilic NOM comprises molecules that do have some affinity
for XAD-4 resin, but that have a low adsorption distribution coefficient. As a result,
adsorbed hydrophilic NOM reaches equilibrium with the influent concentration after only
a few bed volumes have been processes, so it is not retained when the system is operated
using large k′ values (50 to 100). The volumes of sample and resin used to collect the
hydrophilic NOM were such that molecules with k' > 5 would be retained with >50%
efficiency. By contrast, in the first exposure of the solution to XAD-4 resin (to isolate the
transphilic NOM), the operational conditions were such that only molecules with k' > 100
would have been retained with >50% efficiency. As a practical matter, it is not possible
to sorb and thereby desalt a significant amount of NOM molecules with k' < 5 efficiently
in this type of column setup. Retention of HPI NOM by XAD-4 resin in the first
processing cycle is also limited by competition with more strongly binding transphilic
NOM molecules for adsorption sites. Ultra-hydrophilic NOM was fractionated and
isolated as shown in the flow chart of Figure 4.3. The fractionation and recovery data for
dissolved NOM from the Suwannee River are shown in Table 4.2.

78
Vacuum-evaporate XAD-8/ XAD-4 effluent (Figure 4.1) at pH 4 to 1.0 L

Acidify to pH 1 with HCl, centrifuge

solids solution

Disperse solids in 0.1 N NaOH, 1. Pass sample through 80 mL


centrifuge, and discard particulates; column and rinse with 200 mL
Solids that dissolve are humic acids. XAD-4 0.1 N HCl
k' =5 2. Desorb with 75% acetonitrile/
Acidify with HCl to pH 1 to precipitate 25% water
humic acid, centrifuge, wash precipitate,
and freeze-dry to isolate ultrahydrophilic Evaporate to 85% of beginning sample
humic acid volume, filter, wash, and discard salts

Repeat steps six times until sample to


Combine extracts and column volume ratio is equivalent to k´ = 5
remove acetonitrile by
Column eluent
evaporation Acidify with from step 6*
HCl to pH 1

80 mL MSC- MSC-1H cation


5. Desorb with
1H cation- exchange
1.0 N NaOH
exchange XAD-4 7. Desorb with
k´ = 5 75% acetonitrile/
100 mL 25% water
Duolite 6. Desorb with Freeze dry to isolate
A-7 anion 1.0 N NaOH hydrophilic acids
exchange
Discard salts
MSC-1H Column removes bleed Non-sorbed material to
cation- from Duolite A-7 resin 8. Remove acetonitrile processing as
exchange by evaporation, ultrahydrophilic
freeze-dry to isolate NOM
hydrophilic bases

Freeze-dry hydrophilic neutrals*


* The mass of hydrophilic NOM slightly exceeded the capacity of the Duolite A-7 resin, so the column
effluent was recycled through regenerated ion-exchange resins, and a hydrophilic acid 2 fraction was
isolated before the isolation of the hydrophilic neutral fraction.

Figure 4.2. Flow chart for fractionation and isolation of hydrophilic NOM (k' = 5-100)
from the Suwannee River

79
Adjust pH to 2.0 of ultra-hydrophilic NOM concentrate

Remove majority of remaining salts by zeotrophic distillation procedure (Aiken and Leenheer, 1993) in
which water from the sample is evaporated from glacial acetic acid; remove inorganic salts by filtration;
ultra-hydrophilic NOM remains in solution

Remove acetic acid by evaporation to dryness

80 mL MSC-1H
cation exchange
resin Neutralize with HCl to pH 7.0

100 mL Duolite
Desorb with
A-7 anion Evaporate to dryness
1.0 N NaOH
exchange resin

20 mL MSC-1H Column is used to remove bleed Methylate dry residue with


cation exchange from Duolite A-7 resin 100 mL of 10% acetyl
resin chloride in methanol

Take effluent to dryness and Evaporate to dryness;


evaporate with methanol to remove Dissolve residue in 50
boric acid. mL H2O
Re-dissolve residue in 10 mL
0.01 N HCl to hydrolyze silicate-
organic esters; filter out silica gel
100 mL XAD-8 Elute w/100%
resin column acetonitrile
Freeze-dry to isolate
ultrahydrophilic neutrals Discard salts in rinse

Evaporate acetonitrile eluent from


XAD-8 resin to isolate methylated
ultrahydrophilic acids

Figure 4.3. Flow chart for fractionation and isolation of ultra-hydrophilic NOM fractions
(k' < 5) from the Suwannee River

80
Table 4.2
Yields and recovery of dissolved natural organic matter (NOM) fractions from the
Suwannee River
Fraction Mass recovered Percent C DOC recovery
(mg) in fraction efficiency (%)
Hydrophobic 26,421 --- 59.92
Neutrals 357 55.8 0.94
Acids 25,969 48.0 58.79
Bases 95 I * (43) I * (0.19)
Transphilic 6,008 -- 12.20
Neutrals 2,523 47.0 5.59
Acids 3,485 40.2 6.61
Hydrophilic 2,649 -- 5.25
Neutrals 90 39.2 0.17
Acids 2,351 43.0 4.77
Acids2 160 35.6 0.27
Bases 48 19.5 0.04
Ultra-hydrophilic 1,140 -- 2.46
Neutrals 107 31.8 0.16
Acids 101 45.3 0.22
(methylated)
Humic 932 47.4 2.08
acid
Total 36,218 -- 79.83
Neutrals 3,077 -- 6.86
Acids 32,998 -- 72.74
Bases 143 -- 0.23
Loss -- -- 20.17
• I = Incomplete data (data in parentheses are estimates).

81
The DOC concentration of the unfractionated sample was 46.8 mg/L, 90% of
which adsorbed on the initial passes through XAD-8 and XAD-4 columns in series.
However, only about 72% of this DOC was recovered by elution of these columns (the
sum of the hydrophobic and transphilic recoveries given in Table 4.2), corresponding to a
loss of 18% of the DOC that was in the original sample. Most of this loss probably
occurred when a freeze-drying flask containing some of the hydrophobic fraction broke.
Of the 4.6 mg/L of the DOC that did not adsorb in these columns, about 80% was
recovered by subsequent processing, i.e., in the hydrophilic and ultra-hydrophilic
fractions, meaning that an additional 2% of the original DOC was lost in these steps.
Therefore, the vast majority (about 90%) of the NOM that was not recovered in any of
the fractions was lost in the processing of the hydrophobic fraction. After the elution
steps, the color of the XAD-8 resins was similar to that of fresh resins, suggesting that
relatively little NOM remained on the resin. Therefore, it is likely that the breakage of the
flask noted above was responsible for most of the DOC loss, and the DOC recovered by
the fractionation is presumed to be representative of the total DOC distribution.

The DOC fractionation data in Table 4.2 show that NOM from the Suwannee
River is predominantly hydrophobic and acidic material. Base fractions are almost
non-existent. This finding is consistent with other studies of DOC from the Suwannee
River (Malcolm et al. 1994**JL this ref not in ref list, Thurman 1985); however, the
DOC fractionation of the Suwannee River is not typical of that from other aquatic
systems (Thurman 1985, Leenheer 1994**JL this ref not in ref list).

South Platte River

The hydrophobic and transphilic fractions of the NOM in the water samples from
the South Platte River were isolated using the same procedures as were used for the
Suwannee, except that the total sample volume was different (848 L) and the evaporative
concentration step applied to solution that passed through the XAD-8 and XAD-4
columns in series was stopped when the volume had been reduced to 3.1 L. Additionally,
no hydrophobic base fraction was isolated from the sample from the South Platte.
82
The process used to isolate the ultra-hydrophilic NOM fractions was somewhat
different from that used for the Suwannee sample and is shown schematically in Figure
4.4. The key differences in the procedures were as follows:

(1) No ultra-hydrophilic humic acid fraction was isolated from the South Platte,
because a large amount of silica gel co-precipitated with this fraction during
vacuum evaporation (the first step in the process; see Figure 4.2).

(2) Two new procedures were tested to isolate ultra-hydrophilic acids from the
South Platte:

(a) co-precipitation with calcium and magnesium hydroxides, and

(b) adsorption on alumina with subsequent removal of phosphate and


sulfate by selective precipitation.

These new procedures for isolating the ultra-hydrophilic acid fraction were
devised to avoid changing the chemical characteristics of this fraction by methylation,
which was part of the isolation procedure for the uHPIA fraction from the Suwannee
River. NOM fractionation and recovery data for the South Platte River sample are
summarized in Table 4.3.

83
Adjust ultra-hydrophilic NOM concentrate to pH = 2.0

Remove majority of remaining salts by zeotrophic distillation procedure (Aiken and Leenheer 1993) in
which water from the sample is evaporated from glacial acetic acid; remove inorganic salts are by
filtration; ultra-hydrophilic NOM remains in solution

Remove acetic acid by evaporation to dryness

Dissolve residue in water, adjust to pH 12 with


NaOH, remove solids (Ca(OH)2 and Mg(OH)2) Solids Dissolve in dilute H2SO4
by centrifugation.
Solution
Zeotrophic
80-mL column distillation with
Desorb with
MSC-1H cation- acetic acid to
1.0 N NaOH
exchange resin Neutralize with HCl to pH 6.0 precipitate CaSO4
and MgSO4

100-mL column Contact with 30 g Al2O3


Duolite A-7 anion- at pH 6 for 12 hours
exchange resin 20 mL (Removes
MSC-1H metals)
Centrifuge to collect Al2O3; discard resin
20-mL column supernatant; contact Al2O3 with pH 12
MSC-1H cation- solution prepared with LiOH for 1 hour
exchange resin

Evaporate and freeze-


Evaporate to reduce volume by
Take effluent to dryness dry to isolate
90%; filter to remove Li3PO4
and evaporate with co-precipitated
methanol to remove ultrahydrophilic
boric acid acids
Adjust to pH 1 with formic acid,
add BaCl2 to precipitate BaSO4
Re-dissolve residue in
10 mL 0.01 N HCl to
hydrolyze silicate- 100 mL (Removes
organic esters; filter out MSC-1H
silica gel Al3+ Ba2+, Li+)
resin

Vacuum evaporate and freeze-dry


Freeze-dry to isolate to isolate ultrahydrophilic acids
ultrahydrophilic
neutrals

84
Figure 4.4. Flow chart for fractionation and isolation of ultra-hydrophilic NOM fractions
from the South Platte River (**MB keep w/fig)

Table 4.3
Yields and recovery of dissolved natural organic matter (NOM) fractions from the South
Platte River
Fraction Mass recovered Percent C DOC recovery
(mg) in fraction efficiency (%)
Hydrophobic 1,562 --- 34.01
Neutrals 110 58.8 2.93
Acids 1,452 47.2 31.08
Transphilic 1,450 -- 26.02
Neutrals 538 48.0 11.71
Acids 912 34.6 14.31
Hydrophilic 339 -- 6.28
Neutrals 52 39.3 0.93
Acids 274 41.0 5.10
Bases 13 I* (43) 0.25
Ultra-hydrophilic 61 -- 1.11
Neutrals 25 I(40) 0.45
Acids 12 I(40) 0.22
(coprecipitated)
Acids 24 I(40) 0.44
(alumina)
Total 3,412 -- 67.42
Neutrals 725 -- 16.02
Acids 2,674 -- 51.15
Bases 13 -- 0.25
Loss -- -- 32.58
* I = Incomplete data, data in parentheses are estimates.

85
The dissolved organic carbon concentration (DOC) of the combined sample was
2.6 mg/L, of which an average of 1.1 mg/L was not adsorbed on the initial passage
though the XAD-8 and XAD-4 columns in series. Therefore, this column series adsorbed
57.7% of the DOC, essentially all of which was recovered (computed recovery of 104%).
Of the 1.1 mg/L of DOC that did not adsorb to these columns, only 18% was recovered
by further processing, so it appears that most of the DOC loss was from the hydrophilic
and ultra-hydrophilic NOM fractions. Possible reasons for this loss include the failure to
recover colloidal ultra-hydrophilic humic acids, failure to recover NOM that
co-precipitated with silica during vacuum evaporation, and/or failure to recover the
hydrophobic bases. Because ultrafiltration was not used in the fractionation scheme, it is
postulated that much of the unrecovered ultra-hydrophilic humic acid fraction consisted
of organic colloids, as was found previously for the Mississippi River and its major
tributaries (Leenheer et al. 1995a, 1995b).

To further investigate the causes for the loss of hydrophilic NOM, the South
Platte River was re-sampled on November 21, 1996, at which time 505 L was collected.
The sample was filtered as before, but a 1.0-µm glass fiber filter was used instead of the
0.3-µm filter because the low-porosity filter was no longer commercially available. A
four-column resin sorption scheme was devised that did not use vacuum evaporation,
thereby avoiding the problem of silica precipitation (Leenheer 1996**JL this ref not in
ref list). A flow chart of this NOM fractionation scheme is shown in Figure 4.5.

86
Filtered water sample

Hydrophobic
neutral fraction

1 L XAD-8 resin Base fraction

1 L MSC-1H
hydrogen form Hydrophobic acid, hydrophilic acid, and
cation exchange ultra-hydrophilic acid fractions
resin

0.5 L AG-MP-1
anion exchange Hydrophilic
resin in borate neutral fraction
form

Water to waste

Figure 4.5. Four-column preparative NOM fractionation scheme

The hydrophobic neutral fraction was isolated as in the previous samples (Steps 2
and 7 in Figure 4.1). Organic bases were eluted from the MSC-1 resin with a combination
of 2 N sodium formate and 2 N formic acid, at pH 3.5. A four-fold excess of sodium to
cation-exchange equivalents was used to maximize the elution efficiency. This
combination of sodium formate and formic acid was found to be especially effective in
eluting highly retained cations such as iron and aluminum that interact with organic
amino acids when high pH solutions are used to elute the resin. The column eluent was
acidified to pH 1, most of the water and formic acid were removed by vacuum
evaporation, and the sample was evaporated to a salt slurry. The precipitated salt
(presumably mostly sodium chloride) was filtered in a funnel with a glass-wool plug and
was leached with a minimum volume of water until free of color. The bases in the
87
leachate were isolated on XAD-4 resin as in Step 7 of Figure 4.3. The acid fractions were
eluted from the Duolite A-7 column with 1.0 N NaOH, and hydrophobic, hydrophilic,
and ultra-hydrophilic NOM fractions were isolated from this anion concentrate using the
procedures detailed in Figures 4.1, 4.2 and 4.4.

The fourth column was added to adsorb carbohydrates by borate complexation


with polyalcohol groups. Khym and Zill (1952) studied the retention of a number of
simple carbohydrates on a borate-form, strong-base anion exchange resin. They reported
that sucrose, the least retained sugar, had a k' of 19 (i.e., it was 50% retained when the
system was operated with k' = 19). Most of the other carbohydrates studied had k' values
>100 when dissolved in deionized water. These k' values are comparable with the
operational k' values used previously for the three-column system for NOM fractionation
(Leenheer and Noyes 1984).

The hydrophilic neutral fraction was eluted from the fourth column with 20%
acetic acid. In this step, both boric acid (formed by protonation of the borate ions that
were used to pre-saturate the anion exchange sites) and silicic acid, which adsorbs on the
resin from the sample, are co-eluted with the NOM. The water and acetic acid were
evaporated, and boric acid was removed as volatile trimethyl borate by evaporation with
methanol. The hydrophilic neutrals were solubilized from the silica residue by repeated
extractions with 0.01 N HCl, which hydrolyzes the silicate esters. The hydrophilic neutral
fraction was recovered after evaporation of water and HCl.

The water sample was divided in half and was passed through the four-column
system of Figure 4.5 in two portions because the conductivity of the sample
(400 µmho/cm) indicated that the ion-exchange capacity would be exceeded if all 505 L
was passed through in one portion. The DOC concentration for this sample was 3.0 mg/L.
The NOM fractionation and recovery data for the sample are shown in Table 4.4.

88
Table 4.4
Yields and recovery of NOM fractions from the second South Platte River sample
Fraction Mass recovered Percent C DOC recovery
(mg) in fraction * efficiency (%)
Hydrophobic 1,082 --- 33.94
neutrals 30 58.8 1.16
acids 1,052 47.2 32.78
Bases 85 I † (40) 2.24
Hydrophilic 402 -- 10.82
neutrals 60 39.3 1.56
acids 342 41.0 9.26
Ultra-hydrophilic 28 40 0.74
acids
Total 1,597 -- 47.74
neutrals 90 -- 2.72
acids 1,422 -- 43.94
bases 85 -- 2.24
Loss -- -- 52.26
* Carbon percentages are based on the values of similar fractions from Table 4.3.
† I = Incomplete data, data in parentheses are estimates.

The NOM loss during processing of the second sample was greater than that for
the first. Since the hydrophobic DOC percentages were very similar for both samples, the
NOM loss for the second sample is again probably hydrophilic NOM. Two of the three
previously hypothesized reasons for NOM loss were discounted by this second
experiment. The base fractions represented only a small percentage of the DOC, so
unrecovered bases cannot account for the NOM losses. Further, the silica remaining after
extraction of the hydrophilic NOM was destroyed with dilute HF, and the fluorosilicates
were removed from the solution by anion exchange. No appreciable hydrophilic NOM
was recovered after silica was removed. This finding effectively rules out complexation
by soluble silica as a possible sink for the unrecovered NOM.

89
Therefore, the most likely explanation for the loss of hydrophilic NOM is that it
was present as colloids that passed through the entire four-column resin adsorption
system. The percentage of the NOM that was colloidal in the second sample might have
been greater than that in the first sample because of the use of a larger porosity glass-
fiber filter to pre-filter the second sample. In a previous study of NOM in the Mississippi
River, Leenheer et al. (1995a,b) found that colloidal organic carbon concentrations in the
main stem of the river and the major tributaries were usually between 0.5 and 1.0 mg/L,
while the DOC varied between 3 and 15 mg/L. Electron microscopy showed that most of
this colloidal organic carbon was bacterial cells. If colloidal organic carbon
concentrations in the South Platte River are in this range and if the colloids all passed
through the resin columns, they could account for 30 to 60 percent of the NOM loss. The
remaining loss is probably mechanical, resulting from separating large amounts of salt
from very small amounts of NOM. Future comprehensive NOM isolation studies should
incorporate an ultrafiltration step to quantify and separate the colloidal NOM component
for recovery calculations.

Comparison of NOM Recovery from the Suwannee and S. Platte Rivers

A comparison of the DOC recoveries for various NOM fractions for the
Suwannee River and South Platte River (first sampling) is shown in Figure 4.6.

90
60

50
Percent of DOC

Suwannee
40 South Platte

30

20

10

0
Hydrophobic Transphilic Hydrophilic Ultra-
hydrophilic

Figure 4.6. Comparison of NOM fractionation in the Suwannee and South Platte Rivers
**MB add column ‘unaccounted for’

The figure illustrates dramatically the result shown in Tables 4.2, 4.3, and 4.4 that
the bulk of the NOM is contained in the hydrophobic and transphilic fractions. These
fractions can be isolated with a modest degree of effort (Figure 4.2). The hydrophilic and
ultra-hydrophilic fractions constitute less than 10% of the recovered DOC, and they can
be isolated only with a substantial amount of effort (Figures 4.3, 4.4, and 4.5).

The South Platte River NOM is significantly more hydrophilic than the Suwannee
River NOM, and the base and neutral fractions represent a greater percentage of the
NOM in the South Platte than in the Suwannee. This difference in NOM fractionation
patterns suggests that the NOM is less degraded (humified) in the South Platte. Such a
difference might be related to the winter sampling of the South Platte when microbial
degradation rates are slow. Further discussion of these differences is provided after the
results from some of the other characterization techniques are presented.

91
CASE STUDIES FOCUSING ON MAXIMAL NOM RECOVERY WITHOUT
FRACTIONATION

Membrane Processes for NOM Recovery from European Waters

The efficiency with which NOM could be collected and concentrated by RO and
NF was compared directly using four surface waters. Most of the experiments were
conducted using a concentration factor (initial volume/final volume) near 10. The results
obtained using different membranes are summarized in Tables 4.5 and Table 4.6.

Table 4.5
Volume and DOC content of the solutions collected from reverse osmosis and
nanofiltration processing of five surface waters
Water Membrane Raw Water Concentrate Permeate NaOH elution
DOC Volume DOC Volume DOC Volume DOC Volume
(mg/L) (L) (mg/L) (L) (mg/L) (L) (mg/L) L
Gartempe TW-30 2514 6.4 10 55.5 1 0.6 9 nd * nd
River TW-30 4040 6.4 740 39.0 110 0.2 630 10.9 40
Thames River TW-30 40401 3.9 200 17.2 32.5 0.4 167 4.3 38
TW-30 40402 3.9 95 8.7 35 0.3 60 1.7 40
Vienne River TW-30 4040 4.9 345 37.4 30 0.4 315 10.5 40
NF-70 4040 4.9 340 36.2 28 1.2 312 8.8 40
Blavet River TW-30 4040 12.0 400 160 27.5 0.2 372 16.3 40
(Winter) NF-70 4040 12.0 310 105 28.0 1.0 281 17.3 41
Blavet River TW-30 4040 6.6 200 41 28 0.3 170 nd nd
(Summer) NF-70 4040 6.6 200 37 27 0.2 170 nd nd
* nd : not determined

92
Table 4.6
Isolation of NOM: Efficiency of RO and NF membranes
Water Membrane Rejection Concentrate Permeate NaOH Elution Total
DOC Rejection or Recovery (%)
Gartempe TW-30 2514 92 87 8 - -
River TW-30 4040 97 90 3 9 102
Thames River TW-30 4040 91 72 9 21 102
TW-30-4040 95 81 5 19 105
Vienne River TW-30 4040 93 65 7 24 96
NF-70 4040 77 60 23 21 104
Blavet River TW-30 4040 98 91 2 13 106
(Winter) NF-70 4040 92 79 8 19 106
Blavet River TW-30 4040 97 87 3 - -
(Summer) NF-70 4040 96 76 4 - -

For all waters, the DOC rejection efficiency ranged from 91 to 98% using RO and
from 77 to 96% using NF. Overall DOC recoveries were generally close to 100%, but the
distribution of the DOC among the concentrate, permeate, and the membrane surface
depended on the water source and the type of membrane used. In the only tests in which
the different sizes of units were compared (using the 2514 and 4040 size membranes to
process Gartempe water), they gave similar results.

The effect of raw water quality on the performance of these systems can be
assessed by comparing the results for the five samples obtained using the TW-30 4040
RO membrane or the four samples concentrated using the NF-70 membrane. The DOC
recovery in the RO concentrate was similar (87% to 90%) for the Gartempe river and the
two samples from the Blavet River. However, for the Vienne and Thames rivers, the
NOM recovery in the concentrate was much lower (65% to 81%), and the recovery in the
NaOH rinse was correspondingly higher. The DOC concentration in the source water
does not seem to be a critical factor controlling the recovery efficiency in the concentrate,
since the Gartempe river and the Blavet River (winter sample) yielded similar DOC
recoveries despite having a large difference in DOC contents (6.4 and 12.0 mg C/L,

93
respectively). Sun et al. (1995) also obtained good recoveries by RO regardless of the
DOC concentration in the source water.

The nature of the NOM, and in particular its aromatic character, might be at least
partially responsible for the different behavior of the different waters. The SUVA254
value was significantly lower for the Vienne and Thames rivers (3.6 and 3.2 L/mg-m)
than for the Gartempe and Blavet Rivers (4.4 to 5.1 L/mg-m), suggesting that hydrophilic
NOM was preferentially retained in or on the membrane. The result might also be related
to the salt content of the source water.

Salt Concentration and Recovery

When water samples are concentrated by RO or NF, inorganic species are


concentrated along with NOM. Because inorganic species may influence NOM properties
and can interfere with some analytical characterizations of the NOM, attention was paid
to monitoring and controlling the major inorganic ions in the samples. Only anions were
taken into account, because most inorganic cations were exchanged for Na+ in the cation
exchange resin prior to membrane filtration. Extensive analysis of anions was conducted
only on samples from the Vienne and Blavet Rivers. The results of these analyses are
presented in Tables 4.7 and 4.8.

94
Table 4.7
Concentration of anions in permeate and concentrate produced by reverse osmosis and
nanofiltration
Raw Concentrate Permeate Ion Rejection (%)
water
TW30 NF70 TW30 NF70 TW30 NF70
Vienne River Vol L - 30 28 315 312 - -
(1)
* HCO 3− mg/L 48.2 315 237 2.6 11.6 95 76

Cl− mg/L 20.2 57.0 36.0 0.6 3.7 97 82


Br− mg/L 0.06 0.16 0.07 <0.03 <0.03 >60 >60
NO 3− mg/L 9.7 23.7 2.5 0.7 2.9 93 70

SO 2−
4
mg/L 6.3 41.5 58 0.1 0.2 98 98

Blavet River Vol L - 27.5 28 372 281 - -


(Winter) HCO 3− mg/L 35.0 220 170 9.0 11.0 74 69

Cl− mg/L 22 190 120 1.1 4.4 95 80


Br− mg/L 0.08 0.58 0.4 <0.03 <0.03 >60 >60
NO 3− mg/L 12.0 90 38.5 1.8 6.9 85 42.5

SO 2−
4
mg/L 8.6 79 68 <0.1 <0.1 >98 >98

Blavet River Vol L 28 27 170 170 - -


(Summer) HCO 3− mg/L 37.0 170 160 4.4 4.8 88 87

Cl− mg/L 19.7 96 70 0.3 1.7 98 91



Br mg/L 0.08 0.5 0.3 <0.03 <0.03 >60 >60
NO 3− mg/L 14.1 56 37 0.8 3 94 79

SO 2−
4
mg/L 7.7 51 38 <0.1 <0.1 >99 >99

* as CaCO3

95
Table 4.8
Anion recoveries for reverse osmosis and nanofiltration
Concentrate recovery Permeate recovery Total recovery
(%) (%) (%)
TW30 NF 70 TW30 NF70 TW30 NF70
Vienne River CF(1) 11.5 12.1 - - - -
HCO 3− 57 40 5 22 63 62

Cl− 27 15 3 17 30 32

Br 23 10 - - - -
NO 3− 21 2 7 28 28 30

SO 2−
4
57 75 2 3 59 78

Blavet River CF * 14.5 11.0 - - - -


(Winter) HCO 3− 43 44 24 28 67 72

Cl− 59 49 5 18 64 67

Br 50 45 - - - -
NO 3− 52 29 14 52 66 81

SO 2−
4
63 71 - - - -

Blavet River CF(1) 7.1 7.3


(Summer) HCO 3− 64 58 10 11 74 69

Cl− 68 48 1.5 7 69.5 55


Br− 87 51 - - - -
NO 3− 56 35 5 18 61 53

SO 2−
4
93 67 1 1 94 68

* CF : Concentration factor (initial volume/final volume)

Both RO and NF concentrated the inorganic salts significantly, with RO being


more efficient than NF. Chloride and sulfate were the most efficiently rejected anions,
with RO rejection coefficients ranging from 95 to 98% and from 98% to >99%,
respectively. For NF, the corresponding values were from 80 to 91% and from 98 to
>99%. Rejection coefficients for bicarbonates and nitrates were slightly lower.

96
Because of the low concentration of bromide in natural waters and because of the
relatively high detection limit (35 µg/L), the Br− rejection efficiency could not be
determined exactly. However, a lower limit of 60% could be estimated for both types of
membranes. The actual value is probably similar to that for chloride.

Overall, anion recoveries for the concentrated waters are higher for the Blavet
River sampled in summer (65 to 90%) than in winter (40 to 60%) and are also higher for
the Blavet than for the Vienne River (20 to 60%). No explanation for this finding is
apparent since the three waters had similar ionic balances.

The most significant result of this part of the work is that total recoveries in the
concentrate plus permeate never even approached 100% for most of the inorganic
species, regardless of the water source. With few exceptions, 30 to 40% (by weight) of
the salt content was not recovered in the combined concentrate and permeate. To test for
analytical accuracy, the chloride concentration in several samples was determined by
addition of AgNO3 and gravimetric analysis of the precipitated AgCl(s). The results
confirmed those obtained by ion chromatography. The most reasonable explanation for
this finding is adsorption of inorganic species onto the polyamide membrane or
precipitation of such species in the membrane module.

An additional experiment was conducted on a synthetic solution containing


NaHCO3, NaCl, NaNO3, and Na2SO4 plus the XAD-8/XAD-4 mixture of NOM isolated
from the Blavet River winter sample. These constituents were all solubilized into MilliQ
water to obtain the following concentrations:

Cl− = 21.2 mg/L; NO 3− = 11 mg/L; SO 2− −


4 = 43 mg/L; HCO 3 = 7.1 mg/L

pH=5.5 or 8; DOC = 5.3 mg/L

Ten liters of this solution was concentrated in the lab-scale RO unit equipped with
the TW-30 RO membrane. The concentrate was recirculated into the feed tank until the
volume of the concentrate was reduced to 1 L, i.e., until the concentration factor was 10.

97
Table 4.9 gives the recovery efficiencies for the anions in this system, based on ion
chromatographic analyses.

Table 4.9
Recovery of anions in the concentrated water produced by RO treatment of a synthetic
solution
Recovery Efficiency (%)
Chloride Nitrate Sulfate Carbonate species
pH 5.5 56 72 64 59
pH 8 55 52 54 62

The concentration of anions in the permeate was near the detection limit, which
was approximately 1 mg/L for all species. However, the overall recoveries did not exceed
70% for any anion, which supports the hypothesis that some of the anions were retained
on or in the polyamide membrane.

Use of XAD-8 /XAD-4 for NOM Recovery from European Waters

XAD-8 and XAD-4 resins were used to collect NOM from the Vienne River and
from winter and summer samples from the Blavet River. In each case, approximately
300 L of water was processed in two columns in series containing 10 L of XAD-8 and
XAD-4 resin, respectively. The size of these columns allowed the total volume of water
to be processed in a single run at a k' of 50.

One hundred mL of effluent was collected from each resin column for every 20 L
of water processed. The average DOC in the effluent from the whole run is presented in
Table 4.10, and the data are presented in various alternative forms in Table 4.11.

98
Table 4.10
DOC content of the XAD-8 and XAD-4 permeates of the three water sources
Raw water XAD-8 Effluent * XAD-4 Effluent *
Volume (L) DOC (mg/L) DOC (mg/L) DOC (mg/L)
Vienne River 270 4.9 2.0 1.1
Blavet River (winter) 300 12.0 2.5 1.25
Blavet River (summer) 290 6.6 2.8 1.7
* blank subtracted, 0.1 to 0.2 mg DOC/L for XAD-8, 0.2 to 0.3 mg DOC/L for XAD-4

Table 4.11
XAD-8/XAD-4 DOC distribution and DOC recoveries
DOC Adsorption (%) * DOC Recovery (%)
XAD-8 XAD-4 Total XAD-8 XAD-4 Total
Vienne River 59 18 77 45 13.5 58
Blavet River (winter) 79 10.5 90 55 6 61
Blavet River (summer) 57 21 78 56 17 73
* after column rinse, NaOH elution, and ion exchange.

The DOC adsorption efficiency indicated in Table 4.11 can be appropriately


compared with the rejection coefficient for membrane processes, whereas the total DOC
recovery efficiency (the sum of the DOC recoveries of the XAD-8 and XAD-4 resins)
might appropriately be compared with either the concentrate recovery efficiency or the
total recovery efficiency for membrane processes, depending on the intended use of the
comparison.

For the three surface waters, 77 to 90% of the DOC adsorbed onto the two resins
and 58 to 73% of the raw water DOC was recovered in the eluents. As was the case for
membrane processes, the highest NOM recovery efficiency was obtained for the Blavet
River sampled in the winter, which is also the sample that had the largest SUVA.

99
Combination of RO with XAD Resin for NOM Recovery from European
Waters

NOM that had been concentrated from the Blavet River winter sample and the
Thames river samples using the larger scale RO unit (TW 4040) was subjected to a
second RO concentration step using the smaller RO unit (TW 2514) and was then
desalted using XAD resins. Highly concentrated NOM solutions were produced in the
second stage of RO. DOC recoveries were similar to those obtained in the first RO
concentration step. Any loss of organic matter can be attributed to adsorption or
precipitation onto the membrane, since the DOC content of the permeate was always
negligible. Extrapolating these results, at least for the conditions tested here, the DOC
recovery from a series of RO concentration steps can be roughly estimated as (0.90)n,
where n is the number of concentration steps. (This estimate assumes that adsorbed
and/or precipitated NOM is not recovered by application of an NaOH rinse.)

After acidification to pH 2, the concentrated NOM solutions were filtered through


XAD-8 or XAD-4 resin using low k' values in order to optimize NOM retention. Typical
breakthrough curves for DOC in the column effluent are shown in Figure 4.7.

100
60
Volume of resin : 250 mL
50 DOCin = 250 mg/L

40
DOC, mg /L

30

20

10

0
0.0 0.5 1.0 1.5 2.0 2.5
Vol. H2O/ Vol. XAD-8, L/L

20
Volume of resin : 730 mL
18
16 DOCin = 272 mg/L

14
DOC, mg/L

12
10
8
6
4
2
0
0.0 0.5 1.0 1.5 2.0
Vol. H2O/ Vol. XAD-8, L/L

Figure 4.7. Breakthrough curves for treatment of concentrated NOM solutions with
XAD-4 resin

101
The experiments confirmed the large adsorption capacity of both XAD-8 and
XAD-4 resins for aquatic NOM (Table 4.12). NOM recovery efficiencies were 87% or
higher when k′ was less than 10. As expected based on its greater hydrophilic character,
the recovery efficiency for NOM from the Thames river was lower than that from the
Blavet.

Table 4.12
Purification of RO-concentrated water: RO and XAD resin desalting in series
Water Procedure Membrane XAD desalting § Total
source concentration recovery
(RO TW 2514)
* † DOC Resin k' DOC ‡ DOC DOC
DOCf Recovery volume Recovery Isolation %
(mg/L) (%) (mL) efficiency yield (%)
(%)
Blavet RO +
River XAD-4 + 120 89 250 9 94 74 61
(winter) dilution
RO +
XAD-4 250 89 730 1.7 97 83 69
RO +
XAD-8 250 89 250 3.2 92 80 66
Thames RO +
River XAD-4 52 80 1,000 6.5 87 70 46
(run #2)
* Final DOC content after the second stage of reverse osmosis.
† DOC recovery from the second stage of RO concentration.
‡ DOC recovery calculated based on the elemental analysis of the NOM fraction.
§ Includes both RO steps.

Following the adsorption step, the columns were rinsed with formic acid to
remove salt from the column void volume before the organics were eluted with a mixed
acetonitrile (75%), MilliQ water (25%) solution. After rotary evaporation of the majority
of the acetonitrile, the eluate was lyophilized. During the lyophilization step, residual
formic acid and acetonitrile are removed. As discussed in the following chapter, the ash
content of the isolated fractions was low and confirmed the efficiency of this desalting
technique. However, the DOC recovery efficiencies ranged from only 70 to 83%,
corresponding to 12 to 20% loss of NOM during the desalting procedure. No significant
difference in this respect was observed between the XAD-8 resin and the XAD-4 resins.

102
The last column of Table 4.12 shows the total NOM recovery efficiency in these
tests. The difference between this value and 100% yields the combined loss of NOM in
the two RO concentration steps and the XAD resin desalting step. For the Blavet, total
recoveries for the three experiments were similar, ranging from 61 to 69%. These values
are close to the DOC recovery achieved by processing the whole water by the XAD-8/
XAD-4 protocol alone. For the Thames river, the total recovery was only 46%. The
reason for this poor recovery is unclear. Since the Thames river NOM was probably more
hydrophilic and probably had a lower average molecular weight than that from the
Blavet, it is possible that some volatile organics were lost during the lyophilization. It is
also possible that colloidal NOM was not recovered, including colloids that might have
formed in the RO concentrate because of its high concentration of salts.

NOM Concentration and Isolation of Judy Reservoir and Tolt River Water
By Reverse Osmosis

The performance of RO for NOM recovery was also tested with water from Judy
Reservoir and the Tolt river. The key results can be summarized as follows:

• the DOC concentration in the permeate was in the range 0.2 to 0.5 mg/L for both
waters, comparable to the results for the European water sources;

• the UV absorbance and fluorescence of the permeate were negligible;

• NOM was prone to become trapped in the RO cartridge during each round of
concentration. Most of the trapped NOM could be eluted with deionized water, as
opposed to the experience with the European waters, in which case more elaborate
techniques were required.

The NOM rejection efficiency by RO ( η RO


DOC ) can be calculated based on the DOC

concentrations in the raw water and permeate using Equation 4.1:

103
( R split + 1) ⋅ DOC raw water − R split ⋅ DOC permeate
η RO
DOC = (4.1)
( R split + 1) ⋅ DOC raw water

where Rsplit is the RO membrane splitting ratio (Vpermeate/Vretentate). η RO


DOC is also the

maximum possible NOM recovery efficiency using RO, for a given water and operating
conditions; the actual recovery efficiency is typically less than η RO
DOC , because some

NOM remains bound to the membrane, rather than being recovered in the concentrate.
For the operational conditions used, η RO
DOC was 87% and 92% for the Tolt River and Judy

Reservoir, respectively. The negligible UV absorbance and fluorescence of the permeates


signifies that humic species were essentially completely rejected or retained by the RO
membrane, so the NOM in the permeate must have been low molecular weight and very
low aromaticity fractions of NOM.

For typical operational conditions, calculations based on Equation 4.1 show that
the DOC retention efficiency by RO is not highly sensitive to the DOC of the permeate or
the splitting ratio. For instance, for permeate DOC concentrations from 0.3 to 0.5 mg/L
and splitting ratios from 2 to 7, η RO
DOC is between 75 and 89% for the Tolt River and

between 88 and 95% for Judy Reservoir. Thus, as is commonly observed, high NOM
recoveries are relatively easy to achieve using RO. However, the amount of concentrate
generated and, correspondingly, the number of RO cycles required to achieve a given
concentration factor, do depend strongly on the splitting ratio selected.

The retention of Ca, Mg, and other inorganic salts by RO is potentially the biggest
problem associated with the use of RO to concentrate NOM from natural waters. These
salts can interfere by forming inorganic or mixed organic-inorganic solids in the RO unit,
potentially causing irreversible blockage of the pores and/or retention of NOM. Off-line
removal of calcium and magnesium by cation exchange (as was done in RO experiments
with Judy Reservoir and Tolt River waters) overcomes these problems, but at the risk of
simultaneously losing the basic fractions of the NOM. The performance of RO is
compared below with that of a few sorption-based techniques.

104
Comparison of NOM Concentration and Isolation Using IOCS, IOCO, and
XAD Resins for Judy Reservoir and Tolt River Water

Retention of NOM from the Pacific Northwest waters by IOCS and IOCO was
compared with that by XAD-8 resin and RO. Typical DOC and A254 breakthrough curves
from columns packed with IOCO, IOCS and XAD-8 and receiving Judy Reservoir water
are presented in Figures 4.8 and 4.9.

3.0
Effluent DOC (mg/L)

2.0

1.0
IOCO
IOCS
XAD-8
0.0
0 50 100 150 200 250 300 350 400
Bed volumes

Figure 4.8. DOC breakthrough curves for treatment of water from Judy Reservoir using
IOCO, IOCS and XAD-8 (influent DOC = 3.6 mg/L).

105
0.5
IOCO
IOCS
0.4
XAD-8
A254 (cm-1)

0.3

0.2

0.1

0.0
0 100 200 300 400
Bed volumes

Figure 4.9. A254 breakthrough curves for treatment of water from Judy Reservoir using
IOCO, IOCS and XAD-8 (influent A254 = 0.62 cm−1)

Equation 2.3 was used to convert the data to values of the average retention
efficiency of DOC and A254, respectively, as a function of the number of bed volumes of
water processed. The results are shown in Figures 4.10 and 4.11. Of the three adsorbents,
IOCS was most efficient at removing both A254 and DOC from solution. The integrated
DOC collection efficiencies of IOCO, IOCS and XAD-8 are similar for the two water
sources and are close to 80% for treatment of fewer than ten BVs. However, for treatment
of ~50 to a few hundred BVs of water, the efficiency of IOCS remains close to 70%,
whereas it decreases to ca. 45% for XAD-8 and to approximately 20% for IOCO. In
terms of retention of A254, the performance of IOCS is close to ideal (ηUV near 90%) for
processing of several hundred bed volumes of water, while for IOCO and XAD-8, ηUV is
approximately 40 and 60%, respectively.

106
100%

DOC retention efficiency


75%

50%

IOCO
25%
IOCS
XAD-8

0%
0 100 200 300 400
Bed volumes

Figure 4.10. Cumulative DOC retention efficiency for treatment of Judy Reservoir water
using IOCO, IOCS and XAD-8

107
100%
UV retention efficiency

75%

50%

IOCO
25% IOCS
XAD-8

0%
0 100 200 300 400
Bed volumes

Figure 4.11. Cumulative A254 retention efficiency for treatment of Judy Reservoir water
using IOCO, IOCS and XAD-8

When water from the Tolt River was processed, the IOCS once again
outperformed the other two media in terms of DOC and A254 retention (Figures 4.12
through 4.15). However, in this case, IOCO outperformed XAD-8.

108
1.5
Effluent DOC (mg/L)

1.0

0.5
IOCO
IOCS
XAD-8
0.0
0 100 200 300 400
Bed volumes

Figure 4.12. DOC breakthrough curves for processing of Tolt River water using IOCO,
IOCS and XAD-8 adsorbents (influent DOC = 1.9 mg/L)

109
0.25
IOCO
IOCS
0.20
XAD-8
A254 (cm-1)

0.15

0.10

0.05

0.00
0 100 200 300 400
Bed volumes

Figure 4.13. A254 breakthrough curves for processing of Tolt River water using IOCO,
IOCS and XAD-8 adsorbents (influent A254 = 0.26 cm−1)

110
100%

DOC retention efficiency


75%

50%

25% IOCO
IOCS
XAD-8
0%
0 100 200 300 400
Bed volumes

Figure 4.14. Cumulative DOC retention efficiency by IOCO, IOCS and XAD-8
processing Tolt River water

111
100%
UV retention efficiency

75%

50%

25% IOCO
IOCS
XAD-8
0%
0 100 200 300 400
Bed volumes

Figure 4.15. Cumulative A254 retention efficiency by IOCO, IOCS and XAD-8
processing Tolt River water

Competition Between Sulfate and NOM for Oxide Adsorbents

The most significant limitation on the use of IOCS for concentrating NOM from
natural fresh waters appears to be competition between NOM and sulfate for the IOCS
surface sites. NOM concentrates obtained by eluting the IOCS or IOCO columns with
sodium hydroxide contained substantial amounts of sulfate, but not nitrate or chloride.
Judy Reservoir water contains comparable concentrations of DOC and sulfate (3.4 and
3.7 mg/L, respectively). When this water was applied to the IOCS column, 90%
breakthrough of DOC occurred at BV≈850, whereas 90% breakthrough of sulfate
occurred at BV≈255. Thus, NOM has an affinity for the IOCS surface that is three to four
times as large as that of sulfate. The relative affinities are close enough that competition
from sulfate could be a serious impediment to the use of IOCS for NOM concentration,
especially for low-NOM, high-sulfate waters. As a rough guideline, IOCS is expected to

112
perform well for SO4/DOC mass ratios below about 2.5, and to perform acceptably for
SO4/DOC mass ratios between 2.5 and about 5. For SO4/DOC ratios >5, the presence of
sulfate will become a substantial interfering factor. The actual performance of IOCS in
high-sulfate waters was not tested in this study. However, it is reasonable to expect that
in such waters use of alternative concentration techniques (XAD-8/XAD-4 and/or
membranes) would be preferable.

Recovery of Various NOM Fractions with IOCO

Several samples of effluent from IOCO processing of Judy Reservoir water were
fractionated using XAD and ion exchange resins. The samples were collected after
various numbers of BVs had passed through the IOCO column and had DOC
concentrations ranging from <1 to >3 mg/L. The volume of sample processed in the
fractionation experiments varied from 500 to 2000 mL depending on DOC, such that the
total load of organic carbon applied to the fractionation columns was always close to
2 mg. Only the neutral and acidic NOM fractions were analyzed, because previous
research suggested that organic bases were present in the raw water in negligible amounts
and because, if any bases had been present, they probably would have been partially
removed by the ion exchange step applied in processing the sample. The results of the
analyses are shown in Table 4.13.

113
Table 4.13
Results for the fractionation of IOCO effluent for treatment of Judy Reservoir water
Effluent #1 Effluent #2 Effluent #3 Effluent #4 raw water
Range of BVs during 6-8 54-56 198-200 396-398 n/a
sample collection
DOC (mg/L) 1.05 1.65 2.30 2.88 3.40
Percentage of DOC in Given Fraction
hydrophobic fractions 31 38 38 38 39
(XAD-8)
transphilic fractions 20 24 23 23 22
(XAD-4)
hydrophilic acids 0 0 0 8 11
(Duolite A-7)
hydrophilic neutrals 50 39 38 30 28

Changes over time in the composition of NOM in the IOCO effluent reflect
progressive saturation of the column with adsorbed NOM. At low bed volumes, the
hydrophilic neutrals account for 50% of the DOC in the effluent compared to only 28%
in the raw water, indicating that the IOCO preferentially adsorbs the hydrophobic and
hydrophilic acidic fractions of NOM. At later times, the IOCO seems to be especially
efficient at collecting hydrophilic acids while not collecting hydrophilic neutrals. Thus,
throughout the run, the IOCO adsorbs acidic fractions of NOM preferentially and
efficiently. In all likelihood, the same result would be obtained for IOCS and other iron
oxide-based adsorbent media (see Korshin et al. (1997) for additional discussion of the
IOCS data).

Comparison of NOM Concentration-Isolation-Fractionation Techniques

This portion of the research evaluated NOM isolation using both traditional and
novel methods (RO, NF, XAD-8/XAD-4, iron-oxide-coated adsorbent media). The
XAD-8 resin protocol developed by researchers at the U.S. Geological Survey (Leenheer
1981, Thurman and Malcolm 1981) has been widely used and is considered the reference
method for the isolation of aquatic NOM. In this procedure, NOM is separated into
hydrophobic and hydrophilic parts. The hydrophobic fraction of NOM is separated by
114
adsorption at pH = 2 onto XAD-8 resin packed in a column, using a pre-defined column
capacity factor k´ (typically in the range 50 to 100). Most (at least 70%, and often >80%)
of the NOM adsorbed on XAD-8 resin can be eluted with 0.1 N NaOH; this fraction is
referred to as the hydrophobic acid (HPOA) fraction. Almost all of the NOM remaining
on the resin can be recovered with acetonitrile and is referred as hydrophobic neutrals
(HPON).

Conventionally, hydrophilic NOM is isolated using a similar process with XAD-4


resin. However, in this study, the NOM that is conventionally defined as hydrophilic was
collected as three portions, which were defined (in order of increasing hydrophilic
character) as the transphilic, hydrophilic and ultrahydrophilic fractions. Elution of
XAD-4 resin with NaOH yields the transphilic acid (TPHA) fraction that typically
comprises 50 to 70% of the NOM adsorbed by this resin. The subsequent elution of
XAD-4 resin with acetonitrile yields the transphilic neutral fraction (TPHN). The NOM
that is not adsorbed is processed further and fractionated by sorption reactions into
hydrophilic and ultrahydrophilic fractions. Although significant effort is required to clean
the resins used in these procedures, the adsorption and elution processes are relatively
simple.

The literature (Martin 1997) and our data indicate that the percentage of NOM
that adsorbs on XAD-8 increases substantially with increasing SUVA254 of the influent
(Figure 4.16). For Suwannee and Blavet (winter) NOM that had SUVA254 values close to
5 L/(mg-m), approximately 80% of the DOC adsorbed. By contrast, for South Platte and
Tolt NOM, with SUVA values <3 L/(mg-m), the adsorption efficiency was <40%.

115
Blavet winter
80
NOM Sorbed by XAD-8 resin (%)

Suwannee
75
70
Apremont
65 summer
Vienne
60 Mayenne
55 Blavet
Apremont summer
50
winter
45 Judy y = 13.2x + 8.6
40 2
Tolt R = 0.67
35 S.Platte
30
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
SUVA254 (L/mg-m)

Figure 4.16. Relation between initial SUVA and XAD-8 resin adsorbability (at pH 2) for
several surface waters

The overall NOM isolation efficiency can be improved by using XAD-8 and
XAD-4 resins in tandem. The efficiency of NOM retention by XAD-4 is, to a first
approximation, inversely proportional to that by XAD-8 (Figure 4.17) Therefore, the
benefits of XAD-4 are more pronounced for low-SUVA254 than for high-SUVA254 waters
(Figure 4.18). Thus, for South Platte NOM, use of XAD-8 and XAD-4 in series increases
the overall DOC retention by 26%, while for Suwannee NOM the increase is only 11%.
In the current research, the total DOC adsorption efficiency using the two resins in series
ranged from 60% (South Platte) to 90% (Suwannee or Blavet (winter)).

116
35
DOC Sorption onto XAD-4 (%)

30 Apremont
Mayenne
winter
25 Blavet
S.Platte
summer
20 Apremont summer

15 Vienne
Suwannee

10 Blavet
y = -0.37x + 41.8 winter
5 2
R = 0.84
0
30 40 50 60 70 80 90
DOC Sorption onto XAD-8 (%)

Figure 4.17. Relative amounts of NOM sorbed onto XAD-8 and XAD-4 resins in series

100
NOM Adsorption onto XAD resin (%)

90

80

70

60

50
XAD-8
XAD-8 + XAD-4
40

30
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
SUVA254, L/mg-m

117
Figure 4.18. Relationship between the adsorption of DOC and SUVA using XAD-8 and
XAD-4 resins in series versus XAD-8 alone

It appears that the affinity of acidic NOM fractions for iron-based adsorbent
media may exceed that for XAD-8 and XAD-4. On the other hand, the neutral and basic
fractions are retained only minimally by iron oxides, probably because NOM-oxide
interactions occur primarily between the carboxyl groups of NOM and associated active
sites on the oxide surface (Parfitt and Russell 1977; Gu et al. 1994, 1995). By contrast,
the retention of NOM by XAD and other similar resins occurs primarily through
interactions of the resin with the hydrophobic core of NOM molecules, and ionization of
acidic groups decreases the tendency for these molecules to be retained. (This is the
reason that fractionation using XAD resins is typically carried out at low pH.) For the
two water sources where the comparison were carried out, IOCS retained NOM more
efficiently than did either IOCO or XAD-8, especially when more than 50 bed volumes
of water were processed. On the other hand, IOCO offers the advantage that it can be
used at pH 7, whereas XAD-8 and IOCS required the sample to be acidified to pH near 2
in order to be effective. However, given the fact that many NOM molecules are likely to
have both acidic and non-acidic, and hydrophobic and non-hydrophobic functionalities,
no global conclusion can be reached favoring one sorbent over the other for maximizing
overall NOM recovery. The results presented here suggest that some combination of
oxide-based and XAD adsorbents might be an attractive option for achieving very
efficient NOM recovery, much as XAD-8 and XAD-4 resins have been used together in
the past.

The efficiency of NOM isolation may be improved through the use of adsorption
media other than XAD resins, such as and iron oxide coated sand (IOCS) or olivine
(IOCO). For instance, the retention of Judy Reservoir NOM increased from 45% using
XAD-8 to 70% using iron oxide coated sand (IOCS). For Tolt River NOM, the
corresponding increase was from 30% to 60%. It is possible that similar results could be
achieved using XAD-8 and XAD-4 in series. Even so, an advantage of using IOCS is that
the retained NOM can be completely recovered by a single elution with sodium
hydroxide. The major disadvantages of IOCS are that this material is not commercially
118
available (although it may be prepared using relatively simple procedures (Benjamin et
al.1993)) and that sulfate co-adsorbs with NOM on IOCS. Adsorption of sulfate may
compromise the efficiency of NOM retention by IOCS in high-sulfate waters. Also, since
sulfate and NOM are co-eluted by sodium hydroxide, sulfate tends to accumulate in the
concentrate from an IOCS process. Therefore, more effort toward the development of
alternative elution techniques and/or of iron oxide surfaces that are less active toward
sulfate is recommended.

In general, 20 to 30% of the NOM in surface waters is not adsorbed by XAD-8 or


XAD-4 resins or by iron-oxide-coated media. This part of NOM includes the compounds
defined in this work as hydrophilic or ultrahydrophilic. The material isolated as
hydrophilic NOM may include both high molecular weight colloidal NOM (probably
associated with polysaccharides and N-acetyl amino sugars that have acidic groups) and
low molecular weight organics (e.g., aliphatic and other acids). More work is needed to
improve the recovery of these NOM fractions. In particular, the use of ultrafiltration
and/or rotary evaporation (for low humic content waters) as a preliminary step to separate
and isolate the colloidal fraction of NOM should be explored.

The isolation of hydrophilic and ultrahydrophilic NOM from the inorganic


constituents present in the sample is particularly challenging. Evaporative concentration
of this NOM with separation of crystalline inorganic salts followed by desalting with
non-ionic and ionic exchange resins was successful for the Suwannee HPI fractions, but
<20% of the South Platte HPI fractions were recovered using this desalting method.
Additional experiments demonstrated that colloidal organic carbon, which might be more
abundant in high conductivity, low SUVA surface waters such as the South Platte, may
account for the major part of the lost NOM due to size exclusion from the adsorbent
media.

Reverse osmosis and other membrane-based techniques offer an alternative


approach for isolating NOM. Compared to other NOM isolation methods, RO is
relatively simple and rapid, but it requires a large capital investment. Ideally, an RO
membrane would be permeable to water molecules but would reject all solutes. For the

119
waters studied, the DOC rejection using RO was usually >90% (87% for the Tolt River).
NF membranes are typically 5 to 10% less efficient than RO membranes for collecting
NOM. Humic species are essentially completely rejected by RO, but highly hydrophilic,
low molecular weight organic compounds can break through the membrane.

The major problems associated with RO are that it concentrates inorganic salts
with high efficiency, along with the NOM, and that some NOM (and, surprisingly,
inorganic salts) might be retained in or on the membrane itself. The accumulation of salts
may cause substantial alteration and/or loss of NOM via interactions with both dissolved
and precipitated inorganic components (e.g., formation NOM-silicic acid complexes).
Unless the concentrated NOM is intended to be used in experiments in which the
presence of salts is not detrimental, desalting procedures must be carried out, and these
procedures themselves can cause loss or alteration of the NOM. The use of NF membrane
reduces the problems associated with salt accumulation, but trapping and/or adsorption of
NOM remains a serious problem.

The loss of NOM on the surface or in the pores of the membrane can substantially
lower the DOC recovery in high conductivity, low SUVA254 waters. In such waters
organic colloids may also account for >10% of the DOC. Some of the trapped NOM and
salts can be recovered by rinsing the membranes with MilliQ water or NaOH solution.
Without the additional effort to release NOM trapped in the cartridges, the efficiency of
RO in isolating NOM is 10 to 20% higher than that of XAD-8 and XAD-4 used in
tandem. Further research into the retention and release of NOM and salts by membranes
is needed.

Ultimately, the selection of an NOM concentration technique should be based on


the experiments expected to be carried out using the NOM and on the amount of effort
that can realistically be invested. The physico-chemical properties of the water source
(e.g., DOC, SUVA254, and conductivity) must also be considered, since they will affect
the performance of the isolation methods. Although improvements in NOM concentration
and isolation are certainly achievable, the goal of ~100% efficiency for NOM isolation
can probably never be achieved. In our study, losses of NOM due to precipitation of salts

120
and experimental errors were only slightly less when the most laborious and efficient
techniques were used than when NOM was collected using XAD-8 and XAD-4 resins or
IOCS, for which the required effort was much less. Further research directed at
improving the isolation and recovery efficiency is therefore of interest, as is research into
analytical techniques that can be carried out without pre-concentration or isolation of
NOM. Some such techniques are discussed in greater detail in subsequent chapters.

121
CHAPTER 5 NOM CHARACTERIZATION: SUWANNEE AND SOUTH
PLATTE RIVERS

INTRODUCTION

Although some significant new NOM fractionation and characterization


procedures were developed in this project, most of the procedures utilized are not new.
Rather, the novel aspects of this project were the comprehensive and integrated use of
multiple procedures and the comparisons among different approaches. In the following
sections, we present the results obtained by applying these procedures to fractionate and
characterize water samples from two sources that are expected to have substantially
different qualities of NOM. The ultimate goal is neither to provide the most
comprehensive description possible of those particular waters nor to provide a technically
advanced and detailed description of how to interpret the data from each analytical
approach. Rather, it is to assess whether and how the procedures can be used in an
integrated fashion to gain insight into NOM structure and reactivity, including insights
into key similarities and differences in NOM characteristics from different samples.

An attempt has been made in the discussion to provide sufficient technical detail
so that the basis for the inferences drawn is clear, while not providing so much detail that
the broader picture is obscured. To this end, the first part of the chapter is organized by
analytical approach. The key information that each analysis provides about each sample
is then discussed, and the various NOM fractions are compared. Then, in the latter
portion of the chapter, the information obtained from all the analytical techniques is
compared and contrasted to provide a more comprehensive picture of the composition
and properties of the NOM in various fractions. In that section, an attempt is made to
highlight the NOM characteristics that are consistent with the data from several analyses.
Similarly, NOM characteristics that seem to be supported by some analyses and not
others are discussed, and possible interpretations or ways to resolve the apparent conflicts

122
are proposed. Finally, the overall characteristics of the NOM from the two sources that
were studied most intensively are compared.

BULK FRACTIONATION AND ELEMENTAL ANALYSIS

Figures 5.1 and 5.2 show the DOC distribution of the Suwannee and South Platte
Rivers among the various fractions collected. As mentioned previously, the 34% loss of
DOC from the South Platte sample is thought to be primarily hydrophilic material. The
elemental analyses of these isolates are presented in Tables 5.1 and 5.2. For some
fractions, the elemental analysis could not be obtained because of the very small quantity
of material isolated.

123
Loss HPOB 0.2 %
HPI fractions 8 % 2% HPON
1%
TPHA 6 %

TPHN
5%
HPOA
77%

HPIB 0.04%
uHPIN 0.2 %
HPIN 0.2 %
uHPIA 0.2 %

uHA 2.1 % HPIA 5%

Figure 5.1. DOC distribution of the Suwannee River (A) all fractions (B) hydrophilic
fractions

124
HPI fractions HPON 3%
6%

HPOA 31 %

loss 34 %

TPHA 14 %
TPHN 12 %

uHPIA HPIN
1.1 % 0.9 %

HPIA 5%

Figure 5.2. DOC distribution of the South Platte River (A) all fractions (B) hydrophilic
fractions

125
Table 5.1
Elemental analysis of the Suwannee River NOM isolates
Fraction C H N O S Ash Total
Fraction of mass (%)
Hydrophobic neutrals HPON 55.8 6.11 1.19 32.6 1.69 0.20 97.6
Hydrophobic acids HPOA 48.0 4.13 0.69 41.3 < 0.30 3.80 97.9
Transphilic acids TPHA 40.2 3.9 0.87 41.7 3.00 4.44 94.1
Transphilic neutrals TPHN 47 4.9 1.58 43.5 0.87 <0.4 97.8
Hydrophilic acids HPIA 43.0 3.99 1.28 46.6 0.39 1.90 97.2
Hydrophilic acids 2 HPIA-2 35.6 4.01 1.77 43.2 0.28 16 100.9
Hydrophilic bases HPIB 19.5 2.32 2.51 17.6 0.50 5.40 47.8
Hydrophilic neutrals HPIN 39.2 5.49 3.74 35.3 0.38 14.5 98.6
Ultrahydrophilic acids uHPIAMe 45.2 4.57 0.97 44.4 < 0.30 <0.10 95.1
Ultrahydrophilic humic uHA 47.4 5.24 3.20 29.6 < 0.30 4.40 89.8
acids
Ultrahydrophilic uHPIN 31.8 4.63 2.16 39.5 <0.30 7.50 95.5
neutrals

Table 5.2
Elemental analysis of the South Platte River NOM isolates
Fraction C H N O S Ash Total
Fraction of mass (%)
Hydrophobic neutrals HPON 58.8 7.33 2.13 24.1 1.41 1.11 94.9
Hydrophobic acids HPOA 47.2 4.96 1.07 36.8 1.44 <0.20 91.5
Transphilic acids TPHA 34.6 4.04 1.95 36.9 3.06 5.04 85.6
Transphilic neutrals TPHN 48.0 6.68 11.91 27.7 1.57 0.20 96.1
Hydrophilic acids HPIA 41.0 4.04 2.74 43.8 1.85 5.13 98.6
Hydrophilic neutrals HPIN 39.3 5.57 4.40 36.1 0.73 10.80 96.9

126
With the exception of the hydrophilic neutrals, the isolated fractions are
characterized by a low ash content, indicating that the isolation protocol was quite
efficient with respect to elimination of inorganics from the sample. For 12 of the 17
isolates, the sum of the elemental masses accounts for 94 to 100% of the total mass of the
isolate. The unaccounted mass is probably a result of analytical errors rather than the
presence of minor elements (e.g., phosphorus and trace metals), which generally
comprise less than 1% of the mass of NOM. For 3 of the 17 isolates (the uHA fraction in
the Suwannee, and the HPOA and TPHA fractions in the South Platte), the sum of the
elemental masses accounts for only 85 to 90% of the total mass of the sample. The
identity of the missing mass is unclear, but once again the source of the discrepancy is
probably analytical error. Although ash can sometimes interfere significantly with the
oxygen determination, the samples for which the mass balance was poor did not have
unusually high ash contents. The analysis of the HPIB fraction from the Suwannee River
was particularly poor (sum of elements equal to 52% of total mass); however 13C-NMR
and FTIR spectra gave no indication of impurities in this sample.

Tables 5.3 and 5.4 give the C/O, C/N and C/H ratios for all NOM fractions.
Higher percentages of carbon in these samples correspond to increased unsaturation and
therefore might correspond to increasing aromaticity.

127
Table 5.3
C/O, C/H and C/N ratios of the Suwannee River NOM isolates
Fraction C/O C/N C/H
Hydrophobic acids HPOA 1.16 69.5 11.61
Transphilic acids TPHA 0.96 46.2 10.3
Hydrophilic acids HPIA 0.92 33.61 10.78
Hydrophilic acids 2 HPIA-2 0.82 20.10 8.87
Ultrahydrophilic acids uHPIAMe 1.02 46.6 9.89
Ultrahydrophilic humic uHA 1.60 14.8 9.04
acids
Hydrophobic neutrals HPON 1.71 46.9 9.03
Transphilic neutrals TPHN 1.08 29.74 9.59
Hydrophilic neutrals HPIN 1.11 10.49 7.14
Ultrahydrophilic neutrals uHPIN 0.8 14.7 6.85
Hydrophilic bases HPIB 1.10 7.75 1.10

Table 5.4
C/O, C/H and C/N ratios of the South Platte River NOM isolates
Fraction C/O C/N C/H
Hydrophobic acids HPOA 1.28 44.1 9.52
Transphilic acids TPHA 0.94 17.7 8.56
Hydrophilic acids HPIA 0.93 14.9 10.13
Hydrophobic neutrals HPON 2.44 27.59 8.01
Transphilic neutrals TPHN 1.73 4.03 7.19
Hydrophilic neutrals HPIN 1.09 8.92 7.05

The following general observations can be made about the elemental ratios in the
isolates. Within any hydrophobic, transphilic, hydrophilic, or ultrahydrophilic fraction,
the acidic molecules have lower C/O ratios than the neutral and basic molecules. This
result reinforces the idea that most of the acidic groups are carboxylic. Correspondingly,

128
the C/N and C/H ratios of the neutral molecules are generally lower than those of the
acidic molecules, consistent with greater aliphatic character of the neutrals, and the basic
molecules have the highest average N content, consistent with the expectation that most
organic bases are related to amino and amide functionality.

Within any acidic or neutral fraction the C/O, C/N, and C/H ratios generally
decrease with increasing hydrophilic character for both acid and neutral fractions
(ignoring the ultrahydrophilic acids, which should not be considered in the comparison
because they were methylated as part of the isolation procedure). Put another way, the
higher the hydrophilic character, the higher the proportions of oxygen, nitrogen and
hydrogen in the NOM fraction, i.e., the more aliphatic it is.

The C/N ratios of the Suwannee fractions are greater than in the corresponding
fractions of the South Platte. The ratios suggest that the NOM in the Suwannee is
primarily allochthonous, whereas that in the South Platte is a mixture of allochthonous
and autochthonous material.

FTIR CHARACTERIZATION

The FTIR spectra for the hydrophilic and transphilic NOM fractions isolated from
the Suwannee River are shown in Figures 5.3, and those for the hydrophilic and
ultrahydrophilic fractions are shown in Figure 5.4. The spectra indicate that the majority
of the fractions are free of detectable inorganic constituents such as carbonates, nitrates,
phosphates, silica, and sulfates. Some silica was present in the ultrahydrophilic humic
acid and hydrophilic neutral fractions, as indicated by peaks at 1050, 800, and 460 cm−1
(spectra D and F, respectively, in Figure 5.4).

129
A

A
C

4000 3000 2000 1500 1000 400


CM-1

Figure 5.3. FTIR spectra of HPO and TPH Suwannee River NOM fractions (A) HPON
(B) HPOA (C) HPOB (D) TPHA (E) TPHN

A D

4000 3000 2000 1500 1000 400


CM-1

Figure 5.4. FTIR spectra of HPI and uHPI NOM fractions from the Suwannee River
(A) HPIA (B) HPIA-2 (C) uHPIA (D) uHA (E) HPIB (F) HPIN (G) uHPIN

All of the spectra shown in Figure 5.3 reflect the predominately acidic character
of the isolates by the carboxyl peak at 1720 cm−1. The hydrophobic fractions (spectra
A-C) have larger aliphatic hydrocarbon peaks (2960, 2920, 1440 and 1380 cm−1) than do

130
the transphilic fractions (spectra D and E). The transphilic neutral fraction (spectrum E)
has carboxyl group peaks that are broadened and shifted compared to the transphilic acid
13
fraction (spectrum D). Differences between the C-NMR spectra of these fractions
(discussed below) were much less noticeable. Bellamy (1960) notes that carboxyl groups
that are clustered close together in organic polyacids often form hydrogen bonds with one
another that are apparent in solid-state spectra, and this produces broadening and shifting
of carboxyl-group peaks.

The spectra in Figure 5.4 have much more diversity than those in Figure 5.3.
Significant alcohol content (from carbohydrates) is indicated by the C-O stretching peak
near 1100 cm−1 in the HPIA- (spectrum B), uHA (spectrum D), HPIN (spectrum F), and
uHPIN (spectrum G) fractions. Significant secondary amide content indicated by the
amide-1 (1660 cm−1) and amide-2 (1550 cm−1) bands from proteins can be seen in the
uHA fraction (spectrum D) and HPIB fraction (spectrum E), and from the N-acetyl group
of amino sugars in the HPIN fraction (spectrum F). The presence of silica is indicated by
peaks at 1050, 800, and 460 cm−1 in the uHA fraction (spectrum D) and the HPIN
fraction (spectrum F); the rest of the fractions have no detectable inorganic constituents.

Anomalous hydrophobic character of the uHA fraction is indicated by the


aliphatic C-H stretching peak near 2920 cm−1 in spectrum D. One possible explanation
for this observation is that this fraction contains hydrophobic colloids whose size
prevents penetration into the resins. A similar observation was made by Aiken et al.
(1979) for a series of polyacrylic acids, some of which were large enough to be regarded
as colloids.

The FTIR spectra of the hydrophobic and transphilic NOM fractions isolated from
the first South Platte River sampling are presented in Figure 5.5. The two neutral
fractions (spectra A and C) have signals indicative of the presence of proteins; in fact, the
TPHN fraction (spectrum C) appears to be almost entirely proteinaceous, based on the
absence of any substantial acidic content (i.e., the absence of a peak near 1720 cm−1).
Consistent with expectations, the TPHA fraction (spectrum D) has more carbohydrate
character (the broad peak near 1100 cm−1) than does the HPOA fraction (spectrum B).
131
The HPON fraction shows pronounced aliphatic hydrocarbon character by its peaks at
2940, 1440, and 1380 cm−1.

B
A

4000 3000 2000 1500 1000 400


CM-1

Figure 5.5. FTIR spectra of hydrophobic and transphilic NOM fractions from the first
sampling of the South Platte River (A) hydrophobic neutrals (B) hydrophobic acids
(C) transphilic neutrals (D) transphilic acids

The spectra of the HPI and uHPI fractions isolated from the same sample after
evaporative concentration are presented in Figure 5.6. Although these NOM fractions
constitute only about 7.4 percent of the DOC (Table 5.2), their spectra are of interest
because these fractions have rarely been isolated and purified from a natural water
sample previously. Spectra 5.6 B, C, and D are particularly unusual because they have a
strong amide-1 peak near 1660 cm−1 without an amide-2 peak near 1550 cm−1. This result
indicates that these fractions are not proteinaceous, but that they might have primary
amide functional groups, possibly formed by the incorporation of ammonia into NOM.

132
A

C
A

4000 3000 2000 1500 1000 400


CM-1

Figure 5.6. FTIR spectra of hydrophilic and ultrahydrophilic NOM fractions from the
first sampling of the South Platte River (A) hydrophilic acids (B) hydrophilic bases
(C) coprecipitated ultrahydrophilic acids (D) ultrahydrophilic acids (E) hydrophilic
neutrals (F) ultrahydrophilic neutrals.

The FTIR spectra of all the NOM fractions isolated from the second South Platte
River sampling using the resin sorption procedures of Figure 4.5 are presented in
Figure 5.7. Fewer NOM fractions were generated from the second South Platte River
sample than from the first. The major difference between the spectra from the two
sampling dates was that proteinaceous NOM was much less apparent in the second
sample than in the first. This difference is likely related to NOM changes in the river,
because the second sample was collected in the fall and the first was collected in the
winter. The remainder of the NOM fractions are similar in the two samples.

133
A

C
A

4000 3000 2000 1500 1000 400


CM-1

Figure 5.7. FTIR spectra of neutrals NOM fractions isolated from the second South Platte
River sampling (A) hydrophobic neutrals (B) hydrophobic acids (C) bases
(D) hydrophilic acids (E) ultrahydrophilic acids (F) hydrophilic neutrals

13
C-NMR CHARACTERIZATION

The 13C-NMR spectra for the various NOM fractions isolated from the Suwannee
and South Platte rivers are collected in Appendix **MB. The integrated areas under
13
various portions of the C-NMR spectral curves can be assigned to specific structural
features using the guidelines provided in Table **MB2.2. The assignments for the
Suwannee NOM fractions are presented in Table 5.5.
The fraction with greatest aliphatic hydrocarbon content (0 to 60 ppm) is the
hydrophobic neutral fraction, whereas the hydrophobic acid fraction has the greatest
aromatic hydrocarbon content (percent aromaticity) and ketone content (190 to 220 ppm).
Consistent with expectations, the fraction with the greatest acid content (160 to 190 ppm)
is the ultrahydrophilic acids, and the fraction with greatest carbohydrate content (90 to
110 ppm) is the ultrahydrophilic neutral fraction.

134
Table 5.5.
Integrated areas of 13C-NMR Spectra of Suwannee River NOM fractions
Fraction Shift (ppm) * Aromaticity
(%)
220-190 190-160 160-110 110-90 90-60 60-0
Relative Area (%)
HPON 2.4 10.4 12.7 3.6 14.8 56.0 14.4
HPOB 1.3 12.1 24.0 5.2 12.5 44.6 26.6
HPOA 4.8 16.7 26.1 7.8 16.8 27.9 30.8
TPIN 3.1 17.3 16.3 8.1 23.8 31.4 19.5
TPIA 4.5 18.6 16.5 10.3 22.3 27.8 20.9
HPIA 2.6 22.2 14.7 9.0 23.6 27.7 18.2
HPIA-2 4.0 20.8 10.4 9.8 33.8 21.1 12.7
uHPIA 0 22.7 9.0 7.9 24.0 36.4 11.2
uHA 1.5 11.4 22.6 8.4 20.4 35.6 27.0
HPIB 3.5 19.6 16.6 3.4 16.2 40.6 18.3
HPIN 2.0 10.2 6.9 10.7 46.9 23.3 8.3
UHPIN 1.3 12.9 6.1 12.5 49.5 17.6 7.5
* % Aromatic = %C110-160 + [%C110-160/(%C110-160 + %C60-90)] x %C90-110

The corresponding data for the NOM fractions from the first and second South
Platte River sampling events are presented in Tables 5.6 and 5.7, respectively.

135
Table 5.6
Integrated areas of 13C-NMR spectra of South Platte River NOM fractions from first
sampling (spectra presented in Figures Error! Reference source not found. and Error!
Reference source not found.)
Shift (ppm)
Fraction 220-190 190-160 160-110 110-90 90-60 60-0
Relative Area (%)
HPON 2.7 8.4 9.3 3.2 15.0 61.4
HPOA 2.0 15.2 11.6 3.4 15.4 52.4
TPIN 0.0 17.9 9.4 1.7 12.7 58.3
TPIA 3.6 19.7 5.3 5.4 22.7 43.3
HPIA 1.5 25.5 9.4 5.9 23.6 34.1
uHPIA 0.0 19.9 8.9 1.9 11.9 57.4
HPIN 0.0 13.2 2.6 8.7 39.2 36.2

Table 5.7
Integrated areas of 13C-NMR Spectra of South Platte River NOM Fractions from second
sampling (spectra presented in Figure Error! Reference source not found.)
Relative Area (%)
Integration 220-190 190-160 160-110 110-90 90-60 60-0
Range (ppm)
Hydrophobic 3.1 6.1 16.8 2.3 14.8 56.9
Neutrals
Hydrophobic 2.2 15.1 13.9 4.6 16.7 47.7
Acids
Bases 0.0 15.5 18.8 3.6 17.1 45.0

Hydrophilic 1.6 19.4 9.1 6.9 25.7 37.3


Acids
Ultrahydrophili 0.0 24.4 6.3 5.4 29.3 34.6
c Acids
Hydrophilic 0.0 13.9 6.8 5.1 33.4 40.8
Neutrals

136
In the spectra from the first sampling, the hydrophobic neutral fraction has the
largest aliphatic hydrocarbon content; the hydrophobic acid fraction has the largest
aromatic carbon content; the hydrophilic acid fraction has the largest carboxylic acid
content; and the hydrophilic neutral fraction has the largest carbohydrate content.

The 13C-NMR spectra of the hydrophobic neutral and hydrophobic acid fractions
from the second sampling are similar to those from the first, except that the aromatic
peak of the hydrophobic neutral fraction is a little greater in the second sampling. In the
spectra from the second sampling, the base fraction shows a distinct C-N peak near
50 ppm, possibly related to amines. The hydrophilic acid fraction has spectral features
that make it appear similar to a combination of the transphilic acid and hydrophilic acid
fractions isolated from the first sampling. The ultrahydrophilic acid fraction appears to be
a polycarboxylic hydroxy acid and has few of the primary amide characteristics found for
this fraction from the first sampling. Lastly, the hydrophilic neutral fraction (isolated, in
this sample, by sorption on the borate-form anion exchange resin) is similar to the
corresponding fraction from the first sample (isolated by evaporative concentration in
combination with desalting by ion exchange and selective precipitation).

The computed aromatic carbon contents of all the fractions with the exception of
the ultrahydrophilic acids were greater in the second sample than in the first, probably
due to the different times and locations of sampling. The hydrophilic and ultrahydrophilic
acid fractions had greater carbohydrate content (spectral areas from 90 to 110 and 60 to
90 ppm) and the hydrophilic neutral fraction had lower carbohydrate content in the NOM
from the second sampling. This difference may be caused by the methodology change: as
noted above, fractionation was by evaporative concentration, ion exchange fractionation,
and selective precipitation during the first sampling, and by resin sorption during the
second sampling. The extensive evaporative concentration required heating an acidified
solution and may have caused polysaccharide hydrolysis in the hydrophilic and
ultrahydrophilic acid fractions, shifting the hydrolyzed neutral sugars into the hydrophilic
neutral fraction during processing of the first sample.

137
1
H-NMR CHARACTERIZATION

Satisfactory 1H-NMR spectra were obtained on only four fractions of the


Suwannee River sample because of solubility problems, rolling baselines, and inadequate
sample sizes of minor fractions. Because of these spectral problems and losses of sample
incurred upon re-processing the NOM fraction during re-isolation, and because the
spectral information was not sufficiently unique to merit further effort, these four
1
H-NMR spectra were the only 1H-NMR data collected in this study. The spectra are
shown and described in the Appendix.

DISSOLVED AMINO ACIDS AND CARBOHYDRATES

Figures 5.8 and 5.9 show the TDCA and TDAA content of the Suwannee and
South Platte River NOM isolates, respectively.

138
TDCA Concentration, µ g C/mg DOC
80
70
60
50
40
30
20
10
0
HPIA

HPOA

HPIB

HPIN
HPIA2

uHPIA
TPHA

uHPIN
TPHN
Fraction
TDAA Concentration, µ g C/mg DOC

160
140
120
100
80
60
40
20
0
HPIA

HPOA

HPIB
uHPIA

HPIA2

uHPIN

TPHA

uHA
TPHN

Fraction

Figure 5.8. TDCA and TDAA contents of the Suwannee River NOM isolates **JP can
you add bars for raw water?

139
TDAA Concentration, µ g C/mg DOC TDCA Concentration, µ g C/mg DOC

0
10
20
30
40
50
60
0
5
10
15
20
25
30
35

HPOA HPIA

TPHN TPHN

HPIA HPOA

140
HPIN TPHA

Fraction
Fraction

TPHA HPIN

HPON HPON

Raw Raw
water water

Figure 5.9. TDCA and TDAA contents of the South Platte River and its NOM isolates
TDCA and TDAA account for only a small percentage (0.2 to 8% and 0.6 to 6%,
respectively **JP see Note 2) of the DOC in the raw water samples, although they
account for a somewhat larger percentage in some of the individual fractions (e.g., 9%
and ~15% of the DOC, respectively, in the uHA fraction from the Suwannee). TDAA
accounted for the majority of the nitrogen in the hydrophobic fractions of the Suwannee
and the HPON fraction of the South Platte, and for about 5 to 20% of the nitrogen in most
of the other fractions.

For the Suwannee isolates, the HPIB fraction is one of the richest in amino acids,
consistent with the expectation that it contains proteinaceous NOM. The uHA fraction
also has a strongly proteinaceous character, which might be taken as support that it
contains hydrophobic colloidal material. Not surprisingly, the neutral fractions (HPIN,
uHPIN) are the richest in carbohydrates.

The total mass of amino acids and carbohydrates in the raw water from the South
Platte exceeded the mass that could be accounted for in the various fractions by a
substantial amount (**JP see Note 3). This observation suggests that amino acids and
carbohydrates were selectively lost during the isolation procedure (in which nearly 35%
of the DOC was not recovered). However, it is also possible that this unexpected result
reflects an analytical error.

Figures 5.10 and 5.11 show the distribution of individual carbohydrates in the
various NOM fractions of the South Platte and Suwannee Rivers, respectively. For both
waters, glucose is the dominant sugar in all isolated fractions, except for the HPON
fraction of the South Platte River, in which galactose was dominant. Sweet and Perdue
(1982) (cited by Thurman (1985)) found that glucose accounted for 75% of the total
carbohydrate of the humic acid fraction in the Williamson river (Oregon). The transphilic
and hydrophilic neutral fractions also contained significant proportions of galactose,
rhamnose, mannose and xylose, as did the uHA fraction from the Suwannee.

141
100
90 HPOA HPON TPHA
TPHN HPIA HPIN
80
Percentage of TDCAs

raw water
70
60
50
40
30
20
10
0

man-xyl
arabinose
rhamnose

glucose
fucose

galactose
glucos-

fructose
amine

Carbohydrate

Figure 5.10. TDCA distribution of the South Platte River and its NOM isolates (**MB
convert to table?)

142
100
90 HPOA uHA TPHA TPHN
HPIB HPIA HPIA2 uHPIA
80
Percentage of TDCAs

HPIN uHPIN
70
60
50
40
30
20
10
0

man-xyl
arabinose
rhamnose

glucose
fucose

galactose
glucos-

fructose
amine

Carbohydrate

Figure 5.11. TDCA distribution of the Suwannee River and its NOM isolates

Figures 5.12 and 5.13 show the distribution of individual amino acids in the
various NOM fractions isolated from the South Platte and Suwannee Rivers, respectively.
Consistent with the results reported by Thurman (1985), Malcolm (1989), and Martin
(1995), aspartic acid, glutamic acid, glycine, alanine, and serine are generally the major
amino acids in the NOM isolates. However, some particularities of the current samples
are worth noting.

143
100
90 HPOA HPON TPHA
80 TPHN HPIA HPIN
raw water
Percentage of TDAAs

70
60
50
40
30
20
10
0

lys
ile
gly
his

ala

leu
glu

met

phe
asp

tyr
ser

arg

orn
thr

Amino Acid

Figure 5.12. TDAA distribution of the South Platte River and its NOM isolates

100
90 HPOA uHA TPHA TPHN HPIB
80
HPIA HPIA2 uHPIA HPIN
Percentage of TDAAs

70
60
50
40
30
20
10
0
lys
ile
gly
his

ala

leu
glu

met

phe
asp

tyr
ser

arg

orn
thr

Amino Acid

Figure 5.13. TDAA distribution of the Suwannee River NOM isolates

144
For the South Platte, the distribution of amino acids in all fractions is quite similar
to that in the raw water; the major exception is the HPIN fraction, in which glutamic acid
is relatively more significant than in the other fractions. The FTIR and 13C-NMR spectra
suggest that this fraction consists of carbohydrates, amino sugars, and carbohydrate acid
lactone moieties.

One very distinctive feature of the TDAA distribution in the Suwannee samples is
the predominance of ornithine in the HPOA, TPHA, and TPHN fractions. A similar result
was found for the IHSS standard fulvic acid. According to Spitzy (1988), the presence of
high levels of ornithine can be an indicator of intense bacterial (**JP algal?) activity in
the system.

PYROLYSIS - GAS CHROMATOGRAPHY - MASS SPECTROMETRY

Only some of the Suwannee and South Platte River isolates were subjected to
Pyr-GC-MS analysis. The pyrochromatograms are shown in the Appendix. Table 5.8 lists
the major compounds identified in the samples and on the chromatograms.

145
Table 5.8
Major compounds identified by Pyr-GC-MS analysis of NOM fractions
Compound * Origin n° † Compound Origin n° †
Acetone 1 Methyl-pyrrole PR 22
Butanone 2 Methyl-furfural PS 23
Methanol 3 Acetophenone 24
Benzene 4 Propenoic acid 25
Acetonitrile PR 5 Acetamide AS 26
Toluene PR 6 Methoxy-phenol PHA 27
Alkylbenzene 7 Levo-glucosenone PS 28
Pyridine and methyl-pyridine PR 8 Phenol PHA 29
Styrene 9 p-cresol PHA 30
Hydroxy-propanone PS 10 m-cresol PHA 31
2-Methylfuran PS 11 Piperidinone 32
2-Methyl-2-cyclopentene- PS 12 Dimethyl-phenol PHA 33
1-one
Methyl-furanone PS 13 Hydroxy-methyl- 34
acetophenone
Furfural PS 14 Indole PR 35
Acetic acid 15 Methylindole PR 36
Furfural (isomer) PS 16 Phthalate 37
Ethyl-hexanol 17 Benzonitrile 38
3-methyl-2-cyclopentene- PS 18 Hydroxy-methyl- PR 39
1-one cyclopentenone
Pyrrole PR 19 1-methyl-naphthalene 40
Cyclo-hexanone 20 Propionic acid 41
Trimethyl-cyclopentenone PS 21 Dimethyl- PR 42
cyclopentenone
Butenoic acid 43
* PR = proteins, PS = Polysaccharides, AS = Amino sugars, PHA = Polyhydroxy aromatics
† Peak number

146
The Pyr-GC-MS chromatogram of the HPOA fraction was typical of those
presented elsewhere for hydrophobic acid fractions (Croue et al. 1993b, Martin 1995,
Harrington et al. 1996). Phenol and cresols were the predominant peaks (ignoring the
CO2 peak), which can be interpreted as a signature of the presence of
polyhydroxyaromatic (PHA) type structures.

The Pyr-GC-MS chromatogram of the HPON fraction is significantly different


from that of the HPOA fraction, with fatty acids (C12, C16) as the most abundant
pyrolysis fragments. Other acids such as butenoic, propionic, and acetic are also present.
The origin of the acids produced by the pyrolysis of NOM is not clear; they might be
produced from sugars or might be an indicator of the presence of lipids.

As in the HPOA fraction, phenol is the most abundant fragment of the


pyrochromatogram of the TPHA fraction, which again is an indicator of the aromaticity
of the NOM. However, the peaks for acetonitrile, pyrrole, cyclopentenone and especially
acetamide are relatively more significant in the TPHA fraction, indicating that proteins,
sugars and amino sugar structures are more abundant in this fraction. The origin of the
acetic acid fragment is not clear, but a large proportion of the sugars in NOM structures
can lead to production of this fragment (Bruchet, personal communication). Gray and
Bornick (1996) also reported that this fragment is not specific for a particular parent
structure, but is typically observed in waters influenced by algal productivity and may be
13
a pyrolysis product of an aliphatic backbone highly substituted with oxygen. C-NMR
spectra also showed that the TPHA fraction contains a higher proportion of carbohydrates
than the HPOA fraction.

13
The TPHN fraction was of particular interest since the C-NMR and FTIR
spectra indicated that this fraction was almost entirely proteinaceous. Its
pyrochromatogram gave a substantial protein signature. Most of the identified pyrolysis
fragments in this chromatogram (acetonitrile, toluene, methyl, dimethyl- and trimethyl-
pyridines, pyrrole, and methyl and trimethyl-pyrroles) are produced from proteins.

147
The analysis of Pyr-GC-MS chromatograms for Suwannee River NOM focused
on a comparison of the hydrophilic NOM fractions (HPIA, HPIB, HPIN). The
pyrochromatogram of the HPIA fraction was quite similar to that of the South Platte
River HPOA fraction and other HPOA fractions reported in the literature, with phenol as
the major fragment, followed by acetic acid. The general hydrophobic character of the
Suwannee River NOM may explain this result.

The pyrochromatogram of the HPIB fraction was different. The abundance and
intensity of fragments such as acetonitrile, pyridine and alkyl pyridines, pyrrole and alkyl
pyrroles are markers of proteinaceous organic structures in the sample. The abundance of
acetamide also indicates the presence of amino sugars in this fraction of NOM.

The pyrochromatogram of the HPIN fraction confirmed the hypothesis based on


13
the C-NMR and FTIR data that this fraction includes a large amount of nitrogenous
species. This fraction includes a large proportion of amino sugars, as indicated by the
exceedingly large acetamide peak. The corresponding peak was never as large in other
pyrochromatograms, confirming the specificity of this fragment as a marker of amino
sugar type structures.

The pyrolysis data can be interpreted using a modified version of the scheme
proposed by Bruchet et al. (1990). The approach groups all well identified pyrolysis
fragments into six classes: polyhydroxyaromatics (PHA), non-substituted aromatics,
proteins (PR), polysaccharides (PS), amino sugars (AS) and unknowns (identified
fragments for which the origin is not well determined). Tables 5.9 and 5.10 indicate the
relative significance of the six classes in the South Platte and Suwannee samples,
respectively.

148
Table 5.9
Relative proportions of biopolymers in South Platte River NOM isolates
Fraction PHA unsubstituted PR PS AS Origin
aromatics unknown
Contribution of given constituent (% of NOM)
HPOA 27 27.3 11.9 14.2 3.6 16
HPON 18 29.6 23 8.2 6.6 14.6
TPHA 19 18.5 19 22 10 11.5
TPHN 7.4 17.2 53.5 3.2 11.8 6.9

Table 5.10
Relative proportions of biopolymers in Suwannee River NOM isolates
Fraction PHA unsubstituted PR PS AS Origin
aromatics unknown
Contribution of given constituent (%)
HPIA 26.5 20.8 12.6 16 2.7 21.4
HPIN 20.7 8.6 15 27.7 18.6 9.4
HPIB 22 14.8 42.2 8.5 10 2.5

The general structural characteristics discussed above are reinforced using this
semi-quantitative approach. For instance, for the South Platte River, the HPOA fraction
has the highest proportion of PHA of the fractions analyzed; the TPHA fraction has a
higher proportion of proteins, polysaccharides and amino sugars than the HPOA fraction;
and the TPHN fraction has the highest protein content.

For the Suwannee River, the HPIB fraction contains a large proportion of proteins
in its structure, while the HPIN fraction has larger proportions of amino sugars and
polysaccharides. The distribution of the biopolymers in the HPIA fraction is similar to
that in the HPOA fraction of the South Platte River. The larger proportion of
unsubstituted aromatic fragments in the Suwannee River NOM may correspond to its
more pronounced terrestrial origin.
149
UV SPECTRA

The UV spectra of selected Suwannee River NOM fractions are presented in


Figure 5.14. The spectra of the NOM fractions do not exhibit any strong distinctive
features compared to spectra of the unfractionated NOM. Most of the spectra contain
broad shoulders at wavelengths between 200 and 250 nm and at >250 nm, and several of
them have an inflection point near 270 nm. The spectrum of the TPHA fraction exhibited
anomalously high absorbance at λ<250 nm which is probably caused by interfering
inorganic species (e.g., nitrate). The UV spectra were analyzed quantitatively to
determine the specific absorbance at 254 nm (SUVA254), and various other spectral
parameters were determined based on the model of Korshin et al. (1996, 1997c). Results
of these analyses are presented in Table 5.11.

150
10.0
Specific absorbance (L/mg·m)
uHA A
8.0
HPOA

6.0
TPHA

4.0 HPON

2.0

0.0
200 250 300 350 400
Wavelength (nm)

3.0
HPIA B
Specific absorbance (L/mg·m)

2.5 HPIB

2.0
HPIA-2
1.5

1.0
uHPIA

0.5 HPIN

0.0
200 250 300 350 400
Wavelength (nm)

Figure 5.14. Specific UV absorbance (SUVA) spectra of Suwannee River NOM fractions
(all spectra normalized to 1 mg/L DOC, cell length 1 cm)

151
Table 5.11
UV and fluorescence parameters for Suwannee River NOM fractions
# sample SUVA254 A350/A380 ∆ET (eV) Fluorescence
L/(mg-m) maximum
(nm)
1 uHA 5.0 0.471 2.51 463
2 HPOA 4.6 0.392 2.25 446
3 HPON 3.0 0.317 2.03 437
4 TPHA 3.4 0.267 1.89 425
5 TPHN 2.9 0.128 1.52
6 HPIA 2.7 0.282 1.93 439
7 HPIA-2 1.8 0.247 1.84 427
8 uHPIA 1.3 0.223 1.78 430
9 uHPIN 0.7 0.128 1.52
10 HPIB 2.3 0.256 1.86 421

Though the shapes of all the UV spectra were fairly similar, SUVA254 of the
different fractions varied substantially, being largest for the uHA fraction and smallest
for the HPIN and uHPIN fractions. The SUVA254 value was well correlated with the
width of the ET band (∆ET) with R2 = 0.89 (Figure 5.15), with the exception of one outlier
which was excluded from the figure. Since SUVA254 has been shown to correlate with
aromaticity, this result suggests that ∆ET can be used to evaluate the aromaticity of NOM
in situ.

152
2.6
2
R = 0.89
ET band halfwidth (eV) 2.4

2.2

2.0

1.8

1.6

1.4
0.0 1.0 2.0 3.0 4.0 5.0 6.0
SUVA254 (L/mg·m)

Figure 5.15. Correlation between SUVA254 and half-width of the ET band for Suwannee
River NOM concentrates (one outlier was excluded)

Fluorescence emission spectra of selected Suwannee River NOM fractions are


shown in in a normalized format (normalized such that the maximum emission intensity
is assigned a value of 1.0 in all the spectra) in Figure 5.16. The intensity and position of
the maximum in the emission spectra vary substantially among the fractions and appear
to depend on both the hydrophilicity of the fractions and their acid-base character. For
instance, the wavelength of maximum emission intensity of the hydrophilic and neutral
fractions is substantially lower than that of the hydrophobic and acidic fractions.

153
1.00
uHA
Normalized emission intensity HPOA
0.75
HPON

HPIA2
0.50
HPIB

HPIN
0.25

0.00
370 410 450 490 530 570
Wavelength (nm)

Figure 5.16. Normalized fluorescence emission spectra of selected Suwannee River


NOM fractions (excitation at 320 nm)

The emission yields for the fractions are shown in Figure 5.17. The uHA fraction
has the lowest emission yield, and its emission maximum is at a much longer wavelength
(463 nm) than that of any other fraction. The neutral fractions also exhibit low emission
yield, although the emission intensity of the HPON fraction is comparable to that of the
HPOA fraction. As the hydrophilicity of NOM fractions increases, so does the
fluorescence yield. The HPIB fraction has much higher emission yield than any other
fraction.

154
40
Fluorescence yield (L/mg)

30

20

10

0
HPOA

HPIA

HPIB
HPIN

HPON

HPIA2

uHPIA
uHA

TPHA
TPHN

Figure 5.17. Comparison of the fluorescence yield of the Suwannee River NOM fractions
(cell length 1 cm, excitation at 320 nm)

The location of the maximum in the emission spectra of NOM correlates quite
strongly with ∆ET in the corresponding UV absorbance spectra (Figure 5.18). As the ET
band becomes broader (i.e., as ∆ET increases), the emission maximum exhibits a red shift.
In terms of current understanding of the UV absorption and light emission processes by
NOM (see the discussion in Chapter 2), this trend in the λmax and ∆ET values can be
interpreted as indicating that high hydrophobicity and high average MW are correlated in
NOM fractions. Put another way, the trend in Figure 5.18 can be ascribed to the
predominance of highly fluorescent, low molecular weight fractions in the hydrophilic
fractions of NOM.

155
470
2
460 R = 0.88

450
λ max (nm)

440

430

420

410
1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6
ET band half-width (eV)

Figure 5.18. Relationship between the half-width of the ET band in the UV absorbance
spectra of the Suwannee River NOM fractions and the position of the maximum in the
fluorescence emission spectra

UV spectra of selected South Platte River NOM fractions are presented in


Figure 5.19, and the numerical values of some important spectral parameters are
presented in Table 5.12. The HPIA, HPON, uHPIA fractions exhibited high absorbance
at λ < 250 nm, probably due to the presence of interfering inorganic salts, so only the data
at λ > 250 nm are shown.

156
3.5
A
3.0 HPOA
Absorbance (L/mg·m)

2.5 HPON

2.0 TPIA

1.5

1.0
TPIN
0.5

0.0
240 260 280 300 320 340 360 380 400
Wavelength (nm)

2.5
B
uHPIA
2.0
Absorbance (L/mg·m)

HPIA

1.5
HPIA2

1.0

HPIN
0.5

0.0
250 275 300 325 350 375 400
Wavelength (nm)

Figure 5.19. Specific UV absorbance spectra of South Platte River NOM fractions

157
Table 5.12
UV and fluorescence parameters for South Platte River NOM fractions
# sample SUVA254 A350/A380 ET band half- Fluorescence
L/(mg-cm) width (eV) maximum
(nm)
1 HPOA 2.9 0.300 1.98 425
2 HPON 1.6 0.238 1.82 419
3 TPHA 1.9 0.166 1.62 421
4 TPHN 0.7 0.201 1.72 400
5 HPIA 1.7 0.172 1.64 419
6 HPIN 0.5 0.261 1.88 419

Most of the fractions have lower SUVA254 values and narrower ET bands than the
corresponding fractions in the Suwannee River NOM, suggesting that the South Platte
fractions are less chemically diverse, more hydrophilic and in many cases less aromatic.
As expected, SUVA254 values of the hydrophilic South Platte fractions are generally
lower than those of hydrophobic and transphilic ones. In contrast to the results for the
Suwannee fractions, SUVA254 and ∆ET were not well correlated for this group of samples
(R2 = 0.11). Nevertheless, the relationship between SUVA254 and ∆ET for the pooled data
from all samples studied in this work was strong, as shown in Chapter 6.

Normalized fluorescence spectra of the South Platte River NOM fractions are
shown in Figure 5.20. The maxima in the emission spectra of all the fractions are at
substantially lower wavelengths (400 to 425 nm) than those in the Suwannee River
fractions (421 to 463 nm), suggesting that the average molecular weight of NOM in the
South Platte River fractions is lower than that in the Suwannee. Further, the maxima in
the emission spectra of the South Platte fractions do not shift to lower wavelengths with
increasing hydrophilicity. In fact, the wavelength of maximum emission of the transphilic
neutrals fraction (TPHN) is much shorter (400 nm) than that of the more hydrophilic
fractions. On the other hand, the emission spectrum of the ultrahydrophilic acids has a
maximum at 423 nm and a secondary maximum at 472 nm, suggesting the presence of
high molecular weight species in this fraction.

158
1.00 A
Normalized emission intensity
HPOA

HPON
0.75
TPHA

0.50

THPN
0.25

0.00
370 420 470 520 570
Wavelength (nm)

1.00 B
Normalized emission intensity

uHPIA
HPIN
0.75

0.50

HPIA
0.25

0.00
370 420 470 520 570
Wavelength (nm)

Figure 5.20. Normalized fluorescence emission spectra of South Platte River NOM
fractions (excitation at 320 nm)

159
In general, the correlations between the major parameters of the UV and
fluorescence spectra of the NOM in the South Platte River seem to be more complex than
those in the Suwannee River NOM fractions. This added complexity might be due to a
substantial impact on the UV and fluorescence spectra of non-humic nitrogen-containing
compounds in the South Platte NOM.

RELATIONSHIP BETWEEN VARIOUS STRUCTURAL AND SPECTRAL


PROPERTIES OF NOM FRACTIONS

Correlations between elemental composition and other properties of the NOM


fractions were explored, but the correlations were usually quite weak. A few of the
results are presented in Table below.

Table ** Correlations between the spectral parameters and the data of 13C CP-
MAS NMR and pyrolysis-GC/MS for non-aromatic functional groups. Data for 27
samples from four sources (Blavet, South Platte, Suwannee and Tolt Rivers)
Method Structural parameter Spectral parameter Linear R2
value
13
C NMR Total carboxyl carbon SUVA254 0.01
∆ET
13
C NMR Total carboxyl carbon 0.06
λmax
13
C NMR Total carboxyl carbon 0.03
13
C NMR Anomeric carbon SUVA254 0.04
∆ET
13
C NMR Anomeric carbon 0.04
λmax
13
C NMR Anomeric carbon 0.00
Pyr/GC-MS Total nitrogenous species SUVΑ254 0.05
Pyr/GC-MS Total nitrogenous species ∆ET 0.03
Pyr/GC-MS Proteins ∆ET 0.02
Pyr/GC-MS Aminosugars ∆ET 0.01
Pyr/GC-MS Polysaccharides SUVA254 0.02
Pyr/GC-MS Aminosugars SUVA254 0.05
Pyr/GC-MS Total saccharides SUVA254 0.00
Pyr/GC-MS Polysaccharides ∆ET 0.00
Pyr/GC-MS Total saccharides ∆ET 0.02
Pyr/GC-MS Polysaccharides λmax 0.14
Pyr/GC-MS Total saccharides λmax 0.00

160
Figure 5.21 plots the relative proportion of proteins as a function of the TDAA
content of the fractions (determined by HPLC). These two parameters are closely related
(R2 = 0.78). While not necessarily surprising, this correlation validates the approach used
to interpret the pyrochromatograms. However, the TPHN fraction that was identified as
almost pure protein using 13C-NMR and FTIR spectroscopy does not fit the relationship
at all. No explanation for this finding is apparent.

60
TPHN
50

40
Proteins, %

South Platte
Suwannee
30 R2 = 0.78

20

10

0
0 10 20 30 40 50 60
TDAA concentration, µ g C/mg DOC

Figure 5.21. Relation between the relative proportion of proteins and TDAA content in
NOM fractions from the Suwannee and South Platte Rivers.

Figure 5.22 identifies a significant relationship between the relative proportion of


polysaccharides and the anomeric (carbohydrate) C content (based on peaks in the
13
C-NMR spectra with chemical shifts from 90 to 110 ppm). No relationship could be
found between TDCA content and the proportion of polysaccharides determined by
Pyr-GC-MS.

161
30
South Platte
25 Suwannee
Polysaccharides, %

20

15

10

5 R2 = 0.70

0
0 2 4 6 8 10 12
Anomeric Carbon Content, %

Figure 5.22. Relationship between the relative proportion of polysaccharides and the
anomeric carbon content in NOM fractions from the Suwannee and South Platte Rivers.

Figure 5.23 presents the correlation between SUVA254 and the aromatic carbon
content. Given the strong linear regression coefficients, SUVA254 appears to be a
reasonably good surrogate parameter for the aromatic carbon content of NOM fractions,
regardless of whether the fraction is identified as hydrophilic, transphilic, or
hydrophobic. Similar correlations were found by Reckhow et al. (1990) for humic and
fulvic acids isolated from river waters and by Martin (1995) for humic, fulvic, and
transphilic acids isolated from river and reservoir waters.

162
30

25
Suwannee 2
R = 0.83
Aromatic Carbon, %

South Platte
20

15

10

0
0.0 1.0 2.0 3.0 4.0 5.0
SUVA254, m-1/(mg/L)

Figure 5.23. Correlation between aromatic C and SUVA in NOM fractions from the
Suwannee and South Platte Rivers.

Figure 5.24 shows SUVA as a function of the PHA content for the NOM fractions
from the two rivers. The linear regression coefficient is R2 = 0.98, confirming the good
relationship between the aromaticity of NOM and the relative amount of PHA fragments
produced during pyrolysis. Martin (1995) and Harrington et al. (1996) published similar
correlations, although their results were limited to hydrophobic and transphilic acids in
the former, and hydrophobic acids in the latter, respectively. It is interesting that this kind
of correlation appears to apply to hydrophilic NOM with low aromatic character as well.

163
3.0
South Platte
2.5 Suwannee
SUVA254, m-1/(mg/L)

2.0

1.5

2
1.0 R = 0.98

0.5

0.0
0 5 10 15 20 25 30
PHA Content, %

Figure 5.24. Correlation between the relative proportion of PHA and SUVA254 in NOM
fractions from the Suwannee and South Platte Rivers.

Aromaticity of the Suwannee River NOM fractions was strongly correlated with
∆ET (Figure 5.25).

164
35

30

25
Aromaticity (%)

2
20 R = 0.74

15

10

0
1.4 1.6 1.8 2.0 2.2 2.4 2.6
ET band halfwidth (eV)

Figure 5.25. Correlation between the aromaticity of the Suwannee River NOM fractions
(based on 13C-NMR data) and ∆ET.

Both the Suwannee HPIB and South Platte TPHN fractions contain high amounts
of proteinaceous species (see Chapter 4), so their spectral properties are likely to be
affected by the presence of highly fluorescent amino acids such as tyrosine and
phenylalanine. However, as was the case for the Suwannee River NOM fractions, the
fluorescence intensity was not strongly correlated with the sum of the tyrosine and
phenylalanine concentrations in these fractions (R2 only 0.27). Although the
concentration of tryptophan, another fluorescing amino acid, could not be determined
because it is hydrolyzed during the analysis, this amino acid is not expected to be found
in concentrations very different from those of tyrosine or phenylalanine. Therefore, even
in protein-rich samples, the fluorescence intensity does not seem to be determined by the
concentration of fluorescing amino acids; rather, it appears to be controlled jointly by the
concentrations of nitrogen-containing species and, much more prominently, by non-
nitrogenous fluorophores. For humic substances, these fluorophores are likely to be
aromatic species whose fluorescence yield depends on their molecular weight and

165
hydrophilicity. Additionally, the fluorescence of both humic substances and proteins is
likely to be much affected by their mutual interactions.

The position of the maximum in the fluorescence emission spectra of the


Suwannee River NOM fraction appears to be correlated with the aromaticity of the
samples evaluated by 13C-NMR or Pyr-GC-MC methods, but additional data are needed
to confirm these apparent correlations. (Figure 5.26 and 5.27).

35

30

25
Aromaticity (%)

2
R = 0.60
20

15

10

0
410 420 430 440 450 460 470
λ max (nm)

Figure 5.26. Correlation between the aromaticity of the Suwannee River NOM fractions
and the position of the maximum in the fluorescence emission spectra. 13C-NMR data.

166
28

Polyhydroxyaromatic carbon (%)


2
R = 1.00

26

24

22

20
410 415 420 425 430 435 440
λ max (nm)
Contribution of species (%)

polyhydroxyaromatic carbon
40 proteinaceous species

2
R =1

25

10
415 425 435
λ max (nm)

Figure 5.27. Correlation between the position of the emission maxima in the fluorescence
spectra of the Suwannee River NOM fractions and the aromatic carbon content of the
sample, based on Pyr-GC-MS data.

167
60

polyhydroxyaromatic carbon
2
Contribution of species (%)
R = 1.00 proteinaceous species
45

30

15
2
R = 0.91

0
400 405 410 415 420 425 430
λ max (nm)

Figure 5.28. Correlation between the position of maximum in the fluorescence


emission spectra and the content of proteinaceous and polyhydroxyaromatic carbon
(based on Pyr-GC-MS analysis) in the South Platte River NOM fractions

The position of the maximum (λmax) in the emission spectrum of the four South
Platte fractions (HPOA, HPOA, TPHA and TPHN) for which the Pyr-GC-MS data were
available is also well correlated (R2=1.00) with the concentration of proteinaceous
species, as evaluated by pyr-GC-MS (Figure 5.28). The maximum in the emission
spectrum shifts toward shorter wavelengths with increasing concentration of proteins. For
the same series of samples, the correlation between λmax and the PHA carbon determined
by Pyr-GC-MS was also very strong (R2 =0.90). However, the correlation between the
impact of proteinaceous species and λmax for the Suwannee River NOM fractions was
practically non-extant (R2=0.14). Thus, the site-specificity of NOM in terms of the
influence of nitrogen-containing species on λmax is obvious, and no generalization
applicable to all NOMs can be provided at this stage. An additional problem here is that
neither the total concentration of dissolved amino acids nor the concentrations of two
individual fluorescing amino acids (tyrosine and phenylalanine, which are likely to

168
contribute predominantly to the emission of proteins) were correlated with λmax. Also, the
fluorescence yield (i.e., the fluorescence intensity per unit DOC) could not be correlated
with the concentration of aromatic carbon or proteins alone. All these issues need to be
studied in more detail.

Comparison of NOM Characteristics between the Suwannee and South


Platte Rivers

Qualitatively, the Suwannee and South Platte rivers are thought to be quite
different in terms of how NOM enters the water and the controls that are exerted on the
NOM. The Suwannee River NOM is thought to be primarily allochthonous, whereas the
NOM in the South Platte is thought to be of mixed autochthonous-allochthonous origin.
The nitrogen content of the TPHN fraction of the South Platte River is very high,
consistent with the suggestion, based on its FTIR and 13C-NMR spectra, that this fraction
is highly proteinaceous., which is consistent with its highly aromatic character (discussed
below).

The NOM fractions isolated from the South Platte River were quite different from
other NOM samples discussed in this report, due mainly to the increased concentration of
nitrogen content and increased hydrophilicity of the South Platte samples. Though the
UV and fluorescence spectra of the South Platte NOM fractions are superficially similar
to those from other water sources examined in this study, the correlations between the
chemical composition of the samples and their spectral responses are markedly different.
Namely, the values of SUVA254 and ∆ET are not well correlated in the South Platte
fractions, though they are correlated in the other waters studied. Also, the aromaticity of
the fractions estimated via 13C-NMR spectroscopy is not strongly correlated with ∆ET or
SUVA254. On the other hand, the correlation between aromaticity as estimated from
Pyr-GC-MS data and the corresponding values of SUVA254 and λmax is very strong.
These correlations may be useful for characterizing NOM samples that have a substantial
non-humic component.

169
The most significant difference in NOM composition between the two rivers is
the greater aromatic carbon percentage in NOM from the Suwannee River. The
hydrophobic acid fraction, which is the largest NOM fraction in both rivers, contains
nearly 31% aromatic carbon for the Suwannee River (Table 5.5), and only 12-14%
aromatic carbon for the South Platte River. This decrease in aromaticity for the South
Platte samples is offset by an increase in aliphatic hydrocarbon content.

Another significant difference between NOM composition in the two rivers is the
presence of significant levels of proteinaceous NOM in the South Platte River, especially
during the first sampling in the winter. Figure 5.29 compares the FTIR spectra of the
transphilic neutral fractions. Based on the position (1732 cm−1) and broadness of the C=O
peaks in these two spectra, the transphilic neutral fraction from the Suwannee River
appears to be a mixture of esters and acids, whereas the C=O peaks for this fraction from
the South Platte River are indicative of proteins.

1732.39 1206.90
3396.73

1398.48

1653.69

A
A

3334.97 1535.54
1238.14
2936.42 1451.50

567.72

4000 3000 2000 1500 1000 400


CM-1

Figure 5.29. Comparison of FTIR spectra of transphilic neutral fractions from


(A) Suwannee River and (B) South Platte River.

170
A final significant difference is that NOM is predominantly hydrophobic in nature
in the Suwannee River, whereas the transphilic and hydrophilic NOM constitutes a
greater percentage of the NOM in the South Platte River.

All of the above NOM differences indicate an allochthonous terrestrial plant


source for the Suwannee NOM with a lack of mineral soil solubility controls that, giving
rise to the high DOC. In contrast, the South Platte River NOM has mineral soil solubility
controls that minimize allochthonous DOC inputs of aromatic phenolic NOM
constituents, and autochthonous inputs from lakes and reservoirs on this river contribute
to the relative abundance of carbohydrates and proteins in this water.

REACTIVITY OF NOM

This section describes reactivity of NOM in two important water treatment


processes and explores the relationship between reactivity and the NOM properties
determined in the characterization studies described above.

Coagulation-Flocculation

Removal of DOC and A254

Tables 5.13 and 5.14 give the removal efficiencies for DOC and A254 by
coagulation of the NOM isolates from the Suwannee and South Platte Rivers,
respectively. All the NOM isolates were studied using the same experimental conditions,
at a pH of 6.5 and with a coagulant dose of 1 mg Al/mg DOC.

171
Table 5.13
DOC and A254 removals of Suwannee River NOM isolates during coagulation-
flocculation with aluminum at pH 6.5.
Fraction SUVA (L/mg-m) DOC removal (%) A254 removal (%)
UHA 5 86.6 ±1.4 99.6 ± 0.8
HPOA 4.6 79.5 ± 2.6 94.6 ± 1
HPON 3 20.2 ±1.5 73.5 ± 1.8
TPHA 3.3 75.5 ± 0.9 92.5 ± 0.5
HPIA 2.6 74.2 ± 2.8 88 ± 2
HPIA-2 1.8 54.4 ± 1.5 66.7 ± 1.3
uHPIA 1.4 35.8 ± 2.5 57.5 ± 1.6
uHPIN 0.7 37.8 ± 0.6 75.5 ± 1.5
HPIB 2.4 36.2 ±2.7 70 ± 5

Table 5.14
DOC and A254 removals of South Platte River NOM isolates during coagulation-
flocculation with aluminum at pH 6.5.
Fraction SUVA (L/mg-m) DOC removal (%) A254 removal (%)
HPOA 2.9 48 ±2 74 ± 3
HPON 1.7 18.7 ± 1.7 43.7 ± 4.5
TPHN 0.9 53 ± 1 39 ± 1.3
TPHA 1.8 54.6 ±1 69 ± 0.9
HPIA 1.9 56 ± 2.3 68 ± 1.3
raw water 2.4 29 ± 1 50 ± 3

For the Suwannee, the acid fractions (uHA, HPOA, TPHA, HPIA, HPIA-2) were
better removed than the neutral (HPON, uHPIN) and basic (HPIB) fractions. The low
DOC removal efficiency for the uHPIA fraction is undoubtedly related to the fact that it
was methylated. Among the six acid fractions, the uHA fraction was best removed (87%
DOC removal), probably because it contained a substantial amount of colloidal material.
172
The HPOA fraction was slightly better removed than the TPHA and HPIA fractions.
HPIA-2, which is more hydrophilic than HPIA was less efficiently removed by aluminum
coagulation (54% compared to 75%).

The DOC removal efficiencies obtained for the South Platte River NOM isolates
were lower than those for the corresponding Suwannee River fractions. Again, the acid
fractions had the highest reactivity with aluminum coagulant (ranging from 50 to 55%
DOC removal). As in the Suwannee fractions, the HPON fraction was poorly removed
(19% DOC removal). However, surprisingly, the TPHN fraction, which appears to be
proteinaceous, was as well removed as the acid fraction.

Jar tests on the South Platte River using similar experimental conditions (pH,
dose) removed only 30% of the DOC. Edzwald (1993) and Croue et al. (1993c) obtained
similar results when treating natural waters with low SUVA254 values (below 3 L/mg-m).
This poor DOC removal suggests that the hydrophilic neutral and base fractions that were
lost during the isolation procedure would have been removed only slightly by coagulation
and flocculation.

The removal efficiency of A254 was higher than that of DOC for all the fractions
studied in both the Suwannee and South Platte, except for the TPHN of the South Platte.
This result further supports the idea that aromatic structures and higher molecular weight
organics are preferentially removed by coagulation-flocculation.

Assuming that the behavior of NOM fractions in a mixture (bulk NOM) is similar
to their behavior alone under similar water quality and treatment conditions, the NOM
remaining in solution after coagulation-flocculation can be expected to be more
hydrophilic and to have increased proportions of neutral and base fractions compared to
the bulk NOM in the raw water. However, for surface waters that contain a ‘typical’
NOM composition, the DOC distribution among hydrophobic, transphilic, and
hydrophilic fractions will be only slightly modified by coagulation-flocculation, as has
been shown by Collins et al. (1986) and Croue et al. (1993c).

173
Figure 5.30 presents correlations between removal of DOC or UV254 and
SUVA254 for all the waters studied in the current research.

174
90
80 Suwannee
South Platte
70
SUVA254, L/mg-m

60 R2=0.63

50
40
30
20
HPON HPON
10
0
0 1 2 3 4 5
PHA Content, %

100
90
80
HPIN
70
A254 Removal, %

60
50
40 2
R = 0.69
30
20 Suwannee
South Platte
10
0
0 1 2 3 4 5
SUVA254, L/mg-m

Figure 5.30. Relationship between DOC or A254 removals and SUVA254

175
As expected, there is a relationship between the removal of NOM by coagulation-
flocculation and SUVA254, which can be considered a surrogate for the aromaticity of the
NOM.

When plotted against the SUVA254 of the raw water, a better relationship is
obtained for removal of A254 (R2 = 0.69) than for removal of DOC (R2 = 0.63), excluding
the uHPIN from the A254 data set and HPON from the DOC data set. The reason for the
unusually efficient removal of A254 associated with the HPIN fraction is unclear. The
poor removal of the HPON fraction from both source waters is not surprising, since it is
the most aliphatic fraction based on the 13C-NMR and FTIR spectra.

Chlorination of NOM: Reactions of Different Fractions and Changes in NOM


Characteristics

DBP formation potentials

Chlorine demand, TOXFP, THMFP, TCAAFP and DCAAFP of the Suwannee


and South Platte River isolates are listed in Tables 5.15 and 5.16, respectively. Table 5.16
also includes the results obtained on raw water from the South Platte.

176
Table 5.15
Chlorine demand and disinfection by-product formation potentials of the Suwannee River
NOM isolates
Fraction Initial Chlorine TOXFP THMFP TCAAFP DCAAFP
SUVA254 demand ⎛ µg Cl ⎞ ⎛ µg CHCl 3 ⎞ ⎛ µg TCAA ⎞ ⎛ µg DCAA ⎞
⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟
(L/mg-m) ⎛ mg Cl 2 ⎞ ⎝ mg C ⎠ ⎝ mg C ⎠ ⎝ mg C ⎠ ⎝ mg C ⎠
⎜ ⎟
⎝ mg C ⎠

HPON 3.0 1.5 182 51 51 24

HPOA 4.6 1.4 268 55 59 25

TPHA 3.4 1.4 224 40 57 23

TPHN 3.0 1.2 204 40 44 22

HPIA 2.7 0.8 171 36 36 22

HPIA-2 1.8 1.1 150 41 34 17

uHPIAMe 1.3 0.78 121 28 30 21

HPIB 2.3 2.5 175 29 31 39

uHA 5.0 2.8 285 63 66 45

uHPIN 0.7 0.8 117 23 26 22

177
Table 5.16
Chlorine demand and DBP formation potential of the South Platte River and its isolated
NOM fractions
Initial Chlorine TOXFP THMFP TCAAFP DCAAFP
Fraction SUVA254 demand ⎛ µg Cl ⎞ ⎛ µg CHCl 3 ⎞ ⎛ µg TCAA ⎞ ⎛ µg DCAA ⎞
⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟
(L/mg-m ⎛ mg Cl 2 ⎞ ⎝ mg C ⎠ ⎝ mg C ⎠ ⎝ mg C ⎠ ⎝ mg C ⎠
⎜ ⎟
) ⎝ mg C ⎠

HPON 1.6 0.83 114 29 16 12

HPOA 2.9 0.95 122 46 28 14

TPHA 1.8 0.81 100 39 21 14

TPHN 0.7 2.3 66 25 12 20

HPIA 1.7 0.86 98 35 24 16

HPIN 0.5 1 81 28 15 19

Raw 2.2 2.2 127 36 15 29

Water

The yields of THMs (chloroform) and TCAA are similar (expressed as µg/mgC)
for the Suwannee isolates, with the exception of the TPHA (TCAAFP>THMFP) and
HPIA-2 (THMFP>TCAAFP) fractions. More TCAA was formed than DCAA in all
fractions except the HPIB. For the South Platte isolates, chloroform was the major
chlorination by-product. More TCAA than DCAA was formed in the acid fractions, but
the opposite trend was observed for two of the three neutral fractions (TPHN and HPIN).

The range of specific THM and TOX yields found with the HPOA and TPHA
fractions of both the Suwannee and South Platte Rivers fall within the ranges reported in
the literature. However, the formation of chlorinated DBPs per gram of carbon was
consistently greater in the Suwannee than in the South Platte, in corresponding fractions.
It is widely reported that the relative production of THMs (mostly chloroform) and
TCAA is pH dependent (production of chloroform increases when the pH increases,
while the production of TCAA decreases) (e.g., Miller and Uden 1983, Arora et al. 1997,

178
**MB refer to authors: Disinfection/Disinfection By-Products 1991). The current study
indicates that the nature of the NOM is also an important factor in this distribution.

In Tables 5.17 and 5.18, THMFP, TCAAFP and DCAAFP are given in terms of
µg Cl/mg C to facilitate a comparison with TOXFP. For some fractions, THMFP’s
expressed in this form include contributions from dichlorobromomethane in addition to
chloroform. In all cases, chloroform was the major THM; dichlorobromomethane
accounted for about 2% of the THMFP (in µg Cl/L) for the Suwannee isolates and
around 10% for the South Platte isolates.

Table 5.17
DBP formation potentials of the Suwannee River NOM isolates
THMFP THMFP/ TCAAFP TCAAFP/ DCAAFP DCAAFP/
Fraction ⎛ µg Cl ⎞ TOXFP (%) ⎛ µg Cl ⎞ TOXFP (%) ⎛ µg Cl ⎞ TOXFP (%)
⎜ ⎟ ⎜ ⎟ ⎜ ⎟
⎝ mg C ⎠ ⎝ mg C ⎠ ⎝ mg C ⎠

HPON 45 25 37 20 13 7

HPOA 49 18 38 14 14 5

TPHA 36 16 37 16 13 6

TPHN 36 18 29 14 12 6

HPIA 32 19 23 13 12 7

HPIA-2 37 25 22 15 9 6

uHPIAMe 25 21 19 16 11 9

HPIB 26 15 20 11 21 12

uHA 57 20 43 15 25 9

uHPIN 21 18 17 14 12 10

179
Table 5.18
DBP formation potentials of the South Platte River and its NOM isolated fractions
Fraction THMFP THMFP/ TCAAFP TCAAFP/ DCAAFP DCAAFP/
⎛ µg Cl ⎞ TOXFP (%) ⎛ µg Cl ⎞ TOXFP (%) ⎛ µg Cl ⎞ TOXFP (%)
⎜ ⎟ ⎜ ⎟ ⎜ ⎟
⎝ mg C ⎠ ⎝ mg C ⎠ ⎝ mg C ⎠

HPON 27 24 10 9 6 5

HPOA 43 35 18 15 8 7

TPHA 37 37 14 14 8 8

TPHN 23 35 8 12 11 17

HPIA 34 35 16 16 9 9

HPIN 26 32 10 12 11 14

Raw 39 31 10 8 16 13

Water

The sum of THMs, TCAA and DCAA accounted for 37 to 52% (average 42%) of
the TOX for the Suwannee isolates, and 38 to 64% (average 55%) of the TOX for the
South Platte isolates. The fact that these three compounds represent a larger proportion of
the TOX in the South Platte isolates than in the Suwannee isolates primarily reflects
differences in the production of chloroform. This representation identifies the South
Platte TPHN and HPIN and the Suwannee HPIB fractions as having somewhat larger
DCAA/DBP ratios than the other fractions. As mentioned previously, the South Platte
TPHN and the Suwannee HPIB fractions are largely proteinaceous, while the South
Platte HPIN fraction has a stronger carbohydrate and amino sugar character. Thus, it
appears that nitrogenous units in the NOM structure (proteins and amino sugars) could be
an important class of DCAA precursor sites. However, it is also possible that the strong
reactivity of the three fractions with chlorine (they had the highest chlorine demands of
all the fractions studied) may have lowered the Cl2/DOC ratio during the course of the
reaction more than in the other fractions, favoring the production of DCAA over more
highly halogenated products. Reckhow (1984) and Croue (1987) demonstrated that for
low Cl2/DOC ratios, DCAA appeared to be more abundant than TCAA.

180
Relationships between DBPFP and Spectral Properties

Figure 5.31 shows the formation potential (FP) for various DBPs as a function of
SUVA for the Suwannee and the South Platte isolates. Good correlations could be
obtained between SUVA and THMFP, TOXFP, or TCAAFP for the isolates from both
rivers, supporting the importance of aromaticity for the production of chlorinated DBPs
regardless of the nature of the fraction. Linear regression coefficients (R2) ranged from
0.8 to 0.97, except for the THMFP data set of the South Platte isolates (R2 = 0.7). The
best linear regression coefficients were obtained for the correlation between TOXFP and
SUVA, probably because TOX is a more general parameter for evaluating the reactivity
of NOM with chlorine than are individual species such as THM or TCAA.

Correlations between DBPFP and the aromatic carbon content or the percent
13
aromaticity determined from C-NMR spectra had lower linear regression coefficients.
For example, the regression coefficient (R2) calculated for the relationships between
TOXFP and the phenolic content, the aromatic C content, the percent aromaticity, and
SUVA in the Suwannee isolates were 0.86, 0.89, 0.91 and 0.97, respectively. Similar
correlations for humic and fulvic acids (Xiong 1990, Croue 1987, Reckhow et al. 1990),
and rarely for transphilic acids (Martin 1995), have been published by others.

The correlation between DCAAFP and SUVA254 was poor for the two series of
isolates, which might indicate that organic structures other than aromatics are responsible
for the production of DCAA upon chlorination. DCAA has been identified as a
chlorination by-product of methionine (Hureiki and Croue 1997**JP provide full ref),
aspartic acid (De Leer et al. 1990), and tyrosine (Horth 1989), and it was the major
identified by-product of proline (De Leer et al. 1990, Hureiki 1993) and cysteine
chlorination (Hureiki 1993). Consistent with the discussion above, these data further
support the possibility that proteins may play a significant role in the production of
DCAA.

For both sources, the more hydrophobic and more acidic fractions provide the
most active precursor sites (i.e., they have the largest formation potential for TOX,
THMs, and HAAs). Because the HPOA and TPHA fractions account for the largest part

181
of the DOC, they comprise the main precursor sites for TOX, THM and TCAA in these
waters.

182
60.0
Suwannee
50.0 South Platte
THMFP, µ g Cl/mg C

40.0

30.0

20.0

10.0

0.0
0 1 2 3 4 5
SUVA254, L/mg-m

300

Suwannee
250
South Platte
TOXFP, µ g Cl/mg C

200

150

100

50

0
0 1 2 3 4 5
SUVA254, L/mg-m

183
45

40 Suwannee
South Platte
35
TCAAFP, µ g Cl/mg C

30
25

20
15

10
5

0
0 1 2 3 4 5
SUVA254, L/mg-m

25
Suwannee
20 South Platte
DCAAFP, µ g Cl/mg C

15

10

0
0 1 2 3 4 5
SUVA254, L/mg-m

Figure 5.31. Relationships between DBPFPs and SUVA254 for NOM fractions from the
Suwannee and South Platte Rivers **MB remove decimal point from axis on top figure

184
The relationship between DBPFPs and SUVA254 for the isolated fractions appears
to differ between the two sites. Reckhow et al. (1990) indicated that the nature of the
aromatic sites, and in particular the relative abundance of activated and non-activated
rings, is one of the major parameters governing the reactivity of NOM with chlorine.

The plots of THMFP vs. SUVA254 are linear with almost identical slopes for the
two sources, but the y-intercept (the extrapolated estimate of THMFP corresponding to
zero SUVA) is greater in the South Platte. The opposite trend is observed in the
relationship between the HAAs and SUVA, i.e., at a given SUVA254, the Suwannee
fractions have a greater HAAFP than the South Platte fractions. The results suggest that
the non-aromatic moieties in the South Platte NOM are somewhat better precursors for
THMs and poorer precursors for THAA than those in the Suwannee.

The best-fit lines in Figure 5.31 have y-intercepts that indicate that even in the
absence of aromatic structures a significant amount of DBPs would be produced, and the
intercept is significantly higher for THMs than for HAAs. Amino acid structures in the
base fraction (HPIB for the Suwannee River) and β−hydroxyacids and β-ketones that
might be predominant in neutral and hydrophilic acid fractions are known to be THM
precursors. Because of the more pronounced hydrophilic character of the South Platte
River NOM, β−hydroxyacids and β−ketones may account for a significant part of the
THM precursors in NOM from this source.

Considering all DBPs together (as TOX), the Suwannee NOM has more Cl-
reactive sites than the South Platte NOM (Figure 5.32). The relative TOXFPs of the two
13
waters are consistent with the C-NMR spectra, which illustrate that the Suwannee
NOM has a greater aromatic carbon content than the South Platte NOM, and also
contains a higher fraction of aromatic phenolic constituents in its structure. Reckhow et
al. (1990) reported that molecules having a high degree of conjugation preferentially lead
to TCAA formation over chloroform formation. The production of TCAA in the two
NOM sources is consistent with this observation. Because the NOM in the South Platte
has a higher THMFP:SUVA ratio and a lower yield of other DBPs than does the NOM in

185
the Suwannee, THMs represent a substantially larger fraction of the TOX in the South
Platte.

300
Suwannee
250 South Platte R2 = 0.74
TOXFP, µ g Cl/mg C

200

150

R2 = 0.62
100

50

0
0 10 20 30 40 50 60
THMFP, µ g Cl/mg C

Figure 5.32. Relationship between TOXFP and THMFP in Suwannee and South Platte
River NOM fractions

SUMMARY AND COMPARISON OF SUWANNEE RIVER AND SOUTH


PLATTE RIVER NOM CHARACTERIZATIONS

Following are brief summaries of Suwannee River and South Platte River NOM
characterizations:

1. DOC Fractionation. Suwannee River NOM is dominated by hydrophobic acids,


whereas hydrophilic and transphilic NOM percentages are much greater in the South
Platte River.

2. Elemental Analyses. The C/N ratios of the Suwannee River NOM fractions are
greater than in the corresponding fractions of the South Platte River, reflecting the
186
greater allochthonous character of the Suwannee River NOM. C/H, C/N, and C/O
ratios decrease as the hydrophilic character of the NOM fraction increases.

3. FTIR Spectra. NOM fraction isolates were obtained that were virtually free of
inorganic constituents except for minor amounts of silica in certain fractions. Almost
all of the NOM fractions have carboxylic acid peaks; amide peaks from proteins and
n-acetylamino sugars are especially noticeable in certain transphilic and hydrophilic
NOM fractions from the South Platte River.

13
4. C-NMR Spectra. As the hydrophilic character of NOM fractions increases, the
NMR peaks generated by aliphatic and aromatic hydrocarbon components decrease,
and the peaks generated by carbohydrates and acids or amides increase. Suwannee
River NOM has more aromatic and phenolic carbon than corresponding fractions
from the South Platte River. C-N peaks from amines and amides are evident in base,
hydrophilic neutral, and transphilic neutral fractions, especially in South Platte River
NOM, and the methyl peak from N-acetyl amino sugars is readily detected.

5. Contributions of Dissolved Carbohydrates and Amino Acids to NOM. TDCA and


TDAA concentrations are greater in the South Platte River than in the Suwannee
River, but they account for only a small concentration of the DOC in both waters.
TDAA accounts for the majority of the nitrogen in the hydrophobic fractions of both
waters, but only 5 to 20% of the nitrogen in the other fractions. Glucose is the
dominant sugar in most NOM fractions. Large ornithine contents in the Suwannee
River NOM distinguish it from South Platte River NOM. The distributions of both
TDAA and TDCA were relatively similar among the various NOM fractions isolated
from the two water sources studied in the current project, and in other water sources
described in the literature. Thus, these parameters do not appear to be specific
indicators of the origin of the water source or its history. The one exception to this
statement is that ornithine seems to be a good indicator of intense microbial activity,
so its concentration might be useful as a tracer of surface water in some
circumstances.

187
6. Pyrolysis Gas Chromatography-Mass Spectrometry. Pyr-GC-MS analysis of NOM
yields valuable information about the NOM composition and origin. Hydrophobic
neutral NOM is characterized by fatty acid fragments; hydrophobic acid NOM is
characterized by polyhydroxyaromatic fragments typical of humic substances; and
transphilic and hydrophilic NOM fractions are characterized by fragments indicative
of carbohydrates, proteins, and amino sugars. Much of the nitrogen in the transphilic
and hydrophilic fractions that cannot be attributed to TDAA is probably present in
amino sugars. The transphilic neutral fraction of the South Platte River NOM is very
high in protein content.

7. Ultraviolet Spectrometry. Specific UV absorbance (SUVA) varied substantially in


Suwannee River NOM fractions, decreasing in the order HA > HPO-NOM >
TPH-NOM > HPI-NOM. Similar variations were found in South Platte River NOM
fractions, but SUVA was much lower than for corresponding fractions from the
Suwannee River. SUVA was well correlated with the width of the ET band for NOM
fractions from both water samples. The state and concentration of aromatic carbon in
the samples largely define their spectral properties. At least two spectral parameters
(SUVA254 and ∆ET) can be used to estimate the aromatic carbon content of the
sample.

Fluorescence Spectrometry. For the Suwannee River NOM fractions,


fluorescence emission yields increase and the maxima of emission spectra shift to lower
wavelengths as the hydrophilic character of the NOM increases. This trend is indicative
of a relative increase in lower molecular weight species as hydrophilic character
increases. Fluorescence data for South Platte River NOM fractions indicate generally
smaller molecular weights than for corresponding Suwannee River NOM fractions. The
fluorescence results suggest that the average molecular weight of NOM fractions tends to
increase as the hydrophilic character of the fraction increases. The fluorescence spectra
also appear to be sensitive to the hydrophilicity and to acidity-basicity of the NOM
fractions. These results might reflect an inverse correlation between the average
molecular weight and the hydrophilicity of the fractions.

188
8. Characterization Correlations. The carbon content of the NOM fractions investigated
13
correlated inversely with carbohydrate content (based on C-NMR), and oxygen
content was directly correlated with carboxylic acids and/or amides (13C-NMR).
Proteins (pyrolysis GC-MS) were correlated with TDAA, but nitrogen content
correlations were weak because of multiple sources of nitrogen from both proteins
and amino sugars. For Suwannee River NOM fractions, aromatic carbon content
(13C-NMR) was directly correlated with SUVA, ∆ET, PHA fragments, and the
fluorescence emission maximum; however, for the South Platte River NOM fraction,
aromatic carbon content correlated only with PHA fragments, indicating the
importance of phenolic aromatics (as compared with total aromatic carbon) on
ultraviolet and fluorescence measurements.

9. Overall NOM Characteristics. Suwannee River NOM is derived from allochthonous


tannins and lignins that give rise to highly colored humic and fulvic acids of high
poly-phenol content and low nitrogen content. Hydrophobic characteristics
predominate. South Platte River NOM is derived from both allochthonous inputs in
which aromatic and acid constituents are depleted by mineral solubility controls and
autochthonous inputs from phytoplankton and bacteria. These autochthonous inputs
contribute proteins, polysaccharides, and amino sugars that result in significant
hydrophilic character for this NOM.

10. Reactivity of NOM

a. Coagulation-Flocculation. Coagulation with alum at pH 6.5 removes acid NOM


fractions better than neutral or base fractions. NOM in the Suwannee River
fractions was removed more efficiently than that in than South Platte River
fractions. Moderate correlations were found between SUVA and DOC removal by
coagulation, and better correlations were found between SUVA and A254 removal.

b. Chlorination. Chlorinated DBP yields were greater in Suwannee River NOM


fraction isolates than in corresponding fractions from the South Platte River. For
Suwannee River NOM fractions, THM and TCAA yields are relatively similar;

189
chloroform was the major chlorination by-product for South Platte River NOM
fractions. SUVA correlated well (and aromatic carbon somewhat less well) with
THMFP, TOXFP, and TCAAFP, but did not correlate with DCAAFP.
Extrapolation of the correlation plots suggests that significant amounts of DBPs
could be produced even from NOM fractions with negligible SUVA or aromaticity.

Overall, the results point to major differences between Suwannee River and South
Platte River NOM characteristics and reactivity. In almost all cases, the various NOM
characterization methods gave supporting and complementary information rather than
contradictory information.

Despite some as yet unexplained findings, the qualitative and semi-quantitative


approaches used for the interpretation of Pyr-GC-MS chromatograms support previous
structural hypotheses for some NOM fractions and confirm the structural differences
13
between hydrophobic and hydrophilic NOM. The combination of C-NMR, FTIR and
Pyr-GC-MS analyses are complementary and can be a powerful tool for NOM
characterization.

190
CHAPTER 6 CHARACTERISTICS OF NOM CAPTURED BY DIFFERENT
TECHNIQUES

NOM from the Vienne, Gartempe, and Blavet Rivers in France, the Thames river
in England, and Judy Reservoir and the Tolt River in Washington State was fractionated
and characterized, though not as intensively as that from the Suwannee and South Platte
rivers. The intent was to determine whether and how different fractionation techniques
alter the NOM and thereby affect the conclusions that one might draw about the NOM in
the water source. Although not all fractionation and characterization tools were used for
all waters, the results of the testing were qualitatively similar in all cases. Of the waters
studied, the Blavet River was investigated most thoroughly. For this reason, and in light
of the large amount of data gathered, the discussion in this chapter focuses primarily on
the Blavet.

Most of the experiments and analyses reported in this chapter were conducted
using NOM isolates that were lyophilized and then, in some cases, re-dissolved.
However, the SUVA of the RO and NF isolates was analyzed on the concentrated waters
prior to lyophilization. Dissolution of the lyophilized sample of non-desalted RO and NF
concentrates was often problematic, yielding solutions that contained some suspended
particulates that were removed by 0.45-µm membrane filtration.

GENERAL CHARACTERIZATION OF NOM IN THE BLAVET RIVER

The Blavet River was sampled twice during the project, once in summer and once
in winter. Inorganic characteristics of the river water were quite similar in the two
samples, except that the pH was higher in the summer than winter (7.8 vs. 7.0). The
difference was probably due to the algal bloom that was in progress during the summer
sampling period (in fact, a few hours after the summer sampling, copper sulfate was
added to the reservoir to reduce the algal population). Bromide was analyzed monthly

191
between September 1995 and October 1996, and its concentration varied from 60 to
120 µg/L (Figure 6.1), with the maximum at the beginning of the summer.

120
Br− Concentration, µ g/L

100

80

60

40

20

0
Jul-95 Oct-95 Feb-96 May-96 Aug-96 Dec-96
Date

Figure 6.1. Bromide concentrations in the Blavet River during a one-year sampling
period

By contrast, the organic composition of the water varied substantially throughout


the year. Figure 6.2 presents the DOC and A254 values obtained in a survey conducted by
the SAUR laboratory between September 1995 and October 1996 on the Blavet River at
the Kerne Uhel reservoir, and Figure 6.3 shows the profile of SUVA254 during the same
period. The winter sample contained 12 mg/L DOC, compared with 6.6 mg/L during the
summer. A254 was also significantly lower during the summer, causing the SUVA254
values to be similar in the two samples (4.8 and 5.1 L/mg-m, respectively). The high
SUVA254 values indicate that the organic material has a pronounced humic nature. The
similarity in the SUVA values in summer and winter is somewhat unexpected, in light of
the algae bloom that was occurring during the summer sampling period.

192
12 5
DOC Concentration, mg/L

4.5
10 4
3.5
8

UV254, m− 1
3
6 DOC
2.5
2
UV
4 1.5
1
2
0.5
0 0

May-96
Mar-96
Oct-95

Oct-96
Sep-95

Dec-95

Aug-96
Feb-96
Jul-95

Jul-96
Date

Figure 6.2. DOC and A254 in the Blavet River during a one-year sampling period

5.0
4.5
4.0
3.5
SUVA254, m− 1

3.0
2.5
2.0
1.5
1.0
0.5
0.0
May-96
Mar-96
Oct-95

Oct-96
Nov-95

Dec-95

Apr-96

Aug-96

Sep-96
Jan-96

Jun-96
Feb-96

Jul-96

Date

Figure 6.3. SUVA254 in the Blavet River during a one-year sampling period

193
DOC concentration and UV absorbance in the Blavet decreased by ~50% during
the first few months of 1996, increased to the earlier values during Spring, 1997, then
decreased to reach relatively stable values in the summer. The increase in Spring
probably reflects an algal bloom. The DOC content of this reservoir also seems to be
strongly related to the local rainfall pattern (greatest in fall and winter), which introduces
soluble terrestrial organic materials from runoff. Turnover of the reservoir may also play
a role.

Other properties of the NOM in the Blavet also differed during the summer and
winter sampling periods. For example, the absolute concentrations of TDAA and TDCA
were substantially higher in the summer sample (Table 6.1). Because DOC was higher in
winter sample, these components comprised a much larger fraction of the DOC in
summer than in winter. These results were undoubtedly influenced by the algal bloom
noted above. Most of the increase in the TDAA content in the summer sample could be
attributed to a large increase in the ornithine concentration, which is a good indicator of
intense biological (**JP algal?) activity (Spitzy 1988). The increase in the TDCA
concentration was more uniformly distributed among all the carbohydrates detected, with
fucose having the largest proportional increase.

Table 6.1
TDCA, TDAA, and BDOC in the Blavet River
Winter Summer
TDAA µg C/L 342 664
µg/L N 133 277
µg C/mg C 28 100
TDCA µg C/L 194 338
µg C/mg C 16 51

The apparent molecular weight (AMW) distribution of the NOM (Table 6.2)
provided further support that the organics in the summer sample differed significantly
from those in the winter. Specifically, the proportion of DOC smaller than 1,000 Daltons

194
increased from 7% in winter to 17% in summer, a change that is consistent with a
somewhat more hydrophilic character of the NOM in summer.

Table 6.2
Apparent molecular weight distribution of the NOM of the Blavet River
Apparent Molecular Weight Distribution Range (amu)
<500 500 to 1000 1000 to 10,000 >10,000
Sampling season DOC A254 DOC A254 DOC A254 DOC A254
(%) (%) (%) (%) (%) (%) (%) (%)
Winter 3.5 1.2 3.5 3.1 47.8 40.9 45.2 54.8
Summer 6.8 1.1 10.5 8.6 36.5 28.1 47.2 62.2

ELEMENTAL ANALYSIS AND SUVA OF THE NOM ISOLATES FROM THE


BLAVET RIVER

Table 6.3 shows the elemental analyses of the NOM isolates from the Blavet
River samples. Although samples that were obtained using the same techniques in the
two sampling periods do not differ dramatically in their elemental composition, apparent
differences in composition are noticeable when dissimilar NOM concentration methods
are compared. All the RO and NF NOM isolates had a substantial amount of ash, causing
the estimate of carbon content to be low and preventing analysis of oxygen. For the
winter samples, attempts were made to remove cations from the RO and NF concentrates
by H+-ion exchange prior to lyophilization. This reduced the ash content of the isolates,
but not enough to allow an accurate elemental analysis to be conducted. For the summer
samples, ash accounted for approximately one-third of the mass of the RO and NF
isolates. HCl acidification and CO2 stripping helped reduce the ash contents to 8 to 15%,
but this was still too high to obtain good results from the elemental analyses of the
organics.

195
Table 6.3
Elemental analysis of the Blavet River NOM isolates
Fraction C (%) H (%) N (%) O (%) S (%) Ash (%)
*w s w s w s w s w s w s
XAD-8/XAD-4 46.1 44.8 4.5 4.4 2.1 1.7 39.9 41.8 1.1 nd 2.4 3.0
mixture
XAD-8 fraction 47.0 46.6 4.6 4.6 2.0 2.1 38.8 38.4 0.5 nd 6.2 2.8
XAD-4 fraction 43.3 41.7 4.6 4.2 2.9 2.5 40.6 44.9 1.6 nd 2.7 3.5
RO isolate 13.0 10.6 1.5 1.2 2.0 2.2 nd nd 1.8 nd 34.3 33.3
NF isolate 15.6 13.2 1.7 1.4 1.4 1.5 nd nd 2.7 nd 20.5 32.5
RO + IX 22.5 - 3.7 - 1.3 - 44.8 - 7.9 - 14.5 -
NF + IX 24.9 - 3.9 - 1.4 - nd - 6.5 - 13.7 -
RO + XAD-8 35.0 - 3.7 - 1.5 - 31.8 - 0.7 - 2.7 -
RO + XAD-4 46.2 - 4.7 - 2.5 - 39.4 - 0.9 - 3.7 -
RO + HCl - 6.9 - 0.8 - 2.2 - nd - nd - 15.2
NF + HCl - 7.7 - 1.0 - 0.9 - nd - nd - 8.7
NF + XAD-4 - 45.0 - 4.4 - 2.6 - 40.4 - nd - 4.2
* w: winter sample, s: summer sample
**JP is this correct: nd: analysis not reliable due to interferences

By contrast, the XAD-8 and XAD-4 desalting procedures were efficient in


removing inorganic species from the membrane-concentrated waters. The elemental
analysis of the RO + XAD-4 isolate was almost identical to that of the XAD-8/XAD-4
mixture, and >97% of the NOM mass in the original sample could be accounted for by
the masses of the elements recovered. On the other hand, only about 75% of the original
mass was recovered in the RO + XAD-8 isolate. No explanation is apparent for this loss
of material.

For both rounds of sampling, the XAD-4 fraction was richer in oxygen and
nitrogen and poorer in carbon than the XAD-8 fraction. In the winter sample, the ash
content was significantly lower in the XAD-4 fraction (2.7%) than in the XAD-8 isolate
(6%), and sulfur was also more abundant in the XAD-4 fraction.

196
Table 6.4 shows the C/O, C/N and C/H ratios (by weight) and SUVA254 values for
the isolates. The elemental ratios support the greater hydrophilic character of the XAD-4
fraction than the XAD-8 fraction. The XAD-4 isolates that have the highest
hydrophilicity according to their C/O, C/N and C/H ratios also have the lowest SUVA254
values. The opposite was observed for the (hydrophobic) XAD-8 fraction. The SUVA254
value of the XAD-8/XAD-4 mixture was comparable to that of the XAD-8 fraction,
because the XAD-8 fraction comprises up to 90% of the isolate. The XAD-8/XAD-4
mixture and the RO + XAD-4 isolate were also similar with respect to both elemental
composition and SUVA254. In the absence of XAD resin desalting, the elemental ratios
calculated for the RO and NF isolates are not necessarily meaningful. The RO
concentrates that were desalted with XAD-4 and XAD-8 have similar elemental ratios,
although this result should be interpreted with caution, given the loss of material in the
RO + XAD-8 sample preparation process.

197
Table 6.4
C/O, C/N and C/H ratios with SUVA of the Blavet River NOM

C/O C/N C/H SUVA


(L/mg-m)
w. s. w. s. w. s. w. s.

XAD-8/XAD-4 1.15 1.07 21.75 26.18 10.27 10.29 4.4 4.1


mixture
XAD-8 fraction 1.21 1.21 23.73 21.76 10.33 10.17 4.3 5.2
XAD-4 fraction 1.06 0.93 14.77 16.74 9.41 9.95 2.6 3.2
RO isolate nd nd 6.45 4.84 8.65 8.66 4.0 5.2
NF isolate nd nd 11.27 9.07 9.09 9.32 4.3 4.16
RO + IX 0.50 - 16.95 - 6.17 - nd -
NF + IX. nd - 17.50 - 6.39 - nd -
RO + XAD-8 1.10 - 22.74 - 9.39 - 4.3 -
RO + XAD-4 1.17 - 18.32 - 9.91 - 3.7 -
RO + HCl - nd - 3.07 - 8.35 - nd
NF + HCl - nd - 8.92 - 7.91 - nd
NF + XAD-4 - 1.11 - 17.23 - 10.31 - 4.3

TOTAL DISSOLVED AMINO ACIDS AND CARBOHYDRATES

The TDAA and TDCA contents of the Blavet River NOM isolates are presented
in Table 6.5, and the distributions of species comprising the TDAA and TDCA are
represented in Figures 6.4 through 6.7. Among the three summer isolates analyzed for
TDAA and TDCA, the RO isolate had the highest concentration of these compounds. The
XAD-8/XAD-4 sample contained less carbohydrates and more amino acids than the
XAD-4 fraction. For the winter samples, the TDCA content decreased in the order
XAD-8 ≈ NF + IX > XAD-8/XAD-4 > XAD-4 ≈ NF + IX. Although it is possible that
the difference between the NF + IX and RO + IX isolates was caused by preferential
adsorption of carbohydrates onto the surface of the RO membrane, no such difference

198
was observed for NOM from the Vienne River (data not shown). The difference might
have been caused by analytical problems.

Table 6.5
TDAA content of the Blavet River NOM isolates (winter sample)
TDAA µg C/mg C TDAA µg N/mg C TDCA µg C/mg C
winter summer winter summer winter summer

XAD-8/XAD-4 25 23 9 9 15 17
mixture
XAD-8 fraction 30 - 11.5 - 21 -
XAD-4 fraction 22 18.4 9 7 10 24
RO+H+cat res. 13 - 5.4 - 8 -
NF+ H+cat res 12.5 - 5 - 19 -
RO isolate - 43 - 16 - 39.4

10
9
µ g AA carbon / mg DOC

8
7
6
5
4
3
2
1
0
met
tyr
ser

thr
arg

phe

orn
lys
gly
glu

ala

leu
ile
asp

his

Amino Acid

Figure 6.4. TDAA distribution of the Blavet River (winter sample)

199
µ g carbohydrate C / mg DOC
2.5

2.0

1.5

1.0

0.5

0.0

man-xyl
glucos-

glucose
rhamnose

galactose
arabinose
fucose

fructose
amine

Carbohydrate

Figure 6.5. TDCA distribution of the Blavet River (winter sample)

40
35
µ g AA carbon / mg DOC

30
25
20
15
10
5
0
met
tyr
ser

thr
arg

phe

orn
lys
gly
glu

ala

leu
ile
asp

his

Amino Acid

Figure 6.6. TDAA distribution of the Blavet River (summer sample)

200
µ g carbohydrate C / mg DOC
14
12
10
8
6
4
2
0

man-xyl
glucos-

glucose
rhamnose

galactose
arabinose
fucose

fructose
amine

Carbohydrate

Figure 6.7. TDCA distribution of the Blavet River (summer sample)

The TDAA in the XAD resin fractions did not follow the general trend discussed
in the literature (Croue et al. 1993a) or observed in the Suwannee and South Platte
Rivers. In this sample, TDAA was higher in the XAD-8 (HPOA) fraction than in the
XAD-4 (TPHA) fraction. Furthermore, less TDAA appeared in the RO + IX and NF + IX
isolates than in the fractions isolated using XAD resins, a result that is almost certainly
attributable to adsorption of amino acids onto the ion exchange resin.

13
C-NMR AND FTIR DATA FOR THE BLAVET RIVER NOM ISOLATES

The FTIR spectra of the Blavet River NOM isolates obtained with XAD resins
did not show any trace of the major anions (bicarbonate, nitrate and sulfate) in either
winter or summer, indicating that the desalting process was very efficient (Figure in
Appendix**MB). Silica could be detected in the XAD-8 and the XAD-8/ XAD-4 winter
isolates (peak at 470 cm−1), but not in the spectrum of the XAD-4 sample from the winter
or in any of the spectra of the summer isolates. In the FTIR spectra of the winter and
201
summer XAD-8 and NF + XAD-4 isolates, there are features associated with phenolic
structures. In the winter samples, these phenolic structures are more prominent in the
XAD-8 and XAD-8/XAD-4 isolates.

The major peaks in the FTIR spectra of the RO and NF concentrates were caused
by the inorganic anions. Not surprisingly, the same is true after each of the concentrates
was treated by H+-cation exchange. These results reaffirm that membrane treatment
followed by cation exchange is not appropriate if a low-salt NOM concentrate is
required. The FTIR spectrum of the RO + XAD-4 isolate had no significant signal from
inorganic species except for a small silica signal. In general, it looked similar to the
spectrum of the XAD-8/XAD-4 mixture.

Partial desalting via H+-cation exchange did not allow complete resolution of the
13
C-NMR spectra of the membrane isolates. As discussed previously, the apparent
increase of the aromatic carbon content and the presence of distinct bands in the C-O and
C-C regions in the membrane concentrates might be explained by the presence of
sulphonated aliphatic and aromatic structures that were produced by lyophilization in the
presence of sulfuric acid. The reaction between sulfuric acid and carbohydrate species
can also lead to the production of furans that increase the intensity of the aromatic carbon
band. Therefore, the higher aromatic carbon content in the membrane samples might be
an artifact caused by interactions between the organic and inorganic components of the
isolates.

The spectra of the winter RO + XAD-8 and RO + XAD-4 isolates look more like
those of ‘normal’ NOM. The major differences between the two spectra are the larger
phenolic content of the RO + XAD-8 isolate and the larger C-O band of the RO + XAD-4
isolate.

The integrated areas of the 13C-NMR spectra in Figures Error! Reference source
not found. and Error! Reference source not found. are reported in Table 6.6. This
semi-quantitative approach could be applied only to the fractions that were desalted on
13
XAD resins. According to the C-NMR integration data for the winter samples, the

202
XAD-4 fraction is the most hydrophilic NOM fraction, with the highest C-O content,
highest anomeric carbon content, lowest aromatic carbon content, and high carboxylic
acid content. The XAD-8 fraction is the most hydrophobic. The three other NOM
fractions have similar carbon distributions that indicate an intermediate hydrophobic (or
intermediate hydrophilic) character. Among these three fractions, the RO + XAD-4
isolate was poorest in aromatic carbon content, as expected from its SUVA254.

Table 6.6
Integrated areas of 13C-NMR spectra of the Blavet River NOM isolates
Integration range (ppm)
0-60 60-90 90-110 110-160 160-190 190-220
Sample w. s. w. s. w. s. w. s. w. s. w. s.

XAD-8/XAD-4 * 34 47 21 20 5 7 21 15 15 10 4 1
mixture
XAD-8 fraction 39 41 13 19 4 5 28 20 13 13 3 2
XAD-4 fraction 38 35 24 25 7 7 13 12 16 19 2 2
RO + XAD-8 36 - 17 - 6 - 22 - 16 - 2 -
RO + XAD-4 37 - 21 - 6 - 17 - 16 - 3 -
NF + HCl - 35 - 21 - 10 - 16 - 17 - 2
NF + XAD-4 - 42 - 20 - 8 - 15 - 13 - 1
RO + HCl - 38 - 31 - 10 - 9 - 10 - 2
13
* Values represent the area under the C-NMR spectral curve in the indicated range as a percentage of
total integrated area of the spectrum.

Based on integration of the NMR spectra in the 110 to 160 ppm range, the
aromaticity of all the summer NOM isolates is low, exceeding 16% only in the XAD-8
fraction (20%). The aromaticity of the XAD-8 and XAD-8/XAD-4 isolates are notably
higher in the winter than the summer. The relatively hydrophilic character of the
NF + HCl isolate is confirmed by its high proportion of alcoholic and carboxylic carbon.
The carbon distribution of the XAD-4 fraction was similar to that of the NF + HCl
isolate, while the NF + XAD-4 isolate was more comparable to the XAD-8/XAD-4

203
mixture. Thus, partial or total desalting of the membrane isolates had a significant impact
on the apparent structural characteristics of the NOM.

PYROLYSIS - GAS CHROMATOGRAPHY - MASS SPECTROMETRY FOR


THE BLAVET RIVER NOM ISOLATES

The relative proportions of biopolymers in the Blavet River NOM isolates are
given in Table 6.7. (This semi-quantitative approach was not utilized on the NF + IX
isolate because of the structural changes noted above.)

Table 6.7
Relative proportions of biopolymers in the Blavet River NOM isolates
Fraction * PHA UA PR PS AS unknown
Fraction of DOC (%)

w† s w s w s w s w s w s

XAD-8/XAD-4 31 21.8 21 14.7 14.6 20.5 19.5 21.4 1.7 6.9 14 14.7
mixture
RO isolate 35.5 15 8.8 8.7 35 31.6 7 9.5 10.5 33.8 2.6 1.4

NF isolate 33.6 - 14.4 - 35 - 6.9 - 8.5 - 1.5 -

RO + XAD-4 19.7 - 7.2 - 26.8 - 11.4 - 7.2 - 13.7 -

XAD-8 - 25.9 - 13.4 - 21.3 - 18.7 - 4.7 - 16


fraction
XAD-4 - 14.4 - 16.3 - 22.6 - 18 - 10.2 - 18.5
fraction
PHA: Polyhydroxyaromatics, UA: unsubstituted aromatics, PR: Proteins, PS: Polysaccharides, AS: Amino
sugars
† w: winter, s: summer

Based on these analyses, the summer XAD isolates had higher proportions of
polyhydroxyaromatics and polysaccharides than the winter sample. The differences
between the XAD-8 and XAD-4 fractions of the Blavet NOM are similar to those that
characterize these fractions in the Suwannee and South Platte Rivers (see Chapter 5).
Phenol and cresol are the major peaks of the XAD-8 fraction, while acetic acid,

204
acetamide (fragment from amino sugars), acetonitrile and methylpyrrole (produced from
proteins), methyl furfural and levoglucosenone (fragments produced from
polysaccharides) are proportionally more abundant in the pyrochromatogram of the
XAD-4 fraction. The XAD-4 fraction was also characterized by its higher proportion of
acetamide and lower proportion of polyhydroxyaromatics the XAD-8 sample.
Qualitatively, the pyrochromatogram of the winter XAD-8/XAD-4 isolate is also a
typical fingerprint of NOM associated with the hydrophobic acid fraction. The pyrolysis
data for the XAD-8/XAD-4 isolate correspond to the results obtained for the two XAD
resin fractions mixed in a 70%/30% mass ratio.

The Pyr-GC-MS chromatograms of the NF and RO isolates were different from


those of the XAD-8/XAD-4 samples, having acetonitrile (a protein pyrolysis fragment)
and acetamide (an amino sugar pyrolysis fragment) as major constituents. In the winter
RO isolate, the signals from acetonitrile and acetamide were higher than even those from
phenol and cresol, which are commonly found PHA fragments. Ethyl hexanol, which is
generally not found as a pyrolysis fragment of NOM, was also identified in these two
chromatograms for the winter RO sample. This was not the case for the summer RO
isolate, whose pyrochromatogram contained butanine, benzene and acetamide as the
major fragments. Phenol was also an important fragment, but most of the other identified
peaks were generated from the pyrolysis of polysaccharides and proteins. The NOM
isolated via RO was enriched in proteins and amino sugars as compared to the resin-
isolated fractions, but it contained lower proportions of polyhydroxyaromatic and
polysaccharide moieties, indicating that it is quite hydrophilic.

In order to determine the origins of ethyl hexanol in the winter RO sample, a


small piece of hexanal tubing that is used in the membrane apparatus was pyrolyzed. The
corresponding pyrochromatogram showed the presence of this compound, but only at a
trace level that could not explain the large signal from this molecule in the RO isolate.
Possible explanations for the presence of ethyl hexanol in the pyrochromatogram include:

• the compound was originally present in the raw water as a contaminant. Ethyl
hexanol is a widely used industrial chemical (mainly for the production of poly(vinyl

205
chloride) plasticizers. Ethyl hexanol is widely diffused in the environment and its
presence has been detected in natural waters (Vitali and Leoni 1993).

• the compound was released to the water from the membrane or from another part of
the membrane unit, or was produced during the pyrolysis of an impurity that came
from them. This possibility is considered unlikely, because the RO and NF isolates
produced with the same unit did not show any trace of ethyl hexanol in their
Pyr-GC-MS spectra.

• the compound was produced during the pyrolysis of NOM structures that were
selectively isolated with the membranes.

The final hypothesis seems to be the most plausible, because RO was the most
efficient technique in terms of overall NOM retention, and trace amounts of organic
species could be retained by this method and lost by others. Nevertheless, the exact
source of the ethyl hexanol in the winter RO concentrate remains unclear.

The pyrochromatogram of the winter RO + XAD-4 isolate was also characterized


by an abundance of pyrolysis fragments produced from proteins (especially pyrrole) and
amino sugars. The identification of sulfur-containing organic compounds in the
Pyr-GC-MS chromatogram of the NF + IX isolate seems to confirm the hypothesis that
the structure of NOM was modified by lyophilization in the presence of sulfuric acid.

The major points established by the table can be summarized as follows:

• with or without desalting, the membrane isolates contain larger proportions of


proteins and amino sugars than the XAD-8/XAD-4 mixture does.

• the XAD-8/XAD-4 mixture contains a larger proportion of polysaccharides than the


non-desalted RO and NF isolates and the RO + XAD-4 isolate.

• the RO + XAD-4 isolate contains a lower proportion of polyhydroxyaromatic


structures than the other three NOM fractions investigated, all of which contain
similar amounts of this structural unit.

206
13
This last point is consistent with the SUVA254 values and C-NMR spectra for
these fractions, which support the idea of a preferential loss of high molecular weight
organics during the back elution of the column with the acetonitrile/ water solution.

CHLORINATION OF THE BLAVET RIVER NOM ISOLATES

Table 6.8 shows the formation of by-products as a result of chlorination of the


Blavet River and its isolates under UFC conditions. The TOX concentrations analyzed in
all solutions were similar, except for the summer RO and NF isolates.

207
Table 6.8
Some chlorinated DBPs formed by chlorination of the Blavet River and its NOM isolates

XAD-8/XAD-4 NF isolate RO isolate Raw water


mixture
w. s. w. s. w. s. w. s.
Cl2 dose 1.4 1.4 1.3 1.1 1.6 1.4 1.6 1.6
(mg/mgC)
Cl2 residual 1.1 0.9 0.8 2.6 1.2 5.3 1.0 0.6
(mg/L)
TOX 143 146 140 80.4 164 78 160 157
(µgCl/mgC)
CHCl3 42.8 39.3 46.6 25.4 46.6 26.7 47.4 44.1
(µg/mgC)
CHCl2Br 0.6 0.9 2.2 2.5 3.2 3.4 4.2 8.7
(µg/mgC)
CHClBr2 nd Nd 0.05 0.1 0.1 0.2 0.2 0.8
(µg/mgC)
TTHM 38.7 35.6 42.9 24.4 43.6 24.0 45.1 45.4
(µgCl/mgC)
DCAA 11.5 17.3 8.1 9.2 8.8 9.6 14.2 19.4
(µg/mgC)
TCAA 20.2 21.8 9.8 13.4 10.1 13.4 19.7 19.7
(µg/mgC)
DBAA nd 0.01 0.9 0.1 0.6 1.5 0.9 0.2
(µg/mgC)
DCBAA 0.1 0.6 0.1 0.7 0.2 0.08 0.1 3.2
(µg/mgC)
DBCAA 0.1 Nd 0.8 nd 0.8 nd 0.08 nd
(µg/mgC)

The results obtained with the XAD-8/XAD-4 mixture were similar to those for
the unfractionated sample. The addition of bromide did not significantly change the yield
and speciation of DBPs.

For all samples, chloroform was the major THM species produced during
chlorination, accounting for about 30% of the TOX. Except in a few cases discussed

208
below, the yield of chloroform was similar in all the solutions studied, ranging from 41 to
48 µg chloroform generated per mg of DOC in the sample. CHCl2Br and CHClBr2 were
detected in all chlorinated solutions, but CHBr3 was below the detection limit. CHCl2Br
was the main brominated THM. The concentrations of brominated THMs were lower for
the NF and RO isolates than for the raw water, probably because of loss of bromide
during the membrane concentration step. The production of brominated THMs was lower
in the XAD-8/XAD-4 mixture than in the other solutions, however the values obtained
with the mixed XAD-8/XAD-4 solution spiked with bromide were significantly increased
and of the same magnitude as the raw water and RO isolate (data not shown? **JP am I
right that we are referring to data here that we are not including? (ok with me, I’m just
checking))

TCAA and DCAA were the main HAAs detected, and the brominated HAAs
were present in relatively minor concentrations. Chlorination of the membrane isolates
generated less TCAA and DCAA than did chlorination of the raw water, while the
concentrations of these by-products formed by chlorination of the XAD-8/XAD-4
mixture were closer to those in the raw water. As was observed for THMs, the addition of
bromide to the mixed XAD-8/XAD-4 solution led to an increase in production of the
brominated HAAs after chlorination.

The chlorine demand of the summer RO and NF isolates was low, as were the
concentrations of DBPs formed in these samples. In fact, the production of TOX, THMs
and HAAs was only about half of the corresponding production in the raw water or the
XAD-8/XAD-4 mixture. It is unlikely that these results were caused by analytical
problems, because the results obtained on the same day for the XAD-8/ XAD-4 mixture
were similar to those for the raw water, as expected based on prior work. The most likely
explanation for the anomalous results appears to be incomplete dissolution of the
lyophilized sample (and low reactivity of the undissolved NOM). Additional evidence for
the erroneous DOC value includes a computed SUVA value that is well out of the
expected range based on the strong relationship previously established between SUVA
and DBP formation or chlorine demand (computed SUVA near 2.0 L/mg-m for both NF
and RO isolates, versus an expected value between 4 and 5). This data set highlights the
209
problems with resolubilizing the non-desalted NOM isolates obtained from RO- and NF-
concentrated waters.

UV AND FLUORESCENCE SPECTRA OF THE BLAVET NOM


CONCENTRATES

UV and fluorescence spectra of the Blavet NOM concentrates were analyzed in


order to investigate the influence of concentration and fractionation methods on its
spectral parameters. The UV spectra of all samples were recorded at a DOC
concentration of 10 mg/L. The UV spectra for selected isolates are presented in
Figure 6.8. Only the data for λ>220 nm are shown, because in some samples (including
almost all those from the summer sampling period) the absorbance was too high at
λ < 220 nm to be measured with sufficient precision, possibly because of the presence of
interfering species. The spectral parameters of the samples are compiled in Table 6.9.

3.0

XAD-8
2.5
NF
2.0
Absorbance

1.5 RO

1.0
XAD-4

0.5

0.0
225 250 275 300 325 350 375 400
Wavelength (nm)

Figure 6.8. Set of UV spectra of NOM concentrates from the Blavet River winter
sampling period (10 mg DOC/L, cell length 5 cm)
210
Table 6.9
UV and fluorescence parameters for NOM samples concentrated from the Blavet River

SUVA254 (L/(mg-m)) A350/A380 ∆ET (eV) λmax (nm)


Sample w. s. w. s. w. s. w. s.
raw water 5.1 4.8 nd nd Nd nd nd nd
XAD-8/XAD-4 4.4 4.1 0.39 0.35 2.23 2.11 438 434.5
XAD-8 4.3 5.2 0.40 0.36 2.26 2.14 437 432
XAD-4 2.6 3.2 0.29 0.28 1.97 1.92 424 423
RO 4.0 5.2 0.38 0.35 2.20 2.11 428.5
NF 4.3 4.2 0.39 0.35 2.24 2.13 431
RO + IX 4.5 0.45 2.42 438
NF + IX 4.6 0.44 2.41 434
RO + HCl 4.1 0.43 2.35
NF + HCl. 4.5 0.43 2.35
NF + XAD-4 4.3 0.34 2.08 427

The SUVA254 values for the raw Blavet River water from both the winter and
summer sampling periods are remarkably high. In fact, these values are larger than the
corresponding value in almost all of the NOM fractions from these samples or the NOM
fractions from the Suwannee or South Platte. In light of the widely accepted correlation
between the aromaticity of NOM and its SUVA254 value, one would conclude that the
aromaticity of the NOM in the Blavet is very high. However, the 13C-NMR data suggest
that the aromaticity of the desalted isolates is in the range 21 to 28% for the winter
samples and 15 to 20% during the summer (Table 6.6), which are not unusually high.
Also, as a point of comparison, the aromaticities of the Suwannee River fractions with
similar SUVA254 (the uHA and HPOA fractions) were ~27 and 31%, respectively.

Pyr-GC-MS and other compound- and structure-specific analyses described in


Chapter 5 and the preceding sections of this chapter suggest that NOM from the Blavet
Rivers is very similar to that from the Suwannee. Thus, it seems unlikely that the
difference in SUVA254 values of the raw waters can be attributed to any specific group of
211
high-absorbance chemicals that are present in the Blavet and absent in Suwannee. Three
explanations for the difference that are considered to be more likely include: (1) the
aromaticity of the Blavet NOM is underestimated by integration of the 13C-NMR spectra;
(2) the isolation and fractionation procedures altered the NOM so that the raw water
could not be represented as the sum of the fractions; or (3) the DOC of the sample was
greater than the reported value, due to some analytical problem.

With respect to the first of the proposed explanations, it is important to recall the
13
limitations of C-NMR as a quantitative tool for estimating the structural identity of
carbon atoms in NOM, as noted in Chapter 2. Correlations of SUVA254 and other spectral
parameters with the NOM aromaticity evaluated using 13C-NMR data acquired at a 5 ms
contact time may be considerably better than those derived using a 1-ms contact time
(which was used in the current study).

The fact that the SUVA254 values for raw Blavet River water prior to NOM
concentration were higher than those of virtually all of the sub-fractions isolated from
those samples is even more difficult to explain than the anomalously high SUVA254 of
the raw water samples. Based on prior studies by the authors and others, the SUVA254
values of the RO and NF concentrates and, even more so, the XAD-8 and XAD-8/
XAD-4 fractions, were expected to be higher than the corresponding values in the raw
waters, since low-absorbing NOM molecules are selectively lost during these processing
steps. The fact that the opposite result was found for the Blavet concentrates might
signify that the concentration and desalting techniques altered the properties of the NOM,
13
a possibility that is supported to some extent by the C-NMR and Pyr-GC-MS data
discussed in the previous sections of this chapter.

A third possible explanation for the results is that the measured DOC
concentration of the raw water samples was less than the true value, so that the correct
value of SUVA254 was smaller than reported. While analytical or human error is always a
possibility, it seems odd that such errors should be manifested in the two raw water
samples, while the results for all the other samples fell within commonly expected

212
ranges, and that no evidence of such errors were apparent in the QA-QC checks
conducted throughout the project. Nevertheless, this possibility cannot be ruled out.

NOM concentration and fractionation method(s), however gentle they may be, are
intended to alter the chemical composition of a very complex solution. As a result, they
are almost certain to alter NOM composition in unforeseen ways that one hopes are not
critical to the evaluation being carried out. The techniques that have been developed to
date for this purpose seem to be quite successful at meeting this goal. Nevertheless, it is
clear that greater attention needs to be paid to development of internal and/or easily
performed checks that can be used to provide an ongoing assessment of whether the
techniques are achieving the desired result (**MB add to exec summary). One such
check should involve careful comparison of characteristics of the NOM (e.g., SUVA254)
before and after each processing step. Alternatively, or in parallel, development and
validation of in situ NOM characterization methods that do not involve any concentration
steps would be extremely valuable. The uncertainty about the source of the anomaly in
the SUVA values of the Blavet raw water and fractions is testimony to the complexity of
the problem and the need for constant vigilance in all aspects of the isolation-
fractionation-characterization process.

CORRELATIONS BETWEEN SPECTRAL AND STRUCTURAL


CHARACTERISTICS OF NOM IN BLAVET RIVER FRACTIONS

The next section of this chapter focuses on evaluation of correlations between the
spectral parameters of the Blavet isolates and their chemical characterization. In
particular, various data associated with the fluorescence and UV absorbance spectra of
the of NOM from the Blavet samples will be discussed. As noted above, isolation
procedures are bound to alter NOM. Analysis of the chemistry of NOM by in situ
measurements provides a valuable adjunct to the information that can be acquired by
application of sophisticated techniques to the concentrated and isolated samples.

213
Generally, the A350/A280 ratios and ET band half-widths (∆ΕΤ) of the samples
follow the same patterns as SUVA254. The correlation between SUVA254 and ∆ET for the
samples is shown in Figure 6.9. This correlation (R2 = 0.63) probably reflects the intrinsic
interdependence between the aromaticity and molecular weight of NOM, on one hand,
and the inter-chromophore interactions on the other. One possible advantage of using ∆ΕΤ
rather than SUVA254 as an indicator of these properties is that ∆ΕΤ can be estimated
directly from the UV absorbance spectrum of the sample, without simultaneous analysis
of the DOC.

2
2.40 R = 0.63
ET band halfwidth (eV)

2.25

2.10

1.95

1.80
2.0 3.0 4.0
SUVA254 (L/mg·m)

Figure 6.9. Correlation between SUVA254 and ∆ΕΤ for NOM concentrates from the Blavet
River (data shown are for both the summer and winter sampling periods)

The fluorescence emission spectra of the NOM concentrates are sensitive to the
concentration method employed (Figure 6.10). The emission intensity of the XAD-4
sample is substantially higher than that of any of the other samples, and the maximum of
its emission band is at shorter wavelengths. Similar results have been reported in the
literature for the transphilic fractions of NOM (Donard et al. 1987, Ewald et al. 1992).

214
The increased fluorescence intensity of the XAD-4 sample is almost certainly related to
its lower molecular weight (compared to the other NOM in the other samples), which
decreases the rate of radiationless losses of excitation.

240

210 XAD-4
180 XAD-8
Emission intensity

150
NF
120 RO

90

60

30

0
375 400 425 450 475 500 525 550
Wavelength (nm)

Figure 6.10. Selected fluorescence emission spectra of NOM concentrates from the
Blavet River, winter sampling period (10 mg DOC/L, cell length 1 cm, excitation at
320 nm)

The emission spectrum of the XAD-4 (transphilic) acids is substantially blue-


shifted compared with that of the RO isolate, whereas the maximum in the XAD-8
concentrates is slightly red-shifted. This comparison is more obvious when the emission
spectra are normalized, as in Figure 6.11. The normalized emission spectra of the RO and
NF concentrates are virtually identical. The position of the maximum in the emission
spectrum of the different concentrates is correlated with ∆ET in the corresponding UV
absorbance spectrum, as shown in Figure 6.12. As the ET band becomes broader, the
emission maximum exhibits a red shift.

215
Normalized fluorescence intensity 1.00
XAD-8

0.75
RO,NF

0.50

0.25 XAD-4

0.00
375 400 425 450 475 500 525 550 575 600
Wavelength (nm)

Figure 6.11. Normalized fluorescence emission spectra of selected NOM concentrates


from the Blavet River winter sample (10 mg DOC/L, cell length 1 cm, excitation at
320 nm)

216
440

435
λ max (nm)

430 2
R = 0.64

425

420
1.90 2.00 2.10 2.20 2.30 2.40 2.50
ET band halfwidth (eV)

Figure 6.12. Correlation of the ET band half-width and the wavelength of maximum
fluorescence emission intensity (NOM samples concentrated from the Blavet River,
winter and summer sampling periods)

Based on these and literature data, as well as theoretical considerations, it is


reasonable to conclude that the UV and fluorescence spectra of NOM isolates are mainly
determined by the aromaticities and MW distribution of the humic species comprising the
NOM. In the next section, we explore whether chemical properties other than aromatic
content might affect the spectral response of NOM in consistent ways.

Potential correlations between the elemental composition of NOM and its spectral
response were explored first. Only the low-ash samples (see Table 6.1), for which the
absolute concentrations of elements and the C/O, C/N and C/H ratios were known most
reliably, were included in the analysis. The carbon mass fraction (%C) in these samples
was well correlated with the SUVA254 (R2 = 0.78) and even more strongly correlated with
∆ET (R2 = 0.90). The total carbon content of the NOM was also correlated with the
percentage of aromatic carbon in the NOM concentrates (as determined by integration of
13
the C-NMR spectra over the range 110 to 160 ppm) and with the PHA carbon (as
217
determined from Pyr-GC-MS analysis) (Figure 6.13). Thus, the correlation between ∆ET
for the Blavet concentrates and %C probably reflects the fact that the NOM
concentration-isolation methods are likely to be most efficient at collecting high-MW and
aromatic molecules; these molecules, in turn, are expected to be less oxygenated and
have a high carbon content than the molecules that are collected less efficiently.

35 35

110-160 ppm aromatic carbon


Pyr-GC-MS
Polyhydroxyaromatic carbon

30
13C NMR data
25 25

(%)
(%)

20 2
R = 0.84
2
R = 0.74
15 15

10

5 5
41 42 43 44 45 46 47 48
Carbon in NOM (%)

Figure 6.13. Correlation between the percentage of carbon in low-ash NOM concentrates
and percentages of aromatic carbon as determined through 13C-NMR and Pyr-GC-MS
data. Blavet River samples (summer and winter sampling periods)

Contrary to the case for carbon content, the nitrogen content of NOM did not
correlate well with any of the major spectral parameters (SUVA254, ∆ET, λmax, and
fluorescence intensity). Attempts were also made to correlate the spectral parameters of
the concentrated NOM samples with the concentrations of specific groups of nitrogen-
containing species. These attempts utilized data from all the samples, regardless of their
ash content. It was hypothesized, for example, that the intensity of NOM fluorescence
might be related to the total concentration of dissolved amino acids, since the amino

218
acids include highly fluorescent species such as tyrosine, tryptophan and phenylalanine.
However, the correlation between the TDAA concentration and the intensity of
fluorescence was relatively weak (R2 = 0.47), and no correlation could be found between
the concentrations of proteins or amino sugars and any spectral parameter. Similarly,
little evidence was found linking the spectra of the NOM samples and their carbohydrate
contents.

Not surprisingly, SUVA254 was correlated with the polyhydroxyaromatic carbon


content, and slightly less strongly with the total aromatic carbon content of the NOM
(Figure 6.14). The correlation of polyhydroxyaromatic carbon with ∆ET was similar or
somewhat stronger than that with SUVA254. The position of the maximum in the emission
spectra was sensitive to the percentage of aromatic carbon (Figure 6.16): as the
concentration of aromatic carbon decreases, the emission maximum shifted toward
shorter wavelengths. This result further supports the assumption that the UV and
fluorescence spectra of the Blavet NOM concentrates are largely controlled by the
concentration and chemical state of aromatic carbon they contain.

219
40

35
Aromatic carbon (%)

30

25
2
R = 0.50
20

15

10

0
2.0 2.5 3.0 3.5 4.0 4.5 5.0
SUVA254 (L/mg·m)

Figure 6.14. Correlation between the values of SUVA254 and the percentage of
polyhydroxyaromatic carbon (Pyr-GC-MS data).

220
45
40
35
Aromatic carbon (%)

30
25
2
20 R = 0.73

15
10
5
0
1.90 1.95 2.00 2.05 2.10 2.15 2.20 2.25 2.30
ET band halfwidth (eV)

Figure 6.15. Correlation between ∆ET and the percentage of polyhydroxyaromatic carbon
in the Blavet NOM fractions based on Pyr-GC-MS data.

221
30

25
Aromatic carbon (%)

20

15 2
R = 0.64

10

5
420 425 430 435 440
λ max (nm)

Figure 6.16. Correlation between the position of maximum in the fluorescence emission
spectra and the percentage of aromatic carbon based on 13C-NMR data.

CONCLUSIONS

The data presented in this chapter illustrate some of the complexity of NOM and
its changes when it is isolated and fractionated. The conclusions drawn from the data
relate to the NOM characteristics in a particular water supply (the Blavet River) and,
more generally, to ways that fractionation-isolation may alter NOM. These conclusions
are summarized below.

NOM from the Blavet River was efficiently bound to XAD-8 resin (79 and 57%
for the winter and summer samples, respectively), and its SUVA254 was comparable to or
higher than that of the HPOA and uHA fractions of the Suwannee River NOM. NOM
from the Suwannee is widely accepted to be highly humified and hydrophobic, perhaps
even representing one extreme on a scale of NOM properties. The Blavet NOM appears
to be similar to the Suwannee NOM in such properties. The differences in the properties

222
of the Blavet NOM between winter and summer are most notably associated with
changes in small, biodegradable constituents such as TCAAs and TDAAs. However,
these chemicals represent a minor fraction of the NOM in either season, so the overall
similarity between Blavet and Suwannee NOM applies to both samples collected. One
possible interpretation is that the highly modified NOM is roughly the same in the two
seasons, and the samples differ primarily in the amounts of degradable molecules that
‘dilute’ the more highly modified molecules.

The summer sample had a higher BDOC and was more hydrophilic: hydrophobic
and transphilic NOM accounted for 57 and 21%, respectively, of the NOM in the
summer, and 79 and 11% in the winter. In terms of this distribution, the NOM in the
Blavet is more akin to Suwannee NOM in the summer than in the winter; disregarding
losses during the fractionation, the hydrophobic and transphilic fractions accounted for
roughly 60 and 21%, respectively, of the NOM in the Suwannee.

Other conclusions derived from the data presented in this chapter are concerned
with the performance of the NOM isolation-concentration methods. As expected, RO was
the most efficient concentration and/or fractionation technique for retaining the TCAA
and TDCA fractions of NOM. However, carbohydrates tended to adsorb on the RO
13
membranes. The C-NMR data also indicate that RO is probably the best method to
isolate the proteinaceous fraction of NOM.

In our study, NF was noticeably less efficient than RO at retaining amino acids
and carbohydrates, although paradoxically, it did retain considerable amounts of salts. In
many cases, the desalting of RO and NF isolates is necessary before the chemical
composition and properties of the collected NOM can be elucidated. However, the
desalting and ion-exchange procedures commonly used for this purpose can alter the
composition and properties of the NOM. For example, amino acids in the NF isolates
were noticeably depleted by ion exchange. As a result, while cation exchange might be
necessary for desalting NOM concentrates collected using membranes, it does have
drawbacks and should be used only if a clear need for desalting exists.

223
Interactions between the inorganic and organic species in highly concentrated and
lyophilized RO and NF isolates might alter the NOM due to the formation of sulfonates
13
and, possibly, furanes that may be detected as aromatic carbon in C-NMR
measurements. The irreversible alteration of NOM by lyophilization is also manifested in
the impossibility of completely re-dissolving lyophilized NF and RO isolates.

224
CHAPTER 7 CORRELATIONS BETWEEN DATA FROM STRUCTURE-
SENSITIVE METHODS AND UV AND FLUORESCENCE SPECTROSCOPY

INTRODUCTION

This study indicates that the majority of the NOM in most water sources can be
isolated, but that limitations exist on the extent and significance of NOM isolation. Even
careful application of the most efficient isolation technique (evaporation) recovers less
than 100% of the NOM in a sample. Furthermore, the NOM that can be isolated may be
altered by any isolation process. Although approaches have been developed to limit the
extent of NOM alteration during processing, the possibility always exists that as yet
unrecognized mechanisms of NOM alteration are active in a given situation. Because of
the inherent limitations of NOM isolation efficiency and the possibility of NOM
alteration during processing, use of characterization methods that do not require NOM to
be isolated and concentrated are attractive. However, the sensitivity and sample
requirements of many available characterization methods do not allow those techniques
to be used on unaltered NOM.

A comparison of analytical methods that do and do not require extensive NOM


pre-processing permits a researcher to judge whether NOM isolation is necessary for a
particular purpose. Thus, one goal of this project was to evaluate the scope of information
on NOM structure and properties that can be extracted using various analytical methods,
alone and in combination. Given the wide range of situations in which information about
NOM might be of interest, it is not possible to recommend a single method of
characterization that would best for all applications. It is useful, nevertheless, to critically
assess the information that can be obtained from different analytical procedures. Such an
assessment is provided here.

For the purposes of this discussion, the analytical techniques of interest are
grouped into two categories. One category includes structure-sensitive methods such as

225
13
C-NMR and Pyr-GC-MS. In general, these analyses require advanced instrumentation,
skilled personnel, and, in many cases, substantial preconcentration of NOM.

13
C-NMR spectrometry has constituted a benchmark in NOM structural studies,
and there is support for the idea that Pyr-GC-MS spectrometry can become a second one.
Interpretation of the data from both of these advanced methods cannot be as
unambiguous as it might be for analysis of individual species. The complexity of NOM
necessitates the use of aggregate parameters (e.g., estimates of organic functional groups
based on the integration of the CPMAS 13C-NMR signal in assigned ranges of chemical
shifts, or quantification of PHA, PR, AS and PS by comparing the intensity of peaks of
signature functional groups in Pyr-GC-MS spectra). The precision of these integration
assignment procedures is limited, and the results are semi-quantitative.

The other category of NOM characterization methods includes UV and


fluorescence spectroscopy, techniques that are easy to use but whose output is related to
the composition and structure of NOM in a more complicated and ambiguous way. These
techniques cannot quantify the presence of specific chemical functional groups as is
accomplished using 13C-NMR and Pyr-GC-MS. At best, they can probe only some of the
aggregate properties of NOM (e.g., aromaticity). However, the spectral measurements are
highly precise, rapid and inexpensive, and they can be carried out in situ on unaltered
NOM.

13
UV and fluorescence data have been compared with results from C-NMR and
Pyr-GC-MS methods in preceding sections of the report, but the comparisons were
carried out separately for NOM samples derived from different sources. In this section, a
more general discussion of these correlations is presented, based on pooled data from
various sites and fractions. Because NOM is known to be at least somewhat site-specific,
the correlations for the pooled data are bound to be weaker than those for more narrow
data sets. However, the analysis might be useful for identifying relationships that deserve
additional study. It might also provide some insight into the type of information that can
be extracted by in situ analyses, as opposed to information that can be obtained only on
isolated, concentrated and/or purified NOM.

226
THE MAJOR PARAMETERS OF UV AND FLUORESCENCE SPECTRA OF
NOM

Data presented in this report indicate that several parameters of the UV


absorbance and fluorescence spectra can be used to characterize NOM. The only spectral
parameter that has been used extensively in previous studies is the specific absorbance at
254 nm (SUVA254), which is widely understood to be related to the aromaticity of NOM
(e.g., Korshin et al. 1997b, Westerhoff et al. 1998, Edzwald et al. 1985).

The half-width of the ET band (∆ET) has been introduced as another parameter of
the UV spectra of NOM in this study. The value of this parameter is a complex function
of the concentration, extent of activation, and mutual interactions among aromatic
chromophores. However, it is easily estimated from the UV absorbance spectrum in a
range where interferences from inorganics are minimal (280 - 350 nm), and it seems to be
related to important characteristics of the NOM.

The fluorescence emission spectra of NOM can also be recorded in a range of


wavelengths (λ > 360 nm) where interferences are minimal. Although several parameters
might be used to characterize NOM fluorescence emission (e.g., the wavelength the
maximum emission intensity (λmax), the fluorescence yield at λmax, the half-width of the
fluorescence spectrum, and others), interpretation of many of these parameters is
complex and speculative, and only λmax will be discussed here.

CORRELATIONS BETWEEN 13C-NMR SPECTROSCOPY AND THE MAJOR


PARAMETERS OF UV AND FLUORESCENCE EMISSION SPECTRA

The ranges of several parameters of interest from 13C-NMR, UV absorbance, and


fluorescence analyses NOM from the Suwannee, South Platte, Tolt and Blavet Rivers are
presented in Table 7.1. The data ranges shown are from analysis of both concentrated
NOM and isolated NOM fractions.

227
Table 7.1
Summary of results from 13C-NMR, UV absorbance and fluorescence emission analysis
for 27 samples of concentrated or fractionated NOM from the Blavet, South Platte,
Suwannee and Tolt Rivers.
Parameter range
Aromaticity (%) 3 to 34
Total carboxyl carbon (%) 11 to 27
Specific absorbance at 254 nm, SUVA254 (L/(mg-m)) 0.6 to 6.2
ET band half-width, ∆ET (eV) 1.52 to 2.51
Position of emission maximum, λmax (nm) 416 to 463

The SUVA254 values for the samples correspond to a range from virtually no light
absorbance at 254 nm to highly absorbing samples (that were also highly colored).
Values of ∆ET have not been reported previously, so the range for these samples cannot
be compared with literature data. The range of emission intensities is wider than those
reported in previous research.

The relationships between the spectral parameters and aromaticity is shown in


Figures 7.1 and 7.2. SUVA254 is correlated with aromaticity, ∆ET, and λmax. The
correlation coefficients for these relationships and several others are summarized in
Table 7.2.

228
7.0
2
R = 0.55 2.5
6.0

ET band halfwidth (eV)


5.0 2.0
SUVA254 (L/mg·m)

4.0 1.5
2
3.0 R = 0.73
1.0
2.0
SUVA254 0.5
1.0
ET band halfwidth

0.0 0.0
0 5 10 15 20 25 30 35
Aromaticity (%)
13
Figure 7.1. Correlation between the aromaticity of NOM estimated using C-NMR
spectroscopy and corresponding values of SUVA254 and ∆ET. Data for Suwannee and
South Platte River fractions and Tolt and Blavet River NOM concentrates

229
470

460
2
450 R = 0.61
λ max (nm)

440

430

420

410
0 5 10 15 20 25 30 35
Aromaticity (%)

Figure 7.2. Data for Suwannee and South Platte River fractions and Tolt and Blavet River
NOM concentrates

230
Table 7.2
External and internal correlations for the UV and fluorescence spectral parameters
and the data of CPMAS 13C-NMR spectroscopy for 27 samples from four sources
(Blavet, South Platte, Suwannee and Tolt Rivers)
13
C-NMR parameter UV/fluorescence parameter Linear R2 value
Aromaticity SUVA254 0.72
Aromaticity ∆ET 0.57
Aromaticity λmax 0.61
Total carboxyl carbon SUVA254, ∆ET, λmax 0.01, 0.06, 0.03
Anomeric carbon SUVA254, ∆ET, λmax 0.04, 0.04, 0.00
Aromatic/carboxyl ratio SUVA254 0.65
Aromatic/carboxyl ratio ∆ET 0.56
Aromatic/carboxyl ratio λmax 0.60
Aromatic/anomeric ratio SUVA254 0.44
Aromatic/anomeric ratio ∆ET 0.39
Aromatic/anomeric ratio λmax 0.28
Correlations among CPMAS 13C-NMR parameters
Aromaticity Total carboxylic carbon 0.00
Aromaticity Anomeric carbon 0.01
Total carboxylic carbon Anomeric carbon 0.00
Correlations among UV/fluorescence parameters
SUVA254 ∆ET 0.72
SUVA254 λmax 0.46
∆ET λmax 0.60

In addition to their association with the aromaticity of NOM, the SUVA254, ∆ET
and λmax values are all inter-correlated. This result reflects the fact that all of these values
are manifestations of the excitation and relaxation of aromatic units caused by irradiation
of NOM with UV or visible light. As a result, any or all of these parameters may be used
to predict and monitor the state and alteration of aromatic moieties in NOM. Though they

231
are partially correlated with one another, the values of SUVA254, ∆ET and λmax are also
partially independent of one another. Combinations of these types of data can potentially
enhance our ability to monitor NOM characteristics and reactions.

CORRELATIONS BETWEEN PYR-GC-MS SPECTROSCOPY AND THE


MAJOR PARAMETERS OF UV AND FLUORESCENCE EMISSION SPECTRA.

Thirteen samples were analyzed using Pyr-GC-MS, and the results of these
analyses can be compared with the corresponding SUVA254, ∆ET and λmax values. Two
samples that were deemed extreme outliers due to their very high concentration of
nitrogenous species (the Suwannee River HPIB and South Platte River TPIN fractions)
13
that were excluded from the analysis of C-NMR data are included in this discussion,
since Pyr-GC-MS provides a direct estimate of the concentration of nitrogenous species
simultaneously with those for aromatic and carbohydrate units. Ranges of the parameters
of interest are summarized in Table 7.3, and the corresponding R2 coefficients are given
in Table 7.4.

Table 7.3
Ranges of major parameters of Pyr-GC-MS, UV absorbance and fluorescence emission
analyses of 13 samples from three sources (Blavet, South Platte and Suwannee Rivers)
Parameter Range
Polyhydroxyaromatic carbon, PHA (%) 7 to 34
Unsubstituted aromatic carbon, UA (%) 8 to 30
Total aromatic carbon, PHA+UA (%) 24 to 54
Polysaccharides, PS (%) 3 to 28
Amino sugars, AS (%) 3 to 34
Total saccharides, PS+AS (%) 15 to 46
Specific absorbance at 254 nm SUVA254 (L/(mg-m)) 0.6 to 4.6
ET band half-width, ∆ET (eV) 1.62 to 2.24
Wavelength of emission maximum, λmax (nm) 400 to 438

232
Table 7.4
Correlations of UV and fluorescence spectral parameters with Pyr-GC-MS results for 13
samples from the Blavet, South Platte and Suwannee Rivers
Pyr-GC-MS parameter UV/fluorescence parameter Linear R2 value
External correlations, aromatic groups
PHA SUVA254 0.37
PHA ∆ET 0.41
PHA λmax 0.65
External correlations, nitrogen-containing groups
Total nitrogenous species λmax 0.55
AS λmax 0.46
Proteins λmax 0.39
Correlations for Pyr-GC-MS vs. 13C NMR data
PHA Aromaticity 0.44
PS Anomeric carbon 0.03
AS Anomeric carbon 0.12
Total saccharides Anomeric carbon 0.00

Several results shown in Table 7.4 are worth noting. First, the SUVA254 and ∆ET
values are correlated only with the concentration of PHA units, and then with R2 values
of only 0.37 to 0.41. By contrast, the emission of NOM is affected by both PHA and
nitrogenous species. The position of the fluorescence emission maximum is related to the
percentage of PHA in the NOM (R2 = 0.65) and to the amino sugar (AS) content
(R2 = 0.46). The relationship between the position of the emission maximum and the
concentration of nitrogenous species that was first noticed in the studies of the South
Platte NOM fractions (Chapter 5) is reinforced by the results presented here.

The correlation between the 13C-NMR and Pyr-GC-MS data is generally weak; R2
is >0.2 only for the relationship between the aromaticity and Pyr-GC-MS
233
polyhydroxyaromatic carbon (R2 = 0.44). There is little correlation between the anomeric
13
carbon and the relevant Pyr-GC-MS carbohydrate data. Thus, it appears that C-NMR
and Pyr-GC-MS may probe somewhat different moieties, or that their calibration and
validation need to be developed in considerably more detail. Note, however, that the
absolute concentrations of various functional groups cannot be directly compared
between the two methods, since the integration is done over all organic carbon atoms in
the sample with 13C-NMR, but only over an indeterminate portion of the organic carbon
with Pyr-GC-MS.

CONCLUSIONS

13
Data from C-NMR, Pyr-GC-MS, UV absorbance and fluorescence emission
analyses are correlated in important ways, primarily because all of these analytical
techniques are sensitive to aromatic carbon in NOM molecules. Specifically, SUVA254,
∆ET, and λmax are all correlated with the aromaticity of NOM quantified by either
13
C-NMR or Pyr-GC-MS. As opposed to SUVA254 and ∆ET (which are affected solely by
the aromatic carbon), λmax is related to both the aromaticity and, in some cases, to the
presence of nitrogen-containing species.

13
While the C-NMR aromaticity is thought to be proportional to the total
concentration of aromatic carbon in NOM, the only parameter of the Pyr-GC-MS
analysis that correlates strongly with spectral parameters is the polyhydroxyaromatic
carbon in the sample. The compatibility and interpretation of these two methods should
be investigated in more detail.

All the spectral parameters (SUVA254, ∆ET, λmax) can be used to monitor the
concentration and transformation of aromatic carbon-rich NOM samples in situ without
preconcentration. At present, ∆ET and λmax are not widely used for this purpose, but use of
SUVA254 is relatively extensive. Information from analysis of these parameters is
somewhat overlapping, but also complementary. The values of ∆ET and λmax can be

234
determined directly from spectral analysis, whereas determination of SUVA254 requires
spectral data and analysis for DOC.

The strength of the correlations between spectral parameters and the aromaticity
13
(from C-NMR) and/or PHA concentration (from Pyr-GC-MS) ranges from weak to
moderately strong. For example, the R2 value for the PHA (Pyr/GC-MS data) vs.
SUVA254 was 0.37, while the R2 for 13
C aromatic carbon vs. SUVA254 was 0.72. Given
the wide range of properties of the NOM samples studied, the amount of scatter observed
in these correlations is not surprising. It is possible that, for a series of NOM samples
derived from the same source but subjected to the types of physico-chemical alteration
expected in water treatment processes, the correlations between the structural and
spectral properties of NOM will be substantially stronger.

235
CHAPTER 8 SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

COMPARISON OF NOM CONCENTRATION METHODS

This research assessed the efficiency and practicality with which NOM could be
concentrated and isolated, using both existing and novel methods (RO, NF,
XAD-8/XAD-4, iron-oxide-coated adsorbent media). The concentration methods tested
are compared in Table 8.1.

236
Table 8.1
Comparison of NOM concentration methods
Evaporation RO NF IOCS IOCO XAD-8 XAD-8/
XAD-4

DOC 80-90 87-98 77-96 70-80 20-50 † 45-50 † 60-75


retention (100*)
efficiency,
%
UV254 100* 100 90-100 85-95 20-70 50-60 80-90
retention
efficiency
(%)
Benefits Theoretically High High High No pH Traditionally Improved
complete efficiency, efficiency, efficiency, lack adjustment is used method, efficiency
recovery of high speed somewhat less of irreversible necessary lack of compared with
non-volatile salts compared adsorption accumulation XAD-8, lack of
organic with RO, high of salts accumulation
speed of salts

Problems Very labor- Entrapment/ Entrapment/ Less efficiency Low Laborious Laborious
intensive, adsorption of adsorption of compared with efficiency, preparation preparation and
precipitation organics by organics by RO, loss of low limited pH and cleaning, cleaning,
of salts, membranes, membranes, molecular range, possible hydrophilic irreversible
desalting is loss of low loss of low weight co- adsorption NOM is lost adsorption on
necessary molecular molecular organics, co- of sulfate, XAD-4, loss of
weight weight adsorption of desalting may low molecular
organics, organics, sulfate, be necessary weight
desalting is desalting is desalting may organics
necessary necessary be necessary
* maximum theoretical efficiency
† typical data are shown, performance widely varies; in some waters 80% of DOC may be retained by XAD-8 while in others <35%.

237
The goal of collecting and concentrating NOM from a natural water with 100%
efficiency is impossible to achieve, and the effort required to carry out the separation and
concentration steps increases dramatically as that efficiency is approached. Even for the
most laborious and efficient techniques currently available (e.g., complete evaporation),
significant losses of NOM can occur due to volatilization, precipitation of salts and
experimental errors.

All the methods investigated have both benefits and drawbacks, and none can be
recommended universally for all waters and applications. Evaporation typically requires
substantial investment of time and effort and produces only a very limited amount of
NOM. However, its importance for the future of NOM studies should not be discounted.
With current technology, just a few milligrams of NOM are sufficient to carry out
13
Pyr-GC-MS examination and, as the sensitivity and performance of C-NMR
instrumentation improves, it is possible that a comparable sample size will be adequate to
conduct that analysis as well. Nevertheless, at present, analyses of 13C-NMR, FTIR and
elemental composition typically require much more sample than evaporation is
realistically able to deliver.

RO and NF require much less time and effort than evaporative concentration.
They afford a possibility of treating large volumes of water, and they are relatively easy
to implement. In this research, RO collected NOM more efficiently than any other non-
evaporative concentration method for all the waters studied. RO retained 90 to 95% of
the DOC and virtually 100% of the UV absorbance in most samples. The NOM that was
not collected appeared to include primarily compounds with low molecular and low
aromaticity that typically fractionate as hydrophilic and ultrahydrophilic species.
Currently, no NOM isolation technique except evaporation isolates these fractions
efficiently. NOM collection efficiency by NF was less than that by RO and was
comparable to that using IOCS or an XAD-8/ XAD-4 combination.

In terms of the NOM recovery, which is the product of the retention efficiency
and the efficiency of subsequent elution of NOM, RO outperformed all other processes

238
studied in this research. The NOM recovery efficiency with RO was 20 to 35% better
than NF, IOCS or XAD-8 and XAD-4 resins used in tandem. Due to the high surface area
of RO and NF membranes, a noticeable amount of NOM (typically about 10% of the
load) and inorganic salts may be trapped or adsorbed within the membrane cartridge.
Most NOM retained by RO and NF membranes can be eluted with sodium hydroxide.
However, some cannot, and the adsorption of biopolymers (proteins and amino sugars)
on the membrane may be associated with their fouling. **MB add a sentence or two
about salt problems

IOCS seems to be an efficient adsorbent for NOM concentration in low


mineralized waters, providing NOM recovery efficiencies around 80%. Although DOC
retention on IOCS is comparable to or slightly worse than by NF, the NOM retained by
the IOCS is virtually completely recoverable by elution with sodium hydroxide. The
major problem associated with the use of IOCS is that moderate to high concentrations of
sulfate can compete effectively with NOM for adsorption sites, compromising the NOM
retention efficiency. No such competitive effect comes into play when NOM is
concentrated using RO, NF, or resin adsorption methods, even when high-sulfate source
waters are processed. Also, since sulfate and NOM are co-eluted by sodium hydroxide,
sulfate tends to accumulate in the concentrate from an IOCS process. Therefore, more
effort toward the development of alternative elution techniques and/or of iron oxide
surfaces that are less active toward sulfate is recommended. IOCO may be used in
situations when no chemical alteration of NOM-containing stream is possible since, as
opposed all other adsorption-based method, IOCO adsorbs a substantial amount of NOM
at pH as high as 7.

The efficiency of NOM retention using adsorption onto XAD-8 and XAD-4 resins
in tandem is somewhat lower than using NF. Elution with an acetonitrile/ water mixture
may improve recovery of NOM retained by XAD resins, and this issue needs to be
explored further. The use of XAD resins always involves some NOM fractionation. For
example, hydrophobic and transphilic acids are selectively bound to XAD-8 and XAD-4,
respectively. Together, these fractions typically constitute 60 to 70% of the DOC in
surface waters. Small molecules and most hydrophilic molecules do not seem to be
239
retained by the resins. The major attractive feature of the use of XAD resins is that they
yield NOM concentrates of high purity. Ash is typically <5% in these samples, making
them amenable to almost any analytical and/or characterization methods. However, XAD
resins are less convenient to use than RO or NF, requiring more time and extensive
repetitive washing of the media to remove small particulates (fines), to rinse out residual
monomers and reagents used to conserve the resins during storage.

COMPARISON OF CHEMICAL COMPOSITION OF NOM ISOLATES

Identification of the types of organic compounds collected by the different


isolation processes is important, because inferences about the entire pool of NOM in the
source water are often drawn based on analysis of the material collected. In this study,
comparisons of the types of NOM collected by RO, NF, and adsorption onto XAD were
made predominantly using water from the Blavet (France). (The co-adsorption of sulfate
and NOM on IOCS/IOCO interfered with characterization of the collected material until
appropriate desalting methods could be developed, which occurred late in the project.)
The characterization of the concentrates was based on several major chemical groups
13
identifiable by Pyr-GC-MS and C-NMR. These include polyhydroxy-aromatic (PHA)
carbon and NOM aromaticity, both of which are associated predominantly with humic
species; and three subclasses of biopolymers, namely proteins, amino sugars and
polysaccharides.

RO concentrates are always richer in amino sugars and proteins than samples
concentrated by XAD resins, while the latter are enriched with polysaccharides
(Table 8.2). The smaller percentages of amino acids and proteins recovered in the XAD
samples is a result of their loss during the adsorption and elution procedures. The loss of
polysaccharides from the RO and NF concentrates may occur because of irreversible
sorption of these molecules on the membranes.

240
Table 8.2
Comparison of distribution of biopolymers in RO and XAD-8/XAD-4 samples, based on
UV and Pyr-GC-MS
Site Method PHA PR (%) PS (%) AS (%) SUVA254,
(%) (L/mg-m)
Vienne XAD-8/XAD-4 22 22 17 4 2.5
RO 16 26 7 14 3.5
Blavet winter XAD-8/XAD-4 31 15 20 2 4.4
RO 36 35 7 11 4.0
Blavet summer XAD-8/XAD-4 22 21 21 7 4.1
RO 15 32 10 34 5.2

The higher efficiency of RO and NF with respect to retention of proteins and


amino sugars reduces the relative significance (on a mass basis) of the PHA carbon in
sample. The high efficiency of RO for retention of proteins is independently supported by
13
the high intensity of C-NMR signal in the range 40 to 60 ppm, which is attributed to
C-N structures in the sample. Work conducted on the South Platte and Suwannee River
waters shows that amino acids are also prominent in the basic and neutral NOM
fractions. These fractions are not eluted from XAD resins by sodium hydroxide and
therefore are depleted in the XAD concentrates. These observations help explain the
trends shown in Table 8.2.

Compared with RO and NF, the XAD resins were noticeably less efficient at
retaining small, highly polar molecules (e.g., aliphatic and amino acids and
polysaccharides). RO and NF retained 80 to 90% of the TDAA in the source water,
compared with 70 to 80% for the XAD-8/ XAD-4 tandem.

The NOM fraction eluted from XAD-8 was typically richer in carbon but depleted
in nitrogen and oxygen compared with the XAD-4 samples, supporting the widely
accepted assumption that XAD-8 selectively retains the more hydrophobic molecules
compared to XAD-4. The hydrophobic character of XAD-8 concentrate is also
manifested by its higher SUVA254 and more intense fluorescence, and also by an intense

241
13
signal from aromatic carbon in the C-NMR spectra and a significant proportion of
PHA-associated structures detected by Pyr-GC-MS. The 13C-NMR spectra of the XAD-4
fractions indicate the presence of carboxylic acids, aliphatic alcohols, ethers and esters in
the samples, along with much less aromatic carbon than in the XAD-8 fraction. These
structural factors support the importance of aliphatic carbon in this fraction and its
corresponding hydrophilic character. The XAD-4 fraction isolated from the Blavet River
also indicated that that site was rich in amino sugars.

A general conclusion is that membrane-based NOM isolation methods are


complementary to those that use XAD resins. The membrane isolates are more useful for
evaluating the presence of specific types of biopolymers in the sample, while XAD
isolates may be more useful for examination of aromatic and hydrophobic humic species.
As a result, it is possible that a combination of membrane- and resin-based NOM
isolation methods might be used successfully to detect the presence of certain urban and
other anthropogenic contaminants in natural streams.

DESALTING OF NOM ISOLATES

Evaporation, RO and NF all retain several inorganic species present in the initial
solution in addition to retaining NOM, and both IOCS and IOCO retain sulfate. The
presence of these inorganic salts may make the characterization of NOM concentrates or
isolates difficult. The key such interferences may be summarized as follows:

• The ash content may be as high as 35% in the membrane concentrates and
>50% in IOCS/IOCO isolates, making it impossible to correctly determine the
percentage of oxygen and other elements in NOM.

• In 13C-NMR, inorganic species and most notably carbonates contribute to the


signal between 110 and 160 ppm, decrease the signal-to-noise ratio, and made
the identification of weak bands very difficult.

• Salts decrease the intensity of the peaks associated with the pyrolysis products
in Pyr-GC-MS analysis of the concentrates, although this does not necessarily
242
affect the shape of the pyrochromatograms (Alcaniz et al. 1989**JP need full
reference).

• The analysis for sugars and amino acids can be seriously impeded by salts,
since the effects of acidic hydrolysis of the sample are difficult to evaluate.

• Nitrates and possibly other salts can interfere with measurements of UV


absorbance and fluorescence.

• Silica can affect the state of NOM in the concentrates, possibly leading to
aggregation or precipitation of NOM, or forming unspecified chemical
complexes with the NOM via polymerization reactions.

Thus, desalting of NOM must be carried out in order to carry out a number of
valuable characterization steps. NOM isolates can be efficiently desalted by a
combination of XAD resin adsorption followed by elution with acetonitrile/ water
mixtures and zeotrophic distillation of the non-adsorbable part of NOM with acetic acid.
However, this technique is very laborious and requires substantial skill to carry out. Also,
the extent of possible NOM alteration caused by this and other desalting procedures
needs to be carefully examined. For example, use of H+-saturated cation exchange resins
to remove cations from RO, NF and IOCS/IOCO isolates can lead to the formation of
sulfuric acid as sodium ions are exchanged for H+ ions in sulfate-rich solutions. As the
concentration of this acid increases during the subsequent lyophilization process,
sulfonation of NOM, degradation of sugars and formation of furan derivatives seems to
occur. The extent to which these and other NOM-altering reactions occur as a result of
desalting procedues is not well understood, and more research addressing this topic is
needed.

NOM concentrates can be desalted using XAD resins. However, use of XAD-4
resin for this purpose may lead to loss of highly aromatic NOM fractions due to their
irreversible adsorption on these styrene-divinylbenzene resins. NOM adsorption on
XAD-8 resin, which is an acrylic polymer, is more easily reversed, but this resin retains
less hydrophilic compounds than XAD-4 does, and its use for desalting of membrane

243
isolates therefore compromises the NOM recovery. NOM isolated using RO or NF can be
efficiently desalted using XAD-8/XAD-4 columns in series (operated with a low k′),
followed by elution with an acetonitrile/ water mixture. However, this approach
decreases the final recovery of NOM to levels comparable to that achieved by using
XAD-8 and XAD-4 resins in series without RO. Nevertheless, membrane isolates
desalted in this way seem to retain some ‘signature’ of the membrane concentration step,
such as relatively high concentrations of proteins and amino sugars and lower
carbohydrate concentrations.

VARIABILITY OF NOM

The variability of the chemical composition of NOM, i.e., its site-specificity, was
addressed in this work using a modified hydrophilic-hydrophobic, acid-base-neutral
fractionation approach. The modifications to the procedure developed during this project
were designed to improve the isolation and recovery efficiencies for the hydrophilic and
ultrahydrophilic fractions of NOM. The approach that was adopted relies on a
combination of non-ionic, cationic and anionic exchange resins, followed by various
evaporation and desalting procedures. In the case of the Suwannee and South Platte
Rivers, this method allowed isolation of almost 100% and 65% of the NOM,
respectively. In the latter case, the lower efficiency of NOM isolation was caused by
losses of hydrophilic and ultrahydrophilic NOM when it precipitated with silica during
rotary evaporation.

The steps in the isolation method are time- and labor-intensive. The method
produces a set of NOM fractions whose distribution and intrinsic properties depend on
the nature of NOM in the raw water. Using the new procedure, four major NOM fractions
were quantified. These fractions were designated the hydrophobic (HPO), transphilic
(TPH), hydrophilic (HPI) and ultrahydrophilic (uHPI) fractions. The hydrophobicity of
the fractions changes in the order HPO>TPH>HPI>uHPI. NOM attributed to each
gradation of hydrophobicity can be further divided into acidic, neutral and basic
fractions.
244
The acidic fractions of NOM are characterized by slightly lower C/O ratios than
the basic and neutral fractions, reinforcing the hypothesis that most of the acidic groups
are carboxylic. The C/N and C/H ratios of the neutral fractions are generally lower than
those of the acid fractions, consistent with the greater aliphatic character of the neutral
NOM fractions. The base fractions typically have the highest nitrogen content, consistent
with the expectation that most organic bases are related to amino and amide functionality.
In general, the C/O, C/N, and C/H ratios decrease with increasing hydrophilic character
for both acid and neutral fractions (the isolated ultrahydrophilic acids do not fit this trend
because they were methylated as part of the isolation procedure). Put another way, the
higher the hydrophilic character, the higher the proportions of oxygen, nitrogen and
hydrogen in the NOM fraction, i.e., the more aliphatic it is.

South Platte NOM is derived from both allochthonous inputs in which aromatic
and acid constituents are depleted by mineral solubility controls, and autochthonous
inputs from phytoplankton and bacteria. These autochthonous processes contribute
proteins, polysaccharides, and amino sugars that increase the hydrophilic character of this
NOM. The nitrogen content of the TPHN fraction of the South Platte River is very high,
consistent with the suggestion, based on its FTIR and 13C-NMR spectra, that this fraction
is highly proteinaceous. The Suwannee NOM is predominantly hydrophobic and is
thought to be primarily allochthonous. It is derived from tannins and lignins that give rise
to highly colored humic and fulvic acids of high poly-phenol content and low nitrogen
content. The C/N ratios of the Suwannee River fractions are greater than these ratios in
the corresponding fractions in the South Platte River.

In almost all cases, the various NOM characterization methods gave supporting
and complementary rather than contradictory information. Similarities and differrences
between Suwannee River and South Platte River NOM, based on the results of various
characterization methods, can be summarized as follows.

• DOC Fractionation. Suwannee River NOM is dominated by hydrophobic acids:


hydrophilic and transphilic species contribute much more to the South Platte River
NOM.

245
• Elemental Analyses. The C/N ratios of the Suwannee River fractions are greater than
these ratios in the corresponding fractions in the South Platte River, reflecting the
greater allochthonous character of Suwannee River NOM. C/H, C/N, and C/O ratios
decrease as the hydrophilic character of the NOM fraction increases.

• FTIR Spectra. NOM fractions can be isolated that are free of inorganic constituents
except for minor amounts of silica in certain fractions. Almost all of the NOM
fractions have carboxylic acid peaks, and amide peaks from proteins and n-
acetylamino sugars are especially prominent in certain transphilic and hydrophilic
NOM fractions from the South Platte River.

• 13
C-NMR Spectra. As the hydrophilic character of the NOM fraction increases, the
NMR spectra have smaller aliphatic and aromatic hydrocarbon peaks and larger
carbohydrate and acid or amide peaks. Suwannee River NOM fractions have greater
aromatic carbon and phenol contents than corresponding fractions from the South
Platte River. C-N peaks from amines and amides are evident in base, hydrophilic
neutral, and transphilic neutral fractions, especially in South Platte River NOM, and
the methyl peak from N-acetyl amino sugars is readily detected.

• Dissolved Amino Acids and Carbohydrates. TDCA and TDAA concentrations are
greater in the South Platte River than in the Suwannee River, but they account for
only a small part of the DOC in both waters. TDAA accounts for the majority of
nitrogen in the hydrophobic fractions of both waters, but only accounts for 5-20% of
the nitrogen in the other fractions. Glucose is the dominate sugar in most NOM
fractions. Large ornithine contents distinguish Suwannee River NOM from South
Platte River NOM.

• Pyrolysis Gas Chromatography-Mass Spectrometry. Hydrophobic neutral NOM is


characterized by fatty acid fragments; hydrophobic acid NOM is characterized by
polyhydroxyaromatic fragments typical of humic substances; and transphilic and
hydrophilic NOM fractions are characterized by fragments indicative of
carbohydrates, proteins, and amino sugars. Much of the nitrogen in the transphilic
and hydrophilic fractions that cannot be attributed to TDAAs is present as amino

246
sugars. The transphilic neutral fraction from the South Platte River is very high in
protein content.

• Ultraviolet Spectrometry. Specific UV absorbance (SUVA) varied substantially in


Suwannee River NOM fractions, decreasing in the order HA > HPO-NOM > TPH-
NOM > HPI-NOM. Similar variations were found in South Platte River NOM
fractions, but SUVA was much lower than for corresponding fractions from the
Suwannee River. SUVA was well correlated with the width of the ET spectral band
for NOM fractions from both water samples.

• Fluorescence Spectrometry. For the Suwannee River NOM fractions, fluorescence


emission yields increase and the wavelength of maximum emission shifts to lower
values as the hydrophilic character of the NOM increases. This trend is indicative of a
decrease in the average molecular weight species as hydrophilic character increases.
Fluorescence data for South Platte River NOM fractions indicate generally smaller
molecular weights than for corresponding Suwannee River NOM fractions, but
molecular weights tended to increase as the hydrophilic character of the fraction
increased. The protein-rich fractions from both NOM sources exhibit strong
fluorescence that is blue-shifted compared with that of highly aromatic fractions.

• Coagulability. Acid NOM fractions were better removed by alum at pH 6.5 than
neutral or base fractions. Suwannee River NOM was better removed by coagulation
than South Platte River NOM. Moderate correlations were found between SUVA and
DOC removal, and better correlations were found between SUVA and A254 removal.

• Characterization Correlations. The carbon content of NOM fractions was inversely


correlated with the carbohydrate content (13C-NMR), and oxygen content was
directly correlated with carboxylic acids and/or amides (13C-NMR). The protein
content of NOM (based on pyr-GC-MS analysis) was correlated with TDAA content.
For Suwannee River NOM fractions, aromatic carbon content (13C-NMR) was
directly correlated with SUVA, ∆ET, PHA fragments, and the fluorescence emission
maximum; however, for the South Platte River NOM fraction, aromatic carbon
content correlated only with PHA fragments which indicates the importance of

247
phenolic aromatics (as compared with total aromatic carbon) for ultraviolet and
fluorescence spectral measurements.

SEASONAL CHANGES OF NOM

Seasonal changes of NOM were studied using NOM extracted from the Blavet
River during summer and winter sampling periods. The NOM was more hydrophobic in
the winter than in the summer (hydrophobic and transphilic NOM accounted for 57 and
21% of the DOC, respectively, in the summer and was 79 and 11% in the winter). In
terms of its hydrophobic-transphilic-hydrophilic composition, the Blavet NOM in the
summer was comparable with Suwannee River NOM. The seasonal changes in the
properties of the Blavet NOM were mainly associated with a considerable increase in
TCAA and TDAA in the summer sample. The properties of the humic component of
Blavet NOM were not significantly affected by seasonal processes.

EFFECTS OF NOM ISOLATION AND CONCENTRATION ON DBP


PRECURSORS

NOM chlorination studies showed that the membrane isolates typically formed
less DBPs than the NOM in the raw water did. However, it is possible that this result was
an artifact related to the difficulty of re-dissolving lyophilized membrane isolates. XAD
isolates of NOM behaved very similarly to the NOM in the raw water in this respect.
Experiments with IOCS isolates carried out using the method of differential UV
spectroscopy (published in detail elsewhere) showed that in terms of halogenation these
isolates were virtually identical with the raw water. Therefore, it is concluded that XAD
resins and IOCS are satisfactory media for isolating the majority of the DBP precursors
in natural samples. Nevertheless, it must be acknowledged that aliphatic structures,
whose retention by non-membrane methods is not necessarily efficient, can affect the
formation of halogenated disinfection by-products in some cases.

248
Chlorinated DBP yields were greater in isolated Suwannee River NOM fractions
than in the corresponding fractions from the South Platte River. For Suwannee River
NOM fractions, THM and TCAA yields were similar, but chloroform was the major by-
product from chlorination of South Platte River NOM fractions. It is hypothesized that
the more hydrophilic nature of the South Platte NOM is responsible for this difference.
Proteinaceous NOM fractions (e.g., Suwannee HPIB, South Platte HPIN and TPHN)
yield considerably more DCAA than do other fractions, showing the importance of
nitrogenous units in the formation of this DBP species.

SUVA254 correlated well (and aromatic carbon somewhat less well) with THMFP,
TOXFP, and TCAAFP, but did not correlate with DCAAFP. Some DBP production can
occur by reactions of chlorine with NOM fractions that have negligible SUVA and
aromaticity.

DBP formation potentials of the NOM fractions are not well correlated with their
SUVA254 values. Although DBP yields increase with SUVA254, the correlations are
stronger when the data for each source are considered separately. This seems to indicate
the importance of NOM origin and its intrinsic properties for halogenation.

COMPARISON OF EX SITU AND IN SITU METHODS FOR NOM


CHARACTERIZATION

CPMAS 13C-NMR

13
CPMAS C-NMR is a powerful analytical tool whose utility is well established
in NOM research. Use of this tool to quantify the abundance of dissimilar types of
organic carbon in NOM is well established, and the data obtained using this technique are
valuable in any study attempting to probe the reactivity of NOM. However, its use is
contingent on the availability of dry, preferably desalted NOM samples from which
inorganic carbonates have been removed. The acquisition of high quality spectra requires
substantial time and highly sophisticated and expensive equipment. Continued
249
development of NMR spectrometers is increasing their sensitivity, improving spectral
quantitation, and decreasing spectral acquisition time and cost.

13
While the value of CPMAS C-NMR data is undeniable, the technique might
have significant limitations that affect its precision in estimating the contribution of
aromatic and carbonyl or carboxyl carbon to NOM. Specifically, the contribution of
aromatic carbon may be underestimated by 20-40% (phenolic by 50%), and that of
carbonyl (carboxyl, ester, amide) carbon by 30-50%. The aliphatic carbon may be
overestimated by comparable amounts. Due to these limitations, 13C-NMR data should be
viewed as providing semi-quantitative information about the NOM, much as is the case
for Pyr-GC-MS data. NOM aromaticity values obtained using a 1 ms contact time should
13
be considered as minimum estimates. Increasing the contact time in CPMAS C-NMR
experiments from 1 to 5 ms is expected to improve the precision of the method.
Correlations of SUVA254 and other spectral parameters with the NOM aromaticity
13
evaluated using C-NMR data acquired at a 5 ms contact time may be considerably
better than the corresponding correlations based on contact times of 1 ms.

Fourier Transform IR Spectroscopy (FTIR)

The presence of non-halide inorganic salts and silica (especially important in


XAD-8 samples) may by ascertained by FTIR analysis of NOM isolates in KBr pellets.
Important organic functional group information, especially the distinguishing of acids,
amides, and esters, can be derived from FTIR spectra if interfering inorganic constituents
have been removed. FTIR is presently a qualitative spectrometric method, but it may
become a semi-quantitative method if it is calibrated with standards that are applicable to
NOM composition. Recent developments in spectral software programs can now
deconvolute complex and broad peaks that are typical of NOM FTIR spectra. It might be
possible to apply these peak deconvolution programs to derive semi-quantitative
information for FTIR spectra as was done for UV spectra in this study.

250
Pyrolysis GC-MS

Pyr-GC-MS analysis requires only a few milligrams of dry sample which can be
obtained by lyophilization or rotary evaporation. Salts in the sample do not seem to
interfere with the pyrolysis process and/or analysis of the fragments, but the available
literature on this subject is limited and more studies are needed. Metals might affect the
pyrolysis fragmentation.

13
Similarly to C-NMR, Pyr-GC-MS is a semi-quantitative analytical tool. Data
interpretation yields information about the distribution of molecules belonging to various
biopolymer classes. The interpretation of NOM pyrochromatograms may be more
subjective than that of NMR spectra, since only a portion of the pyrolysis fragments are
generally used for the interpretation. Furthermore, the interpretation is complicated by
the fact that some pyrolysis fragments (e.g., phenol, cresol) may have several origins, and
others can be produced through secondary reactions (e.g., defunctionalization or
cyclization). Previous studies of the NOM “fingerprint” defined by the pyrolysis
fragments have led to the conclusion that proteins and carbohydrates are major
constituents of humic substances. This inference is in conflict with evidence from
spectroscopic data and other analyses for specific constituents in NOM, which suggest
that proteins and carbohydrates are only minor constituents of NOM. Thus, more work is
needed to reconcile these conflicting pieces of data.

Despite the limitations imposed by the relative paucity of Pyr-GC-MS data in the
literature, this technique is already a valuable tool for understanding of NOM. It provides
researchers with a fingerprint of NOM that is distinct from that obtainable by other
techniques. Our results show that each NOM fraction isolated from two source waters
generated a unique pyrochromatogram, with some resemblance between fractions
obtained using the same isolation protocols. In some cases, good correlations were found
between NOM characteristics identified by other analytical techniques and Pyr-GC -MS
chromatograms. Also, some aspects of the pyrochromatograms reinforce the
13
interpretation of C-NMR, FTIR and UV or fluorescence spectra, in particular with
respect to the presence of nitrogenous moieties in NOM.
251
Total Dissolved Amino Acids and Carbohydrates

Total dissolved amino acids and carbohydrates (TDAA and TDCA, respectively)
represent the sum of the monomers analyzed by HPLC after acidic hydrolysis of NOM.
The analyses can be conducted on natural or treated waters and on NOM isolates. State-
of-the-art instrumentation requires only a few milligrams of NOM for TDAA and TDCA
analyses. To ensure adequate accuracy of trace-level analyses in natural waters, an ion
exchange resin column, fluorimetric and pulsed amperometric HPLC detectors and in
most cases dedicated equipment need to be used. The TDAA and TDCA analytical
methods have been validated using known pure biopolymers. However, since the
structure of NOM is not well understood, the reliability of the techniques for analyzing
natural samples, especially with regard to the efficiency of the acidic hydrolysis step, is
uncertain.

Amino acids and carbohydrates comprise a relatively minor fraction of the DOC
of surface waters. They exist as free amino acids and monosaccharides and in bound
forms (as polypeptides or proteins, polysaccharides and/or as monomer units
incorporated into humic substances). The concentrations of free amino acids and
monosaccharides are typically much less than those of the corresponding bound forms.
As a result, polysaccharides and bound (combined) amino acids represent the major
fraction of TDCA and TDAA in natural waters. This research and literature data indicate
that the distributions of monomeric species in TDAA and TDCA are not very site-
specific and/or indicative of the NOM generation processes. This was found to be case
for the Suwannee and South Platte NOM fractions. The only exception is the
concentration of ornithine, which seems to be a good indicator of microbial (**JP algal?)
activity.

In tandem with other methods such as FTIR, TDCA and TDAA data may provide
useful information to support conclusions regarding the molecular structure of selected
NOM fractions. For instance, the neutral NOM fractions were found to be richest in
sugars, while the basic fractions were richest in amino acids. However, because amino
acids and carbohydrates represent a small part of the bulk NOM, and analyses for these
252
components are difficult, the evaluation of TDAA and TDCA should not be a high
priority for structural characterization of NOM. These analyses are certainly more useful
in NOM biodegradability studies since the associated organic compounds are rapidly
assimilated by microorganisms, and amino acids and carbohydrates represent a
significant part of the BDOC fraction of NOM.

Elemental Analysis

Elemental analysis can be reliably conducted only on dry NOM ash-free isolates.
If the ash content of a dry sample is > 5% by mass, significant errors occur in the
evaluation of organic oxygen. Thus, elemental analysis may be a good indicator of the
“purity” of NOM and the efficiency of isolation and desalting protocols.

Elemental analysis does not appear to be a very specific NOM characterization


tool, since the elemental composition of NOM is similar even for highly divergent
fractions (e.g., Suwannee River HPOA and South Platte TPHA). Thus, elemental
analyses are not sensitive enough to characterize or distinguish among NOM samples
from various sources. However this analysis does provide important information such as
elemental ratios that allow NOM fractions isolated from the same source to be compared.
For instance, the C/O ratio is indicative of the concentration of oxygenated functional
groups in the sample, the C/N ratio is indicative of the concentration of nitrogenous
functional groups, and the C/H ratio is indicative of the degree of unsaturation of the
NOM. The C/N ratio also seems to be a good indicator of the extent to which a given
sample of NOM is derived from autochthonous material.

UV Spectroscopy

The research led to several important findings relevant to the use of UV


spectroscopy in NOM-related research and in the water treatment practice. In this report,
only the data of conventional UV spectroscopy are discussed. The fundamentals and

253
applications of differential UV spectroscopy for studying NOM reactions are discussed in
separate publications (Li et al. 1998, Korshin et al. 1997a, 1996).

Of all types of organic carbon in NOM, only the aromatic moiety in NOM has
been unambiguously shown to affect its UV absorbance. SUVA254 is a good indicator of
13
NOM aromaticity quantified by either CPMAS C-NMR or by Pyr-GC-MS. A
noticeable exception from this correlation was found with the Blavet isolates, which have
a high SUVA254 and only moderate aromaticity. The probable reason for this deviation is
13
an underestimate of the aromaticity based on the C-NMR data. It is expected that
13
improvement in the C-NMR data acquisition methods in NOM research will improve
these relationships. An auxiliary hypothesis that nitrogen bases (e.g., purine, pirimidine)
and tryptophane associated with algal activity might affect SUVA254 has been proposed
but has not been experimentally tested.

SUVA254 is not the only indicator of intrinsic NOM properties that can be probed
by UV spectroscopy. An alternative parameter is the width of the composite electron-
transfer band (∆ET), which is correlated to both NOM aromaticity and, probably, its
molecular weight. The manifestations of the latter in UV spectra of NOM need to be
investigated in more detail. Other information that can be derived from UV spectral
analysis of NOM might also be useful. For instance, the A252/A202 ratio might be
indicative of the extent of activation of aromatic units. This and other parameters have
been associated with NOM coagulability, and the corresponding data are discussed
elsewhere (Korshin et al. 1996).

Although UV spectroscopy does not convey information on abundance of specific


types of organic carbon of structural units in NOM, it is a powerful tool for predicting
NOM reactivity and for probing NOM reactions in situ. It is especially efficient in water
with high concentrations of humic species. On the other hand, this analysis is not
sensitive to the presence of some important groups of NOM molecules, such as those that
are responsible for the BDOC. Further studies of the UV spectra of NOM and their
changes in response to various physico-chemical processes used in the potable water
industry can enhance the usefulness of UV absorbance to the drinking water community.

254
Advances in this area might require the use of more sophisticated instrumentation for
collection and analysis of UV (and fluorescence) spectra than that deemed to be adequate
at present.

Fluorescence

NOM fluorescence is an extremely sensitive method that permits investigating


NOM in situ at DOC concentrations < 1 mg/L. The current study supported the widely
accepted hypothesis that the fluorescence emission of NOM is predominantly governed
by aromatic functionality in NOM molecules. However, in several important cases, the
nitrogenous fluorescing species associated with proteins and free amino acids also
contribute noticeably to the emission. In those cases, the emission is blue-shifted
compared with that of predominantly aromatic NOM. Thus, NOM fluorescence may be
useful as an in situ probe to investigate the predominance of aromatic or biopolymeric
species in NOM and therefore may be employed to monitor and predict biological
processes.

It is also clear that the fluorescence emission of aromatic units in NOM molecules
is substantially affected by their molecular weight and conformation. This fact may allow
fluorescence to be used to track the reactions of NOM in water treatment processes or,
alternatively, to probe the origin of NOM and to probe mixing processes in water
distribution systems, if sources with dissimilar NOM are blended. The amount of high
quality data currently available on the relationship between average molecular weight
and/or conformation of NOM and the intensity and shape of emission spectra does not
permit adequate quantification of this relationship. However, these effects are strong, and
they merit further state-of-the-art exploration. There is also no unified mathematical
theory of NOM emission. These and other issues need to be addressed in order for this
method to be used at its full potential in NOM-related research and practice.

255
Relationship Between Data From in Situ and ex Situ Analyses

13
Data from C-NMR, Pyr-GC-MS, UV absorbance and fluorescence emission
analyses (parameterized by SUVA254, ∆ET, and λmax) are correlated, since all of the
analytical tools are sensitive to aromatic structures in NOM. Correlations between
spectral parameters and the 13C-NMR aromaticity and/or PHA concentrations as inferred
from Pyr-GC-MS data are noticeable but not exceedingly strong. Given the wide range of
the properties of the NOM samples and the semi-quantitative nature of the aromaticity or
PHA estimates by 13C-NMR and Pyr-GC-MS, the scatter observed in these correlations is
not surprising. We believe that SUVA254, ∆ET, and λmax might be useful for monitoring
the concentration and transformations of the aromatic moieties of NOM in situ. At
present, ∆ET and λmax are not widely used for this purpose, but use of SUVA254 is
extensive. The use of other proposed spectral parameters may augment the predictive
capabilities of UV spectroscopy.

No functionality other than the aromatic and/or PHA moiety seems to affect the
UV spectrum of NOM. However, the fluorescence emission (quantified by λmax) is
sensitive to both the aromaticity and, in some cases, the presence of nitrogen-containing
species in NOM. The abundance and reactions of fluorescing nitrogenous moieties in
NOM may also potentially be tracked in situ by fluorescence spectroscopy, but
substantially more research is necessary to achieve this goal. The development of
numerical methods to process UV and fluorescence spectra of NOM and improvements
13
in the precision and interpretability of CPMAS C-NMR and Pyr-GC-MS spectra will
enhance the versatility and predictive capacity of both in situ and ex situ methods.

256
APPENDIX

257
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