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JERRY LEENHEER
Dissolved
Characterizing
Aquatic
ORGANIC MATTER
Understanding the
unknown structures is
key to better treatment
of drinking water.
JERRY A. LEENHEER
U.S. GEOLOGICAL SURVEY
JEAN-PHILIPPE CROUÉ
UNIVERSITY OF POITIERS (FRANCE)
© 2003 American Chemical Society JANUARY 1, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY ■ 19 A
where the greatest per- centrations range from
centage of the organic 0.1 milligrams per liter
material has been char- (mg/L) in groundwater
acterized at the com- to 50 mg/L in bogs (2).
pound class level. DOC concentrations not
only depend on the na-
Defining organic matter ture of the watershed,
DOM can range in mol- but are also influenced
ecular weight from a few by seasonal variations
hundred to 100,000 dal- and POC inputs such as
tons (Da), which is in runoff or algae bloom.
the colloidal size range. Most of the NOM is con-
Humic substances have sidered to be refractory
been regarded as macro- to rapid biodegradation.
molecular, but recent Biodegradable organic
studies of aqueous hu- matter (BOM) can be
mic extracts from soil (3), measured based on op-
lignite (4), and water (5, erationally defined pro-
6) found relatively small tocols, such as biodeg-
primary molecular struc-
tures (100–2000 Da) with
DOM is a complex mixture radation over a given
time. BOM is expressed
macromolecular charac- as biodegradable dis-
teristics resulting from
of aromatic and aliphatic solved organic carbon
aggregates formed by hy-
drogen bonding, non-
hydrocarbon structures that (BDOC) or assimilable
organic carbon (AOC)
polar interactions, and that results in biomass
polyvalent cation inter-
have attached functional production, depending
actions. DOM is a com- on the analytical proto-
plex mixture of aromatic
groups. col used. Surveys con-
and aliphatic hydrocar- ducted on rivers in the
bon structures that have United States and France
attached amide, carbo- showed that the BDOC
xyl, hydroxyl, ketone, and various minor functional content ranged from a few to about 40% (8). The
groups. Heterogeneous molecular aggregates in nat- BDOC content of rivers varies with the origin of the
ural waters increase DOM complexity. NOM. Autochthonous NOM, which is produced from
Certain investigators have concluded that humic macrophites, algae, and bacteria, is more biodegrad-
substances cannot be characterized at the molecular able than allochthonous NOM, which has a pedo-
level (7). However, recent findings that show small genic origin. Chemical and biological treatments
primary structures compose the majority of DOM applied to drinking water production significantly in-
raise molecular characterization to a tractable prob- crease or decrease the biodegradable fraction of NOM,
lem. DOM research can be divided into two cate- depending on the treatment (9, 10).
gories: whole water studies, in which DOM is DOM profiling by resin sorbents. DOM is com-
characterized in water and its inorganic constituents, monly characterized by fractionating it into distinct
and studies of DOM fractions isolated from water and categories with resin sorbents. A protocol with
inorganic constituents. XAD-8 resin has been widely used to isolate humic
substances (humic and fulvic acids [11]) and is the
Whole water characterizations basis of a simple DOC analysis that determines the
TOC, DOC, and BOM analyses. Total organic carbon so-called humic/nonhumic distribution (DOC pro-
(TOC) is the most comprehensive measurement used filing) of raw and treated waters. Another fraction-
to quantify the presence of organic matter (OM) in ation approach requires a serial two-column array
aquatic systems. TOC is often synonymous with nat- of resins (12), in which the nonhumic DOC fraction
ural organic matter (NOM) because organic contam- is operationally defined as “transphilic DOC”. Two
inants in natural systems generally represent an small, serial resin columns requiring only a few hun-
insignificant fraction of the TOC. dred milliliters of water have been used to deter-
The first characterization of NOM can be based mine DOC distribution between operational
on the subdivision of TOC into operationally defined categories based on polarity (13, 14). The terms “hy-
fractions such as dissolved organic carbon (DOC) and drophobic”, “hydrophilic”, “acidic”, “basic”, and “neu-
particulate organic carbon (POC). POC is the fraction tral” refer to the predominant property of a DOM
of the TOC that is retained on a 0.45-micrometer (µm) fraction while recognizing that many DOM mole-
porosity membrane. DOC is the organic carbon small- cules are both amphiphilic and amphoteric. A third
er than 0.45 µm in diameter. POC generally repre- anionic exchange resin column can further frac-
sents a minor fraction (below 10%) of the TOC, tionate the hydrophilic DOC into charged and neu-
according to Thurman (2). The proportion of POC in- tral DOC (15). In addition, spectrophotometric
creases with a river’s size and flow rate. DOC con- measurements, such as ultraviolet (UV) absorbance
FIGURE 1
Classifying DOM
Resolving the detailed components of dissolved organic matter (DOM) can be difficult and expensive.
Acid/neutral/
base Acids Neutrals Bases Acids Neutrals Bases 100% $300
Organic and
inorganic Cupric ion <1%
complexes complex
based on their polarity (hydrophobic/hydrophilic), DOC fractionation (39), and, depending on the or-
acid/neutral/base properties, compound-class char- ganic matter concentrations and how much water (20
acteristics, specific compound characteristics, and or 200 L) must be processed, $3000–6000 for a prepar-
compound complex characteristics (37). This frac- ative DOM fractionation with infrared and 13C-NMR
tionation approach was improved into a prepara- spectral characterizations of the fractions. Examples
tive fractionation method in which DOM fractions of DOM fractionations of natural and wastewater
can be quantitatively isolated as desalted, freeze- samples with spectral characterization and fraction
dried preparations and subsequently characterized reactivity studies are shown in the Supporting
(38). This preparative fractionation method led to Information.
the isolation of bacterial cell-wall peptidoglycan col- As indicated in Figure 1, specific compound char-
loids that constitute significant percentages of DOC acterization of natural DOM typically can identify
in most surface water samples. The DOM classifi- only 1–10% of the DOM. Mixture complexity and
cation scheme is presented in Figure 1. molecular complexity have defeated previous at-
These DOM fractions are operationally defined. tempts to resolve natural DOM into discrete com-
For a previously filtered water sample, the hy- ponents with high-resolution chromatography.
drophobic base and neutral fractions are first isolat- However, multistage mass spectrometry coupled with
ed by selective sorption/desorption on Amberlite electrospray ionization is showing promise in giving
XAD-8 resin. Colloids are next isolated by dialysis of molecular-level structural information about DOM
the evaporated concentrate through a 3500-Da mem- components (6).
5 340.90
322.90
354.90
310.90
4
298.90
Intensity × 10 4
3 400.90
412.90
250.90
2
203.00
484.90
1 169.10
626.60
790.40
941.80
0
100 200 300 400 500 600 700 800 900 m/z
1000 222.80
328.90 400
Figure 2 presents multistage mass spectra of tra. Synthesis is a costly process, so the major value of
Suwannee River fulvic acid. Figure 2a is a mixture of multiple mass spectral studies of DOM is in postulat-
singly and multiply charged ions, as well as molec- ing structures that yield additional insights into the na-
ular aggregates and fragments of the original mole- ture and properties of DOM. According to Figure 1, the
cular species. Therefore, it cannot be regarded as a most specific level of DOM characterization is the
molecular-weight distribution. This mixture com- structural determination of its inorganic and organic
plexity in Figure 2a can be resolved into multiple complexes. The nature and measurements of these
mass spectra shown in Figures 2b–d. The spectrum complexes are given in the Supporting Information.
in Figure 2b is still a complex mixture, but further
simplification in Figure 2c suggests a single precur- Research challenges
sor compound can be postulated from the hypo- Extending DOM characterization from the com-
thetical fragmentation pathway shown in Figure 3. pound-class level to the specific compound level
The fragmentation pathway is based on multiple and synthesizing standards are the present research
mass spectrometric studies of model compounds with challenges. Molecular characterization of DOM will
similar structures. Confirming parent DOM structures give specific information about precursors (lignins,
and fragmentation pathways depends on synthesiz- tannins, terpenoids, proteins, amino sugars), the
ing the parent structure for confirmatory mass spec- processes that produce DOM (biodegradation, con-
Fragmentation pathway
This scheme describes a hypothetical parent compound for dissolved organic matter based on mass spectrometric
results from Figure 2. Using mass spectral results is much less expensive than relying on synthesis. Molecular
masses are in parentheses.
OH OH O O O O
–
COOH – –
OOC OOC O OOC O O
O
HOOC COOH HOOC HOOC
–H2O O –H2O O –H2O O
O
OH OH OH O–
OH OH OH OH
–3CO2
(329) (311) (293) (275)
–2CO2 CO
O –2CO2
CO 2C
C
O
COO–
O O–
O– OH
OH OH (109)
(205) O
(165)
densation reactions, photolysis), and reactive struc- structural information that will result in a better un-
tures in DOM (disinfection byproduct [DBP] pre- derstanding of NOM structure and reactivity.
cursors, metal-binding sites, surfactant structures). These are a few of the research challenges; there
Anthropogenic components of DOM, such as poly- are undoubtedly more. DOM research is rapidly ad-
carboxylic acid metabolites of various surfactants in vancing with many exciting and useful applications.
wastewater, can also be distinguished from NOM at As the need for water resources and recycling con-
the molecular level. tinues to increase, so will the need for understand-
The costs associated with the compound-class ing the nature and properties of DOM.
characterizations presented in Figure 2 are too ex-
pensive for DOM monitoring, but continued advances Jerry A. Leenheer is a hydrologist at the U.S. Geological
in pyrolysis/GC/MS and thermochemolysis/GC/MS Survey in Denver, Colo. Jean-Philippe Croué is a re-
have the potential for inexpensive characterization. search scientist in the Laboratory of Water and
To be relevant and comprehensive, these techniques Pollution Chemistry at the University of Poitiers in
need to be calibrated with model compounds that Poitiers, France. Address correspondence to Leenheer at
are closely related to DOM structures. Thus, the in- leenheer@usgs.gov.
formation from expensive DOM fractionations with
spectral characterizations transfers to calibration and References
interpretations of inexpensive methods. (1) Stevenson, F. J. Humus Chemistry, 2nd ed.; Wiley & Sons:
New York, 1994.
Understanding the structural chemistry of these hy- (2) Thurman, E. M. Organic Geochemistry of Natural Waters;
drophilic DOM components, and especially the dis- M. Nijhoff and W. Junk Publishers: Dordrecht, the
solved organic nitrogen (DON), can be useful in Netherlands, 1985.
designing new water treatment processes to remove (3) Simpson, A. J.; et al. Naturwissenschaften 2002, 89, 84–88.
(4) Piccolo, A.; Conte, P.; Trivellone, E.; Van Lagen, B.;
these components from drinking water. It has been
Buurman, P. Environ. Sci. Technol. 2002, 36, 76–84.
generally accepted that hydrophobic DOM represents (5) Leenheer, J. A.; Brown, P. A.; Noyes, T. I. In Aquatic Humic
the major source of DBP precursor sites, but certain hy- Substances, Influence on Fate and Treatment of Pollutants;
drophilic DOM components not removed by conven- Suffet, I. H., MacCarthy, P., Eds.; Advances in Chemistry
Series 219; American Chemical Society: Washington, DC,
tional water treatment have been found to react with 1989, pp 25–39.
disinfecting agents to produce undesirable DBPs (40). (6) Leenheer, J. A.; Rostad, C. E.; Gates, P. M.; Furlong, E. T.;
There is some evidence that the nitrogen-rich con- Ferrer, I. Anal. Chem. 2001, 73, 2461–2471.
stituents (e.g., proteinaceous-type structures) repre- (7) MacCarthy, P. The Principles of Humic Substances: An
Introduction to the First Principle. In Humic Substances,
sent an important class of the problematic hydrophilic
Structures, Models, and Functions; Ghabbour, E. A., Davies,
NOM fraction. Macromolecular polysaccharide com- G., Eds. Royal Society of Chemistry: Cambridge, U.K.,
ponents of DOM also clog membranes used in water 2001, pp 19–30.
treatment. HPLC/SEC technology with multiple de- (8) Kaplan, L. A.; Reasoner, D. J.; Rice, E. W. J.—Am. Water
Works Assoc. 1994, 2, 121–132.
tectors (UV, fluorescence, DOC, and DON) may be used
(9) Malley, J. P.; Eighmy, T. T.; Collins, M. R.; Royce, J. A.;
in combination with advanced characterization tools Morgan, D. F. J.—Am.Water Works Assoc., 1993, 75 (12),
(electrospray/MS, pyrolysis GC/MS) to obtain detailed 47–57.