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to anion attraction in the FA and HA. Thus, as ionization of functional groups such as
hydroxyls, phenolic hydroxyls, and, probably most importantly, carboxyl groups proceeds,
the effect of static quenching with cations likely increases.
Work with amino acids like tryptophan (Trp) has shown that a quenching effect is deter-
mined by accessibility of the fluorophore to quenchers in solution. Trp residues on the
surface of macromolecules (or by extension bound to surfaces of DOM or clays) will be
exposed to the aqueous environment containing the quenchers and thus will be quenched
to a much greater degree than Trp contained within the unexposed interior of the macro-
molecular structure (Lakowicz, 2006 ). For natural DOM, it is most likely the fluorophores
on macromolecular surfaces that are most effectively quenched in aqueous solutions. This
phenomenon has been used to determine molecular conformation by fluorescence (Eftink,
1991 ).
7.3 Effects of Molecular Weight and Fluorophore Size
DOM is operationally defined as organic molecules less than 0.2-0.7 μm in size (equiva-
lently, 200-700 nm). Most fluorescence analyses have thus been measured on DOM because
this size separation is easily achieved via filtration. Colloidal organic matter (COM) is gen-
erally defined as particles from 1 to 1000 nm in size (from measurements of hydrodynamic
radius) and so DOM less than 200 nm and COM may operationally overlap. In relation
to the ultrafiltration methods of separating COM, a 1 nm pore size approximates 1000
Daltons (Da, measurement of molecular weight) cutoff (Benner, 2002 ). Aquatic colloids
have been isolated using cross-flow (or tangential flow) ultrafiltration, producing COM
of sizes greater than 1000 Da (and roughly equivalent to 1 nm spherical diameter; Floge
and Wells, 2007 ), though other size separations are achieved using different ultrafilters
(Mopper et al., 1996 ; Guo and Santchi, 1997 ; Wells, 2002 ), low-ield-low fractionation
(FlFFF; e.g., Zanardi-Lamardo et al., 2002 ; Boehme and Wells, 2006 ), and split-flow thin
cell fractionation (SPLITT; e.g., Lead et al., 2006 ). FlFFF uses a flow field to separate
masses via diffusion (Zanardi-Lamardo et al., 2002 ), whereas SPLITT achieves separation
via gravitational force. Both FlFFF and SPLITT are advantageous over other separation
methods as they do not require ultrafiltration and can perhaps avoid artefacts caused by
the aggregation of colloids, a possible interference in ultrafiltration methods (Lead et al.,
2006 ).
Colloids can be a significant fraction (10-40%) of marine (Benner et al., 1992 ; Wells,
2002 ) and (>65%) of freshwater DOM (Liu et al., 2007 ). However, COM represents only
a fraction of the DOM pool, and making interpretations on total DOM biogeochemical
reactivity using COM fluorescence therefore requires some caution. Mopper et al. ( 1996 )
conducted a comprehensive survey of COM isolated by ultrafiltration (1 kDa filters) from
marine samples and suggested that COM (>1 kDa) accounted for roughly 50% of the ambi-
ent DOM based on fluorescence quantum yields (fluorescence divided by absorption) data.
However, coastal mixing of COM does not appear to be uniform across different regions,
possibly due to varying COM sources. For example, Boyd and Osburn ( 2004 ) examined
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