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were measured at increasing concentrations and the HIX EM was found to increase with
concentration in all three samples. This was due to the greater absorption, and thus greater
sensitivity to concentration effects, in the 300-345 nm region. Ohno ( 2002a ) made two
recommendations for avoiding the inner filter effect in this situation. The first was to dilute
all solutions to a fixed absorbance (<0.3 cm -1 ) and the second was to calculate the HIX EM
as a ratio of the area of the fluorescence intensity under 435-480 nm divided by the sum
of the areas under 300-345 nm and 435-480 nm). Zsolnay ( 2002 ) commented that the
concentration error in HIX EM would be on the order of only 10% in dilute samples and
Ohno ( 2002b ) further emphasized that reducing concentration to limit the inner-filter effect
allows a more accurate measure of DOM quality. Because HIX EM is a ratio of two regions
that are not equally affected by concentration, it should be used with caution, and concen-
tration and inner-filter effects could have a large impact if not corrected. The original cal-
culation proposed by Zsolnay et al. ( 1999 ) is most commonly used in the literature, and can
be accurately used to compare samples. This is provided they are diluted to an absorbance
no greater than 0.3 cm -1 at 254 nm to minimize inner-filter effects due to concentration and
allow for inner-filter corrections to be effective (Ohno, 2002a ).
9.2.3 Freshness Index to Identify Microbial Material in Marine
DOM (the “β/α” and “BIX” Index)
Parlanti et al. ( 2000 ) reported fluorescent components and EEMs in filtered water samples
collected from marine and freshwater end members in a coastal shelf environment. Peaks
C, A, M, B, and T of Coble (1996) were renamed in Parlanti et al. ( 2000 ) where peak C
is α , peak M is β , and peak B is γ (see Table 9.2 ). The α peak (max intensity within ex
330-350 nm, em 420-480 nm) is associated with humic-like components, the β peak (max
intensity within ex 310-320 nm, em 380-420 nm) with marine humic-like components,
and the γ peak with tyrosine-like, protein-like components. Observations of the β peak co-
occurring with protein-like peaks supported the hypothesis of it originating from biological
activity ( Figure 9.5 ). The ratios of the fluorescence intensity of these three peaks were
reported for both the marine and freshwater surface samples, and for a laboratory degrad-
ation experiment with blue-green algae. Parlanti et al. ( 2000 ) demonstrated that peaks γ and
β both originate from recent biological activity and could be used as markers of biological
activity in coastal zones. They found the ratio of these peaks to peak α (humic-like) pro-
vided an index of biologically produced to terrestrially derived fluorescent organic matter
in coastal environments. They named this β / α ratio the freshness index, as it was indicative
of the amount of organic matter recently produced from biological activity.
Huguet et al. ( 2009 ) further developed the β / α ratio, renamed BIX. This index accounts
for the presence of the biological activity- based β peak causing a broadening of the humic
portion of the emission spectrum. BIX is the ratio of the emitted fluorescence intensity at
380 nm (max of the β band) to the emitted fluorescence intensity at 430 nm (max of the α
band) when excited at 310 nm. This is essentially a single line scan version of the β / α ratio,
where an increase in BIX is related to an increase in the intensity of fluorophore β . Huguet
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