1. Fluorescence (source) index
ex 370
((McKnight et al., 2001)
Freshness index
ex 310
((Parlanti et al., 2000)
Humification index
ex 254
((Zsholnay et al., 2001)
Table 1. Fluorescence Intensity Fluorescence Indices
Size Particle
Fluorescence
Effects
Peak A Peak C
Humification
Index
Fluorescence
Index
Freshness
Index
Influence of
filtration
Unchanged
percentage 10-20% 15-25% 20-40% 1.5-3%
INFLUENCE OF FILTRATION AND SIZE OF NATURAL ORGANIC MATTER ON
FLUORESCENCE CHARACTERIZATION
Wellemeyer, B., Mladenov, N.
References:
Fellman, Jason Hood, Eran Spencer,Robert G.M. (2010). Fluorescence spectroscopy opens new
windows into dissolved organic matter dynamics in freshwater ecosystems: A review. Limnology and
Oceanography, 55(6), 2452-2462. doi:10.4319/lo.2010.55.6.2452
Natural organic matter (NOM) is a major source of biologically available carbon for
aquatic ecosystems.
• NOM contributes to color and acidity of surface water,
• NOM reacts with sediments and metals,
• NOM also reacts with disinfectants to produce carcinogenic byproducts.
Results
Methods
Summary:
1. Exclusion of large, more complex humic NOM molecules by filtration influenced the fluorescence
intensity, humification index, and freshness index.
 Fluorescence intensity decreases with size fraction
 Humification index decreases with size fraction
 Freshness index increases with size fraction
2. Filtration does not significantly affect fluorescence index in these samples
Recommendation: Filtration effects should be considered when reporting Freshness index and HIX.
Fig. 1. Illustration of FS from
http://scienceprojectideasforkids.com/
Acknowledgments:
Thanks to the KSU Civil Engineering Undergraduate
Research Assistantship for funding this project. Sincere
thanks to my advisor Dr. Natalie Mladenov for her
guidance.
gFig. 5. Representative EEM spectra
from New 3 sites. Amino acid
fluorescence is associated with tyrosine
and tryptophan-like compounds. The
Humic and Fulvic regions contain 2
distinctive Peaks A and C, respectively.
Contact: barretw@ksu.edu
Amino acid
region
Humic
region
▲Fig. 3. Filtration vs various indices
Introduction
Conclusion
Way forward:
• Larger volume samples will be filtered. For each size fraction molecular weight and specific compounds
analyzed with GC-MS or HPLC techniques to explain trends above.
R² = 0.7344
R² = 0.8598
R² = 0.2255
0.5
0.51
0.52
0.53
0.54
0.55
0.56
0.000.501.001.50
FreshnessIndex
Filtration (micrometers)
R² = 0.3331
R² = 0.6098
R² = 0.5831
1.2
1.25
1.3
1.35
1.4
1.45
1.5
1.55
1.6
0.000.501.001.50
FluorescenceIndex
Filtration (micrometers)
R² = 0.1744
R² = 0.8681
R² = 0.9219
1
2
3
4
5
6
7
0.000.501.001.50
HumificationIndex
Filtration (micrometers)
New
3
New
11
Palm
4
Fluorescence spectroscopy (FS) is a technique
that has been shown to rapidly and reliably
characterize NOM, including its source, redox
state, and biological reactivity in natural and
polluted waters. In characterizing dissolved organic
matter with fluorescence, often different research
goals necessitate the use of filters of different
sizes.
Objective: To assess the changes in
fluorescence properties that describe source,
age, and transformation of NOM for different
NOM size fractions.
Fluorescence indices:
nm
nm
I
I
FI
520
470

Filtration. Samples were filtered
sequentially through pre-rinsed 0.8, 0.45,
and 0.2 μm filters. 0.8 μm removes
suspended particles, 0.45 μm is the
smallest dissolved organic matter, and 0.2
μm is sterile removing most of the
bacteria.
Fluorescence analysis.
Horiba Aqualog fluorometer with
simultaneous UV-vis absorbance
acquisition and 1.5 second integration
time. Spectra were inner-filter effect
corrected, raman normalized, and blank
subtracted prior to calculation of
fluorescence indices.
)435420(
380
):(
nmnm
nm
I
I



 nm
nm
nm
nm
I
I
HIX 445
300
480
435
Fig. 2. Unfiltered samples collected from
Okavango River Delta, Botswana
Fig. 3. Filtration setup with syringe and
nylon filter shown
▲ Fig. 4. Similar relationships in all 3
indices for New 3, New 11, and Palm 4
samples.
Peak C
Peak A
Fulvic
region