1. Measuring the Binding of Carbamazepine to Aquatic Humic Substances by Fluorescence
Spectrophotometry
Daniel Cairnie, Department of Chemistry and Biochemistry, George Mason University
Introduction
Listed by the World Health Organization (WHO) as an essential medicine [1],
carbamazepine (CBZ) has been widely used since its synthesis in 1953 to treat epilepsy and
neuropathic pain [2]. However, as time progressed along with research, CBZ has been detected
in wastewater effluent and freshwater systems, with some concentrations as high as micrograms
per liter [3,4]. CBZ is unique among many pharmaceutical and personal care products (PPCPs)
detected in the aquatic environment in that it is unreactive through the wastewater treatment
process and is efficiently transmitted through wastewater treatment plants leading to its
widespread detection in rivers and streams. The presence of carbamazepine, along with other
PPCPs, in the aquatic environment can be largely attributed to their inherent physiochemical
properties and interactions involving aquatic humic substances (AHS). AHS are an important
reservoir of natural organic carbon in the aquatic environment and are critically linked to the
transport and fate of PPCPs in aquatic systems. CBZ is composed of a dibenzoazepine ring
system with a carboxamide functional group attached to the central nitrogen atom of the system.
Its structure provides many potential mechanisms of binding to AHS.
Since CBZ has both polar (carboxamide) and non-polar (dibenzoazepine) structural
components, it is useful for environmental chemists to measure its distribution ratio, known as
Kd [5], between AHS and the amount of unbound carbamazepine in solution (i.e., dissolved in
water). More specifically, my primary interest is to evaluate the equilibrium distribution constant
(Kd) for CBZ in AHS in laboratory studies [6,7]. Shown below is a mathematical representation
of Kd:
𝐴𝐻𝑆 + 𝐶𝐵𝑍 → [ 𝐴𝐻𝑆 − 𝐶𝐵𝑍] ( 𝐸𝑞. 1)
𝐾𝑑 =
[ 𝐴𝐻𝑆 − 𝐶𝐵𝑍]
[ 𝐴𝐻𝑆] [ 𝐶𝐵𝑍]
( 𝐸𝑞. 2)
where [AHS-CBZ] is the equilibrium concentration (micrograms per kilogram) of bound CBZ,
[AHS] is the equilibrium concentration (milligrams per liter) of free or unbound AHS in water,
and [CBZ] is the equilibrium concentration (micrograms per liter) of free CBZ in water [6]. The
Kd is a critical parameter needed to assess and model the distribution of chemicals in the aquatic
environment, and Kd is best evaluated through measurement.
AHS are colloidal-sized particles (i.e., <1 micrometer in spherical diameter) dispersed in
surface waters, and are extremely difficult to measure directly. For example, AHS cannot be
filtered from water to isolate them for further study. Thus, I will employ a spectrophotometric
method to evaluate the binding of CBZ to AHS, which does not require the AHS phase to be
isolated for Kd measurement. Fluorescent spectrophotometry has recently been shown to be an
effective analytical tool for measuring the binding of small molecules such as CBZ to colloidal
particles [6]. My study hypothesis is that the Kd of CBZ can be quantitatively evaluated by using
fluorescent spectrophotometry. Fluorescent spectrophotometry acts by measuring the quenching
of fluorescence promoted by the binding of CBZ to AHS. AHS are fluorescent-active substances
and can be easily measured with a fluorometer [6,7].

2. Process
To measure the Kd of CBZ in AHS, a Model RF-6000 Shimadzu Spectrofluorometer will
be employed, an instrument recently purchased by the Mason Chemistry & Biochemistry
Department. The instrument is located in Planetary Hall room 402A adjacent the Foster research
laboratory (room 406). AHS will be obtained from the International Humic Substances Society
in the form of Mississippi River Aquatic Humic Substances, a commercial source of AHS that is
available for research study.
Initial experiments will be conducted with Mississippi River AHS (MRAHS) at a
concentration of 10 milligrams per liter in distilled water to optimize the excitation and emission
wavelengths of MRAHS in the spectrofluorometer. A calibration curve will be developed to
measure a dilution curve of MRAHS in water for quantitative assessment of MRAHS
concentrations. Subsequently, CBZ at a total concentration of 10 micrograms per liter will be
added to varying MRAHS concentrations, ranging from 1 to 100 milligrams per liter initially,
and later optimized for the Kd determination. Fluorescence data (wavelength versus emission
intensity) from the RF-6000 will be downloaded and evaluated mathematically by parallel factor
analysis [6] using Matlab software (available through the Mason mainframe) in conjunction with
a DOMFluor subroutine obtained from Stedman and Bro. [8]. The Kdmay be evaluated via the
following relation [6]:
𝐼 = 𝐼0 + ( 𝐼 𝑀𝐿 − 𝐼0)(
1
2𝐾𝑑 𝐶 𝐿
) (1 + 𝐾𝑑 𝐶 𝐿 + 𝐾𝑑 𝐶 𝑀 − [(1 + 𝐾𝑑 𝐶 𝐿 + 𝐾𝑑 𝐶 𝑀)2
− 4𝐾𝑑
2
𝐶 𝐿 𝐶 𝑀]
1
2⁄
)( 𝐸𝑞.3)
I = Fluorescent intensity at the CBZ concentration CM
I0 = Fluorescent intensity without adding CBZ
IML = Limiting value below which the fluorescent intensity does not change due to CBZ addition
Kd = Distribution coefficient
CL = Total ligand concentration
CM = Carbamazepine Concentration
The Kd values obtained from my experiments will be repeated in triplicate to determine the
associated uncertainties in the spectrophotometric method. Three different total-CBZ
concentrations will be tested to evaluate the sorbate concentration effect on the Kd estimate.
Timeline
January–February 2017
- Procure needed materials, prepare control solutions. CBZ and MRAHS
- Conduct preliminary assays to verify actual concentration of control solutions
- Conduct initial experiments on control solutions to optimize FS excitation and emission
values

3. February–March 2017
- Conduct further experiments with progressive CBZ additions to control solutions
- Conduct further experimentation to optimize Kd
- Data analysis and computations using Matlab software and DOMFluor subroutine
April-May 2017
- Prepare project report, submit draft for peer review, and finalize
- Present final report and oral presentation of research
Expected Outcomes
The purpose of my research project is to develop a spectrophotometric fluorescence
analytical method for measuring the binding of pharmaceutical chemicals in natural organic
matter, specifically aquatic humic substances found in surface waters. The method will be used
to quantitatively assess the AHS-water distribution constant (Kd) for carbamazepine in colloidal
organic matter. The quantitative determination of the binding of pollutant chemicals to colloid-
sized particles is currently of great concern in the environmental chemistry community because
little is known on the subject due to the difficulty of accurately measuring binding constants. My
hope is to provide a clear picture of the role colloids have on the fate and transport of organic
pollutants in the aquatic environment.
Budget
Supplies and/or Materials: $1,000 of the budget will be spent mostly on attaining IHSS
aquatic humic substances, cuvettes, weigh boats, sample vials for binding experiments, and
additional supplies needed for spectrophotometric operation. The remaining $500 will be kept in
reserve for any additional software needed to perform parallel factor analysis computation on the
fluorescence data; otherwise these funds will be used for the above consumable supplies as
needed.

4. References
[1] World Health Organization. “World Health Organization List of Essential Medicines.” WHO
International. Accessed November 3, 2016.
http://apps.who.int/iris/bitstream/10665/93142/1/EML_18_eng.pdf?ua=1.
[2] “DailyMed - CARBAMAZEPINE- Carbamazepine Capsule, Extended Release.” Accessed
November 3, 2016. https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=7a1e523a-b377-
43dc-b231-7591c4c888ea.
[3] Brown, DelShawn, Daniel Snow, George A. Hunt, and Shannon L. Bartelt-Hunt. “Persistence
of Pharmaceuticals in Effluent-Dominated Surface Waters.” Journal of Environment Quality 44,
no. 1 (2015): 299. doi:10.2134/jeq2014.08.0334.
[4] Feitosa-Felizzola, J., and S. Chiron. “Occurrence and Distribution of Selected Antibiotics in a
Small Mediterranean Stream (Arc River, Southern France).” Journal of Hydrology 364, no. 1–2
(January 15, 2009): 50–57. doi:10.1016/j.jhydrol.2008.10.006.
[5] Tinsley, Ian. Chemical Concepts in Pollutant Behavior. 2nd ed. Oregon State University: A
John Wiley & Sons, Inc., 2004.
[6] Wang, Ying, Manman Zhang, Jun Fu, Tingting Li, Jinggang Wang, and Yingyu Fu. “Insights
into the Interaction between Carbamazepine and Natural Dissolved Organic Matter in the
Yangtze Estuary Using Fluorescence Excitation-Emission Matrix Spectra Coupled with Parallel
Factor Analysis.” Environmental Science and Pollution Research International 23, no. 19
(October 2016): 19887–96. doi:10.1007/s11356-016-7203-2.
[7] Tsuda, Kumiko, Morimaru Kida, Suzuka Aso, Taku Kato, Nobuhide Fujitake, Masahiro
Maruo, Kazuhide Hayakawa, and Mitsuru Hirota. “Determination of Aquatic Humic Substances
in Japanese Lakes and Wetlands by the Carbon Concentration-Based Resin Isolation Technique.”
Limnology 17, no. 1 (May 8, 2015): 1–6. doi:10.1007/s10201-015-0455-6.
[8] Stedmon, Colin A., and Rasmus Bro. “Characterizing Dissolved Organic Matter
Fluorescence with Parallel Factor Analysis: A Tutorial.” Limnology and Oceanography:
Methods 6, no. 11 (November 1, 2008): 572–79. doi:10.4319/lom.2008.6.572.