Calcein Research Thesis- Anthony Wright

The Effect of Halide Ion
Concentration on Fluorescence
of the Calcein-Calcium Complex:
Fluorescence Quenching by the
Halide Ion
Anthony Wright, Sean Carrigan
Department of Natural Sciences;Piedmont College;
165 Central Avenue, Demorest Ga 30535
Awright0722@lions.piedmont.edu
ABSTRACT
Image 1
Abstract. Calcein is widely used to quantitatively measure Ca2+ concentrations in both chemistry and biological
systems. A beneficial characteristic of calcein is the ability to form a calcein-calcium complex with fluorescent
properties. In previous studies, halide ions’ effect on fluorescence quenching was investigated. In this study, the
halide ions bromide and iodide were investigated to determine the effects on the calcein-calcium complex. Each trial
utilized varying concentrations of halide ions and analyzed the corresponding effect on the calcein-calcium complex.
The Perkin-Elmer LS-50B luminescence spectrophotometer was used to measure each solution’s fluorescence
intensity. Using the intensity data, Stern-Volmer plots were prepared for each set of solutions. The average slope of
Br-
was 1.1860. The average slope of I-
was 4.4734. ANOVA, an analysis of variance statistical test, was used to
analyze the data. With a P-value of <0.0001, the results showed a significant difference between the quenching
effects of Br-
and I-
. The data indicated that fluorescence quenching by I-
was significantly greater than quenching by
Br-
. These results were in agreement with the ∆E, ionization energy, radii, and oxidation potential of the oxidation
reactions of these two ions. These results suggest that halide ions' interference of calcein fluorescence may affect
quantitative measurements of Ca2+
.
Introduction.
The purpose of this study was to test the quenching
effect of bromide, Br-, and iodide, I-, on the fluorescence of
the calcein-calcium complex and to determine which halide
ion was the more effective quencher.Fluorescence
spectroscopy is a widely used and sensitive analytical
method.1 Fluorescence quenching,in which the emission
intensity of a fluorescent compound is reduced, has been
reported for a wide variety of chemical species, including
halide and pseudohalide ions.2 Fluorescence occurs when a
ground state electron absorbs energy in the form of light,
hv, and becomes excited. Then, when the electron relaxes,
energy in the form of a photon,is released. When
quenching occurs,the quencherdonates an electron to the
ground state orbital and accepts an electron from the

excited state orbital; thereby, preventing electron relaxation
and the release of energy in the form of a photon.2
Fluorescence spectroscopy is used to investigate
problems in chemical and biological sciences by
determining the distance between molecule sites,
conformational changes,and binding interactions. It is also
used to study interactions of solvent molecules with
fluorophores, cellular imaging, and single molecule
detection. Utilizing fluorescence spectroscopy,calcein
substrate is used to measure the concentration of calcium in
joint fluid, urine, plasma, and milk. Calcium is used as a
tracer to determine distances between sites on molecules,
conformational changes,and binding interactions. Calcein
is the substrate used to detect the calcium during these
processes.3-7
Image 1 is the chemical structure of calcein. When the
carboxyl groups are deprotonated,calcein will bind to one
calcium ion much like the chelating effects of EDTA. All
four deprotonated carboxyl groups bind to the calcium ion,
bending the calcein molecule around it. Because the calcein
molecule must be deprotonated,the solution must be
alkaline to remove the hydrogen atoms. This calcein-
calcium complex is the fluorescing compound studied in
this experiment. The excitation reaction for the calcein-
calcium complex is shown below, where S represents the
calcein-calcium complex, hv represents energy in the form
of light, and S* represents the excited calcein-calcium
complex.
S + hv  S* [Reaction 1]
There are three ways for S* to relax.10 Radiative
relaxation is shown below. This is simply the emission of
energy in the form of light, a photon, hv.
S*  S + hv [Reaction 2]
Next is the non-radiative relaxation, or quenching where
Q is the quenching molecule.
S* + Q  S-Q* [Reaction 3]
The final relaxation reaction is collisional relaxation,
where the excited calcein-calcium complex relaxes and
releases the energy in the form of heat instead of a photon.
S*  S + heat [Reaction 4]
Halide ions have been shown to be quenchers of the
antimalarial drug quinine, coumarin fluorescence is
quenched by Br- and I- but not by Cl-, and 5,6-
benzoquinoline fluorescence is quenched by Cl-, Br-, I-, and
SCN-.8, 9, 1 Awareness of chemical agents capable of
hindering the studies and processes involved in the calcein-
calcium complex is important for accuracy and error
prevention. If quenching molecules capable of affecting the
fluorescence of the calcein-calcium complex are present,
fluorescence will decrease and measurements of calcium
and othercalcein-calcium complex processes will be
hindered.
In this study,the quenching halide ions Br- and I- were
investigated.The ions’ quenching effects on the calcein-
calcium complex were studied using a spectrophotometer.
The raw fluorescence data obtained was used to plot I0/I
versus [Q], utilizing the Stern-Volmer equation,as shown
below.6
I0/I = (kq / ke + kd)[Q] + 1 [Equation 1]
Iₒ is the fluorescence intensity measured when there is no
quencherpresent,I is the intensity measured with
quencher. kq, the quenching constant,ke, the emission
constant,and kd, the deactivation constant,are all constants
and can be written as kꞌ, shown in the equation below.12
I0/I = kꞌ[Q] + 1 [Equation 2]
The plot of I0/I versus [Q] produces a linear plot. When
[Q] equals 0 (no quencher present),I0/I equals 1. As the
[Q] increases from one solution to the next, the data points
of I0/I form a positive slope that equals kꞌ. This is shown in
the example below.
In this study,it is hypothesized that the quenching of the
calcein-calcium complex by I- is significantly different than
fluorescence quenching by Br-. The null hypothesis is that
the quenching of the calcein-calcium complex by I- is not
significantly different than fluorescence quenching by Br-.
Iodide is predicted to be the more effective quenchersince
I- is a strongerreducing agent.1
Methods.
Quartz cuvettes and class A volumetric flasks were used
for all solutions.A Perkin-Elmer LS-50B luminescence
spectrophotometerwith an excitation wavelength of 495
nm and an emission wavelength of 511 nm was utilized.
Reagents used included: calcein (a fluorescent dye used for
the detection of calcium in alkaline solution), calcium
(found abundantly in nature), KOH (the basic solution
needed to form the calcein-calcium complex), and NaBr
and NaI (the two quenching halide ions). The reagents were
used to make the following stocksolutions in 500 mL
volumetric flasks: 6.26x10-5 M calcein, 40.0 ppm calcium
(from CaCO3 dissolved in solution by nitric acid, HNO3),
1.00 M KOH, 0.100 M NaBr, and 0.100 M NaI. These
stocksolutions were used for all dilutions.
Seven 50 mL volumetric flasks were obtained and
labeled 1-7. In each volumetric flask, 5.0 mL calcein, 2.0
mL calcium, and 10.0 mL KOH were added. Next, the
halide solution was added. In the first three flasks, 0.0 mL
of halide, 2.0 mL of halide and 4.0 mL of halide were
added, respectively. This pattern continued throughout the
seven flasks with 12.0 mL of halide added to the seventh
flask. Deionized water was then added to all seven flasks to
dilute each solution to the mark, each solution was corked,
and mixed well.
To record the intensity of each solution,starting with the

solution containing 0.0 mL of halide, the solution was
pipetted into the quartz cuvette and placed into the Perkin-
Elmer LS-50B luminescence spectrophotometer.The
intensity of each solution was measured and recorded.
Three sets of solutions were made for a total of three runs
and three graphs,using the Stern-Volmer Equation 2, for
each halide, Br- and I-.
An ANOVA statistical analysis was applied to the
intensity measurement raw data and a P-value was
obtained. The P-value was used to determine if a
significant difference existed between the quenching
effects of Br- and I-. Additionally, raw data slopes were
graphed and compared using the ANOVA analysis.
The slope was obtained from each Stern-Volmer plot
and used to compare the mean of slopes for Br- and I-. The
standard deviation and 95% confidence interval were also
determined. This was used to determine which halide ion
was the more effective quencher.
Results.
The following six graphs were obtained using the Stern-
Volmer Equation 2.

Below is a table showing the summary of the slopes
obtained from the Graphs 1-6. The slopes,mean of the
three slopes for Br- and I-, along with the standard
deviations and 95% confidence interval are included in the
table.
Summary of Graphs
Iodide Bromide
Slope: Run 1 4.8478 1.0332
Slope: Run 2 4.5661 1.3104
Slope: Run 3 4.0064 1.1243
Mean of Slopes 4.4734 1.1860
Standard Deviation 0.4283 0.1408
95% Confidence Interval ± 3.4094 - 5.5374 0.8363 - 1.5357
Graph 7 shows the raw data for Intensity for Br- and I-
versus the mL of halide.
From the ANOVA statistical analysis, a P-value of
<0.0001 was obtained.
Discussion.
An accepted P-value of <0.05 indicates significant
difference at the 95% confidence interval. The P-value of
<0.0001, obtained from the raw data ANOVA analysis,
indicates a significant difference between the quenching
effects of Br- and I-.
Graphs 1-6 are notably linear with R2 values of 0.9504-
0.9988. Additionally, all graphs display positive slopes.
Graphs 2 and 4 contain data points suggestive of
experimental procedure errors. In Graph 2, data points 3
and 4 appearequal, possibly indicating that the third
solution’s intensity was measured twice and recorded for
both solutions 3 and 4. In Graph 4, the solutions used for
data points 2 and 3 appear switched.
From the Summary of Graphs table, the mean of slopes
for Br- equals 1.1243 and for I- equals 4.0064. The 95%
confidence interval ± for Br- equals 0.8363 - 1.5357 and for
I- equals 3.4094 - 5.5374. These do not overlap; thereby,
demonstrating that the slopes are significantly different. In
addition, the slopes for I- clearly demonstrate that I- is the
more effective quencher. As the concentration of halide
ion increased, fluorescence decreased more in solutions
containing I- than in solutions containing Br-.
Graph 7 shows the slopes of raw intensity data versus
the mL of halide for both I- and
Br-. Graph 7 provides a visual display of the quenching
effect differences between the two halide ions. The I- slope
is more negative; thereby, indicating that I- is the more
effective quencher. Ideally, the graph would plot the same
intensity for Br- and I- as the first data point (0.0 mL of
halide). However, that did not occur in this study because
the calcein solution slowly underwent photodegradation.
All three runs for I- occurred one day while the three runs
for Br- occurred a few days later. During that time interval,
the calcein solution underwent photodegradation.The
photodegradation accounts forthe Br- and I- starting
intensity differences. However, the starting intensity
differences do not affect the overall effects of the halides’
quenching.For future studies,all intensity measurements
should be obtained on the same day or one run of each
halide ion should occur on the same day.
Oxidation Reaction and Important Values
Oxidation
Reaction
∆E (kJ/
mol)
Ionization
Energy
(kJ/mol)
Radius
of Ion
(pm)
Oxidation
Potential
(V)
Br-  Br + e-
325 1140 114 -1.07
I-  I + e-
295 1008 206 -0.54
The table above shows the oxidation reactions for
Br- and I- and important values involved with the oxidation
of each halide ion. The lower the ∆E, the less energy is
required for the oxidation reaction to occur.13 The
ionization energy is the energy required to remove an
electron from a neutral atom.13 The more positive the
oxidation potential, the more energetically favorable the
reaction.14 The ∆E and ionization energy for I- are lower.
The oxidation potential for I- is more positive. These results
are supported by structure and radii difference between I-
and Br-. I- has the larger radius; consequently,the
outermost electrons receive less proton pull and are more
easily removed. Therefore, when compared to Br-, I- is
more likely to donate an electron, making I- a better
quencher.
The study’s hypothesis is accepted and the null
hypothesis is rejected. The calcein-calcium complex
quenching by I- is significantly different than fluorescence
quenching by Br-. As predicted, I- is the more effective
quencher. The conclusions are clearly supported by the
Summary of Slopes table and Graph 7. Additionally, the
conclusions are further supported by the Oxidation
Reaction and Important Values table.
For future studies,the application of the modified Stern-

Volmer equation can be used.This modified equation is
valid because the quenchermust be within a certain space
and not all quenchers will be available in this space.This
space is called the active sphere around the fluorescent
group. When the group is within the active sphere,
instantaneous quenching occurs.The probability that the
quencheris not in the sphere equals e-V[Q], where V is the
volume of the active sphere.I0 is reduced to I0e-V[Q] and I0e-
V[Q] / I = kq[Q] +1. The modified form allows the rate
constant for quenching,kq, and the lifetime of the excited
state,, to be determined from the slope.1 Future studies
could continue this investigation using other known
quenchers,such as SCN-, O2, and Cl-. Future studies
should endeavorto obtain paired sets of halide
measurements in order to minimize the photodegradation
effects of calcein. In addition, the calcein solution could be
wrapped in aluminum foil to prevent exposure to light.
References.
1. Carrigan, S.; Doucette, S.; Jones, C.; Marzzacco, C. J.; Halpern, A. M. The fluorescence quenching of 5, 6-
benzoquinoline and its conjugate acid by Cl-, Br-, SCN-, and I- ions. Journal of Photochemistry and Photobiology. A:
Chemistry. 1996, 99, 29-35.
2. Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: Baltimore, 2007; pp 277.
3. Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: Baltimore, 2007; pp 1-3
4. Yavorskyy, A.; Hernandez-Santana, A.; McCarthy, G.; McMahon, G. Detection of calciumphosphate crystals in the
joint fluid of patients with osteoarthritis – analytical approaches and challenges. Analyst , [Online] 2008, 133, 302-318.
http://pubs.rsc.org/en/content/articlehtml/2008/an/b716791a
5. Pybus, J. Determination of calcium and magnesium in serum and urine by atomic absorption spectrophotometry.
Clinica Chimica Acta [Online] 1969, 23, 309-317. http://www.sciencedirect.com/science/article/pii/0009898169900461
6. Lewin, M. R.; Wills, M. R.; Baron, D. N. Ultramicrofluorimetric determination of calcium in plasma. Journal of
Clinical Pathology [Online] 1969, 22, 222-225. http://jcp.bmj.com/content/22/2/222.abstract
7. Ntailianas, H. A.; Whitney, R. McL. Calcein as an Indicator for the Determination of Total Calcium and
Magnesium and Calcium Alone in the Same Aliquot of Milk. Journal of Dairy Science [Online] 1964, 47, 19-27.
http://www.journalofdairyscience.org/article/S0022-0302(64)88575-1/abstract
8. Gutow, J. H. Halide (Cl-) Quenching of Quinine Sulfate Fluorescence: A Time-Resolved Fluorescence Experiment
for Physical Chemistry. Chem. Educ. 2005, 82 (2), 302.
9. Fluorescence quenching of coumarins by halide ions. Elsevier. 2004, 60 (4), 757-763.
10. Harris, D. C. Quantitative Chemical Analysis, 8th ed.; Freeman: New York, 2010; pp 433-434.
11. Harris, D. C. Quantitative Chemical Analysis, 8th ed.; Freeman: New York, 2010; pp 436.
12. Brown, T. L.; LeMay, H. E., Jr.; Bursten, B. E.; Murphy, C. J.; Woodward, P. M. Chemistry: The Central Science,
12th ed.; Prentice Hall: Boston, 2012; pp 257, 261, 264, 1064.
13. Brown, T. L.; LeMay, H. E., Jr.; Bursten, B. E.; Murphy, C. J.; Woodward, P. M. Chemistry: The Central Science,
12th ed.; Prentice Hall: Boston, 2012; pp 263.
14. Harris, D. C. Quantitative Chemical Analysis, 8th ed.; Freeman: New York, 2010; pp 298.

Calcein Research Thesis- Anthony Wright

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