The Effect of Halide Ion Concentration on Fluorescence of the Calcein-Calcium Complex: Fluorescence Quenching by the Halide IonAnthony 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 quencher donates 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 à 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* à S + hv [Reaction 2]

Next is the non-radiative relaxation, or quenching where Q is the quenching molecule.

S* + Q à 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* à 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 other calcein-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 quencher present, 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 quencher since I- is a stronger reducing agent.1

Methods. Quartz cuvettes and class A volumetric flasks were used

for all solutions. A Perkin-Elmer LS-50B luminescence spectrophotometer with 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 stock solutions 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 stock solutions 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 GraphsIodide Bromide

Slope: Run 1 4.8478 1.0332Slope: Run 2 4.5661 1.3104Slope: Run 3 4.0064 1.1243

Mean of Slopes 4.4734 1.1860Standard Deviation 0.4283 0.140895% Confidence Interval

±3.4094 -

5.53740.8363 -

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 appear equal, 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 for the 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- à Br + e-325 1140 114 -1.07

I- à 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 quencher must 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 quencher is 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 = kqt[Q] +1. The modified form allows the rate

constant for quenching, kq, and the lifetime of the excited state, t, 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 endeavor to 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 1-34. Yavorskyy, A.; Hernandez-Santana, A.; McCarthy, G.; McMahon, G. Detection of calcium phosphate 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.