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Fluorescence Lab

Fluorescence is a type of photoluminescence spectroscopy. Fluorescence occurs when a photon is emitted from a molecule as it moves from a higher excited state to a lower excited state with in the same spin. A molecule normally is in its ground state energy. When a light source puts off an electromagnetic energy, the molecule can move to a higher more excited energy. When the molecule transitions down in energy, it usually is released as heat. Conversely, if the molecule is subject to high energy the molecule can transition down to its ground state by fluorescence.

This emission of a photon creates wavelengths that are captured by spectrophotometric methods. Fluorescent intensity is represented by the equation F=k C. Where F is the fluorescent intensity measured by these wavelengths, C is the concentration of the sample and k is a constant based on the power of the light source, the molar absorptivity of the molecule and the quantum yield. The intensity of fluorescence emission can increase when k increases by increasing either the power of the light source, the molar absorptivity or the quantum yield.

The intensity also increases when more electron donating groups are on the molecule being determined, and decrease when the molecule has more electron withdrawing groups. The intensity also decreases when the temperature increased. This is because as the temperature increases more collisions can occur between the molecule and the solvent, which transfers more energy to another component of the matrix without releasing a photon. This means that it does not create fluorescence. For a fluorescent molecule the emission wavelength is generally higher than the excitation wavelength.

This is because when the molecule is transitioning back to its ground state, it can loose some of its absorbed energy to vibrational energy rather than fluorescence energy. Therefore, the magnitude the absorbed energy is greater than the magnitude of the fluorescence energy, but is equal to the fluorescence energy transitions plus the vibrational energy transitions [1]. The main purpose of this experiment was to understand spectroscopic methods, specifically fluorometric spectroscopy. Quinine is a molecule, shown in figure 1, with three aromatic rings, an -OH group, a CH3O- group and two amino groups.

These functional groups are electron-donating groups that increase the intensity, so the fluorescence intensity of quinine is very strong because of them. Another molecule that is highly fluorescent is tryptophan. Tryptophan, shown in figure 2, also has electron-donating groups such as the aromatic ring, the –OH and the amino groups. These electron-donating groups increase the electron density, which increases the fluorescence because more electrons have been excited and are transitioning back to ground state and therefore emitting photons.

The purpose of this experiment was to determine the amount of quinine, a flavoring in tonic water, using fluorescence. Quenching occurs when a molecule in an excited state moves to a less excited state and loses energy to the matrix forcing fluorescence to not occur. The purpose was also to investigate the quenching effect of halides by determining the effects of the halide chlorine on the fluorescence of quinine. Another purpose was to determine sample’s matrix effect by determining the effects of a yellow dye sample matrix on the fluorescence of quinine.

Lastly, the purpose was to be able to determine the relationship between voltage and the fluorescence intensity of quinine. The basic theory behind fluorescence can relate to the Stern-Volmer equation which is F0/F=1+KSV[Q]. To break it down, the F0/F is the ratio of the intensities. F0 is the fluorescence intensity without the halide, which is the number of times that a photon is emitted when the molecule transitions down energy levels. And F is the fluorescence with the halide. [Q] is the concentration of the halide only and the KSV is known as the quenching constant.

When you graph the intensity ratio versus the concentration of the halide the graph created is linear. The linear fit generated from this graph correlates to the Stern-Volmer equation. The slope of the linear fit is how we interpreted our results. The slope is the produced quenching constant, KSV, for the specific halide chlorine and the molecule quinine. The methods used to collect data to generate our graphs used to understand fluorescence include using the Perkin-Elmer fluorometer. The fluorometer collects wavelength measurement of the sample being analyzed.

The Perkin-Elmer fluorometer design is so that the sample is analyzed by providing light source, the source of radiation at a narrow band, and then the sample emits fluorescent radiation in all directions. The detector that collects that wavelength data is 90o to the light source that way it only reads the fluorescent radiation and not the light source radiation [1]. The solutions for the samples analyzed in this lab were made during this class period. A similar study conducted on fluorescence and the effect of quenching was performed by Lukasz Lesin? ski and Jorg Duschmale.

In part of their experiment the effects of the halides chlorine, bromine, iodine and fluorine was investigated. From their data it was seen that fluorine does not quench at all, and that iodine was a relatively strong quencher. The trends in their data indicate that the heavier the halide the more quenching that can occur, decreasing the amount of fluorescence emitted [2]. In another experiment conducted by Andy Baker took advantage of emission spectroscopy as well as absorption spectroscopy and looked at the effects of sewage treatments on rivers using a Perkin-Elmer instrument.

Rivers with high sewage concentrations demonstrate high fluorescence intensities while rivers treated have lower fluorescence intensity. By taking samples from the rivers upstream and downstream from the sewage treatment works, he was able to track the flow pattern of the sewage as it has been treated through the rivers. He was able to do this by looking at tryptophan and fulvic fluorescence in the water samples. This is an example of how fluorescence can be used in analyzing biological settings [3]. Briefly, the results from our data revealed the amount of quinine present in the tonic water was 34. 3 ppm.

It also was determined that the quenching constant for the halide chlorine on quinine was 124. As well, the data shows that the most efficient matrix for quinine was the sample matrix with no color. Lastly, it was concluded that as the voltage of the power source is increased, the fluorescent intensity increase. These results are more closely discussed in the results and discussion section. Table 1 shows the intensity data collected from the prepared concentrations of qunine. Using this data, a calibration curve was created. Graph 1 is the calibration curve.

The linear fit line was found to be y = 41. x – 1. 86. The correlation coefficient was found to be 0. 996. The linear fit line is the equation that was used to extrapolate the concentration of quinine in our tonic water sample. The tonic water sample was diluted 4 times then the intensity was collected and found to be 354 nm. This intensity and the linear fit line were used to determine the concentration of quinine as 34. 3 ppm. The completed calculation can be found on lab notebook page 14. This is comparable to the legal limit of 83 ppm and suggests that the tonic water is in compliance with the regulation.

Results of the Quenching Effect of the Halide Chlorine Table 2 shows the calculated concentrations of the halide in molarity and the calculated intensity ratio of fluorescence. The completed calculations can be found on lab notebook page 14. Using this data, graph 2 was generated showing the relationship between the intensity ratio and the concentration of chlorine. Relating to the Stern-Volmer equation of F0/F=1+KSV[Q] and the linear line equation of y = 124x + 1. 08, the KSV is the slope of the equation 124. Compared to the literature value of KSV of 184, the relative error as a percent was calculated to be 32. % [2].

This was calculated by finding the difference between the measure value and the literature value, divided by the literature value then multiplied by 100. The completed calculation can be found on lab notebook page 15. Heavy atoms in the solution promote intersystem crossing. Intersystem crossing is when a molecule has been excited and resides into the lowest vibrational energy level of a high transitional energy state, and then transitions to a high vibrational energy level of a lower transitional energy and does not produce a photon.

This is occurs often when heavy halides are in the solution because they increase vibrational relaxation [1] Concentration of NaCl (mols/L) Intensity Ratio of Fluorescence of Quinine with no halide/ Fluorescence of Quinine with halide Results of the Sample Matrix Effect on Fluorescence of Quinine The sample’s matrix effect was evaluated by determining the fraction of fluorescence maintained in different solutions. Three samples with different matrixes were made and the intensities were measured and are reported in table 3.

In order to determine the fraction of fluorescence maintained, the calculation of the difference in intensity between the sample B and sample C divided by A was preformed. The completed calculation can be found on lab notebook page 15. The fraction of fluorescence maintained was computed to be 0. 403. The difference in fluorescence for quinine in water and in the colored soda can be explained by this fraction of fluorescence maintained. This suggests that in the colored soda matrix the fluorescence intensity of quinine was only occurring 40% of the number of photons emitted that it would in sample A’s matrix.

This is because the quinine in the colored soda is not absorbing the same initial energy as the quinine in the clear water. Meaning, that the number of photons it initially absorbs is less, so there are less excited molecules and therefore, less deactivation of these molecules. Results of the Voltage Effect on the Fluorescence of Quinine The voltage effect on the fluorescence of quinine was examined at two different voltages, 3. 19 and 3. 77 volts. The relative fluorescence of a 20 ppm sample of quinine was measure and is recorded in table 4. When the voltage was increased to 3. 7 volts, the relative fluorescence intensity increased by a factor of 4. 73.

The completed calculation can be found on lab notebook page 15. The data shows the trend that increasing the voltage increases the fluorescence. The trend can be explained by the equation F=k C, referring back to the introduction k is a constant based on several aspects. One of these aspects that k is reliant on is the power of the light source. If the power is increased, k is increased and then based on the equation we can see that the fluorescence would therefore increase.

Perkin-Elmer Fluorometer versus MeasureNet Fluorometer 0. 1 ppm quinine is below the detection limit of the MeasureNet fluorometer. The reading measured when this concentration is used is not greater than three times that of the noise reading. While 0. 1 ppm quinine for the Perkin-Elmer fluorometer is within the limit of detection because the signal produced was greater than 3 times that of the noise. A reasonable limit of detection for the Perkin-Elmer fluorometer could be 2. 37 x 10-3 ppm. A reasonable limit of detection of the MeasureNet Fluorometer would be 0. 346 ppm.

The completed calculations can be found on page 16. When comparing the MeasureNet fluorometer with the Perkin-Elmer fluorometer, the Perkin-Elmer fluorometer has a better detection limit for quinine. This is because the Perkin-Elmer fluorometer uses a high power Xe lamp light source. This light source provides more energy radiation than the MeasureNet fluorometer is able to. Therefore, less energy is lost as heat and more energy able to transition down to ground state and emit a photon producing longer wavelengths than the MeasureNet fluorometer is able to produce.

Conclusion From our results, we learned that the concentration in our tonic water sample of quinine was 34 ppm which is with in the legal limitations that it should follow. We also learned from our results the significance that halides had on the quenching of fluorescence intensity of quinine. When chlorine was present in the quinine solution, the fluorescence intensity decreased. As well as the more concentration of chlorine, the more quenching that occurred.

The halide prevents the emission of a photon, fluorescence, and the heavier the atom the more quenching that occurs. The significance of the sample matrix was also learned based on our results. It was determined that the matrix’s ability to absorb energy from a light source based on its color, affected it’s ability to emit a photon and produce fluorescence intensity. Lastly, the significance of the voltage on the amount of fluorescence intensity was studied. From the equation F = k C, it was understood that increasing the voltage increases the fluorescence.

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