Francis Gerald C. Talip Activity No. 8 Fluorescence Analysis by Chelation Abstract In fluorescence spectroscopy, the analysis of the fluorescent properties of a sample is involved. During this experiment fluorescence analysis by chelation was used. Chelation is simply the bonding of ions or molecules to metal ions. With the aid of a fluorescence spectrophotometer, intensity measurements were made for varying volumes of a ππ2+ solution. These measurements were made in three cycles at πππ₯ =557 nm and πππ =575 nm. The corrected intensities were then plotted against the increasing concentrations of the ππ2+ solution to generate a calibration curve. The calibration curve provided data such as a slope value of 39.514 and a y-intercept of 4.1959. These were then used to calculate the unknown concentration of a ππ2+ solution. The calibration curve also showed excellent linearity with a coefficient of determination (π 2 ) of 0.9988. The generated curve proved that the intensity and concentration has a linear relationship, as it was observed that as the concentration of ππ2+ solution was increased the intensity measured also increased. The unknown concentration was found out to be 5.40 ppm. Introduction Fluorescence spectroscopy is a kind of electromagnetic spectroscopy that provides analysis of the fluorescent properties of a sample. Using a beam of light, the electrons of the molecules are excited which results to the emission of light. In this experiment, fluorescence analysis by chelation is utilized. Chelation is the bonding of ions or molecules to metal ions. The process involves the formation or presence of multiple separate dipolar bonds between a polydentate (a ligand that has multiple bonded atoms) and a central metal atom. A fluorescence spectrophotometer was used to generate intensity measurements for varying volumes of a ππ2+ solution. Eight solutions were made and the measurements were performed at these specific concentrations 0, 1, 2, 3, 4, 5, 8 πππ of ππ2+ . The gathered intensity measurements were then plotted against the concentration to generate a calibration curve excluding the blank solution. As the initially generated calibration curve was undesirable, a number of measurements were removed to achieve ideality. Figure 1. Calibration Curve for Intensity vs. Concentration of ππ2+ solution Intensity vs. Concentration Intensity 400 y = 39.514x + 4.1959 R² = 0.9988 300 200 100 0 0 2 4 6 8 10 Concentration (ppm) This experiment aims to utilize fluorescence analysis through chelation to generate data that will be used to calculate for an unknown concentration of the ππ2+ solution. This unknown concentration was calculated using this formula. πΆπ’πππππ€π(πππ) = ππππ πΌππ‘πππ ππ‘π¦π’πππππ€π − π π Equation 1. Formula for calculating the unknown concentration of ππ2+ solution in ppm (Gomez, 2022) All gathered data in this experiment was presented using three tables and a figure. Table 1 contained the various concentrations of ππ2+ solution together with the mean intensities measured from each varying concentration. In table 2, the corrected data used to generate the ideal calibration curve is presented and in table 3, the different blank intensity measurement together with the identified unknown concentration is shown. It is expected in this experiment, that the generated calibration curve will show the linear relationship between the intensity and concentration of the solution and that the data from intensity measurements of the unknown will be used together with the slope, and the y-intercept will be utilized to calculate the unknown concentration. Methodology PREPARATION OF REAGENTS The materials and chemicals used is indicated on the appendices section. ο· Preparation of 10 ppm ππ2+ solution 0.6546 g of πππΆπ2 was obtained using an analytical balance. It was then dissolved in a beaker using a minimal amount of ethanol. After the solvation process, the solution was then transferred to a 100 ml volumetric flask and diluted to mark using ethanol. This resulted in the production of 783 ppm of ππ2+ solution 1.277 ml of this solution was then obtained and transferred to another 100 mL volumetric flask and diluted to mark to obtain 10 ppm ππ2+ solution. ο· Preparation of the unknown concentration of ππ2+ solution An unknown concentration of ππ2+ solution was provided for by the lab technician. ο· Preparation of 100 ppm 8-hydroxyquinoline 10 mg of 8-hydroxyquinoline was weighed using an analytical balance. It was then transferred to a beaker and dissolved using a minimal amount of ethanol. After the solvation process, the solution was then transferred to a 100 mL volumetric flask and diluted to mark using ethanol. ο· Preparation of alcohol ππ2+ 8-hydroxyquinoline solution and Intensity Measurements Using 10 mL volumetric flasks, varying volumes of the ππ2+ solution was transferred specifically 0, 100, 200, 300, 400, 500, and 800 πL . 6 mL of the 8-hydroxyquinoline solution was then added to each flask and diluted to mark using ethanol. Each solution was then transferred to their corresponding test tubes. The flask that contains no ππ2+ solution was used as the blank for this experiment. Using a fluorescence spectrophotometer, intensity measurements were performed at each solution with varying ππ2+ concentrations. These measurements were done at πππ₯ =557 nm and πππ =575 nm and in three cycles. Intensity measurements were then corrected and were then plotted against the concentration of ππ2+ to generate a calibration curve. ο· Intensity Measurement and Calculation for the Unknown Concentration of ππ2+ solution in ppm For measuring the intensity of the unknown concentration of ππ2+ solution, the first step was to measure the intensity of a blank solution. The intensity of the unknown is measured after. After the intensity measurement was corrected, the data provided by the calibration curve was utilized to calculate for the unknown concentration. Specifically, the slope and the y-intercept (calculation is displayed on the appendices section). Results Table 1. Concentration of ππ2+ solution and its Measured Intensity Concentration of ππ2+ solution ( ppm) 0 1 Mean Intensity Corrected Intensity (Standard-Blank) 53.82 96.52 0.00 42.70 2 3 138.20 183.30 84.38 129.48 4 245.00 191.18 5 285.43 231.62 8 371.43 317.62 Table 2. Corrected data for Ideal Linear Curve Concentration of ππ2+ solution ( ppm) 0 1 2 3 8 Corrected Intensity 0.00 42.7 84.38 129.48 317.62 Figure 2. The Generated Calibration Curve Intensity vs. Concentration Intensity 400 y = 39.514x + 4.1959 R² = 0.9988 300 200 100 0 0 2 4 6 8 10 Concentration (ppm) Table 3. Corrected Intensity of the Unknown and the Calculated Concentration Concentration of ππ2+ solution ( ppm) 5.40 Intensity 217.38 Discussion Table 1 shows all the gathered data used to generate the initial calibration used to test for linearity. Table 2 contains the data used to generate the ideal linear curve. The generated curve is shown in figure 2 which yields a slope of 39.514 and a y-intercept of 4.1959. It is also shown in the curve a coefficient of determination (π 2 ) of 0.9988. Table 3 is simply the one containing the calculated concentration of the unknown with its measured intensity. Possible errors in this experiment could be the spilling of minimal volumes of solution due to transferring from one container to another. Dilution errors could have also occurred stemming from the inaccuracy in diluting the solution to mark, which can affect the concentration. The experiment can be further improved through repetition of the steps, which can help in improving the accuracy of the measurements. Conclusion Figure 2.0 shows the calibration curve equation for the fluorescence spectrophotometer experiment, y= 39.514x +4.1959, which has a coefficient of determination (π 2 ) of 0.9993. It also depicts that the relationship between intensity and concentration is linear as the intensities increased in proportion to an increase in concentration. In this experiment, it is found out that the intensity of the unknown and the data gathered from the generated calibration curve such as the slope and y-intercept can be utilized in calculating the unknown concentration of ππ2+ solution which is 5.40 ppm. Reference https://en.wikipedia.org/wiki/Chelation Appendices Materials: 100 mL volumetric flask 10 mL test tubes Beaker Chemicals: 100 ppm ππ2+ solution 100 ppm 8-hydroxyquinoline 99.9 % ethanol Excel Calculation: πΆπ’πππππ€π(πππ) = 217.38 − 4.1959 = 5.40 πππ ππ2+ 39.514 Table 3. Table of All Gathered Data Trial Blank 1 2 3 4 5 6 Blank Unknown Concentration of ππ2+ solution ( ppm) 0 1 2 3 4 5 8 0 ? Intensity per Cycle Number 1 2 3 Mean Intensity Corrected Intensity 53.69 96.33 138.4 183.4 246.7 285.9 371.2 29.41 247.3 53.88 96.65 138.2 183.4 244.1 285.0 371.5 28.98 246.5 53.88 96.57 138 183.1 244.2 285.4 371.6 29.57 246.3 53.82 96.52 138.20 183.30 245.00 285.43 371.43 29.32 246.7 0.00 42.70 84.38 129.48 191.18 231.62 317.62 0.00 217.38