Department of Chemistry
College of Science and Mathematics
Mindanao State University – Iligan Institute of Technology
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 𝑀𝑔2+ solution. These measurements were made in three cycles at
𝜆𝑒𝑥 =557 nm and 𝜆𝑒𝑚 =575 nm. The corrected intensities were then plotted against the increasing
concentrations of the 𝑀𝑔2+ 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 𝑀𝑔2+ solution. The calibration curve also showed excellent linearity with a
coefficient of determination (𝑅 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 𝑀𝑔2+ 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 𝑀𝑔2+ solution. Eight solutions were made and the measurements were performed at these specific
concentrations 0, 1, 2, 3, 4, 5, 8 𝑝𝑝𝑚 of 𝑀𝑔2+ . 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 𝑀𝑔2+ 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 𝑀𝑔2+ solution. This unknown concentration was
calculated using this formula.
𝐶𝑢𝑛𝑘𝑛𝑜𝑤𝑛(𝑝𝑝𝑚) =
𝑀𝑒𝑎𝑛 𝐼𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦𝑢𝑛𝑘𝑛𝑜𝑤𝑛 − 𝑏
𝑚
Equation 1. Formula for calculating the unknown concentration of 𝑀𝑔2+ 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 𝑀𝑔2+ 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.

Preparation of 10 ppm 𝑀𝑔2+ solution
0.6546 g of 𝑀𝑔𝐶𝑙2 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 𝑀𝑔2+ 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 𝑀𝑔2+ solution.

Preparation of the unknown concentration of 𝑀𝑔2+ solution
An unknown concentration of 𝑀𝑔2+ solution was provided for by the lab technician.

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.

Preparation of alcohol 𝑀𝑔2+ 8-hydroxyquinoline solution and Intensity Measurements
Using 10 mL volumetric flasks, varying volumes of the 𝑀𝑔2+ solution was transferred specifically
0, 100, 200, 300, 400, 500, and 800 𝜇L . 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 𝑀𝑔2+ solution was used as the blank for this
experiment. Using a fluorescence spectrophotometer, intensity measurements were performed at
each solution with varying 𝑀𝑔2+ concentrations. These measurements were done at 𝜆𝑒𝑥 =557 nm
and 𝜆𝑒𝑚 =575 nm and in three cycles. Intensity measurements were then corrected and were then
plotted against the concentration of 𝑀𝑔2+ to generate a calibration curve.

Intensity Measurement and Calculation for the Unknown Concentration of 𝑀𝑔2+ solution in ppm
For measuring the intensity of the unknown concentration of 𝑀𝑔2+ 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 𝑀𝑔2+ solution and its Measured Intensity
Concentration of 𝑀𝑔2+
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 𝑀𝑔2+
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 𝑀𝑔2+
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 (𝑅 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 (𝑅 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 𝑀𝑔2+ 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 𝑀𝑔2+ solution
100 ppm 8-hydroxyquinoline
99.9 % ethanol
Excel Calculation:
𝐶𝑢𝑛𝑘𝑛𝑜𝑤𝑛(𝑝𝑝𝑚) =
217.38 − 4.1959
= 5.40 𝑝𝑝𝑚 𝑀𝑔2+
39.514
Table 3. Table of All Gathered Data
Trial
Blank
1
2
3
4
5
6
Blank
Unknown
Concentration
of 𝑀𝑔2+
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