Fluorescence Quenching of Fluorescein
Name: ______Date: ______
Partner: _if applicable______
Use complete sentences, use the proper number of significant figures, and include units. We completed parts of the Theory, Procedure, and Results to help get you started. Remove all the italicized prompts in your final report.
Introduction: (State the purpose of the experiment and the principle means that you are going to use to achieve that purpose. The introduction should be just two sentences.)
Theory: The mechanism of quenching was determined by comparing the absorbance spectrum of the dye with and without quencher to determine the involvement of the ground state. A Stern-Volmer plot was used to determine the effectiveness of the quenching. (Give the Stern-Volmer equation and identify each variable in words (Io, I, KQ, and [I–]).)
Procedure: A solution of fluorescein in pH 7.4 buffer was prepared using the dye from a commercial highlighter. (Give the manufacturer of the highlighter and model name.) The absorbance spectrum of the dye without quencher and with 0.5 M I– were determined. A Stern-Volmer plot was constructed with four KI concentrations from 0.1 to 0.4 M in 20% ethanol.”1 (List any changes that were made to the written procedure in the lab write-up. State the manufacturer and model of the spectrophotometer. Report the excitation wave length for the fluorescence measurements.)
Results: (Present the results of your calculations as requested in the Results section of the write-up:)
The spectra of fluorescein without I– and with 0.5 M I– in 20% aqueous ethanol are shown in Figure 1. The results of the quenching experiment are given in Table 1 and Figure 2. The corresponding Stern-Volmer plot is shown in Figure 3.
Table 1: (Provide caption)
Volume KI (mL) / [I–] (M) / Intensity, I / Io/I(If there were student mistakes, discuss them here. Give the remaining results as requested in the Results section of the write-up)
Discussion: (a). Purpose accomplished: Essentially restate the purpose of the experiment, but as completed goal in a sentence or two:
(b). Final Results: State your conclusion on the interaction of the quencher with the ground state of the molecule and report the quenching constant:
(c). Factors that influenced the precision and accuracy of the data:
i. Give a measurement that is predominant source of random error. Does this predominant error have an effect on the accuracy or precision of the final result?
ii. Give a possible source of systematic error in the quenching constant with evidence:
(d). Answer questions 1-4:
For question 5: Draw the quantitative energy level diagram on the grid below. The vertical energy axis is shown in both joules and kJ/mol. Label the axes in regular intervals; use at least four tic marks for each axis. The intervals you choose need not coincide with the scale lines (the tic marks can be between the grid-lines if easier). Include all observed excited states as determined from the absorption spectrum. Include an arrow for the excitation using the 405 nm source, a dotted arrow for the internal conversion, and an arrow for the fluorescence transition to the ground state (where should the fluorescence transition start?). The plot can be hand drawn. The ground state is shown to help get you started. Complete the figure in pen, by hand.
The energy level diagram for the excitation and fluorescence transitions of fluorescein is shown in Figure 4.
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Figure 4: Energy level diagram for the excitation and fluorescence transitions of fluorescein.
Literature Cited
1. (Give the reference to the lab write-up and the corresponding Web address (URL). Give a last accessed date. See last week’s lab report for the format.)
Please attach a copy of your absorption spectra with a caption as Figure 1.
Please attach a copy of your fluorescence spectra with a caption as Figure 2.
Please attach a copy of your Stern-Volmer plot with a caption as Figure 3.
Checklist:
Use complete sentences and provide the proper number of significant figures and units.
All Figures and Captions must have captions.
Acknowledge any data that were not taken by you and your partner (if you had a partner).
Captions start with Figure # or Table # and then a concise description of the contents.
You can write the captions by hand in black pen on attached sheets.
Answer all the questions in the Discussion section of the write-up.
Remove all the italicized prompts in your final report. The report should then read smoothly.
Pay attention to the parts of this report that we wrote. You will be on your own in subsequent reports.