Learn what is fluorescence quenching of fluorescein and explore how determine it with theory, procedure. Quenching decreases the fluorescence or glow intensity of given substance.
Determination of fluorescence quenching of fluorescein?
What is fluorescence quenching?
Fluorescence quenching is a process where the glow (fluorescence) of a substance decreases or disappears when it’s exposed to another substance.
It used to detect and measure of the presence of specific molecules in various applications like environmental monitoring, drug discovery and DNA analysis.
By observing how the fluorescence changes, we can gain important information about the interactions and concentrations of molecules. Before fluorescence quenching, we have to know about fluorescence.
What is fluorescence?
Fluorescence is a mechanism where a substance absorbs light energy (usually from ultraviolet or visible light) and then it reemits that absorbed energy as visible light.
This light creates a distinctive glow and allow us to study and detect specific substances, such as fluorescent dyes or certain molecules.
What is fluorescein?
Fluorescein is a synthetic organic compound used as a fluorescent tracer or dye. It exhibits strong fluorescence when exposed to ultraviolet or blue light and emits green light. This property used for tracking and labeling of biological molecules, detecting pH changes and visualizing tissues and structures.
Today we will explore determination of fluorescence quenching of fluorescein.
Requirement
Chemical
- Fluorescein
- Potassium iodide (KI)
- Ethanol 95%
- 0.1 M pH 7.4 buffer 11.03 g Sodium phosphate (Dibasic Na2HPO4)
- 3.05 g Potassium dihydrogen phosphate (KH2PO4) in 1 L water.
Apparatus
- Plastic cuvettes
- Plastic fluorescence cuvettes
- Volumetric flasks
- Automatic pipettor
- Beaker.
- Fluorescence Spectrometers (Vernier Spectro Vis Plus)
Theory
Quenching is a process which decreases the fluorescence or glow intensity of given substance. It may occur due to various factors like pH, temperature, viscosity, complex formation. Oxygen is a particularly good quencher.
So, it is necessary to remove the oxygen from solution before measuring fluorescence spectra. Fluorescent probes are used in biochemistry to study the binding sites in large macromolecules.
Procedure to determine fluorescence quenching of fluorescein
1)Prepare stock solutions of fluorescein
- Take a 150-mL beaker.
- Put a yellow paper strip in it.
- Add about 100 mL of pH 7.4 buffer to the beaker.
- Stir with a magnetic stir bar for a few minutes until the dye comes off the paper.
- Make sure the absorbance or fluorescence intensity isn’t higher than 0.2 for even fluorescence.
- Use a frosted-sided cuvette to measure the absorbance of your fluorescein stock solution.
- The absorbance should be between 0.4 and 0.7 at around 450 nm.
- If it’s too concentrated, dilute the stock solution with buffer.
- Remember that this stock solution will be diluted by ten times for the final solutions to hit the target absorbance range.
2)Prepare stock solutions of 1.0 M KI: Prepare a 1.0 M KI solution in reagent grade water in a 25-mL volumetric flask.
3)Absorbance Measurements
- Take a plastic cuvette (with a frosted side).
- Use an auto-pipettor to add 1 mL of stock dye solution and 1 mL of water to the cuvette.
- Take another cuvette.
- Add 1 mL of stock dye solution and 1 mL of 1.00 M KI to this second cuvette.
- Cover each cuvette with plastic wrap or parafilm.
- Shake both cuvettes to mix the solutions well.
- Follow the attached instructions to determine the absorbance spectrum of the two solutions.
- Observe any significant changes in the absorption spectrum observed upon addition of iodide ion.
- Small changes in intensity might result from the relatively crude manner of solution preparation.
4)Determination of the Excitation Profile at 405 nm:
- Switch to fluorescence mode by going to the Experiment menu, selecting “Change units” and “Spectrometer 1,” and choosing “Fluorescence 405 nm.”
- Determine the excitation wavelength range by making a slightly turbid suspension with coffee creamer and transferring it to a frosted-sided cuvette.
- Follow attached instructions to measure the spectrum.
- Compare the dye’s absorption spectrum with the excitation profile at 405 nm.
- Check for significant overlap between the molecule’s absorption and the fluorescence excitation.
- Clear all data by going to the Data menu and selecting “Delete All.”
5)Fluorescence Measurements
- Prepare six solutions in 10-mL volumetric flasks:
- Add 1.00 mL of fluorescein stock solution.
- Include 2.00 mL of ethanol in each flask.
- Keep the first solution unchanged.
- In the remaining five flasks, add 1, 2, 3 and 4 mL of KI stock solution separately.
- Fill each flask to the mark with reagent grade water, using a plastic dropper to align with the calibration mark.
- Use fluorescence cuvettes with four transparent sides:
- If you have six cuvettes, fill each with one of the prepared dye solutions.
- Overlay spectra for comparison by selecting “Store the previous run” after clicking the green Start button in Logger Pro.
- Now you have only one cuvette:
- Rinse it thoroughly with the new solution before measuring the spectrum.
- Dry the cuvette’s outer surfaces with Chem Wipes or soft tissue to prevent scratches.
- Never introduce stirring rods or spatulas inside a cuvette to avoid scratching.
- Make a Stern-Volmer plot to determine the quenching constant, KQ.
Observation
- Find the absorption spectrum of fluorescein in both water and the I- solution to check for interactions in the ground state.
- Establish the wavelength range of the 405 nm light source used for fluorescence excitation.
- Record the fluorescence intensity of the dye by itself and as you increase the concentration of I- as the quencher.
Determination result
- Write down two absorption spectra with and without quencher.
- Take a spectrum of the fluorescence without quencher and with varying amounts of quencher.
- Write the results of the calculations for the Stern-Volmer experiment in a table.
- Draw Stern-Volmer plot Io/I vs I-
- Give the slope and intercept of plot and the corresponding quenching constant, KQ. Make sure that each table and figure have captions and that each table or figure is referenced in the body of the text. The captions should summarize the content of the table or figure. The captions on the spectra can be handwritten.
(Precautions: Dispose of all dye solutions in the aqueous waste container. Do not to scratch the surfaces of cuvettes while cleaning the cuvettes).
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