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Student ID #



Fluorescence Quenching

Teaching Assistant: TA Name

Chem 375-003

Group #5

2/13/97



Lab Partners:

Name 1

Name 2



Introduction & Theory

When an atom absorbs a photon, the energy associated with that photon causes in increase in the energy level of the atom. The electrons of this excited atom move briefly into a higher energy state, then jump back down to ground state and emit a photon. This photon is of a lower wavelength than the incident photon, and may be observed as fluorescence or phosphorescence. As the quantum electrical surroundings of an atom changes, so do the conditions under which it may absorb or emit a photon. As a result, the wavelengths of absorption and emission are characteristic of a compound.

Phosphorescence is not investigated in this lab, but occurs when excitation causes an electron to become unpaired in its higher energy state, forming a triplet state. This triplet state decays slowly, and will continue to emit photons for a time after the incident photon source is removed. Molecules containing heavy atoms have a large spin-orbit coupling, making the formation of triplet states more likely.(1)

In fluorescence, the excited electron is paired and forms a singlet state. This state decays rapidly, and persists only while photons are incident on the sample.

Goal

The goal of this experiment is to measure the effect of quenching molecules on the fluorescence of acridine ions.

Apparatus(2)

Fluorimeter, fluorescence curvette, curvette holder, 3 pipettes (1,2, and 5 mL), 10 mL graduated pipette, 10 volumetric flasks (10 mL), pipette bulbs.

10-4 M Acridine in 10-2 M H2SO4, 2x10-3 M NaBr or NaI, 4x10-2 M CoSO4, 2.9 g SDS (Sodium Dodecyl Sulfate)



















Experimental Observables

The fluorescence spectrum of acridinium ions will be recorded using a fluorimeter.

Experimental Method

The quenching effect of NaBr or NaI and SDS micelles on acridine ions will be determined by recording emission and excitation scans on soutions of various concentrations. The scans will be made using a fluorimeter, which measures the amount of light being emitted from a compound as a result of excitation from a monochromatic light source. The detection of fluorescence is done at a right angle to the incidence angle of the light source to ensure that only fluorescence is measured and not light from the source.

Experimental Procedure

Make up ten solutions of 1 mL acridinium solution in NaBr or NaI, using from 0 to 9 mL of salt in 1 mL increments. Dilute each solution to 10 mL total with distilled water.

Using an excitation wavelength of ~ 360 nm, record an emission spectrum for the solution containing no salt over the range 400 to 650 nm. This sample serves as a baseline throughout the experiment, so save 8 mL of the sample and rescan using the same parameters in the middle of the experiment and at the end to test for drift. Ensure that spectral bandpass, monochromometer step size, PMT voltage, integration time, and other experimental parameters are recorded and kept constant throughout the experiment.

Set the fluorimeter's excitation monochromator to MASTER, and detector A to the wavelength of maximum intensity found in the emission spectrum. Scan the region from 340 to 400 nm and use the wavelength with maximum intensity throughout the rest of the experiment.

Record the spectrum of each of the previously made samples and save the results to floppy disk.

In order to find possible interference due to the other chemicals besides acridine present in the curvette, perform a few scans of solution without acridine with various concentrations of salt. Note the conditions and results if any large contributions are found.

Record emission scans for six samples of the following compositions:

Sample mL AH+ mL dH2O mL CoSO4 mL SDS mL Salt
1 1 1 0 8 0
2 1 9 0 0 0
3 1 4 5 0 0
4 1 0 1 8 0
5 1 0 0 8 1
6 1 0 0 6 3

Table 1 - Sample Compositions

If time permits, prepare samples identical to the first ten using CoSO4 instead of salt. Otherwise, obtain a given data set for data analysis.

The following data analysis may be performed using Spectra Calc. Using the Starn-Volmer technique, construct plots of (I0/I) vs [Salt] and [CoSO4] for the samples not containing SDS. Use the intensity obtained for the solution containing no salt for I0.

Ensure that spectral plots for the salt samples (with blank), drift samples, solutions containing SDS, and the excitation spectrum of fluorophore are obtained.

Preliminary Calculations

The given procedure calls for several solutions of known concentration. The mass of compound x (g) that needs to be dissolved in V liters of solvent to produce a solution of concentration c (M) is given by

where MW is the molecular weight of the compound.

Safety(3)

Acridine is a strong irritant, avoid contact with skin and mucous membranes. In the event of skin contact, flush thoroughly with water. If ingested, seek medical attention immediately. Contact with eyes should be treated by flushing of affected area with copious amounts of water for at least 15 minutes.

Cobolt Sulfate is an irritant, avoid contact with skin and mucous membranes. In the event of skin contact, flush thoroughly with water. If ingested, seek medical attention immediately. Contact with eyes should be treated by flushing of affected area with copious amounts of water for at least 15 minutes.

Sulfuric acid is corrosive, avoid contact with skin and mucous membranes. Inhalation of vapors may cause severe irritation of the respiratory tract, as well as cause tissue damage if inhaled in high concentrations. In the event of skin contact, flush thoroughly with water. If ingested, seek medical attention immediately and do not induce vomiting. Contact with eyes should be treated by flushing of affected area with copious amounts of water for at least 15 minutes. Avoid uncontrolled contact with water, as reaction is very exothermic. Avoid uncontrolled contact with metals, as reaction may produce explosive hydrogen gas.

Sodium Bromide is an irritant, avoid contact with skin and mucous membranes. In the event of skin contact, flush thoroughly with water. If ingested, seek medical attention immediately. Contact with eyes should be treated by flushing of affected area with copious amounts of water for at least 15 minutes.

Sodium Iodide is an irritant, avoid contact with skin and mucous membranes. In the event of skin contact, flush thoroughly with water. If ingested, seek medical attention immediately. Contact with eyes should be treated by flushing of affected area with copious amounts of water for at least 15 minutes.

Sodium Dodecyl Sulfate (SDS) is an irritant, avoid contact with skin and mucous membranes. In the event of skin contact, flush thoroughly with water. If ingested, seek medical attention immediately. Contact with eyes should be treated by flushing of affected area with copious amounts of water for at least 15 minutes.

References(4)(5)(6)(7)



1. P. Atkins, Physical Chemistry, 5th ed., p. 600, W. H. Freeman and Company (1994).

2. Fluorimeter design taken from the internet at "http://www.turnerdesigns.com/fluor.htm."

3. Safety data taken from the internet at "http://www.enviro-net.com/technical/msds",

"http://www-chem.uscd.edu/Courses/CoursePages/Uglabs/MSDS".

4. S.B. Brown, An Introduction to Spectroscopy for Biochemists, Academic Press (1980).

5. N.J. Turro, Modern Molecular Photochemistry, Benjamin/Cummings (1978).

6. J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum (1983).

7. D.A. Skoog, Principles of Instrumental Analysis, 3rd ed., Saunders College Publishing (1984).