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Fluorescence Quenching

Teaching Assistant: TA Name

Chem 375-003

Group #5

3/6/97

Performed: 2/13/97



Lab Partner: Name



Abstract

The quenching effect of NaBr and CoSO4 on acridinium ions was investigated. In addition, the effect of SDS micelles on the effectiveness of the quenchers was investigated. Measurements were made with a computerized fluorimeter.

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.

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.

If the energy of the dissipated state is removed before the electron is able to jump to ground state and release a photon, quenching occurs and the observed fluorescence is reduced. The primary method by which this happens is intermolecular collisions within the sample. The effectiveness of the quenching depends largely upon the concentration, weight (and subsequent size), and proximity of the quenching molecule. Large, heavy molecules are more likely to collide with the fluorescing species, making them more effective quenchers. This is referred to as the heavy atom effect. The proximity of the quenching molecule to the fluorescing species can be affected by static electric fields brought about by micelles. Micelles are spherical groups of polarized molecules that group in such a way that the negative ends of the molecules are facing outward. This attracts positively charged ions in the solution and repels negatively charged ions. In the case of AH+ molecules, the acridine would be attracted to the micelle, along with Co2+, while Br- would be repelled. As a result, miscelle increase the effectiveness of quenchers with a positive ionic charge and reduce the effectiveness of those with negative ionic charges.

Another mechanism that reduces the measured fluorescence of a substance is resonant energy transfer. This occurs when the photons emitted as fluorescence can be reabsorbed by the solution. This phenomenon can be expected when the absorption bands overlap with the emission bands.

Experimental

The following samples were made:

Sample mL AH+ mL dH2O mL NaBr mL CoSO4 mL SDS
1 1 9 0 0 0
2 1 8 1 0 0
3 1 7 2 0 0
4 1 6 3 0 0
5 1 5 4 0 0
6 1 4 5 0 0
7 1 3 6 0 0
8 1 2 7 0 0
9 1 1 8 0 0
10 1 0 9 0 0
11 1 1 0 0 8
12 1 9 0 0 0
13 1 4 0 5 0
14 1 0 0 1 8
15 1 0 1 0 8
16 1 0 3 0 6

Table 1-Sample Compositions

An emission scan from 400 to 650 nm was run on sample 1 using an excitation wavelength of 360 nm, yielding a maximum emission at 474.75 nm. Next, an excitation scan from 340 to 400 nm was run, and the wavelength of peak intensity (343.75 nm) was used through the rest of the experiment as the excitation wavelength. Emission spectra for solutions 1 to 10 were taken, the blank (Sample 1) re-measured, the spectra for solutions 11 to 16 were taken, and the blank was measured again at the end of the experiment.

Results

Average intensity of four runs of blank standard cell (I0) = 9.48x105.

Sample [NaBr] [CoSO4] [SDS] Max Intensity I0/I
1 0.00e+00 0.00e+00 0.00e+00 9.59e+05 98.87%
2 1.15e-04 0.00e+00 0.00e+00 8.95e+05 105.95%
3 2.31e-04 0.00e+00 0.00e+00 8.56e+05 110.72%
4 3.46e-04 0.00e+00 0.00e+00 8.18e+05 115.94%
5 4.62e-04 0.00e+00 0.00e+00 7.72e+05 122.88%
6 5.77e-04 0.00e+00 0.00e+00 7.67e+05 123.55%
7 6.92e-04 0.00e+00 0.00e+00 7.35e+05 129.05%
8 8.08e-04 0.00e+00 0.00e+00 7.26e+05 130.58%
9 9.23e-04 0.00e+00 0.00e+00 6.97e+05 136.00%
10 1.04e-03 0.00e+00 0.00e+00 6.61e+05 143.42%
11 0.00e+00 0.00e+00 4.03e-02 7.93e+05 119.56%
12 0.00e+00 0.00e+00 0.00e+00 9.72e+05 97.58%
13 0.00e+00 5.10e-05 0.00e+00 9.03e+05 104.95%
14 0.00e+00 1.02e-05 4.03e-02 8.23e+05 115.22%
15 1.15e-04 0.00e+00 4.03e-02 8.07e+05 117.50%
16 3.46e-04 0.00e+00 3.02e-02 1.24e+06 76.64%

Table 2-Conditions and readings



















By linear regression, the coefficient of correlation for the first ten samples is 0.9918 and the gradient of I0/I is 394.1.

If I is the intensity, I0 is the intensity with no quencher, [Q] is the concentration of the quencher, kq is a second order rate constant for quenching, and k0 is a rate constant for all other methods of deactivation, then



Quencher Average kq/k0
NaBr 439.0
CoSO4 4329
SDS only 4.854
NaBr and SDS (4.03x10-2 M) 1522
NaBr and SDS (3.02x10-2 M) -675.1
CoSO4 and SDS 14920

Table 5-Comparison of quenching effects

Discussion

The value of kq/k0 represents the amount of deactivation due to quenching vs. The amount of deactivation due to all other processes. With the equations available, it is not possible to calculate k0 alone as the concentration of quencher would then be zero and lead to a division by zero. As a ratio however, it is useful in comparing the effectiveness of quenchers nonetheless as k0 should be constant.

It can be seen from Table 5 that CoSO4 is much more effective quencher than NaBr, presumably due to the heavy atom effect. With the addition of SDS (which has a negligible quenching effect of its own), CoSO4 becomes increasingly more potent due to collisions at the surface of the micelles.

The values seen for NaBr with SDS are somewhat puzzling. At the lower concentration of SDS, the negative value indicates that k0 is actually reduced. If this is correct, the mechanism by which this occurs is not clear. In addition, at the higher concentration the effectiveness of NaBr is dramatically increased, a result the conflicts with the model of AH+ and Br- being separated by the static field produced by the SDS micelles.

Possible sources of error in this experiment include temperature changes, instrumental drift, contamination of sample, inaccuracies in solution preparation, degradation by exposure to ambient light, and contamination of the curvette faces.

Temperature changes were not monitored throughout the experiment, but could be caused by changes in ambient temperature as well as radiation from the xenon lamp. In addition, the solutions were covered to protect them from exposure to ambient light whenever possible in order to minimize possible degradation.

Drift in the instrument could be caused random error, or power fluctuations. Inconsistencies in the way the sample blank was measured could also cause apparent instrumental drift. As shown by the attached sheet entitled Emission (Std), the spectrum of the standard blank did change slightly over the course of the experiment.

The most likely source of sample contamination was in the repeated measurements of the standard sample. The solution had to be reused, and the amount of standard available made rinsing of the cell impossible. As a result, the standard become contaminated by residue in the sample cell with each reading.

The solutions prepared in this experiment were 10 mL in size, and calibrated 1 and 5 mL pipettes were available. As a result, some solutions were made of up to 5 different additions. The error in each of the five measurements was cumulative, and solutions requiring 1mL dH2O were filled strictly with pipettes and compared to the 10 mL mark on the volumetric flasks they were prepared in. As quantitative deviations cannot be found from volumetric flasks, it is estimated that some samples were up to 0.5 mL away from the intended level, casting an uncertainty into the concentrations of the solutions of no more than 5%.



Contamination of the curvette faces by fingerprints, droplets of solution, dust, or lint from kimwipes could cause error in several ways. Liquid residue could refract the light source, resulting in an incident wavelength other than the intended. In addition, absorption of incident and fluoresced light is possible, as well as scattering of incident light in such as way that it is "seen" by the fluorescence detector. This is not believed to be a factor in the results, as the curvettes were cleaned and inspected before each spectra was taken.

References(1)

1. Ebeid, El-Zeiny M., Fluorescence Quenching of Acridinium Ions in Sodium Dodecyl Sulfate Micelles, p 164-5, vol 62, no. 2, Journal of Chemical Education (February 1985).