# Fluorescence study of dyes Essay

Fluorescence Study of the

Kinetics of Energy

Transfer Between Dyes

1. Introduction

Coumarin 1 and Sodium fluorescein are two dyes which absorb and emit light in the visible region. By using a spectrophotometer we are recording an absorption spectrum and then determining the molar decadic absorption coefficients, which will be used later to interpret and analyse the fluorescence spectra.

For fluorescence spectra, there are two kinds, the excitation and the emission spectrum.

The excitation spectrum is obtained by measuring the intensity of the emission as the excitation wavelength is altered by scanning a monochromator. The emission spectrum is obtained by measuring the emission intensity as a function of wavelength for excitation at a fixed wavelength.

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An absorption spectrum and an excitation spectrum are by their nature actually equivalent.

The two dyes exhibit energy transfer properties. The donor is coumarin and the acceptor the fluorescein. When the donor is excited, it is naturally decaying to the ground state. However in presence of an acceptor, this process is enhanced trough energy transfer. We will distinguish between collisional energy transfer and dipole-dipole transfer. From here we will try to determine the quenching constant and the distance at which decay and energy transfer are equally probable, as well as prove that Stern-Volmer’s law and Frster’s theory are obeyed.

2. Results

2.1. Electronic Absorption Spectra

We made up stock solutions for both salts and diluted them down to use in the UV/vis spectrometer.

Coumarin 1

C = 8 x10-5 mol dm -3

We obtained a spectrum with a maximum at 376.5nm at an absorbance of 1.4573, using Beer-Lambert’s law, we deduced the molar decadic absorption coefficients.

Sodium fluorescein

C = 2.425 x10-5 mol dm -3

Here max was 500.5 nm at an absorbance of 2.0922

Summary of the results obtained.

Dye

max (nm)

max (m2 mol-1)

(m2 mol-1)

‘ (m2 mol-1)

D (Coumarin)

376.5

1.82 x 109

—

1.25 x 106

A (Fluorescein)

500.5

5.17 x 107

1.41 x 106

8 x 106

2.2. Fluorimetry

a) Perylene standard

/nmI

Emission Spectrum4384.063EX = 434nm

4672.620

Excitation Spectrum4102.755EX = 438nm

4374.208

These are the values for the maxima in both spectrum, for the graphs, see attached sheet.

b) Coumarin 1 C = 4 x10-6 mol dm -3

/nmI

Emission Spectrum3741.050EX = 377nm

4434.081

Excitation Spectrum3734.156EX = 443 nm

4461.026

b) Sodium fluorescein C = 1.212 x10-6 mol dm -3

/nmI

Emission Spectrum5167.453EX = 501nm

Excitation Spectrum5017.516EX = 516 nm

On the graphs of the standard and the two dyes, we can nicely see that the excitation and emission spectra are mirror images of each other overlaid.

The excitation wavelength in one is the highest emitting one in the other.

2.3. Energy Transfer

Stern-Volmer equation

0 /= 1 + K A(1)

with 0 /being the ratio of quantum yield and

K being the Stern-Volmer quenching constant

a) Experimental study

To test the Stern-Volmer equation, as well as to prove the dipole-dipole transfer, we did fluorimetric measurements with mixtures of different concentrations of D and A.

D

A

I

m

ratio

0.003 070.238 1.001188 0.9883

0.003

4.042E-04

49.052

1.002212

1.4288

0.003 8.084E-04 38.766 1.003237 1.806

0.003

1.213E-03

31.416

1.004262

2.2263

0.003 1.617E-03 25.816 1.005285 2.7064

Table 1: Results from D-A mixtures experiments.

withIbeing the intensity of light emitted

m being a geometric correction factor

and ratio the ratio of the quantum yields

Those values were used to fit the model expression (1),

using the following program

* NonLinear Regression.

MODEL PROGRAM K=1000000 .

COMPUTE PRED_ = 1+K*conc.

NLR ratio

/OUTFILE=’C:win95TEMPSPSSFNLR.TMP’

/PRED PRED_

/SAVE PRED RESID

/CRITERIA SSCONVERGENCE 1E-8 PCON 1E-8 .

Non-linear Regression

Iteration Residual SSK

1.0089160488 1000000.00

1.1.0031167306 1034392.20

2.0031167306 1034392.20

2.1.0031167306 1034392.20

Run stopped after 4 model evaluations and 2 derivative evaluations.

Iterations have been stopped because the relative difference between

successive parameter estimates is at most PCON = 1.000E-08

Nonlinear Regression Summary StatisticsDependent Variable RATIO

SourceDF Sum of Squares Mean Square

Regression 1 18.55731 18.55731

Residual43.116731E-037.791826E-04

Uncorrected Total 5 18.56043

(Corrected Total) 4 1.79521

R squared = 1 – Residual SS / Corrected SS =.99826

Asymptotic 95 %

AsymptoticConfidence Interval

ParameterEstimateStd. ErrorLowerUpper

K1034392.1957 12606.399781 999391.21871 1069393.1726

The crosses are marking the values obtained and the line is the fit based on (1).

So the best fit value for K is 1.034×106, with at confidence limit of +/- 3.5×104.

So K , the Stern-Volmer quenching constant is 1.034×103 mol-1 dm 3.

Parent variance 2ratio = 2.43×10-4.

The mean square residual s2ratio is 7.79×10-4

Reduced chi-square 2v = 3.20.

A value under 3 indicates a good fit, hence this shows that our data, is not really fitted onto the model.

b) Energy transfer rate constant

For coumarin, =0.64

Its intrinsic fluorescence lifetime is 0s = 1/(1.822×108)

Therefore

ktots = 1/ (0s)

=2.846 s-1

and

ket = K ktots

= 2.943x1011mol-1 dm 3 s-1

This value as an error estimation of +/- 9.961 x109mol-1 dm 3 s-1

And as ket kdiff , we can see that 98% of the total energy transfer is due to dipole-dipole transfer.

c) Frster energy transfer

A1/2 = 9.67 x10-4 mol dm -3

Transformation of the raw data through SPSS gives us the following graph.

Values obtained:

JDA = 6.897×1031 nm mol -1

R0 = 0.14 nm

(R0)eff = 0.584 nm based on equation (A3-13)

So we see that those are very clearly quite different, and that could show that it does not obey to Forster’s theory.

3. Conclusion

So we have shown that the quenching of Coumarin 1 by Sodium fluorescein is obeyed by Stern-Volmer kinetics, that there is mainly dipole-dipole transfer, but could not agree with Forster’s theory.