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Fluorescence study of dyes

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Fluorescence study of dyes

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.

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 F'rster'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.


max (nm)

max (m2 mol-1)

(m2 mol-1)

' (m2 mol-1)

D (Coumarin)


1.82 x 109


1.25 x 106

A (Fluorescein)


5.17 x 107

1.41 x 106

8 x 106

2.2. Fluorimetry

a) Perylene standard

/nm I

Emission Spectrum 438 4.063 EX = 434nm

467 2.620

Excitation Spectrum 410 2.755 EX = 438nm

437 4.208

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

/nm I

Emission Spectrum 374 1.050 EX = 377nm

443 4.081

Excitation Spectrum 373 4.156 EX = 443 nm

446 1.026

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

/nm I

Emission Spectrum 516 7.453 EX = 501nm

Excitation Spectrum 501 7.516 EX = 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 ...

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Keywords: fluorescence studies of dyes, which dye is used in fluorescence microscopy, what is the process of fluorescence, what is fluorescence in chemistry, what is fluorescence analysis

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