Energy Transfer Lab Script

Abstract
A kinetic study of the energy transfer between two dyes, coumarin 1 (D) and sodium fluorescein (A), was carried out using a UV/visible spectrometer and a fluorimeter. The rate constant for energy transfer was found to be 3.0 x 1011 dm3mol-1s-1.

Introduction
Monitoring and quantifying the energy transfer between species is an important technique, as energy transfer is a vital process in many reactions, for example, the mechanism of light absorbance and subsequent energy trapping by chlorophyll during photosynthesis. The process of energy transfer is often reliant upon the energy overlap of the donor emission and the acceptor absorbance ranges, which can be observed by combining UV/Vis spectra and fluorescence spectra. In this case, the aim of the experiment was to use the magnitude of Stern-Volmer quenching of coumarin 1 by fluorescein to observe the energy transfer between them and obtain the rate constant for quenching, as well as the critical energy transfer distance, where the probability of energy transfer occurring is equal to the probability of any other deactivating process occurring.

Experimental
UV/Vis spectroscopy was performed on coumarin 1 in ethanol (25 cm3, 6 x 10-5 moldm-3) and fluorescein in ethanol (25 cm3, 1.6 x 10-5 moldm-3) over a 300 600nm range and max was obtained for both solutions. The solutions were then diluted by a factor of 20 and fluorescence spectroscopy was performed. Fluorescence spectroscopy was then performed on a number of mixtures of coumarin 1 (3 cm3, 3.0 x 10-3 moldm-3), fluorescein (0 4 cm3, 0 1.6 x 10-3 moldm-3) and ethanol (3 7 cm3).

Results and Analysis

Excitation and emission analysis Table 1: Raw UV/Visible absorption data for D and A max / nm Abs. at max Abs. at 375 nm 375 1.468 1.468 500 1.199 0.029

Dye D A

Abs. at 449 nm 0 0.029

Table 1 gives the absorbance data for the excitation spectra of D and A. Using these values of absorbance it is possible to use the Beer-Lambert Law (A = cl) to produce absorption coefficients for D and A. To do this, it is required to know the concentrations of D and A used: [ ]

[ ] With these values it is now possible to calculate the absorption coefficients for D and A. For example, using the absorbance of D at 375nm and a cell path length of 1.0cm:

The rest of the values for the absorption coefficients for D and A are shown in Table 2 below. Table 2: Values of absorption coefficients for D and A Dye D A max / nm 375 500 max / dm3mol-1cm-1 25000 75000 / dm3mol-1cm-1 25000 1800 ' / dm3mol-1cm-1 0 1800

Table 3 below shows the raw data for the emission spectra of D and A. Table 3: Raw excitation and emission data for D and A ex / nm em / nm Iex 375 449 76 500 521 102

Dye D A

Iem 74.5 102

To monitor and quantify the energy transfer between the dyes they need to be combined in a mixture. By measuring the Donor intensity (the intensity of D) when the concentration of the acceptor (A) is varied, it possible to obtain ratios of the emission intensities. Table 4 shows the emission data for the mixtures used.

Vol. D / cm3 3 3 3 3 3

[D] / moldm-3 0.003 0.003 0.003 0.003 0.003

Table 4: Emission data for D/A mixtures [A] / Vol. A / cm3 moldm-3 Vol. basic EtOH / cm3 0 0 1 0.0004 2 0.0008 3 0.0012 4 0.0016

Donor intensity 7 6 5 4 3 757.9 544.0 438.2 354.2 319.3

I0, the peak height of the donor fluorescence without acceptor, is shown to be 757.9. I0 can also be expressed as a function of [A] and I: [ ] The ratio of quantum yields, 0/, is proportional to the corresponding ratios of the emission intensities, I0/I. However, a correction factor m is required to allow for the absorption of exciting light and fluorescence. Therefore:

m can be calculated using the equation: ( [ ] [ ] [ ] [ ] [ ] [ ] )

For example, when 1cm3 of A is used, m will equal:

The rest of the values of m, as well as the corresponding values of 0/ are given below in Table 5.

[D] / moldm 0.003 0.003 0.003 0.003 0.003

-3

Table 5: Values for the ratio of quantum yields [A] / moldm-3 m 0.0000 1.000 0.0004 1.017 0.0008 1.034 0.0012 1.050 0.0016 1.066

0/ 1.00 1.37 1.67 2.04 2.23

Plotting a linear regression of 0/ against [A] it is possible to observe the value of the Stern-Volmer quenching constant, K:

Figure 6: Relationship between quantum yield ratio and [A]

2.5 2 0/ 1.5 1 0.5 0 0 0.0002 0.0004 0.0006 0.0008 [A] / 0.001 moldm-3 0.0012 0.0014 0.0016 0.0018 y = 780.55x + 1.0373 R = 0.9903

The value of K is equal to the slope of the linear regression, which is shown to be 780.55 dm3mol-1. In theory, the value of the intercept should be equal to exactly 1. In this case, it is equal to 1.0373. However, this difference is very small, so the value of the intercept compares well with the theoretical value. The R2 value of 0.9903 is very close to 1, showing that the data fits the trend well. Using the Stern-Volmer quenching constant, it is possible to calculate the value of the rate constant of energy transfer. We know that:

And that:

For coumarin 1 in ethanol, we have been told that the value of 0 is equal to 0.64. The value of can be estimated using:

Therefore:

And:

When you compare this to a diffusion controlled rate constant, whose value is typically around 6 x 109 dm3mol-1s-1, it is plain that the kinetics of energy transfer are not limited by diffusion. The

remaining 102 dm3mol-1cm-1 of the energy transfer will most probably occur via dipole-dipole energy transfer. From the Stern-Volmer quenching constant it is possible to calculate [A]1/2, where the level of coumarin 1 fluorescence has decreased to of its original value. [ ] Using [A]1/2 it is possible to determine the distance at which there is equal probability of deactivating coumarin 1 via energy transfer or via unimolecular processes. This distance is known as the critical transfer distance, and is expressed as: ( [ ] )

Where L is Avagadros constant. Therefore: ( [ ] ) ( )

R0 can also be calculated theoretically, using the value of JDA for the overlap between coumarin 1 fluorescence and fluorescein absorption:

In this case, JDA = 1.14 x 1029 nm6mol-1, 2 (the orientation factor) is equal to 2/3, and n (the refractive index of the solvent) equals 1.36. Thus:

This value gives a reasonably similar (same order of magnitude) answer to the experimentally determined value, helping to validate the experimental value.

Discussion
For this experiment, the main results were the values of the kinetic rate constant, 2.98 x 1011 dm3mol-1s-1, the Stern-Volmer quenching constant, 780.55 dm3mol-1 and the experimentally obtained critical energy transfer distance, 5.32 nm.

When the excitation and emission spectra are combined they would be expected to give near mirror images, with the emission peak appearing to be very similar in shape and size to the excitation peak, but at a lower energy. This is due to the fact that when the dye is excited, it is not only excited to a higher electronic energy state, but to a higher vibrational energy state. The excited species will then undergo vibrational relaxation to its vibrational ground state, before undergoing an electronic transition to the ground state. This means that a lower energy photon is emitted than the one absorbed. This is demonstrated in the Jablonski diagram below.

This matches up well with the excitation and emission spectra observed, which give excellent near mirror images, with a small energy difference between then, and some overlapping. The mechanism by which energy transfer between the two species occurs is most likely a long-range Coulombic interaction. This is because, as shown on Figure 5, there is a poor overlap between the donor emission energy and the acceptor absorption energy, ruling out any radiative process. Also, the rate constant obtained is greater than the diffusion controlled rate constant would be, showing that collision between the species is not required, which rules out any short-range electron exchange, leaving only long-range Coulombic as a valid energy transfer mechanism. Additional proof lies in that the wavelength of light emitted by coumarin 1 (450-550 nm) is exactly that required to excite fluorescein, giving an excellent overlap of donor fluorescence and acceptor absorption, so there would be an efficient energy transfer with a long-range Coulombic interaction. During the taking of the fluorescence spectra it was important to control the configuration of the cell holder, as for this experiment we were using strongly absorbing species, with concentrations of around 1 x 10-3 moldm-3. Therefore, to observe intensities within a reasonable range, the cell holder was placed to perform right angle illumination. There are two main sources of non-human error in this experiment; the measuring of solutions during the creation of suitable concentration solutions to perform spectroscopy and any machine error in the recording of the spectra. However, both these sources of error are broadly unavoidable, and must simply be taken into account when performing analysis

Conclusions
The main conclusions from this experiment were that energy transfer between coumarin 1 and fluorescein occurs via a long-range Coulombic interaction, with a kinetic rate constant of 2.98 x 1011 dm3mol-1s-1 and a Stern-Volmer quenching constant of 780.55 moldm-3. Furthermore, the critical energy transfer distance was found to be 5.32 nm.