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Fluorescence has many practical applications such as mineralogy and chemical sensors. In this experiment, the fluorescence spectrum of an organic dye, Fluorescein was obtained and compared with its absorption spectrum. It was found that Fluorescein has a maximum absorption wavelength at (486±2) nm and maximum fluorescence emission at (517±2) nm. The cause in the shift of the wavelength between the absorbed and the emitted photons, known as the stroke’s shift, was caused by the collisional and vibrational non-radioactive decay in which some of the energy from the absorbed photon is converted into heat to the surrounding molecules. Hence the emitted photons have less energy and longer wavelength as:
E=hv (1)
These processes occur before the fluorescence because they have a much shorter lifetime (10-12 s) compared to the lifetime of the fluorescence (10-8 s) and thus competes effectively with fluorescence.
The aim of this experiment is to obtain the absorption and fluorescence spectrum in the organic dye molecule, Fluorescein.
First, the spectrum of a xenon lamp was obtained using a monochromator combined with a photomultiplier tube detector. Then a fluorescence dye was inserted between the xenon lamp and the detector in order to obtain the absorption spectrum of the fluorescein dye. The equipment set up was then altered to investigate the fluorescence spectrum of the dye. The absorption spectrum and the fluorescence spectrum was plotted and compared.

Fluorescence corresponds to the relaxation of the molecule from the singlet excited state to the singlet ground state with emission of light.1 In this experiment, the Fluorescence spectrum of a synthetic organic dye, Fluorescein was obtained and investigated. Fluorescein has the following molecular structure as figure [1] shows:

Figure [1]2

There are mainly three stages that lead to the fluorescence by a molecule which are excitation, non-radioactive decay and emission. This process is shown clearly on a Jablonsiki diagram above in Figure 2. When a photon with energy hν strikes a molecule, the molecule gets excited to one of the electronic excited state. The electronic excited molecule is subjected to collisions with the surrounding molecule where it loses energy (collisional relaxation) and move down in the vibrational levels and eventually reaches the minimum vibrational energy level of the lowest electronically excited molecular state. Due to the energy difference between the ground state and the excited state is much greater than that of the vibrational energy levels, it now becomes difficult for the neighbouring molecules to lower the molecule to the ground state by colliding. Therefore, the molecule would have sufficient time to undergo fluorescence or the relaxation by the emission of a photon to cool back to the ground state.
Overall the energy balance for fluorescence process can be written as:
EFluro=Eabs-Evib.relax-Esolvent.relax4 (2)

In this experiment, a Xenon lamp was used as the source to provide photons to excite the Fluorescein dye because Xenon lamp has a maximum emission at around 485nm which is very close to the maximum absorption wavelength of Fluorescein.1 By using a monochromator combined with a photomultiplier tube connected to an ocillioscope, the absorption and the fluorescence spectrum can be measured and recorded over a range of wavelength from 330nm to 650nm. Fluorescein has a relatively high quantum yield, around 79%, compared to the average efficiency of fluorescence molecules.1 However, in order to obtain a signal with sufficient amplitude on the oscilloscope without increasing the terminating resistance to a very high value, the dye with 20mg/L concentration was used. (Please refer to lab book page 90-95 for method to obtain the normalised absorption spectrum of the dye.)
By plotting a graph of amplitude obtained from oscilloscope against the wavelength (adjusted by the monochromator), the maximum absorption and fluorescence wavelength can be found and compared. (Please refer to lab book page 96)
Experimental methods:
A xenon lamp was placed at 90 degrees angle to a monochromator/photomultiplier assembly. A cell containing the Fluorescein dye solution, Fluorescein of 20mg/L concentration, was placed in front of the monochromator. The xenon lamp was switched on and shined on to the cell containing the dye. The re-emitted light by the dye caused by fluorescence was observed on an oscilloscope and the amplitudes were recorded over a range of wavelength from 330nm to 650nm by adjusting the filter on the monochromator. The resulting spectrum was plotted and analysed with the absorption spectrum of the same dye with lower concentration. (Please refer to page 96 on the lab book for setup diagram)

Figure [3]

Figure [4]
Figure [3] shows that the maximum absorption wavelength of the Fluorescein is at (487±2)nm whereas in Figure [4] the Fluorescence spectrum of the same dye shows that the maximum emission wavelength is at (517±2) nm. This implies that the emitted photons has less energy than the photons absorbed. This shift in the wavelength will be discussed further in the Discussion section. (Data of graph from page 95 and 99 in the lab book)

Discussion and analysis:
The theoretical value of excitation transpires at 495nm and the emission at 521nm. The values obtained in the experiment which were (486±2) nm and (517±2) nm which were very close to the theoretical value if the systematic error of the monochromator of ±5nm were taken into account. The calibration error of the monochromator was checked by using the known wavelength of the Mercury lines.
Throughout the experiment, a black cloak was used as a cover to prevent light other than the light source to enter the detector.
The largest source of error was present in taking the reading from the oscilloscope. Due to the large fluctuation of the signal, it was difficult to find the wavelength that produced the largest amplitude. This was solved by increasing the gain and decreasing the increment of wavelength from 5nm to 0.2nm around the peak.
The shift of the maxima observed in the two graphs is known as the Stroke’s shift which describes the difference in wavelength between the position of the band maxima of the absorption and emission spectra of the same electronic transition.1 Clearly the emitted photons at the maxima have less energy (by comparing their wavelength) than that of the absorbed photons which implies that there is an energy loss or non-radioactive decay before the fluorescence process as mentioned in the theory section. This is associated with Kasha’s law. When a photon with an appropriate energy strikes a molecule, the molecule will be excited from the electronic ground state E0 to one of the electronic excited states En or one of the vibrational sub-levels in En. However, instead of dropping from En to E0 with an emission of a photon, Kasha’s law states that the emission of the photon is only expected from the transition from the lowest possible excited state E1 to E0. Therefore, the emission wavelength is independent of the excitation wavelength.
The question of why fluorescence does not occur immediately after the excitation can be further explained by considering the lifetime of the molecule in the excited state. As soon as the molecule is in the excited state, relaxation can occur in several different ways other than collision deactivation mentioned before. For instance, the internal conversion or vibrational relaxation which is a very rapid process that occurs in 10-12 s will competes effectively with fluorescence process with a lifetime of around 10-8 s.1 Thus, molecules in the excited states usually undergo complete vibrational relaxation before reaching the lowest possible excited state because a significant number of vibration cycles transpire during the lifetime of molecule in the excited states. This explains why the molecule would undergo non-radioactive day to reach the lowest excited state before the fluorescence process. (Please use figure [2] for reference)
In addition, as the Figure 3 and 4 shows, the maximum amplitude of the fluorescence spectrum is much less than that of the absorption spectrum. This suggests that the actual quantum yield of the dye is much lower than the theoretical value of 79%. This might be caused by ‘quenching’. As discussed in the theory section before, the process of collisional relaxation suggests that the intensity of the fluorescence will depend on the ability of the solvent molecules to accept the electronic and vibrational quanta. For solvents that consist of molecules with sufficient spaced vibrational levels, such as water, can often accept the large quantum of electronic energy and therefore lower the molecule to the ground state and terminate the process of fluorescence, a process known as quench.1 This may explain the low quantum yield obtained in the fluorescence spectrum.
It was found that the fluorescence spectrum of the organic dye, Fluorescein has a maximum emission at (517±2) nm. The absorption spectrum of the same dye was found to be (487±2) nm. The stroke’s shift was explained by Kasha’s law and the low yield of the dye might be caused by the quenching effect of the solvent. The results were consistent with the theoretical results of maximum wavelength of absorption and emission if the calibration error of the monochromator was taken into account.
Possible further work:
Further work can be done on investigating how the fluorescence process of the dye will behave when the solvent of the dye is changed to check if there is a relationship between the intensity of the fluorescence emission of the dye and the solvent molecules. This would be important because acting as a chemical sensor in medical applications, it would be useful to know the behaviour of the spectrum in different pH of solvent as the dye might need to go through parts of human body that contains non-neutral pH solvents. By finding the peak emission wavelength of the dye under different pH value would contribute to designing a more sensitive detector built specifically to detect a certain range of wavelength of light which would not only improve the role of dye as an chemical sensor but also has the potential to obtain better image in mineralogy and gemmology.

2., accessed on 9/12/12

3., accessed on 9/12/12

4. J. E. O'REILLY, “J. CHEM. ED”. (1975)…...

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