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Sultan Qaboos University
College of Science
Department of Physics
PHYS5505
Spring 2015
Name: Osama Hashil Al-Bahri
ID: 92891
Advisor: Dr. Abey Issac

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Contents
Part Page
Acknowledgement……………………………………………..…….. 3
Abstract………………………………………………………………...…. 4
Introduction………………………………………………………..….… 4
Theory……………………………………………………………………... 5
Experimental Methods
a) Calibration of emission monochromator…...
b) Calibration of excitation monochromator....
c) Detection of fluorescence spectrum of
fluorescein dye ……………………..…………………..
7
8
9
Results……………………………………………………………………... 10
Discussion
a) Spectrometer characteristics………………………...
b) Emission spectrum of fluorescein dye………..…
12
12
Conclusion……………………………………………………………….. 13
References………………………………………………………………...
Appendix...……………….……………………………………………....
13
14

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Acknowledgements
I take this opportunity to express my profound gratitude and deep regards to my
advisor Dr. Abey Issac for his exemplary guidance, monitoring and constant
encouragement throughout the course of this project. I would like to thanks Dr. Osama
Abou-Zied from the Chemistry department for the kind supply of fluorescein dye
solution which was used in the spectroscopy measurement. Finally, I am very grateful to
the department of Physics, Sultan Qaboos University for giving me this opportunity to
do this project.

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Abstract
We developed a fluorescence spectrometer using two grating monochromators and a
CMOS camera which is used as the photo detector. The calibration of the pixel number
of the camera to nm was performed using a He-Ne laser. The relative accuracy of the
wavelength counter on both monochromators was tested by selecting light of different
wavelength of a tungsten lamp using the excitation monochromator and detects the
same wavelength using the emission monochromator. Finally, the setup was used to
record the emission spectrum of fluorescein dye molecules in water.
Introduction
Fluorescence spectrometer is an optical device used to measure the intensity
distribution of a light signal versus the wavelength. They are designed to operate over a
wide range of wavelengths, from gamma rays and x-rays to the far infrared. Typically
fluorescence spectrometers are used in the near UV to near IR range.
Fluorescence spectrometers are used in many fields. For example, they are used
in astronomy to analyse the radiation from astronomical objects and deduce chemical
composition by their characteristic spectral fingerprints. In chemical and physical
science, electronic transitions of chromophores or inter-band relaxation of excitons can
be studied using fluorescence spectroscopy [1,3].
In this work, we built a fluorescence spectrometer using two monochromators.
One unit serves as the excitation wavelength selector and the other unit spectrally
resolve the fluorescence signal. Using this home built spectrometer we recorded the
emission spectrum of fluorescein dye in water.

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Theory
a) Spectrometer
Resolution of a spectrometer is defined as the ability to resolve two close
wavelengths. It is usually expressed in full width at half maximum (FWHM). Resolution
depends on the focal length of the monochromator (greater focal length give better
resolution), slit width used (narrower slit width give better resolution. If a ccd or cmos
camera is used as the detector, then the pixel size of the chip define the resolution),
groove density of the grating and the diffraction order (usually first order is taken).
The FWHM of a spectrum recorded using a monochromator is the convolution
of the broadening due to a) the finite slit width of the monochromator and linear
dispersion of the grating, d(slit) b) the limiting resolution of the spectrometer which
incorporates system aberrations and diffraction effects, d(resolution) and c) natural line
width of the spectral line being probed, d(line). The equation can be written as [2,3]
2
)(
2
)(
2
)(
2
lineresolutionslit dddFWHM   [1]
fwawRd dslit /)(  [2]
Where w is the slit width, a is the inverse of the number of lines/nm of the grating and f
is the focal length of the monochromator. In this equation the term Rd is the reciprocal
linear dispersion of the spectrometer. Since the linewidth of the laser is very small,
broadening of the spectrum due to the term d(line) can be neglected. Now, the
resolution of the spectrometer can be written as
2
)(
2
)( slitresolution dFWHMd   [3]
b) Optical absorption and emission process

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UV-Vis absorption and fluorescence are referred to the optical transition
between the electronic states of a chromophore. These electronic transitions are
between the highly occupied molecular orbital (HOMO) and least unoccupied molecular
orbital (LUMO). Typically transition between HOMO and LUMO levels of an organic dye
molecule is between its  and * orbitals. Jablonski diagram which explains absorption
and emission process is shown in figure 1. Photon of appropriate energy excite the
molecule from the electronic ground state (S0) into certain vibrational level of the higher
singlet electronic excited state, say for example the first excited state (S1). Rapid
vibrational relaxation (in ps time scale) of the excited state results electron population in
the lowest vibrational level of S1. Fluorescence is the radiative transition from the lowest
S1 state to the vibrational manifold of the electronic ground state S0. Typically the time
scale of fluorescence is 1-10 ns. For most of the dye molecules, (0-0) transition appears
as the most intense band and (0-1) or (0-2) transitions appears as vibrational side bands
[1].
Figure 1: Jablonski diagram of absorption and fluorescence process between electronic ground
(S0) and first excited (S1) state of a chromophore.
The excited state of a molecule, 𝑆1 can relax by various competing pathways. It can
undergo non-radiative relaxation in which the excitation energy is dissipated
as heat (vibrations) to the solvent or relax via inter system crossing to a triplet state,

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which may subsequently relax via phosphorescence or by a secondary non-radiative
relaxation step. Energy transfer between different types of molecules is another type of
relaxation process. In most cases, the emitted light has a longer wavelength (lower
energy), than the absorbed radiation. This is known as Stokes shift. Stokes shift is an
advantage to spectrally isolate the fluorescence spectrum from the excitation light. This
is schematically shown in figure 2.
Figure 2: Schematic representation of Stokes shift between the absorption (blue) and emission
(green) spectra.
Experimental Methods
a) Calibration of emission monochromator
He-Neon laser is used to calibrate the emission monochromator. Collimated laser line
passed was through a solution of SnO2 in water which acts as a scattering medium. The
scattered light was collected at 900 configuration and focussed at the entrance slit
(width 100 m) of the emission monochromator, see figure 3. At the entrance port
(which is not a slit but an open strip), a CMOS camera was installed. The wavelength
selector wheel of the monochromator was set at 633 nm. In this configuration we were
able to see the first order diffraction of the laser line on certain pixel of the camera. We
note down the pixel number. Now, we turned the grating by 20 nm and note down the
new pixel number on which laser spot is observed. Then the difference in these two

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pixel number corresponds to 20 nm. In this way the pixel number to wavelength in nm
calibration was done. The results are shown in figure 6.
Figure 3: Schematic diagram of the experimental setup used for the calibration of the emission
monochromator.
b) Calibration of excitation monochromator
Calibration of excitation monochromator can also be performed in a similar way as
explained above. However, for the detection of fluorescence signal, we need to place
this monochromator at a different position, i.e. next to the halogen lamp. To avoid any
error in calibration due to a different optical path of the white light, we carried out the
calibration in a different way. We focus the white light at the entrance slit of the
excitation monochromator. The entrance and exit slit of the monochromator were set
to 100 m. Using the wavelength selector wheel we position the grating at 450 nm.
Then the light exit from the slit was collimated and scattered by the SnO2 particle in
water. The scatted light was then focused at the entrance slit of the emission
monochromator which has the same settings as the excitation monochromator, see
figure 4. Since the emission monochromator was already calibrated, we can do

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calibration of the excitation monochromator. This procedure was repeated for other
two wavelengths (550 nm and 650 nm). We found an offset of 5 nm for the excitation
monochromator which was corrected during the data analysis.
Figure 4: schematic diagram of the experimental setup used for the calibration of the excitation
monochromator.
c) Detection of fluorescence spectrum of fluorescein dye
After the calibration of both monochromators, the excitation monochromator was set to get
440 nm light from the white light continuum of the halogen spectrum. Fluorescein dye was
dissolved in distilled water and taken in a 1 cm cuvette. The fluorescence of the dye was
collected at 900
configuration and focussed at the entrance slit of the emission
monochromator, see figure 5. The fluorescence spectrum was recorded using the camera with
an integration time of 1 s.

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Figure 5: schematic diagram of the spectrometer developed for the acquisition of fluorescence
spectrum of fluorescein dye in water. Excitation was 440 nm.
Results
He-Ne laser line recorded on the CMOS camera is shown in figure 6a. Note that
the horizontal axis is in pixel. After the calibration of pixel into nm, the spectrum was re-
plotted and shown in figure 6b.
The emission spectrum of Fluorescein dye in water is shown in figure 7. For the
detailed analysis the spectrum was fitted with two Gaussians, one for the S0  S1 (0-0)
transition and the other for the (0-1) transition, see figure 8. The fit parameters are
shown in the inset.

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Figure 6: The spectrum of He-Ne laser recorded using the home built spectrometer. a) spectrum
in pixel number and b) calibrated spectrum in nm.
Figure 7: The emission spectrum of fluorescein dye in water recorded using the home built
spectrometer. The excitation was at 440 nm.

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Peak Center
(nm)
width
(nm)
1 519 22
2 548 36
Figure 8: The emission spectrum of fluorescein dye in water (black line) is fitted with the sum of
two Gaussian functions (red solid line). Individual fits (red dotted line) and the fit parameters
shown.
Discussion
a) Spectrometer characteristics
The emission monochromator was equipped with a 600 lines/mm grating. The focal
length of the monochromator is 300 mm. Using equation 2, we find the reciprocal linear
dispersion 5.6 nm/mm. A slit width of 100 m results )(slitd of 0.56 nm. Taking the
FWHM of the laser line from the recorded spectrum, we find the resolution of the
spectrometer )(resolutiond using equation 3 as 3nm. The resolution can be improved by
lowering the slit width.
b) Emission spectrum of fluorescein dye
The fluorescence spectrum shows (0-0) transition cantered at 519 nm whereas
the (0-1) transition at 548 nm. The (0-0) transition and is narrower (22 nm) than the (0-

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1) transition (36 nm). The features of the spectrum is consistent with the spectrum
recorded using a commercial spectrometer.
Conclusion
In conclusion we developed a fluorescence spectrometer. We used a halogen
lamp as the light source and two monochromators in the excitation and detection path.
A cmos camera was used as a detector. The obtained spectral resolution is 30.4 nm.
Using this home built spectrometer we studied the emission spectrum of fluorescein
dye in water.
References
[1] Joseph R Lakowicz, ‘Principles of Fluorescence Spectroscopy’, 3rd
edition, Springer 2006
[2] Eugene, ‘Optics’, 4th
edition, Addison Wesley 2002
[3] Bernard Valeur, Mário Nuno Berberan-Santos, ‘Molecular Fluorescence: Principles
and Applications’, 2ed
edition, Wiley-VCH 2013

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Appendix
Figure A1: Left) Partially developed spectrometer. The halogen light source is on the left side,
excitation monochromator at the middle and emission monochromator on the right side. The
USB CMOS camera is attached at the exit port of the emission monochromator. Right)
Diffraction pattern of the halogen lamp observed inside the excitation monochromator.
Figure A2: Left) Fluorescence of the dye molecule solution taken in a cuvette with 440 nm
excitation. Right) Image of the fluorescence spectrum of the dye molecules in the camera chip.