Index

Expt. Aim Of The Experiment Page No.

No.

1. Determination of the Quantum Yield of known 1

samples using steady-state spectroscopy.

2. To calculate the lifetime of 7-azaindole from 12

absorbance and emission data.

3. To measure the non-radiative decay rate of 7- 18

azaindole with standard solution of quinine
sulphate.

4. To measure the fluorescence spectra of 26

Anthracene.
 EXPERIMENT-1

Aim Of The Experiment:-Determination of the Quantum Yield of known

samples using steady-state spectroscopy.

Apparatus Required:-Origin Software(Any Version), Datadigitizer

Software, Virtual Labs Setup containing Solution of Rhodamine B and
Rhodamine 6G, pipette, cuvettes, spectrophotometer, spectrofluorimeter
etc.

Theory:-

1.1. Quantum Yield

The Quantum Yield ( ) is a ratio that expresses the number of species that
fluoresce relative to the total number of species that were excited. Earlier we
said that anything that reduces the number of excited state species that undergo
fluorescence is said to quench the fluorescence. The expression for the quantum
yield will depend on the rate constants for the different processes that can occur
for excited state species[1].
The quantum yield of a system (such as a fluorescent molecule) is determined
by the balance between the radiative and non-radiative transition rates within
it[2].
The radiative transition rate ( ) denotes radiative (light emitting) processes
such as fluorescence and phosphorescence whereas the sum of non-radiative
rates, includes processes such as internal conversion, intersystem crossing, and
energy transfer. The quantum yield is therefore the probability that a system in
the excited state deactivates through a radiative process to its ground state[2].

1
 Fig 1.1:- Quantum yield and radiative and non-radiative decay processes .
Image Courtesy:- Edingurgh Instruments

1.2. Relative Quantum Yield Method

In the relative method, the quantum yield of the sample interest is calculated by
comparing its photoluminescence emission to that of a reference standard of
known quantum yield. In conventional fluorescence spectrometers only a
certain fraction of the emitted light is collected and detected; with the size of
the fraction depending on numerous factors[2].
Formula is given as follows:-

( )

1.3. Why we use Relative Quantum Yield Method?

These factors include; the angular distribution (solid angle) of the emitted
photons, the refractive index of the solvent, wavelength, the scattering
properties of the sample and sample geometry. The fraction is, therefore,
impossible to accurately quantify which prevents direct measurement of the
quantum yield. The relative method overcomes this problem by using a
reference standard of known quantum yield and similar optical properties to

2
those of the sample. The emission spectra of the sample and reference standard
are measured under identical excitation conditions and the ratio of the
integrated emission used to calculate the quantum yield of the sample[2].

Procedure:-

1. Virtual Lab Procedure for extracting emission curves

i) At first step, you have open the given link of virtual labs pointed in
[3].
ii) Then at the simulator part, you will get an window containing a
setup of photofluorimeter, cuvettes and solution of Rhodamine B &
Rhodamine 6G.
iii) In this experiment of quantum yield, we have taken Rhodamine B as
Sample solution and Rhodamine 6G as reference solution. Both are
dissolved in Ethanol.
iv) Starting with absorption experiment of Rhodamine B, We sort the
Rhodamine B solution in pipette and pour it in quartz cuvette.
v) After that, we put the cuvette in spectrophotometer for absorption
measurement and absorption curve.
vi) After absorbance, we move forward for fluorescence measurement
and pour the solution in another cuvette and put it in
spectrofluorimeter and we get the emission curve of given solution.
vii) Similarly we have to perform steps (iv) to (vi) for Rhodamine 6G.
viii) Finally we have absorption and emission graphs for both the
solution.

2. Area Finding Procedure:-

3
i) After getting curves, we feed the emission curve of Rhodamine B in
given software[4] for extraction of XY data points.
ii) In the given software, we have to first set the point markers defining
lower and upper limit of X and Y axes.
iii) After defining limits, we put points corresponding to the curve and
extract each X & Y value.
iv) After extracting points, we have a set of data points and exported in
.CSV file.
v) CSV files are then opened in Origin Software and design the curve
according to given data points.
vi) NOTE:- Make sure that your previous emission graph should
resemble with the created graph by the origin.
vii) Now by integrating the curve at peak limits, you get the integrated
area and FWHM value.
viii) Similarly perform steps (i)-(vii) for calculation of Rhodamine 6G.

3. Final Process

i) Now you have values of area of both the curves (Sample and
Reference).
ii) Now you need absorbance values, Both Rhodamine B and Rhodamine
6G are excited at same wavelength i.e, 535 nm. So the ratio of
absorbance is unity.
iii) For refractive indices of solvent, Both the solutions are dissolved in
Ethanol. Hence we have taken refractive index of ethanol as 1.36 at
both the cases. Therefore the ratio of indices we got is unity.
iv) So by the help of relative quantum yield formula, we calculate the
value of quantum yield of Rhodamine B where the quantum yield of
rhodamine 6G is 0.95.

4
Observations:-

Fig 1.2 :- Virtual Lab Setup For Quantum Yield Calculation

Image Courtesy:-MFS Virtual Lab Simulation Website

Fig 1.3:- Absorption Spectrum Of Rhodamine B

5
 Fig 1.4:- Emission Spectrum Of Rhodamine B

Fig 1.5:- Emission Spectrum Of Rhodamine 6G

6
Calculations & Tables:-

Fig 1.6:- Snapshot for procedure of data points

collection.

Fig 1.7:- Data Points of Emission Curves

Left:- Rhodamine B Right:- Rhodamine 6G

7
 ---- Rhodamine B

Intensity (in a.u.)

Wavelength (in nm)

Fig 1.8:- Area Calculation Of Emission Curve

Of Rhodamine B

---- Rhodamine 6G
Intensity (in a.u.)

Wavelength (in nm)

Fig 1.9:- Area Calculation Of Emission Curve

Of Rhodamine 6G

8
 Absorbance
Rhodamine B Rhodamine 6G
0.032 0.032

Table 1.1:- Absorbance Of Rhodamine B and Rhodamine 6G

Given in Virtual Labs Setup

Area
Rhodamine B Rhodamine 6G
3.95617 5.42395

Table 1.2:- Area Of Emission Curve Of

Rhodamine B and Rhodamine 6G

Refractive Index
(Ethanol)
Rhodamine B Rhodamine 6G
1.36 1.36

Table 1.3:- Refractive Index Of Ethanol (Rhodamine B and Rhodamine 6G

Both are dissolved in Ethanol)

( )

On putting above values (from table 1.1 to

1.3) we get

0.69

9
 Quantum Yield
Rhodamine B Rhodamine B
(Calculated) (Actual)[5]
0.692919643 0.7

Table 1.4:- Quantum Yield of

Rhodamine B and Rhodamine 6G

Results & Conclusions:-

Here we have used relative quantum yield method to find the quantum yield of
Rhodamine B with the help of reference as Rhodamine 6G whose quantum yield
is known as 0.95 at high accuracy.
Absorbance values of both the solutions are given as 0.032.
After applying formula with curve areas and refractive indices, we calculated
area of emission curve is as follows:-
Rhodamine B:- 3.95
Rhodamine 6G:- 5.42
After calculating areas, we have calculated the quantum yield of Rhodamine B
as 0.69 with an error of 1%.

Precautions:-
i) 3ml solution should be precisely filled in pipette ( if doing experiment
in physical lab)
ii) Precisely calculate the curve points by the given online software.
iii) Area should be extracted from Emission curves.
iv) Must to take refractive index of solvent not the solutions.

10
References:-
[1] [Link]
[2][Link]
d_Atomic_Spectroscopy_(Wenzel)/3%3A_Molecular_Luminescence/3.5._Quan
tum_Yield_of_Fluorescence_((varphi_ce_F))
[3] [Link]
[4] [Link]
[5] Divya, Kizhmuri P., Sivaraman Savithri, and Ayyappanpillai Ajayaghosh. "A
fluorescent molecular probe for the identification of zinc and cadmium salts by
excited state charge transfer modulation." Chemical Communications 50.45
(2014): 6020-6022.

11
 EXPERIMENT-2

Aim Of The Experiment:- To calculate the lifetime of 7-azaindole from

absorbance and emission data.

Materials Required:- Origin Software (for Graph analysis), Absorbance and

Emission (PL) Data/Graph of 7-Azaindole of dissolved in water having
refractive index 1.33, Cuvettes (1cm *X 1cm).

Theory
Fluorescence Lifetime
Fluorescence lifetime is the mean time elapsed between the activation of
fluorophore and emission of a photon from the fluorophore. The fluorescence
lifetime is the characteristic time that a molecule remains in its excited state
before returning to the ground state. During the excited state lifetime, a
fluorophore can undergo conformational changes, interact with other molecules,
and rotate and diffuse through the local environment [1].

Stricler Berg Equation is used for calculating Fluorescence Lifetime and

radiative rate [2]:-
∫ ( ) ( )
∫ ( )
∫ ----- (1)

Where = Radiative Rate

 = Fluorescence Lifetime

 = Wavenumber (in )

() = Molar Extinction Coefficient.

12
7-Azaindole

7-Azaindole is the chromophoric moiety of the nonnatural amino acid, 7-

azatryptophan. The photophysics of 7-azaindole were originally studied by
Kasha and coworkers in nonpolar hydrocarbon solvents where it was suggested
to dimerize by forming two NIH--N7 hydrogen bonds. The major nonradiative
decay pathway of these dimers was shown to be a very rapid excited-state
tautomerization producing two NI-HN~ hydrogen bonds. An intriguing
characteristic of the emission of 7-azaindole in water is that only a smooth band
is detected and the fluorescence lifetime is single exponential when emission is
collected over the entire band over most of the pH range. 7-Azaindole exhibits a
single-exponential fluorescence decay of 910 ps in water at neutral pH and 20
OC if emission from the entire band ( 320 nm) is collected. The
fluorescence decay, however, deviates from single exponential if emission is
collected with a limited bandpass, 450 nm, a single exponential does
not provide a satisfactory fit . An acceptable fit is obtained using two
exponentially decaying components and indicates that about 20% of the
fluorescent emission decays with a time constant between 40 to 100 ps
(depending on the full-scale time base chosen for the experiment)[3]

Fig 2.1:- Structure Of 7- Azaindole

Image Courtesy:- Sigma Aldrich Website

13
Procedure:-

i) First step is to arrange absorbance and photoluminescence data of 7

Azaindole.

ii) Square the refractive index value of solvent i.e, here water.
iii) After squaring, convert the wavelength (given in nm) into wavenumber
(in ).

iv) After 3rd step, calculate the  and take the mean of all calculated
values.

v) From mean, inverse it to get the Fluorescence intensity.

vi) Now for integration part, you can take area under the absorbance curve
or extinction coefficient at maximum absorbance (Optical Density).

vii) Here taken optical density for better accuracy.

viii) After compiling all the numerical values from step (ii), (v) & (vii) and
putting it in formula given in , you can get the radiative rate.

ix) After inversing radiative rate, you will get fluorescence lifetime of that
particular fluorophore.

14
 Observations:-

Absorbance (in arb. Units)

Wavelength (in nm)

Fig 1.2:- Absorbance vs Wavelength Graph ( Here we have taken only that peak at which fluorescence takes place)
Image Courtesy:- Captured from Origin Software
Fluorescence Intensity

Wavelength (in nm)

Fig 1.3:- Fluorescence Intensity vs. Wavelength Graph ( Here we have taken only that peak at which fluorescence takes place)
Image Courtesy:- Captured from Origin Software

15
Calculations:-
Stricler Berg Equation is given as :

∫ ( ) ( )
∫
∫ ( )

After putting the values like:

( )

∫ ( )
∫ ( )

( )

After Putting all values in the given formula

&
ns

16
Results & Discussions:-
After analyzing both the absorbance data and photoluminescence data of 7 Azaindole
with concentration dissolved in water, we came up with following
conclusions:-

i) The maximum peak of absorbance graph is came as 0.2012 at 288 nm and

corresponding extinction coefficient is 20120.
ii) The maximum peak of emission graph is at 391 nm
iii) Mean value of Fluoresence Intensity is came as .

And finally we have calculated the following two important aspects:-

i) Radiative Rate:-
ii) Fluorescence Lifetime ( )

Precautions:-
i) Carefully plot the absorbance graph with an appropriate peak value
ii) Calculation of fluorescence Intensity part to be done with greater accuracy.
iii) Solvent should be selected appropriately.

References:-

[1] Ryohei Yasuda, in Neurophotonics and Biomedical Spectroscopy, (2019)

53-64.
[2] Strickler, S. J., and Robert A. Berg. "Relationship between absorption
intensity and fluorescence lifetime of molecules." The Journal of chemical
physics 37.4 (1962): 814-822.
[3] Smirnov, Alexandre V. et al. “Photophysics and Biological Applications of
7-Azaindole and Its Analogs.” Journal of Physical Chemistry B 101 (1997):
2758-2769.

17
 Experiment No. 3
Aim Of The Experiment:- To measure the non-radiative decay rate of 7-
Azaindole with standard solution of quinine sulphate.

Apparatus Required:- Origin software (any version), Absorbance and

Emission (PL) data/graph of 7-Azaindole of 10-5 M dissolved in water having
refractive index 1.33, Absorbance and Emission (PL) Data/Graph of Quinine
Sulphate of 10-5 M dissolved in H2SO4 having refractive index 1.43.

Theory:-
1.1. QUANTUM YIELD

The Quantum Yield ( ) is a ratio that expresses the number of species that
fluoresce relative to the total number of species that were excited. Earlier we
said that anything that reduces the number of excited state species that undergo
fluorescence is said to quench the fluorescence. The expression for the quantum
yield will depend on the rate constants for the different processes that can occur
for excited state species[1].
The quantum yield of a system (such as a fluorescent molecule) is determined
by the balance between the radiative and non-radiative transition rates within
it[2].

Non Radiative Transition:-

The total decay rate can be determined form the conductivity decay rate and the
radiative decay rate can be determined from the total photoluminescence decay
rate. The nonradiative rate is the difference between the total and the radiative
one. The sum of non-radiative rates, includes processes such as internal
conversion, intersystem crossing, and energy transfer.
1

18
i) Internal Conversion:- Internal conversion is the radiation
less transition between energy states of the same spin state. Internal
Conversion occurs because of the overlap of vibrational and electronic
energy states. As energies increase, the manifold of vibrational and
electronic eigenstates becomes ever closer distributed. At energy levels
greater than the first excited state, the manifold of vibrational energy
levels strongly overlap with the electronic levels. This overlap gives a
higher degree of probability that the electron can transition between
vibrational levels that will lower the electronic state[3].

ii) Intersystem Crossing:- Intersystem crossing is a radiationless

transition between different spin states. This where the electron changes
spin multiplicity from an excited singlet state to an excited triplet state.
It is indicated by a horizontal, curved arrow from one column to
another. This is the slowest process in the Jablonski diagram, several
orders of magnitude slower than fluorescence[3].

iii) Vibrational Relaxation:- the most common of the three--for most

molecules, occurs very quickly (<1 x 10-12 seconds) and is enhanced by
physical contact of an excited molecule with other particles with which
energy, in the form of vibrations and rotations, can be transferred
through collisions. This means that most excited state molecules never
emit any energy because in liquid samples the solvent or, in gas phase
samples, other gas phase molecules that are present "steal" the energy
before other deactivation processes can occur[3].

Internal conversion occurs in the same time frame as vibrational relaxation,

therefore, is a very likely way for molecules to dissipate energy from light
perturbation. However, due to a lack of vibrational and electronic energy state
overlap and a large energy difference between the ground state and first
excited state, internal conversion is very slow for an electron to return to the
ground state. This slow return to the ground state lets other transitive
processes compete with internal conversion at the first electronically excited
state. Both vibrational relaxation and internal conversion occur in most
perturbations, yet are seldom the final transition[3].

19
 Fig 3.1:- Demonstration of non-radiative processes
Image courtesy:- Sam Houston State University Website

Time Scales:-
It is important to note that a Jablonski diagram shows what sorts of transitions
that can possibly happen in a particular molecule. Each of these possibilities is
dependent on the time scales of each transition. The faster the transition, the
more likely it is to happen as determined by selection rules. Therefore,
understanding the time scales each process can happen is imperative to
understanding if the process may happen. Below is a table of average time scales
for basic radiative and non-radiative processes.

Transition Time Scale Radiative Process?

Internal Conversion 10-14 - 10-11 s no
Vibrational Relaxation 10-14 - 10-11 s no
Absorption 10-15 s yes
Phosphorescence 10-4 - 10-1 s yes
Intersystem Crossing 10-8 - 10-3 s no
Fluorescence 10-9 - 10-7 s yes

Table 3.1:- Time scale of each radiative & non-radiative process.

Data courtesy:- Chemistry Libretext Website

20
Procedure:-

i) First of all you have to extract absorption and photoluminescence data

of 7-Azaindole (sample) & Quinine Sulphate (reference solution).

ii) For calculation of non-radiative decay rate, you need quantum yield and
intrinsic lifetime of the sample as given in the formula in appendix.

iii) From the two solutions, first take data of Quinine Sulphate and extract
the following values:-

a) Quantum Yield (To be extracted online as it is known)

b) Absorbance (From Absorption Data)
c) PL area (From PL data) (Plotted Graph---- Integrate-- Area)
d) Refractive index of H2SO4 (Solvent)

iv) As calculated in step (iii), extract same parameters for 7-Azaindole

dissolved in water and calculate the quantum yield of the respective
sample by the formula given in equation (i).

v) Lifetime calculation of 7-Azaindole can be done by using Stricler Berg

equation (already done in Exp 2).

vi) By the help of calculated values in step (iv) and (v), you can measure
both radiative rate and non-radiative rate given in equation (ii) and (iii).

21
Observations:-

Area=1.05515E8 PL 310
FWHM=80.90619
1400000

1200000

1000000

800000
B

600000

400000

200000

300 350 400 450 500 550 600 650

A
Fig 3.2:- Area Calculation Of Emission Curve
Of Quinine Sulphate.
Image courtesy:- Origin software

Area=9.32415E6 PL
FWHM=74.43659

120000

100000

80000
B

60000

40000

20000

300 350 400 450 500 550 600

A
Fig 3.3:- Area Calculation Of Emission Curve
Of 7-Azaindole.
Image courtesy:- Origin software 5

22
Calculations:-
Formulas are:-

( )

Sr. No. Parameters Values

1. 0.546
2. Absorbance 0.057
3. PL Area 1.08*10^9
4. Refractive Index (H2SO4) 1.43
Table 3.2:- Parameters Of Quinine Sulphate

Sr. No. Parameters Values

1. Absorbance 0.2012
2. PL Area 9.33*10^6
3. Refractive Index (Water) 1.33
Table 3.3:- Parameters Of 7-Azaindole

After putting all the values in formula (i) given below

( )

And Lifetime is 0.89 ns (Calculated in previous experiment by Stricler Berg

equation)
After putting the values in formula (ii) given below

23
 ⇒

Results & Conclusions:-

After performing the experiment and extracting all the values of absorbance,
refractive index (both for reference & sample) etc, we have successfully
calculated the following parameters:-

i) PL area of Quinine Sulphate is and 7- Azaindole is

ii) The calculated quantum yield of 7-Azaindole is calculated as 0.0118

iii) Finally, we have calculated the non radiative rate of 7-Azaindole
and value came up as .
iv) From the formulas given in appendix, we can calculate the radiative
rate also.

Precautions:-
i) Area of peak should be calculated precisely.
ii) Take care of units in every calculation.

References:-
[1] [Link]
[2][Link]
d_Atomic_Spectroscopy_(Wenzel)/3%3A_Molecular_Luminescence/3.5._Quan
tum_Yield_of_Fluorescence_((varphi_ce_F))
[3][Link]
_Textbook_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistr
y)/Spectroscopy/Electronic_Spectroscopy/Jablonski_diagram

24
Appendix:-
Derivation of radiative rate and non radaitive rate formulas

We Know , -------(iv)

And (for natural lifetime, –(v)

(for radiative and non radiative decay) ---(vi)

Hence, ( ) ⇒ ------(vii)

⇒ -----(viii)

Subtract (iv) from 1,

 ( ( )) -----(ix)

 ( )

 ( )

 ------(x)

25
 Experiment No. 4
Aim Of The Experiment:- To measure the fluorescence spectra of
Anthracene.

Apparatus Required:- Virtual Labs Setup containing solution of

Anthracene (1.3×10-4 M) dissolved in cyclohexane, pipette, cuvettes,
spectrophotometer, spectrofluorimeter etc.

Theory:-
In fluorescence, first the light (photon) absorption takes place leading to the
electronic excitation and then part of the acquired (absorbed) electronic
energy is lost via the emission of a new photon. Therefore, light absorption,
excitation and emission are three important processes in fluorescence
spectroscopy. These events are experimentally captured as
absorption/excitation spectra and emission spectra and provide important
information about the electronic distribution in ground and excited states of
the molecules, any modification of the electronic distribution in these states
due to charge transfer, solvent relaxation, etc. The absorption energy is
higher than the emission energy and the emission energy is independent of
the excitation one.
What is fluorescence spectrum?
A fluorescence spectrum is generally a plot of the fluorescence intensity as
a function of wavelength (expressed in nm). In fluorescence spectra, when
spectral distribution of emission as a function of wavelength is scanned by
holding the excitation wavelength constant (at a wavelength at which the
molecule absorbs), an emission spectrum is obtained. Similarly, scanning of
the excitation spectral distribution by holding emission wavelength fixed
gives excitation spectrum[1].

26
Difference between emission spectra and absorption spectra?
The absorption/excitation spectra originate from the ground state and
therefore, these spectra provide information regarding the electronic
distribution in this state. Emission originates from excited states, and so the
emission spectra reflect the electronic distribution within the excited states.
If any modification of the electronic distribution in these states occurs such
as due to a charge transfer that will modify the corresponding spectra. The
width of a band in the absorption and emission spectra of a chromophore
located in a solvent is a result of two effects: homogeneous and
inhomogeneous broadenings. Homogeneous broadening arises due to the
existence of a continuous set of vibrational sub-levels in each electronic
state [1].
Mirror Image Rule
Fluorescence emission spectrum is often the 'mirror image' of the excitation
spectrum. This is known as the 'mirror image rule'. The absorption of
radiation promotes the molecule mainly from the v=0 level of the ground
electronic state, S0, to different vibrational levels in upper electronic state,
S1. The vertical transition has the highest transition probability and
intensity and the transitions to other vibrational levels occur with lower
intensity. Therefore, instead of an electronic absorption occurring at a
single, sharp line, electronic absorption consists of many lines each
corresponding to the stimulation of different vibrations in the upper state.
This gives rise to vibrational structure (or progression) in an electronic
transition. Therefore, the excitation (absorption) spectrum structure reflects
the vibrational levels of the upper S1 state. Because the fluorescence
emission involves transition from v=0 of S1 state to various vibrational
levels of S0 state, emission spectrum will also show a similar vibrational
structure but reflecting the vibrational levels of the ground S0 state. Since
the vibrational levels in the excited states and the ground states are similar,
the vibrational structures in the excitation and emission spectra appear as
mirror image of one another[1].

27
Procedure:-

1. To take a particular solution, first click on the appropriate concentration

on the concentration selection bar and then on the volumetric flask.
2. Carry out absorption and emission measurements as follows (Fig 4.1)
3. Take a quartz cuvette (path length, 1 cm x1 cm) by clicking on it for
spectrophotometric measurement. Quartz cuvettes for
spectrophotometric measurements are transparent only on two opposite
sides, unlike the all-side transparent quartz cuvettes used for
fluorescence measurements.
4. Click on the 5 mL capacity pipette to collect 3 mL of the experimental
solution which will be transferred into the quartz cuvette.
5. Click on the pipette to draw the solution into it
6. Click on the pipette to take it out of the volumetric flask and transfer the
solution into the cuvette.
7. To start the absorption spectral scan, click on the pop-up Start
Absorption Measurement
8. Turn on the spectrophotometer clicking on the power button. In real
operation, it takes approx. 30 min for initialization of the instrument.
9. Open the lid of the sample chamber of the spectrophotometer by clicking
on the lid for placing the sample in the cell-holder.
10. Click on the cuvette to place it in the sample holder. One has to use
pure solvent as the sample blank or reference in this measurement. Here
a double beam spectrophotometer is shown.
11. Close the chamber lid by clicking on it.
12. Open the measurement set-up screen by clicking on the absorption
measurement icon on the computer monitor.
13. On the screen, enter the wavelength range: Start: 620 nm End: 260
nm. In real operation, the wavelength range of incident light for the
sample is chosen and the wavelength scan is run via the accompanied
computer software. One can run the scan in absorbance (A) or
transmittance (%T) mode.
14. Click on the green 'Start' button on the measurement set-up screen to
run the wavelength scan. Observe the wavelength scan (Fig. 4.2)
15. Click on 'Close' button when spectral scan is complete. In real
operation, the scan data are stored in the computer. The instrument stores

28
 data and therefore asks for the Sample File name. One enters a file name
to save the data.
16. To take the cuvette out of the sample chamber, first click on the
sample chamber lid to open it and then on the cuvette.
17. Close the sample chamber lid by clicking on it.
18. Click on the pop-up: Start Fluorescence Measurement .
19. Turn on the spectrofluorimeter by clicking on the power button. In
real operation, it takes approx. 30 min for initialization of the instrument.
20. Click on the spectrophotometric quartz cuvette to transfer its content
into an all-side-transparent quartz cuvette of path length 1 cm x1 cm for
the fluorescence measurement.
21. For placing the sample in the instrument, open the lid of the sample
chamber of the spectrofluorimeter by clicking on the lid.
22. To place the cuvette in the sample holder of the spectrofluorimeter,
click on the cuvette.
23. Close the lid of the sample chamber by clicking on the lid.
24. Open the instrument set-up screen by clicking on the fluorescence
icon on the computer monitor.
25. Select the Emission Scan Mode on the screen.
26. On the screen, enter the excitation wavelength: 350 nm, emission start
wavelength: 360 nm and emission end wavelength: 650 nm. One
chooses the excitation slit(nm) and emission slit(nm) values (here 5 nm)
and the scan speed value (here medium ) also.
27. To run the wavelength scan for emission spectrum, click on the 'OK'
button on the set-up screen (Fig. 4.3).
28. Click on 'CLOSE' button when spectral scan is complete. In real
operation, the scan data are stored in the computer. The instrument stores
data and therefore asks for the Sample Filename. One enters a file name
to save the data.
29. To take out the cuvette out of the sample chamber, first click on the
sample chamber lid to open it and then on the cuvette.
30. Close the lid of the sample chamber by clicking on the lid.
31. Click on 'Reset' button to start over the measurements.
32. Discuss how the maximum emission wavelengths and the intensities
of the shortest wavelength emission bands of Anthracene change with
increasing concentration of Anthracene (Fig 4.4).

29
Observations:-

Fig 4.1:- Experimental setup for fluorescence spectra simulation

(Virtual lab courtesy:- MFS Lab, IIITH)

Fig 4.2:- Observation of absorption spectrum

30
 Fig 4.3:- Observation of emission spectrum

Fig 4.4:- Respective absorption and emission spectrum of

anthracene dissolved in cyclohexane

31
Results & Discussions:-
After performing the experiment of extracting absorption spectra and
emission spectra of anthracene dissolved in cyclohexane, we can conclude
that:-
i) In fig 4.4(a), there are three peaks distinctly visible in absorption
spectra i.e., at around 325 nm, 350 nm and 375 nm. Out of these
three, maximum absorption is seen at 350 nm which will be our
excitation wavelength for emission purpose.
ii) In fig 4.4(b), by taking excitation wavelength of 350 nm, we get an
emission spectrum which contains three emission peaks present at
range of around 375 nm to 425 nm.

References:-
[1] [Link]

32