BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE PILANI

Pilani Campus, Chemistry Department

Instrumental Methods of Analysis, CHEM F313, Semester I, 2022- 23
Laboratory Manual

Experiment 02
Spectrofluorimetry: Determination of unknown concentration of Quinine Sulfate solution
Introduction
Fluorescence is the result of rapid emission of light energy from a molecule, which was excited by
light absorption. Fluorimetric methods are valuable for investigating many aspects of molecular
structure and have greatly enhanced the knowledge of mechanisms involved in excited state
reactions. Fluorimetric methods have also been used extensively for analytical purposes, particularly
by scientists in biological sciences. As an analytical method, fluorescence has an importance for its
high sensitivity and selectivity.
Photophysical processes in excited electronic states
When a suitable quantum of light falls on a molecule, the molecule goes to one of the vibrational
levels of its electronically excited state. (Franck-Condon Principle) The molecule which is present in
the higher vibrational level of the higher excited state (say S2) dissipates its excess vibrational energy
as thermal energy and goes to zero vibrational level of the S 2 state. The energy gap between the
higher electronic states is generally smaller than the energy gap between S1 and S0 states. For this
reason molecule arrives at S1 state quickly by internal conversion followed by vibrational relaxation
to v=0 level of S1 state. Now if there is an overlap between the zero vibrational level of S1 state and
higher vibrational level of S0 state, molecule comes back to the S0 state by internal conversion. It can
also release its excitation energy as photon. This radiative process is called fluorescence.

Figure 1. The Jablonski diagram of fluorophore excitation, radiative decay and nonradiative decay
pathways. S0 is the ground singlet electronic state; S1 and S2 are the successively higher energy
excited singlet electronic states. T1 is the lowest energy triplet state. (Figure taken from Principles of
Fluorescence Spectroscopy by Joseph R. Lakowicz, Springer, New York, 3rd Edition, 2006).

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 BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE PILANI
Pilani Campus, Chemistry Department
Instrumental Methods of Analysis, CHEM F313, Semester I, 2022- 23
Laboratory Manual

Substituent effect in fluorescence

Substituents can bring a substantial change in the absorption and fluorescence spectra of parent
molecules. With increase in conjugation, both the absorption and fluorescence band maxima and
intensities of transition increase. E.g., benzene, naphthalene, anthracene. The substitution of alkyl
group in the aromatic ring results in red shift in absorption and fluorescence spectra. The substitution
of electron donating groups such as –OH, NH2, etc results in large red shift whereas the presence of
halogen atoms on aromatic molecule decreases the fluorescence quantum yield. Electron
withdrawing groups decrease the fluorescence intensity. Molecular rigidity also increases the
fluorescence intensity.
Solvent effect
The environment around the molecule affects the electronic spectrum. The environment thus alters
the position and intensity of absorption and fluorescence spectra. There are two types of solvent
interaction. Dispersive interaction is dependent on the polarity of both solvent and solute. Specific
interaction is dependent on hydrogen bonding and complex formation between the solute and the
solvent. When the polarity of the solvent increases, the fluorescence band maxima gets red shifted,
whereas it is a blue shift when the polarity of the solvent decreases.
Fluorescence quenching
One difficulty that is frequently encountered in fluorescence is that of the quenching of fluorescence
by many substances. There are substances that, in effect, compete for the electronic excitation
energy and decrease the quantum yield. Iodide ion is an extremely effective quencher. Iodide and
bromide substituent groups decrease the quantum yield. Similarly, halogenated hydrocarbon
solvents and oxygenated solvents are also known to quench the fluorescence.
Instrumentation
Figure 2. A typical fluorometer design. LS is the
light source, EXO is the excitation optical train, SC
is the sample chamber, EMO is the emission
optical train, and DET is the optical detector. Both
the excitation and emission optical trains contain
beam-shaping and collimation optics. For
wavelength-resolved measurements, spectral
selection optical components such as
monochromators and filters are included in the
EXO and EMO. For polarization measurement,
polarizers are added to EXO and EMO. For lifetime
measurement, a laser light source is often used
and high-speed electronics are integrated into the
detector subsystem.

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 BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE PILANI
Pilani Campus, Chemistry Department
Instrumental Methods of Analysis, CHEM F313, Semester I, 2022- 23
Laboratory Manual

Determination of unknown concentration of quinine sulfate by fluorimetry

Principle: The fluorescence intensity is proportional to the concentration and from Lambert- Beer’s
Law,
If = 2.303 Φf Io ε c l
Where Φf is the fluorescence quantum yield, l is the path length, ε is the molar absorptivity and c is
the concentration.
Experimental procedure:
Reagents :
[1] 1000 mL 0.1 N sulfuric acid solution
0.1 N sulfuric acid to be prepared from concentrated sulfuric acid (98%). Show calculations. Use
distilled water for the solution.
(CAUTION!! HAZARDOUS, CORROSIVE) Take out slowly (from a measuring cylinder) and make up
the volume to 1 litre and mix well. Add ~ 200 mL of distilled water in the flask first to avoid sudden
heating]
[2] Quinine sulfate solution
Stock solution A: Weigh 10 mg of quinine sulfate powder and transfer to a 100 mL standard flask.
Add about 50 mL of 0.1 N sulfuric acid and shake well to dissolve. Make up the volume to 100 mL
with 0.1 N sulfuric acid, mix well. Dilute this solution whenever is required depending on the
experiment.
Stock solution B: Pipette out 10 mL of the above solution to another 100 mL standard flask and dilute
to 100 mL with 0.1 N sulfuric acid. This is 10 ppm solution of quinine solution.
Stock solution C: From the above, take 10 mL and dilute to 100 mL (with 0.1 N sulfuric acid) in another
standard flask. This is 1 ppm solution.
[3] Making experimental samples
Using stock solution C, prepare standard solutions of known concentrations as given below.
TWO unknown solutions would be provided to determine their concentrations.

Test Tube No. 1 2 3 4 5 6 7 8 9 10

Stock C (mL) 0 1 2 3 4 5 6 7 8 9
0.1 N Sulfuric Acid Solution
10 9 8 7 6 5 4 3 2 1
(mL)

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 BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE PILANI
Pilani Campus, Chemistry Department
Instrumental Methods of Analysis, CHEM F313, Semester I, 2022- 23
Laboratory Manual

Making acrylamide solution for quenching experiment: Prepare 10 mL of 5.0 M acrylamide solution.

Test Tube No. 1 2 3 4 5 6

Stock B (mL) 0.24 0.24 0.24 0.24 0.24 0.24
0.1 N Sulfuric Acid Solution (mL) 2.76 2.61 2.46 2.16 1.86 1.56

1 M Acrylamide Solution (mL) 0.00 0.15 0.30 0.60 0.90 1.20

Experiment
1. Record absorption spectrum of Stock solution A. Use proper reference solution. Determine max,abs
from the absorption spectrum.
2. Record fluorescence emission spectra of the prepared solutions [Test tube no. 1, 2, 3, ....] by
exciting at max,abs.
3. Record fluorescence emission spectra of one of the solutions used in step 2 by exciting at
wavelengths ±10 nm of max,abs (at least four such emission spectra).
4. Record the fluorescence spectra of quinine sulfate solution in presence of quencher, acrylamide.
Observations:
1. Record your observations by tabulating the concentration (0-0.9 ppm) versus fluorescence
intensity.
2. Plot a graph of the observed fluorescence intensity versus the concentration. Plot the absorption
spectrum and emission spectra together to get an understanding of the shifts in the position of the
maxima due to fluorescence.
4. Using regression analysis, plot the best fit and get the best-fit equation with the correlation
coefficient for both calibration graph and quenching plot.
5. Find the concentration of the unknown quinine sulfate with reference to the calibration graph.
6. Determine Stern-Volmer constant.
REPORT every experimental details (except procedure) along with necessary precautions and notes
in the Laboratory notebook/ record-book
…………………………

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BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE PILANI
Pilani Campus, Chemistry Department
Instrumental Methods of Analysis, CHEM F313, Semester I, 2022- 23
Laboratory Manual

Notes

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