Birla Institute of Technology and Science, Pilani (Raj)

I Semester 2013-14
CHEM F313 Instrumental Methods of Analysis
Experiment 3
Spectro-fluorimetry
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 S2
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 a
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.
E denotes the energy scale; 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.

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. Eg., 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.
Flourescence 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
specimen 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 monochrometers 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 highspeed electronics are integrated into the detector subsystem.

Determination of unknown concentration of quinine sulfate by fluorimetry

Principle: The fluorescence intensity is proportional to the concentration and from LambertBeers Law,
If = 2.303 f Io c l
Where f is the fluorescence quantum yield, l is the path length, is the molar absorptivity
and c is the concentration.

Experimental procedure:
Reagents :
0.1 N sulfuric acid:
Take about 300 ml of distilled water in a 1 litre flask add 3 ml of concentrated Sulfuric acid
(98%) (Caution! Hazardous) slowly (from a measuring cylinder) and make up the volume
to 1 litre and mix well.
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 0.1N 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 100ml 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 100ml (with 0.1N sulfuric acid) in
another standard flask. This is 1 ppm solution.
Using stock solution C, prepare standard solutions of known concentrations as given below.
Test tube No.
Stock C (mL)
0.1 N Sulfuric acid (mL)
0.1 N KI solution

1
0
10
0

2
1
9
0

3
2
8
0

4
3
7
0

5
4
6
0

6
5
5
0

7 8 9 10
6
6
4
2
0
2

Experiment
1. Record absorption spectrum of Stock solution A. 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 excitation spectra of Stock solution A and compared the result
with absorption spectrum.
Observations:
1. Record your observations by tabulating the concentration (0-0.6ppm) versus fluorescence
intensity.
2. Plot a graph of the observed fluorescence intensity versus the concentration.
3. Find the concentration of the unknown quinine sulfate with reference to the calibration
graph.
4. Using regression analysis, plot the best fit and get the best-fit equation with the correlation
coefficient.

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