Lecture Notes - Handouts

10/4/2013

UV/VIS Excitation - Emission Spectroscopy

Organic Molecular Fluorescence Spectroscopy

UV/VIS Emission Spectroscopy

Organic Molecular Fluorescence Spectroscopy

CLASSIFICATION OF METHODS

History and Background

FS OMFS

Theory and Principle of OMFS

Multivariate FS Based on chemometric methods PLS, ANN

Instrumentation

Total Luminescence Synchronous Fluorescence

Sources

CWSF

CESF

Detectors

FP

Measurement of Fluorescence Spectra

FRET

Analytical applications of OMFS

FFS FCS
Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

UV/VIS ES OMFS - Background

4 October 2013

OMFS
Background

Types of Light Emission

Ultraviolet (180-380nm) or visible (380-750nm) radiation interacts

with matter which causes electronic transitions (From ground state to
a high energy state).

Luminescence

Fluorescence Spectroscopy (FS) deals with the study or measurement

of emitted radiation by fluorescing species. The emission
(fluorescence) spectra generally obtained by passing UV radiation
(wavelength 260 to 380 nm) on organic or inorganic samples.

Chemiluminescence

Radioluminescence

Organic Molecular Fluorescence Spectroscopy (OMFS) is the study of

optical emission of organic molecules that have been excited to higher
energy levels by absorption of electromagnetic radiation. The main
advantage of fluorescence detection compared to absorption
measurements is the greater sensitivity achievable because the
fluorescence signal has in principle a ZERO BACKGROUND.

Photoluminescence

Fluorescence

Phosphorescence

Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

Senthil Raja A, Dept of Pharma, IIT (BHU) VNS.

Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

Lecture Notes - Handouts

10/4/2013

OMFS - Principle

OMFS - Principle

Absorption of UV radiation by a molecule excites it from a

vibrational level in the electronic ground state to one of the many
vibrational levels in the electronic excited state.

This excited state is usually the first excited singlet state. A

molecule in a high vibrational level of the excited state will quickly
fall to the lowest vibrational level of this state by losing energy to
other molecules through collision.

Fluorescence occurs when the molecule returns to the electronic

ground state, from the excited singlet state, by emission of a
photon.

The electronic states of most organic molecules can be divided into

singlet states and triplet states;

Singlet state: All electrons in the molecule are spin-paired

Triplet state: One set of electron spins is unpaired

Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

Fig 1a. Jablonski energy diagram of emission spectroscopy

OMFS - Principle

Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

OMFS - Principle

Principle of OMFS

Following absorption, a number of vibrational levels of the
excited state are populated.

Molecules in these higher vibrational levels then relax to the
lowest vibrational level of the excited state (vibrational
relaxation).

From the lowest vibrational level, several processes can
cause the molecule to relax to its ground state. The most
important pathways are:

Fig 1b. Jablonski energy diagram of emission spectroscopy

Fig 1c. Transition between

molecular electronic energy
states
Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

Senthil Raja A, Dept of Pharma, IIT (BHU) VNS.

Collisional deactivation (external conversion) leading

to non-radiative relaxation.

Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

Lecture Notes - Handouts

10/4/2013

OMFS - Principle

OMFS - Principle

Intersystem Crossing (10-9s): In this process, if the

energy states of the singlet state overlaps those of the
triplet state, vibrational coupling can occur between the
two states. Molecules in the single excited state can
cross over to the triplet excited state.

Phosphorescence (10 to 100 sec): This is the

relaxation of the molecule from the triplet excited state
to the singlet ground state with emission of light.
Because this is a classically forbidden transition, the
triplet state has a long lifetime and the rate of
phosphorescence is slow.
Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

Fluorescence(~10-8 sec): Corresponds to the relaxation of the

molecule from the singlet excited state to the singlet ground
state with emission of light.

Fluorescence has short lifetime so that in many molecules it can

compete favorably with collisional deactivation, intersystem
crossing and phosphorescence.
The wavelength (and thus the energy) of the light emitted is
dependent on the energy gap between the ground state and the
singlet excited state.

Internal Conversion(10-12 sec): Direct vibrational coupling

between the ground and excited electronic states (vibronic
level overlap) and quantum mechanical tunneling (no direct
vibronic overlap but small energy gap) are internal conversion
processes.

This is a rapid process relative to the average lifetime of the lowest

excited singlet state (10-8 sec) and therefore competes effectively with
fluorescence in most molecules.

Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

OMFS Principle - QE

4 October 2013

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OMFS Principle - QE

Other processes, which may compete with fluorescence, are

excited state isomerization, photoionization, photodissociation
and acid-base equilibria. Fluorescence intensity may also be
reduced or eliminated if the luminescing molecule forms ground
or excited state complexes (quenching).

Factors of affecting FE or FQY

Chemical Structure

The quantum yield or quantum efficiency for fluorescence is

therefore the ration of the number of molecules that luminesce
to the total number of excited molecules.

Chemical Environment

The quantum yield/efficiency () for a compound is determined

by the

Relative rate constants (kx) for the processes which deactivate

the lowest excited singlet states, namely, fluorescence (kf),
intersystem crossing (ki), external conversion (kec), internal
conversion (kic), predissociation (kpd), and dissociation (kd).
Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

Senthil Raja A, Dept of Pharma, IIT (BHU) VNS.

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Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

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Lecture Notes - Handouts

10/4/2013

OMFS - Instrumentation

OMFS - Instrumentation

Fluorescence Spectrophotometer - Instrumentation

Fluorescence Spectrophotometer - Instrumentation

There are two major types of fluorescence instruments

1. Filter fluorometers (use filters to isolate the incident light
and fluorescent light)
2.
Spectrofluorometers
(use
monochromators to isolate the
fluorescent light)

diffraction
grating
incident light and

Fig. 2. Flow Diagram of a Spectrofluorometer

Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

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OMFS - Instrumentation

Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

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OMFS - Instrumentation
SAMPLE COMPARTMENT Sample cells

SOURCE OF LIGHT
Two types of light sources:

Wide spectrum (tungsten halogen lamp)

Series of discrete lines (mercury lamp)

DETECTOR USED - PMT

EXCITATION & EMISSION FILTERS / MONOCHROMATORS

Fluorometer: Filters used for fixing the ex and emission

wavelengths

Spectrofluorometer : Monochromators are used in place

of filters

Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

Senthil Raja A, Dept of Pharma, IIT (BHU) VNS.

15

Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

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Lecture Notes - Handouts

10/4/2013

OMFS Measurement of Spectra

OMFS Measurement of Spectra

Fluorescence spectroscopy can yield low detection limits,

high sensitivity and high specificity.

The high specificity is largely due to the fact that
fluorophores exhibit specific excitation (absorption) and
emission (fluorescence) wavelengths.

These wavelengths can be determined via the collection of
two spectra, an excitation spectrum and an emission
spectrum.

Although the approximate excitation and emission
wavelengths for many molecules are known, these
wavelengths should generally be optimized for the
specific conditions employed.
Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

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OMFS Measurement of Spectra

6. Always orient cells in the same direction in the cell holder.

When using a matched pair of cells, always use the same cell
for the reference.
7. For the disposable plastic cells, solvents like methanol and
ethanol can be contained for a maximum time of 5 min. Never
use the plastic cells for toluene.

Senthil Raja A, Dept of Pharma, IIT (BHU) VNS.

2. Handle cells only at the top portions of the side plates that
do not face the optical axis.
3. When filling cells with sample solutions, a dropper, or
preferably a pipette, should be used rather than direct
pouring/ simple transfering from a beaker or test tube.
4. Rinse the cell with several portions of the solution before
filling. Avoid overfilling the cell.
Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

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Measurement of EMission (fluorescence) spectrum

5. Do not spill liquid on the outside of a cell. Before inserting a

cell into the holder, carefully wipe the cell windows with a clean
lens tissue or suitable absorbent lint-free disposable wiper.

4 October 2013

1. Never touch the optical surfaces of the cell. Contact with

the skin will invariably leave a film that, though invisible to
the eye, will change the light transmission and reflection
characteristics of the cell windows, especially in the ultraviolet
region.

OMFS Measurement of Spectra

Handling rules for sample cuvettes (Contd)

Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

Handling rules for sample cuvettes

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Emission spectrum is measured by keeping the excited

light at a constant wavelength and measuring the
different frequencies of fluorescent light emitted by a
sample.
Measurement of EXcitation spectrum
Excitation spectrum is measured by recording the sum
of the fluorescent light emitted at all frequencies as
function of the frequency of the monochromatic
incident light.
Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

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Lecture Notes - Handouts

10/4/2013

OMFS APPLICATIONS

OMFS APPLICATIONS
Example 1. Analysis of Fluorescein (QE, almost unity)

Applications of OMFS

For the analysis of

Organic fluorophores

Bulk flourophoric drugs

Flourophoric drugs in formulations

Biochemical fluorophores and flourophoric drugs and
their metabolites

Absorption Max: 498nm

Fluorescence Max: 518nm

For protein studies

Qualitative and quantitative studies of

Small peptides

Large polypeptides

Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

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Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

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OMFS APPLICATIONS
Example 2. Analysis of Quinine - Calibration Curve Method

Note: For the quinine analysis, the plastic disposable cuvettes

may be used.

A calibration curve of quinine will be constructed using a series

of standard solutions.

The concentrations of these solutions usually are 0.1, 0.2, 0.3,

0.4, 0.5 and 0.6 ppm.

The excitation and emission maximums for quinine are 350

and 445 nm respectively.

The exact maximums may differ from those given above. You
may want to determine what they are exactly under our
experimental conditions.

Senthil Raja A, Department of Pharmaceutics, IIT (BHU), Varanasi

4 October 2013

Senthil Raja A, Dept of Pharma, IIT (BHU) VNS.

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