Contents • 3.2 Molecular Fluorescence and Phosphorescence Spectroscopy • (04L) • 3.2.1 Theory of Molecular Fluorescence and Phosphorescence • Spectroscopy. • 3.2.2 Jablonski Diagram of Energy Levels. • 3.2.3 Relationship between Fluorescence intensity and concentration, • Factors affecting Fluorescence and Phosphorescence. • 3.2.4 Instrumentation and Applications of Fluorimetry and Phosphorimetry. • 3.2.5 Comparison ofFluorimetry and Phosphorimetry. • 3.2.6 Comparison of Fluorimetry with Absorption methods.

Introduction • The term ‘fluorescence’ was coined by G. G. Stokes in 1852 on the name of the mineral fluorspar (CaF2) that emits visible light on illumination with the UV light.

Basic Terms • Emission: When substance heated to about 725 K temperature ,it emits radiation in the form of ultraviolet or visible energy is called as Emission. • Photoluminescence: A body emits previously absorbed radiations even below this temperature is called as Photoluminescence or cold light. • When such Photoluminescent substance is excited, it reemits radiations of the same wavelength or longer wavelength. • If the emission takes place in a time of approximately10-8 sec. or less after absorption process is termed as Fluorescence.

Fluorescence :If the emission takes place in a time of approximately 10-8 sec. or less after absorption process is termed as Fluorescence. Re-emission of Radiation in less than 10-8 sec. U. V. Light Molecule

Phosphorescence:If the time elapsed between the absorption and re-emission of radiation becomes more than 10-8 sec. is termed as Phosphorescence Re-emission of Radiation in more than 10-8 sec. U.V.Light Molecule

What is a vibration in a molecule? Any change in shape of the molecule- stretching of bonds, bending of bonds, or internal rotation around single bonds.

154 pm 10 pm 10 pm 4o What is vibration stretching vibration For a C-C bond with a bond length of 154 pm, the variation is about 10 pm. bending vibration For C-C-C bond angle a change of 4o is typical. This moves a carbon atom about 10 pm.

Bond length 154 pm, 10 pm. For a C-C bond with a bond length of 154 pm, the variation is about 10 pm.

C C 10 pm 4o C For C-C-C bond angle a change of 4o is typical. This moves a carbon atom about 10 pm.

How does the mass influence the vibration? H2 I2 MM =2 g/mole MM =254 g/mole The greater the mass - the lower the wavenumber

Theory:Energy Level of Electron in a molecule • To begin with, the molecule is in the electronic ground state. In this state, the molecular orbital's are occupied by two electrons. You would recall from the knowledge about Pauli’s principle, the spins of the two electrons in the same orbital must be antiparallel. • This implies that the total spin, S, of the molecule in the ground state is zero [½ +& -½)].

Total spin, S, of the molecule in the ground state is zero [½ + ( ½)]. • This energy state is called “singlet state” and is labeled as S0. singlet state S0.

Excitation of Electron in a molecule • When the molecule is excited by U.V. light The electron spins in the excited state is called a singlet (antiparallel) state. • It is represented by S1.(S0—S1)

Excited singlet S1 Singlet (antiparallel) S1 S1 S0

Excitation of Electron in a molecule • Similarly excited electrons will be in parallel situation this state is called as triplet (parallel) state. • It is represented by T1.

Triplet (parallel state) T1 Excited state T1 Ground state S0 Excited Triplet T1

Deactivation • Excited molecule can undergo a number of relaxation processes during the time it spends in the excited state ,this may be called as vibrational relaxation. (S1 higher to S1 lower) Note: Ground state Singlet, excited singlet and Triplet state consist of number of vibrational levels . • S0=V1,V2,V3,V4------(Ground state Singlet) • S1=V1,V2,V3,V4-----(Excited state Singlet) • T1=V1,V2,V3,V4 ----(Excited state Triplet)

Vibrational relaxation • When the molecule in the excited state (S1) relaxes down to the lowest vibrational level it may emit a photon and come down to the electronic ground state (S0). This process is called fluorescence and takes about 10-9 s.

Intersystem Crossing • In this , the molecule in the vibrational states of a singlet excited state may cross over to a vibrational level of a triplet state if the two have same energy. This process is called intersystem crossing. (S1 to T1)

PHOSPHORESCENCE • In this relaxation process the excited molecule that had crossed over to the triplet excited state by intersystem crossing and has relaxed to the vibrational ground state in the triplet excited state. • In such a case the molecule emits a photon and comes down to a vibrational mode of the electronic ground state, S0. This phenomenon is called phosphorescence. • As the transition from a triplet state to a singlet state is difficult because both electrons have same spin (parallel) it take time to emit light.

Fluorescence is instant phenomenon while phosphorescence is delayed fluorescence • Thus, the fluorescence emission can take place within 10-9 − 10-6 seconds, (the transition from S1—S0). • The transition from an excited triplet state to the ground state in case of phosphorescence requires at least 10-4 seconds and may take as long as 10-2 seconds.

Excited Singlet state S1 Vibrational relaxation Intersystem crossing ENERGY Excited Triplet state S1 Fluorescence Phosphorescence Ground state singlet SO

INSTRUMENTATION • Instruments for the measurement of fluorescence are known as fluorimeters or spectrofluorimeters. • The essential parts of a simple fluorimeter are as follows;

1. Radiation Source: A light from a mercury-vapour lamp (or other source of ultraviolet light) can work as source of radiation in this technique. Other lamps such as xenon lamp can also work as source of radiation. U.V.Light Visible light

2. A primary filter: A primary filter transmits the part of beam which can cause excitation of atoms to induce fluorescence.It select only U.V. Light but absorbs Visible light Primary filter U.V.Light & visible light U.V.Light

3. Sample Cell /Sample holder • Cells are usually made of silica or glass, • In practice fluorescence cells are normally • transparent on all four faces, so that except for work of the highest precision it does not matter much which way round the cell is placed into the sample holder.

4. A Secondary filter: It allows only fluorescent radiation and absorbs Visible light. Secondary filter fluorescent radiation & U.V. light fluorescent radiation Sample cell

5.Detector • Photocell can be used as detector in this technique. • As sample emits fluorescent radiation in all direction, to measure the intensity, • Detector is placed at right angle to that of incident radiation. • At the other angles scattering from solution cell walls can cause error.

6.Readout Device • The output from the detector is suitably amplified and displayed on a read out device like a meter or digital display. • The sensitivity of the amplifier can be changed so as to be able to analyse samples of varying concentrations.

Instrument: Single Beam Fuorimeter Sample Cell U.V. Light & visible light Primary filter Secondary filter Photocell /PMT detector Read Out Device

Single Beam Fluorimeter Primary filter Secondary Filter

Double Beam Fluorimeter Sample cuvette 0.002 Mercury vapour lamp Primary filter PMT detector Read Out Device Secondary filter Blank cuvette

Double Beam Fluorimeter Sample cuvette 0. 0 0 0 Mercury vapour lamp Primary filter Read Out Device PMT detector Secondary filter Blank cuvette

The fluorescence spectrum and intensity of a molecule often depend strongly on the molecule’s environment.

Temperature: • As temperature increases fluorescence decreases. • Due to change in temperature viscosity of the medium changes.(less Viscosity) • Change in viscosity increases the number of collisions of the molecules of the fluorophore with solvent molecules. • No. of collisions increases the probability for deactivation by internal conversion and vibrational relaxation. • To overcome this , it is recommended to use thermo stated cell holders.

pH • Relatively small changes in pH can cause considerable changes in the fluorescence intensity and spectral characteristics of fluorescence. • The molecules containing acidic or basic functional groups undergoes ionisation due to the changes in pH of the medium. • It may affect the extent of conjugation or the aromaticity of the molecule which affects its fluorescence. • For example, aniline shows fluorescence while in acid solution it does not show fluorescence due to the formation of anilinium ion. • Therefore, pH control is essential while working with such molecules and suitable buffers should be employed

Dissolved Oxygen • Oxygen and many transition metals with unpaired electrons are paramagnetic which decrease fluorescence and cause interference in fluorimetric determinations. • The paramagnetic nature of molecular oxygen promotes intersystem crossing from singlet to triplet states in other molecules.(phosphorescence) • Presence of dissolved oxygen influences phosphorescence too and causes a large decrease in the phosphorescence intensity. This is actually the oxygen emission and not the phosphorescence. • Therefore, it is advisable to make phosphorescence measurement in the absence of dissolved oxygen.

Solvent: • The changes in the “polarity” or hydrogen bonding ability of the solvent affect the fluorescent behaviour of the analyte. • Solvent viscosity and solvents with heavy atoms also affect fluorescence and phosphorescence. • A higher fluorescence is observed when the solvents do not contain heavy atoms while phosphorescence increases due to the presence of heavy atoms in the solvent.

Inorganic analysis

Fluorescent Indicators

Organic analysis

Enzyme Assay and Kinetic study

Phosphorimetry • Instrumentation: • Sample Cell: The sample cell is a narrow quartz tube of an internal diameter of 1 to 3 mm. • Phosphoroscope : The sample tubes are placed in liquid Nitrogen held in a quartz Dewar flask which is then placed in the sample holder called Phosphoroscope • Rotating- can Phosphoroscope: It is Hollow cylinder having two slits and rotated by variable speed motor.

Contents

Contents

Presentation Transcript

Contents

Contents

Contents

Contents

Contents

Contents

Contents

CONTENTS

Contents

Contents