FLUORIMETRY

FLUORESCENCE  It is a phenomenon of emission of radiation when the molecules are excited

by radiation at certain wavelength.

FLUORIMETRY: - It is measurement of fluorescence intensity at a particular wavelength with

the help of a filter fluorimeter or a spectrofluorimeter.

PRINCIPLE: - Molecule contains electrons, electrons and nonbonding (n) electron. The

electrons may be present in bonding molecular orbital. It is called as highest occupied

molecular orbital (HOMO).It has lest energy and more stable. When the molecules absorbs

radiant energy from a light source, the bonding electrons may be promoted to anti bonding

molecular orbital (LUMO). It has more energy and hence less stable.

The process of promotion of electrons from HOMO to LUMO with absorption of energy is called

as excitation.  Singlet state:-a state in which all the electrons in a molecule are paired  

Doublet state:- a state in which un paired electrons is present  or   Triplet state:- a state in

which unpaired electrons of same spin present   Singlet excited state:- a state in which

electrons are unpaired but of opposite spin like  (un paired and opposite spin)

When light of appropriate wavelength is absorbed by a molecule the electrons are promoted

from singlet ground state to singlet excited state. Once the molecule is in this excited state
relaxation can occur via several process. The process can be the following  1) Collisional

deactivation  2) Fluorescence  3)Phosphorescence.

Collisional de activation: - In which entire energy lost due to collision de activation and no

radiation emitted.  Fluorescence:-excited singlet state is highly unstable. Relaxation of

electrons from excited singlet to singlet ground state with emission of light. 

Phosphorescence:-At favorable condition like low temperature and absence of oxygen there is

transition from excited singlet state to triplet state which is called as inner system crossing. The

emission of radiation when electrons undergo transition from triplet state to singlet ground

state is called as phosphorescence.

 Fluorescence intensity is proportional to concentration of substance only when the absorbance

is less than 0.02 A=log IoIt or A= abc Io=intensity of incident light a= absorptivity of constant b=

Path length c= concentration Concentration:-

()=NUMBER OF PHOTONS EMITTEDNUMBER OF PHOTONS ABSORBEDS It Is Always Less Than

1.0 Since Some Energy Is Lost By Radiation less Pathways (Collisional, Intersystem Crossing,

Vibrational Relaxation)
  Quenchers: -  Quenching is the reduction of fluorescence intensity by the presence of

substance in the sample other than the fluorescent analyte.  Quenching is following types:-

INSTRUMENTATION

Typically fluorometers utilize a double beam. These two beams work in tandem to

decrease the noise created from radiant power fluctuations. The upper beam is passed

through a filter or monochromator and passes through the sample. The lower beam is

passed through an attenuator and adjusted to try and match the fluorescent power

given off from the sample. Light from the fluorescence of the sample and the lower,

attenuated beam are detected by separate transducers and converted to an electrical

signal that is interpreted by a computer system.

Within the machine the transducer that detects fluorescence created from the upper

beam is located a distance away from the sample and at a 90-degree angle from the

incident, upper beam. The machine is constructed like this to decrease the stray
light from the upper beam that may strike the detector. The optimal angle is 90

degrees. There are two different approaches to handling the selection of incident light

that gives way to different type’s fluorometers. If filters are used to select wavelengths

of light, the machine is called a fluorometer. While a spectrofluorometer will typically

use two monochromators, some spectrofluorometers may use one filter and one

monochromator. Where, in this case, the broad band filter acts to reduce stray light,

including from unwanted diffraction orders of the diffraction grating in the

monochromator.

Light sources for fluorimeters are often dependent on the type of sample being

tested. Among the most common light source for fluorometers is the low-

pressure mercury lamp. This provides many excitation wavelengths, making it the

most versatile. However, this lamp is not a continuous source of radiation. The  xenon

arc lamp is used when a continuous source of radiation is needed. Both of these

sources provide a suitable spectrum of ultraviolet light that

induces chemiluminescence.

Glass and silica cuvettes are often the vessels in which the sample is placed. Care

must be taken to not leave fingerprints or any other sort of mark on the outside of the

cuvette, because this can produce unwanted fluorescence. "Spectro grade" solvents

such as methanol are sometimes used to clean the vessel surfaces to minimize these

problems
 APPLICATIONS

Determination of inorganic substances.

Determination of thiamine Hcl.

Detemination of phenytoin.

Determination of indoles, phenols, & phenothiazines

Determination of napthols, proteins, plant pigments and steroids. .

Determination of ruthenium ions in presence of other platinum metals