Fluorimetry

Factors affecting fluorescence intensity

1- Type of transition
2- Molecular structure
3- Temperature
4- Solvent viscosity
5- Presence of heavy atoms in solvents
6- pH
7- Dissolved oxygen
 Fluorimetry

1 - Effect of transition type on fluorescence

1- Fluorescence is seldom seen in σ* → σ and σ* → n transition.
2- It occurs with * →  and * → n, but the fluorescence of * →  is
more than * → n transition, this is due to
The probability for intersystem crossing is smaller for * →  than for
* → n transition.
 Fluorimetry
2- Effect of Molecular structure on fluorescence
1- Most efficient fluorescence is found in aromatic compounds.
2- Compounds containing aliphatic and alicyclic carbonyl groups with
conjugated double bond structure may also exhibit fluorescence.
3- Fluoresce of unsubstituted aromatic hydrocarbons increases with
increase number of fused ring. Anthracene is a good example.

4- Fluoresce of substituted aromatic hydrocarbons decreases with

presence of electron withdrawing groups (halides, COOH, SO3H and
NO2) and increases with presence of electron donating group (as –NH2
and –OH).
 Fluorimetry
5- Heterocyclic as pyridine, thiophene, pyrrole and furan do not
fluoresce (The lowest transition is *-n system which rapidly
converted to triplet state and prevents fluorescence). But fusion of
benzene rings with the heterocyclic rings decreases the life time of
the excited state for these compounds thus fluorescence is
observed as quinoline, isoquinoline and indole.

O N S N
H
Furan Pyridine Thiophene Pyrrole

H
N N
N

quinoline Isoquinoline Indole

 Fluorimetry

6- Rigidity of the structure

Fluorescence increases in molecules that have rigid planar structure.
For example, fluorene fluoresce more intense than biphenyl due to
rigidity caused by methylene group in fluorene.
Increase fluorescence of certain chelating agents when complexed
with metal ion is due rigidity e.g. fluorescent intensity of 8-
hydroxyquinoline is increased when it forms zinc complex.

O
Zn
N
C
H2 2
Biphenyl Fluorene Zinc complex with
8-hydroxy quinoline
 Fluorimetry
3- Effect of temperature on fluorescence
Fluorescence decreases by increasing temperature due to collision.

4- Effect of solvent viscosity on fluorescence

Fluorescence decreases by decreasing the solvent viscosity.
Temp↑ (collision ↑) or solvent viscosity↓ → probability of collisional
relaxation ↑ → quantum efficiency of fluorescence decrease.

5- Presence of heavy atoms in solvents

Fluorescence decreases in solvents containing heavy atoms as CBr4
or ethyl iodide CH3CH2I due to increase the rate of intersystem
crossing.
 Fluorimetry
6- Effect of pH on fluorescence
Fluorescence of aromatic ring with basic or acidic substituent is pH
dependent. Since emission wavelength and intensity of ionized and
unionized form will be different.
H
H H H H H H H H
N N N N

Aniline in alkaline medium anilinum ion

resonone forms of aniline acid medium

Fluoresce intensity of aniline increases in neutral or in alkaline

medium due stability by resonance.
 Fluorimetry
7- Effect of dissolved oxygen on fluorescence
Presence of oxygen decreases the intensity of fluorescence due to
1- Molecular oxygen may promote intersystem crossing and
conversion of excited state to triplet state.
2- Oxidation of fluorescent species.

Quenching : It is the decrease of fluorescence

There are some compounds that are very effective quenchers and
hence interfere with fluorescence emission. Quenchers induce a non-
radiative deactivation of the excited atoms and hence very small
number of photons is emitted.
 Fluorimetry
1-KI known as strong quencher for quinine.
2-Oxygen is serious quencher for some fluorescent aromatic
hydrocarbons and it is sometime necessary to deoxygenate the
solutions.
3- Heavy metals are also considered as quenchers.
 Fluorimetry
Components of fluorometer or spectrofluorometer

1- Source of radiation (light source)

The light source should deliver highly intense, continuous, constant
and uniform radiation. There are two types of lamps that are commonly
used
a- Mercury – arc lamp: It is used for measurement in visible region.
b- High pressure xenon lamp: It is used for measurement in UV region.
 Fluorimetry

2- Monochromator

-Two monochromators are used at the same time which may be filter

or grating type.

- If it is filter, the instrument is called fluorometer.

-If it is grating, the instrument is called spectrofluorimeter.

- The first one is aligned between the light source and sample which is

used for selection of excitation wavelength.

- The second is aligned between the sample and the detector and it is

used for selection of the most intense emitted wavelength to pass.

 Fluorimetry

3- Sample cell
It is transparent from all sides. It is either glass (visible) or quartz
(UV). Present at right angle with respect to the source and detector
because when the selected wavelengths pass through the sample,
fluorescent radiations are emitted in all directions but is most
conveniently observed at right angles to the excitation beam.
4-Detector
Photomultiplier tube is used as the intensity of emitted radiation is
small.
5- Recorder or read out unit
 Fluorimetry

Application of fluorescence
1- Direct analysis
It is used when the compound has native fluorescence such as
phenobarbitone, cinchonine, reserpine, quinine, uric acid, vitamin A
and vitamin B.
2- Indirect analysis:
It is used when the compound does not have native fluorescence.
The compound is allowed to react with a fluorometric reagent forming
a product that has fluorescent properties. Examples of indirect
analysis are
 Fluorimetry
1- Inorganic ions
- 8-hydroxyquinoline for Al and Zn
- Benzoin for Zn
- Flavanol for Zr
2- Determination of primary and secondary aliphatic amines through
reaction with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) to give
yellow fluorescence.
 Fluorimetry

or through reaction with1-dimethylaminonaphthalene-5-sulphonyl

chloride (Dansyl chloride) to give yellow fluorescence.
 Fluorimetry

3- Enzyme: such as creatine phosphokinase can be determined by using

it to catalyse formation of creatine from phophocreatine.

enzyme +ninhydrine
Phosphocreatine creatin fluorescent product

4- Hormones: such as adrenaline

Adrenaline + K3Fe(CN)6 at pH 6 then alkaline ascorbate

greenish yellow fluorescence

 Fluorimetry
5- Measure the decrease in fluorescence (quenching) when the analyte
is added to a solution containing a fluorescent molecule. NO3- ions
can be determined through its quenching effect on fluorescence of
fluorescein
 Fluorimetry
Chemiluminescence
Chemiluminescence is produced when a chemical reaction yields an
excited compound, which emits light as it returns to its ground state.
A+B C* + D
C* C + h
Where C* represent the excited state of the compound C
The source of radiation is the chemical reaction between the analyte
and reagent.
 Fluorimetry
Applications of chemiluminescence
Chemiluminescence is highly sensitive and the typical detection limits
lie in the parts per billion to parts-per-million range.
A- Analysis of gases
1- Chemiluminescence is used for determination of atmospheric
pollutants such as ozone and oxides of nitrogen and sulfur
compounds.
Nitrogen monoxide “NO” was determine according the following

2- Strong oxidizing agents such as oxygen or hydrogen peroxide can

be react with luminol in presence of -OH to produce active
compound according the following equations
 Fluorimetry
NH2 O NH2

C COO
NH
+ 2OH + O2 N2 + 2H2O + + h
NH
C COO

Luminol 3-aminophthalate ion

B- Analysis for organic compounds

A number of organic compounds have catalytic or inhibiting
effects on the luminol reaction with hydrogen peroxide or oxygen,
thus permitting their determination. Among these are amino acids,
and benzene derivatives containing – NO2 - NH2 and -OH groups.
Important application of this effect is in the identification of blood
stains e.g. hemoglobin has a strong catalytic effect on the oxidation
of luminol.