Phosphorimetry & Fluorimetry

• Some chemical species are photoluminescent.

i.e. they can absorb electromagnetic radiation and
reemit the radiation at the same or longer different
wavelength.

• This process of emitting radiation is collectively

called “LUMINESCENCE”
Luminescence is of two types :

Categorized according to the life time of excited

state

1. Fluorescence

• The life time of the excited state ~ 10-4 to 10-8 sec.

• Fluorescence stops immediately after irradiation
is stopped.
• Fluorescence is usually observed in liquid
solutions at moderate temperatures.
2. Phosphorescence

• Lifetime of the excited states falls in the range

from 10-4 to 100 s or longer and is seen in rigid
media at low temperatures

• i.e. It is observed after a considerable delay.

Compared with Fluorescence
 Physical Process
• Theory of Fluorescence & Phosphorescence
S2 T2
R

S1 VR ISC T1

F P

S0
Energy Level Diagram of a Diatomic Molecule
• ‘S’ represent singlet states, a state where all electrons in
the molecules are paired.

• ‘T’ represent a triplet state, a state where two electrons

are unpaired.

• ( Usually unpaired electrons have lower energy than

paired.)

• So here represent the ground state.

• By absorbing radiation the molecule is excited to the S2

exited state. (singlet). The molecule can come to the
ground state so by releasing a photon with energy equal
to that absorbed. Then is called “ RESONANCE
FLUORESCENCE”
• But mostly in solids and liquids the molecules can come to
lower vibrational levels & then dexcites by releasing energy
(vib) of the molecules in the medium as heat energy
(represented log VR

•Also the molecules change their singlet states by shifting to

another singlet which is lower in energy than the previous one.
Here S2 S1

• This is called Internal Conversion (IC)

Molecules in these levels too quickly deactivate through
vibrational levels to the lowest vibrational level of that
state releasing heat energy.
• From these lowest vibrational state of the excited electronic
state & the molecule can relax to ground electronic state S0,
by relaxing photons.

• This fluorescence is called Stokes stepwise

fluorescence.

• For stokes stepwise fluorescence λfe > λex

Γ ~ 10-9 s to 10-7 s
• An alternative process also occurs from excited (s1) molecules
can shift to excited triplet (T1)
i.e. S1 T1

• This is known as Inter System Crossing (ISC)

• But this is forbidden, therefore it occurs slowly

• Then, transition from triplet state to ground state (S0) also

occurs though forbidden, Since forbidden, the life time of the
excited state T1 is higher

• This entire process of forbidden transition is called

PHOSPHORESCENCE
 Fluorimetry and Phosphorimetry in
Quantitative Analysis
• Intensity of fluorescence depends upon
concentration
Po C P
b

P = P0 e - εbc
• The fraction of radiation absorbed will be
Po - P = (1 - P / P0)
Po
 ( 1 – P / P0) = 1 - e –εbc
Rearranging the equation,
( P0 – P ) = P0 (1 – e -εbc)

• The fluorescence intensity, F, is equal to = Φ (P0 - P)

Quantum efficiency for Fluorescence

i.e. F = Φ P0 (1 – e -εbc)
‘F’ can be expressed
F = K Φ P0 2.3 ε b c

Where, K is the instrumental constant

 Instrumentation

• An intense source, such as Hg arc or Xe arc are used

• Monochromator to select the proper wave length of

excitation

• Luminescence is measured using a photosensitive detector

kept at

• If a filter is used to isolate radiation, the instrument is

called a fluorimeter or phosphorimeter

• If a monochromator is used it is called a

spectrofluorimeter or spectrophosphorimeter
 Source

Recorder
Monochromator
or filter

Sample Cell Monochromator )

or filter
Detector
Fluorescence Instrument categorized into
• Filter Fluorimeter
• Spectro Fluorimeter
• Compemating Fluorimeter

Sources
• High pressure DC Xenon arc lamp (300 – 1300 nm)
• Xenon flash lamp
• Low pressure Hg vapour lamp
• Laser
 Phosphorimeter
Note :
1. Sample cell mostly maintained at liquid N2 (-196 0C)
2. A rotating shutter device called phosphoresce….. is
required to control the time of measuring the emission by
delaying the measuring time, fluorescence can be avoided

Note :
• A mixture of ethyl ether isopentane and ethanol called (EPA)
volume ratio (5:5:2) is used to dissolve analytes; Where
frozen, this makes a glass like rigid structure
Applications

1. In the field of nuclear research, Uranium is

determined by this technique

Evaporated Fused with

U sample HNO3 ( ) NaF NaF, UF fluorescent

cool

sole…….. a glass
LOD ~ (5 x 10-9 g / 1g sample)
2. Determination of transition metals (using
chelates)

(a). Rh in the presence of Pb (group)

5 methyl-1,10- phenolphthalein

Rh
pH = 6

Complex

LOD ~ 0.3 to 2.0 μg / ml

(b) Al(III) alloys

Dye, pH = 4.8
Al3+
Pontachrome

• LOD ~ 0.2 to 25 μg in a volume of 50 ml

• Generally ~ 1 ppm

Benzoin
(c) B in steel Complex

(d) Cd2+, Ca2+ Complexes

3. Fluorescent Indicators
The intensity and colour of many fluorescent of compounds
depends on pH of the medium
Used in acid / base titrations in titrations of coloured solutions

Indicator pH range Colour change

Eosin 3-4 Colourless to green

Fluorescin 4–6 Colourless to green
Quinine………. 3–5 Blue to violet
4. Fluoremetric Reagent

8-OH quinoline Benzoin

*
*
 Ion Reagent Fluorescence Sensitivity
reagent μg / ml
• Al3+ Alizarin garnet B 500 0.007
• Sn4+ Flavanole 470 0.1
• Zn2+ Benzoin green 10

5. Determination of Vit B1 (Thiamin)

6. Determination of Vit B2 (Riboflavin)

7. Organic analysis