Molecular luminescence

F L U O R i M E T RY A N D P H O S P H O R I M E T RY

Radiation due to temperature = incandescence

All other form of light emission is called as luminescence

luminescence

chemoluminescence photoluminescence radioluminescence

phosphorescence fluorescence

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 What makes a compound UV active?

• Ones that do are said to be 'UV-active' and

ones that do not are 'UV-inactive'.
• To be UV-active compounds must possess a
certain degree of conjugation, most
commonly aromatic compounds.

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 What are fluorescent compounds?

• A fluorophore (or fluorochrome, similarly to a chromophore)

is a fluorescent chemical compound that can re-emit light upon light
excitation.

• Fluorophores typically contain

• several combined aromatic groups,
• or planar
• or cyclic molecules with several π bonds

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 Principles of Fluorescence

•Energy level diagram (Jablonski diagram)

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 Energy level diagram
(Jablonski diagram)

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 FLUOROSENCE
QUENCHING

•Self quenching or concentration quenching:

•Chemical quenching : (due to pH,O2,halides, heavy
metals)
•Static quenching : (complex formation, caffeine as quencher for
riboflavin)
•Collision quenching:(Halide, heavy metals, Increase in
temperature, decrease in viscosity)

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 Factors affecting the fluorescence
1. Nature of the molecule: able to absorb uv or visible radiation
2. Nature of the substituent‘s (electron donating group,
electron donating group, SO3H, NH4, alkyl group OH NH2 &
withdrawing gp COOH,NO2, Halides, C=O)

3. Polarity
4. Effect of PH;
•(ANILINE Alkali pH; ---visible fluorescence, acidic—uv
fluorescence
• PHENOLS: alkali: fluorescence, acidic : no fluorescence,
5. Effect of dissolved gasses (it oxidizes fluorescent to non
fluorescent )

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6. Temperature
7. Effect of structural rigidity
8. Quantum efficiency
9.Atomic number( phenol=18, toluene=17, benzene = 10)
10. Photochemical decomposition (due to
polychromatic light, no fluorescence)

11. Formation of metal complex

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 Nature of the substituents

N S O N

Pyrrole Thiophene Furan Pyridine

N
N
Quinoline Isoquinoline

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 . Effect of structural rigidity

CH2
BIPHENYL FLUOREN

• QUANTUM EFFECIENCY= 0.2 1.0

• QUANTUM EFFECIENCY = NUMBER PHOTON EMITTED

• ------------------------------------------
• NUMBER OF PHOTON ABSORBED

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 Quantum efficiency
•Quantum yield:

• the ratio of the number of molecules that luminescence

to the total number of excited molecules.
• f = kf/ (kf + ki + kec + kic + kpd + kd)
•kf: Fluorescence constant
•ki: Intersystem crossing constant
•kec: External conversion constant ( deactivation of excited electronic state ,
b/w solvent & solutes)

•kic: Internal conversion constant ( intermole process mole—lower electronic state

without emission of radiation)

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•kpd: Pre dissociation constant ( due to internal
conversion electrons move from higher ES to upper vibrational state of
lower ES where energy is low)

•kd: Dissociation constant ( after absorption chromophoric

electrons directly to higher vibrational level----- rupture of bonds)

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 Formation of metal complex

•Increase in fluorescence of chelating agents, when they

complexed with a metal due to rigidity:

•Ex: 8 hydroxy quinoline AND hydroxy quinoline zinc complex

O O
Zn
N
N
2

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 Fluorescence measurement

Filter fluorimeters
Spectrofluorometers
Fluorescent microscopes
Fluorescent scanners &
Flow cytometry

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 instrumentation

•Components of a fluorometer:
•Light sources; (mercury and xenon lamp)
•wavelength selection: ( filters and monochromators)
•detectors; photomultiplier tube (PMT)
•sample cell.
•Recorders;

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 Block diagram of fluorometer

excitation sample
source
monochromator

emission
monochromator

detector

Read-out

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 Spectrofluorometer

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 Spectrofluorometer

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 source
•Mercury vapour lamp: metallic arc they are designed to
operate at high temperature and low pressure the emission of
the lamp is at wavelength at 365, 398, 436, 546,579,690 & 734
nm
•Low pressure lamps: equipped with a silica envelop provide
the additional radiation at 254nm

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•Xenon lamp : produce a radiation by the passage of
current through an atmosphere of xenon.
•Tungsten lamp: to produce a strong excitation band
above 450
•Hydrogen & deuterium arc lamp: not employed
because they are not intense
•
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•Filter and monochromators: (absorption + interference
filters + gratings)
•Detector: PMT
•Cells cell compartment: cylindrical, rectangular, made up
of silica or glass, they are designed to reduce amount of
scattered radiation reaching the detector
•Baffles: to prevent the scattered light
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Components of a fluorometer of a spectro-
fluorometer.

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 typical fluourometer

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 spectrofluorometer.

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 application

•Determination of uranium salts by fluorometer

•Determination of inorganic salts
•Fluorescent indicators
•Fluorometric reagent
•Aromatic structure
•Organic analysis
•Pharmaceutical analysis
•Determination of thiamine

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•Determination of riboflavin
•Determination of quinine sulphate
•Determination of tetracycline in serum
•Special application:
•Investigation of chemical structure and process
•Chemical analysis
•Detection of impurities
•Estimation of single component

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•Determination of uranium salts by fluorometer
•Uranium + HNO3 -------- oxidation + NaF
•Determination of inorganic salts ( transition elements)
•Not exhibit the fluorescence with non fluorescence organic
molecule :
•Ex:, ruthenium ions + 5 methyl 1,10 PTL ---- complex -----
fluorescence
• cadnium + 2 OH phenyl benzoxazole + glacial acetic acid --
blue fluorescence

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•Fluorometric reagent:
• used for cation metal ion analysis Ex: , flavanol

OH
O

•Aromatic structure: determination of two or more functional

group
•Ex: benzoin and 8 hydroxy quinoline
OH
O OH

C C N

BENZOIN 8-HYDROXY QUINOLINE

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•Fluorescent indicators:
•Used in acid base titration specially for coloured substances.

•Eosin : pH: 3-4 : color less to green

•Fluorescein : pH: 4-6 : color less to green
•Quinine sulphate: pH: 3-5 : blue to violet
•2-napthaquinone pH: 4.4-6.3 :blue to colour less

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Organic analysis:

Determination of many organic compounds present in cigrate, smoke,

automobile exhaust.
Ex: benzopyrene

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•Pharmaceutical analysis:
•Compound converted to fluorescent products by chemical
reaction.

• excitation emission
1.hydrocortisone: ethanol : 460 520
2.Nicotinamide : cyanogen 250 430
3.Reserpine : oxidation 250 430

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 Determination of thiamine vitamin B1
( it is non fluorescent on oxidation produce florescence)
Method: sample is treated with phosphatase ------- brings the
(hydrolysis of PO4 ester of thiamine) ------ filtration
( remove PO4 + insoluble matter) ------- dilute to known volume with
water -------
divide into 2 group-----
one is used as blank -----
to another add NaOH + alcohol ------ + potassium ferricyanide
( oxidizing agent) + on shaking

( alcoholic extraction is separated from aqueous layer) ------ examined in

fluorometer

H3C N N
S
N N
CH2CH2OH
H CH3 CH3

THIAMINE

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•Determination of riboflavin
1. Treat with acid + KMNO4 (OA) ----- FA
2. Add sodium dithionate ----- measure the fluorescence intensity (FB)
• standard substance ---- measure the fluorescence intensity (FC)
1. Measure the concentration of fluorescent material
•FB-FA mx
•--------- = -------- = mass of riboflavin in sample + standard
•FC-FA mx+ ms

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•Determination of quinine sulphate
• quinine sulphate in 0.1N H2SO4 --- on serial dilution ---
measure the fluorescence--- by plotting the graph

• fluorescence v/s concentration

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•Determination of tetracycline in serum
• Tetracycline is converted to anhydrotetracyclin
------------fluorescence (quantum yield is less)----- on
subsequent complexation with aluminum ----
fluorescence with increase sensitivity

•Special application:
•Investigation of chemical structure and process
•Hydrogen bonding , cis-trans isomers,
polymerization, tautomersim

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Chemical analysis: quantitative and qualitative analysis
Detection of impurities:
Cyclohexane ------ purified until it shows no trace of benzene at 2600AO

Estimation of single component ----- vitamin A

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 Comparison of Triplet and Singlet

Singlet Triplet
magnetic effect diamagnetic paramagnetic
electron transition for more probable less probable
emission (unlikely)
radiation induced more probable less probable
excitation
luminescence Fluorescence Phosphorescence
life time short, < 10-5 s to long, 10-5 s to several
10-9 s seconds or longer

Fluorescence and Phosphorescence

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 Comparison of Fluorescence and Phosphorescence

Fluorescence Phosphorescence
life time short, < 10-5s long, several seconds
electron spin no yes
excited states singlet triplet
quantum yield high low
temperature most temperature low temperature more likely

Resonance fluorescence: absorbed radiation is re-emitted without a

change in frequency.

Stokes shift: molecular fluorescence bands are shifted to wavelengths that

are longer than the resonance line.

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 Luminescence:

1. Energy diagram and basic concepts

2. The factors affect fluorescence

3. Excitation and emission spectra

4. Instrumentation

5. Applications

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Effect of Concentration on Fluorescence Intensity
Power of fluorescence emission F

F = K’ (I0 –I)
I0 and I are the intensities of excitation lights before and after absorbed by
the analytes. K’ is the constant
related to the quantum yield

I/I0 = 10-ebc
F = K’ I0 (1–10-ebc)
F = 2.3 K’ I0 ebc, (when ebc<0.05)

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