PHAR2010

Fluorescence Spectroscopy

Dr New Siu Yee

School of Pharmacy
University of Nottingham Malaysia Campus
SiuYee.New@nottingham.edu.my

SWD | B32C05 | L 7 | 1.
 Aim and objectives
By the end of this session you will be able to

• explain the theoretical basis and applications of

fluorescence spectroscopy

• describe the instrumental set up of a spectrofluorometer

• explain effects such as two-photon excitation and

quenching

SWD | B32C05 | L 7 | 2.
 Fluorescence
• Some compounds are colourless
– Anthracene absorbs in the UV region
– however, when viewed under UV light it EMITS blue visible light
• Fluorescence is light that is emitted (at a longer wavelength) from a
molecule after the molecule has absorbed light of a shorter
wavelength.
• Fluorescence stops when the irradiating light is removed.

SWD | B32C05 | L 7 | 3.
 Energy Level

From UV-vis

photon

Translational < Rotational < Vibrational < Electronic

SWD | B32C05 | L 7 | 4.
 Franck-Condon Principle
• During an electronic transition nuclei do not
move position. Electron redistribution results in
excitation of vibrations.

Energy
Excited electronic
state Nuclei separation (r)

Ground state

r
Vibrational levels

SWD | B32C05 | L 7 | 5.
 Process of Fluorescence

Jablonski diagram
Radiationless Decay

S1 Vibrational levels

huA huF

Radiationless Decay
S0

SWD | B32C05 | L 7 | 6.
 Process of Fluorescence
• Absorption of radiation huA by the ground state
molecule leads to promotion of 1-electron from
S0 to one of the vibrational levels of S1 (or
higher)

• Rapidly deactivates in solution through

radiationless process (vibrational relaxation
due to solvent collisions) to the lowest
vibrational sublevel of S1.

• For the electron to return from S1 to S0 it emits

radiation (huF) of lower energy (longer
wavelength) than huA

SWD | B32C05 | L 7 | 7.
SWD | B32C05 | L 7 | 8.
 Phosphorescence
• Alternative path is S1 to T1 to S0
• Different spin orientation
• Triplet states more stable (longer lived) than singlet
states

Intersystem Crossing
S1
T1
huA huF
huP

S0

SWD | B32C05 | L 7 | 9.
 Phosphorescence

SWD | B32C05 | L 7 | 10.

 Luminescence

Emission of light following excitation of the molecule

Fluorescence
– Excitation by light
– Spin stays paired (anti-parallel)

Phosphorescence
– Excitation by light
– Spin changes (parallel)

Chemiluminescence
– Excitation by chemical reaction
– Bioluminescence (e.g.firefly)
https://www.glofish.com/meet-glofish/glofish-videos/
SWD | B32C05 | L 7 | 11.
 Instrumentation

Detector
Excitation
Filter Emission
Filter
Detector

• Similar to single beam UV

• Measure absorption spectrum as with UV spectroscopy
(in line with source
– excitation spectrum
• Measure fluorescence at 90o to irradiation source
– emission spectrum
SWD | B32C05 | L 7 | 12.
 Fluorescent Molecules
• Difficult to predict if a molecule is fluorescent
• Often have rigid molecular structures with electrons
delocalised over entire structure
• Example molecules
– anthracene
– chlorophyll
– fluorescein
– 4,4’-diaminostilbene
– poly(vinyl pyrolidine) PVP
Quinine
– Quinine
– Noradrenaline
– Ethinylestradiol (BP uses fluorescence for tablet concn)
– Procaine
– Chlorpromazine
– Pentobarbitone

SWD | B32C05 | L 7 | 13.

 Biological & pharmaceutical applications

• Determination of fluorescent drugs in low-dose

formulations

• Bioanalysis – small amounts of drug

• Studying drug-protein binding

SWD | B32C05 | L 7 | 14.

 Two Photon Excitation
• If intensity of light is strong enough (e.g. tightly focussed laser beam) -
increased probability that the energy of two photons may be
absorbed simultaneously to promote the single electron ‘higher’.

• Energy absorbed is twice h

- fluorescent light now has a shorter wavelength than originally applied
• 2 x 640 nm (red) photons
• Excite in 320 nm (UV region)
• Fluoresces at longer (blue/green) λ

• Used in high-resolution optical imaging

SWD | B32C05 | L 7 | 15.

 Quenching
• Light emitted from a luminescent molecule
relates to a possible absorption band of that
molecule

• Luminescence light may be re-absorbed

• Probability of re-absorption is concentration

dependant - if high concentration then light will
not leave the cell and reach the detector.

SWD | B32C05 | L 7 | 16.

 Summary
• Fluorescence is of longer wavelength than absorbed
light.

• Lower energy gap for emission than absorption due to

vibrational relaxation.

• Note: - Two photon excitation effect results in a shorter

wavelength emitted than applied.

• Fluorescence involves singlet states (paired spins) –

short lived.

• Phosphorescence involves triplet states (unpaired

spins) – long lived.

• Instrumentation as UV-Vis, but with detector at 90o.

SWD | B32C05 | L 7 | 17.