9. Fluorescence Spectroscopy

8/10/2019 9. Fluorescence Spectroscopy

http://slidepdf.com/reader/full/9-fluorescence-spectroscopy 1/30

Fluorometry

PHRM 309



Emission spectroscopy

Emission spectroscopy is a spectroscopic

technique which examines the wavelengths of photonsemitted by atoms or molecules during their transition

from an excited state to a lower energy state.



LuminescenceLuminescence is the emission of light from any

substance, and occurs from electronically excited states.

Luminescence is divided into two categories-

fluorescence and phosphorescence.

The emission rates of fluorescence are typically

108 s –1, so that a typical fluorescence lifetime is near 10

ns. The emission rates of phosphorescence are slow

(10

3

to 100 s

–1

), so that phosphorescence lifetimes aretypically milliseconds to seconds.

Fluorescence is much more widely used for chemical

analysis than phosphorescence.



The first observation of fluorescence from a

quinine solution in sunlight was reported by Sir John

Frederick William Herschel in 1845.

QuinineThe quinine in tonic water is excited by the

ultraviolet light from the sun. Upon return to the

ground state the quinine emits blue light with a

wavelength near 450 nm.



Theory of molecular fluorescence

Molecular fluorescence is measured by exciting

the sample at the absorption wavelength, also calledthe excitation wavelength, and measuring the emission

at a longer wavelength called the emission or

fluorescence wavelength.

For example, the reduced form of the coenzyme

nicotinamide adenine dinucleotide (NADH) can absorb

radiation at 340 nm. The molecule exhibits fluorescence

with an emission maximum at 465 nm.




In a non-flurescent molecule when an electron is

excited to the electronic excited state, it return back tothe ground state by losing the energy it has acquired

through conversion of the excess electronic energy into

vibrational energy.

If a molecule has a rigid structure the loss of

electronic energy through its conversion into vibrational

energy is relatively slow and there is a chance for the

electronic energy to be emitted as ultraviolet or visible

radiation.



The energy emitted is of lower energy lower

energy than the energy absorbed because the excited

electron moves to the lowest energy vibrational state in

the excited state in the excited state before returning to

the ground state.

Thus fluorescence emission is typically shifted by

50-150 nm (Stokes shift) to the longer wavelength in

comparison to the wavelength of the radiation used to

produce excitation.




Relaxation processes

Once the molecule is excited to E 1 or E 2 several

processes can occur that cause the molecule to lose its

excess energy. Two of the most important of these

mechanisms, nonradiative relaxation and fluorescence

emission are illustrated in Figure b and c.

The two most important nonradiative relaxation

methods that compete with fluorescence are illustrated

in Figure b.





Energy-level diagram shows some of the processes that

occur during (a) absorption of incident radiation. (b)

nonradiative relaxation,and (c) fluorescence emission by

a molecular species.

Absorption typically occurs in 10-15 s while vibrational

relaxation occurs in the 10-11 to 10-10 s time scale.

Internal conversion between different electronic states

is also very rapid (10-12 s), while fluorescence lifetimes

are typically 10-10 to 10-5 s.



Vibrational relaxation involves transfer of the excess

energy of a vibrationally excited species to molecules of

the solvent. This process takes place in less than 10-15 s

and leaves the molecules in the lowest vibrational state

of an electronic excited state.

Vibrational relaxation depicted by the short wavy

arrows between vibrational energy levels. takes place

during collisions between excited molecules and

molecules of the solvent.



Internal conversion is a type of relaxation that involves

transfer of the excess energy of a species in the lowest

vibrational level of an excited electronic state to solvent

molecules and conversion of the excited species to a

lower electronic state.



Relationship between excitation spectra and

fluorescence spectra

Because the energy differences between

vibrational states is about the same for both ground and

excited states, the absorption spectrum, or excitation

spectrum, and the fluorescence spectrum for a

compound often appear as approximate mirror images

of one another with overlap occurring near the origin

transition.



Fluorescence spectra for 1

ppm anthrecene in alcohol:

(a) excitation spectrum

(b) emission spectrum



Fluorescent species

It is not entirely possible to predict how strongly

fluorescent a molecule will be.For example adrenaline and noradrenaline differ

in their structure by only a single methyl group but nor

adrenaline exhibits fluorescence nearly 20 times more

intensely than adrenaline. Generally, flurescence is

associated with an extended chromophore or

auxochrome and a rigid structure.

NoradrenalineAdrenaline



- Compounds containing aromatic rings give the most

intense and most useful molecular fluorescence

emission.- Few aliphatic and alicyclic carbonyl compounds as well

as highly conjugate double-bonded structures.

The simplest heterocyclics, such as pyridine, furan,

thiophene and pyrrole, do not exhibit molecular

fluorescence, but fused-ring structures containing these

rings often do for example quinoline, isoquinoline,indole.





Substitution on an

aromatic ring causes

shifts in the wavelengthof absorption maxima

and corresponding

changes in the

fluorescence peaks.



The effect of structural rigidity

Experiments show that fluorescence is particularly

favored in rigid molecules. For example, under similarmeasurement conditions, fluorene is more fluorescent

than biphenyl.

The difference in behavior is a result of the increased

rigidity provided by the bridging methylene group in

fluorene. This rigidity lowers the rate of nonradiative

relaxation.

Fluorene Biphenyl



The influence of rigidity also explains the increase in

fluorescence of certain organic chelating agents when

they are complexed with a metal ion. For example, thefluorescence intensity of 8-hydroxyquinoline is much

less than that of the zinc complex.





Fluorescence spectroscopy

Fluorescence spectroscopy (fluorometry or

spectrofluorometry), is a type of electromagnetic

spectroscopy which analyzes fluorescence from a

sample.

It involves using a beam of light, usually ultraviolet light,

that excites the electrons in molecules of certain

compounds and causes them to emit light of a lower

energy, typically, but not necessarily, visible light. This

shift to longer wavelength is called the Stokes shift.

Devices that measure fluorescence are called

fluorometers or fluorimeters.



Instrumentation

The light from an excitation source passes through a

filter or monochromator, and strikes the sample. A

portion of the incident light is absorbed by the sample,

and some of the molecules in the sample fluoresce. The

fluorescent light is emitted in all directions. Some of this

fluorescent light passes through a second filter ormonochromator and reaches a detector, which is

usually placed at 90° to the incident light beam to

minimize the risk of transmitted or reflected incident

light reaching the detector.





Schematic of a fluorometer with 90° geometry utilizing a Xe light source



Schematic of a fluorometer with 90° geometry utilizing a Xe light source

900

http://www.nationmaster.com/encyclopedia/image/Image:Fluorimeter.png





Light source

Xenon lamps

Filters and/or monochromatorsThe most common type of monochromator utilizes a

diffraction grating.



Application

1. Determination of fluorescent drugs in low-dose

formulations in the presence of non-fluorescent

excipients.

2. In carrying out the limit tests where the impurity is

fluorescent.

3. Useful for studying the binding of drugs to componentin complex formulations.

4. Widely used in bioanalysis for measuring small

amounts of drug and for studying drug-protein binding.



Factors interfering with fluorescence intensity

1. If the concentration of a solution prepared forfluorescence measurement is too high, some of the light

emitted by the sample as fluorescence will be reabsorbed

by other unexcited molecules in solution.

For this reason, fluorescence measurements are bestmade on solutions with an absorbance less than 0.02, i.e.

solutions of a sample 10-100 weaker than those which

would be used for measurement by UV-VIS spectroscopy.



2. Heavy atoms in solution quench fluorescence by

colliding with excited molecules so that energy is

dissipated, e.g. chloride or bromide ions in solution cause

collisional quenching.

3. Formation of chemical complex with other molecules

in solution can change fluorescence behavior, e.g. the

presence of caffeine in solution reduces the fluorescenceof riboflavin.

4. In most molecules, fluorescent property decreases

with increasing temperature because the increased

frequency of collision at elevated temperatures increases

the probability of collisional relaxation. A decrease in

solvent viscosity leads to the same result.

Documents

9. Fluorescence Spectroscopy