Fluorescent Spectroscopy

Fluorescence means the emission of light by a substance that has

absorbed light or any other electromagnetic radiation. In most
cases the light which is emitted has longer wavelength than the
absorbed light (it means the emitted light has lower energy than
the absorbed light). Most common example of fluorescence is
the absorption of light in ultraviolet region and emission of
visible light and this gives a fluorescent substance a distinct color
that can only be seen when exposed to ultraviolet light.
Fluorescent stops to glow immediately after radiation source
stops while in contrast phosphorescent materials continue to
emit light sometime after. Spectroscopy is the study of
interaction between matter and some electromagnetic
radiation.

Principle:
Fluorescent spectroscopy is also known as fluorimetry or
spectroflurometry. This technique involves using a beam of light
mainly ultraviolet light that excites electrons in the molecule of
certain compounds and these electrons upon de-excitation emit
mainly light in the visible region of the spectrum. The device in
which this technique is used is known as fluorescence
spectrofluorometer.
The relaxation process of molecules is brought about either by
collisional energy transfer to other molecules in the solution or
through emission of light. The emitted light has two
characteristics it is of longer wavelength and it has lower energy
than the absorbed light this due to the fact that a part of energy
is lost in the form of heat energy. The emitted light is composed
of many wavelengths which results in fluorescence spectrum.
This is due to the fact that fluorescence from a particular excited
molecule will return the molecule to one of many vibrational
levels (this term is commonly used for the energy level of
electrons in the atom, ion or molecule) in the ground state and
the wavelength of maximum fluorescence is observed.
Fluorescence intensity is the intensity of emitted light and the
fluorescence is often defined in terms of Quantum yield which
is the ratio of number of photons emitted to number of photons
absorbed.

Instrumentation:
The instrument generally used for measuring fluorescence is
known as spectroflurometer. It contains following components
 A light source.
 Two monochromators.
 A sample holder.
 Detector

Light source:
A light source used mostly is a xenon arc lamp that emits
radiation in the ultraviolet, visible and near-infrared regions (200
to 1400 nm).
Monochromators:
Two monochromators are used in this instrument one for the
selection of excitation wavelength and the other one for the
selection of emission wavelength.

Sample holder:
A sample holder is used to hold the sample in fixed position
during analysis process.

Detector:
A photomultiplier tube serves as detector to measure the
intensity of light. A PHOTOMULTIPLIER TUBE is a sort of vacuum
tube. It works on the principle of photoelectric effect and
secondary emission in photoelectric effect when light falls on
some surface electrons are emitted from that surface these
electrons in turn fall on electron multipliers which are dynodes
and cause emission of many more electrons and this is known as
secondary emission and this would also result in amplification of
original signal to about million times. The output current from
the photomultiplier tube is fed into some measuring device that
indicates the extent of fluorescence. The final readout is not in
the form of quantum yield but in units of photomultiplier tube
current(microamperes) or in relative units of percent of full
scale. That’s why a scale should be standardized with the known.

Process:
Light from the light source first passes through excitation
monochromator and then through sample. Sample is placed in
special fluorescence cuvettes with four translucent and quartz
sides. Light emitted at right angle to incoming beam is analyzed
by emission monochromator. After passing through emission
monochromator light is detected by photomultiplier tube which
serves as a detector.

Application:
Fluorescent Spectroscopy has many applications.
Determination of inorganic substances:
 It can be used to determine aluminum in alloys.
 Determination of ruthenium ions in the presence of
other metals.
Organic Analysis:
Qualitative and quantitative analysis of organic aromatic
compounds present in cigarette smoke, air pollutants and
in automobile exhaust.
Chromatography:
  Fluorescence is an important method of determining
compounds as they appear at the end of
chromatogram or capillary electrophoresis.
 Also detects compounds from HPLC flow.
Pharmaceutical analysis:
 Useful for studying the binding of drugs to
components in complex formulation.
 Widely used in bioanalysis for measuring small
amounts of drugs and for studying drug-protein
binding.
 Analysis of drugs such as vitamin B2, B6, B12 and
catecholamines such as dopamine and
norepinephrine.
Determination of disease:
Many natural compounds like NADH, FADH, vitamin A,
riboflavin and porphyrins show fluorescence but there
concentration is altered in diseases like fluorescence
intensities from NADH and elastin or collagen are used
for early detection of oral cancer. Similarly, oral, cervix
and breast cancer can be determined.
Environmental significance:
To detect environmental pollutants such as:
 Polycyclic aromatic hydrocarbons.
 Pyrene.
 Benzopyrene.
 Organothiophosphorous pesticides.
 Carbamate insecticides.
Geology:
 Many types of calcite and amber will fluoresce
under shortwave UV.
 Rubies and diamond exhibit red fluorescence
under shortwave UV. Diamonds also emit light
under X-ray radiation.
Microbiology:
 Techniques like fluorescence microscope can be
used to study various bacteria.
 Many bacterial and virus proteins have tryptophan
which can produce fluorescence.

Advantages:
 It is a rapid and sensitive technique.
  Allows non-destructive analysis of the sample.
 Inexpensive.
 Can be used for both qualitative and
quantitative analysis.
 Easy and quick to perform analysis.
Disadvantages:
 Not all compounds fluorescence.
 Contamination can quench the fluorescence and
give false or no result.
 Temperature maintenance is required.