MOLECULAR LUMINESCENCE SPECTROSCOPY
Luminescence
spectroscopy is a technique which studies the fluorescence, phosphorescence,
and chemiluminescence of chemical systems. The analyte or its reaction product
needs to be luminescent. The relative luminescence intensity is related to
analyte concentration as will be seen shortly.
In favourable cases, luminescence methods are amongst some of the most
sensitive and selective of analytical methods available. Detection Limits are
as a general rule at ppm levels for absorption spectrophotometry and ppb levels
for luminescence methods. Collectively, fluorescence and phosphorescence are known as
photoluminescence. A third type of luminescence - Chemiluminescence - is based
upon emission of light from an excited species formed as a result of a chemical
reaction.
Flourimetry is the most commonly used luminescence method. Phosphorimetry usually requires at liquid
nitrogen temperatures (77K). The terms flourimetry and fluorometry are used
interchangeably in the chemical literature. Two wavelength selectors are required filters
(in fluorimeters) and monochromators (in Spectrofluorimeters).
Singlet and
Triplet States
Electrons in
molecular orbitals are paired, according to Pauli Exclusion Principle. When an
electron absorbs enough energy it will be excited to a higher energy state; but
will keep the orientation of its spin. The molecular electronic state in which
electrons are paired is called a singlet transition. On the other hand, the
molecular electronic state in which the two electrons are unpaired is called a
triplet state. The triplet state is achieved when an electron is transferred
from a singlet energy level into a triplet energy level, by a process called
intersystem crossing; accompanied by a flip in spin.
Energy Level
Diagram for Photoluminescent Molecules
The following
diagram represents the main processes taking place in a photoluminescent
molecule when it absorbs and emits energy.
Figure 1: Fluorescence emission showing singlet and triplet
state
The different processes will be
discussed below:
1. Absorption
The absorption of
UV-Vis radiation is necessary to excite molecules from the ground state to one
of the excited states. Absorption of radiation promotes electrons in chemical
bonds to be excited. However, we have seen earlier that not all transitions have
the same probability and while certain transitions are practically very
important, others are seldom used and are of either no or marginal importance. There
are four different types of electronic transitions which can take place in
molecules when they absorb UV-Vis radiation. A σ−σ* and a n−σ* are not useful
while the n−π* transition requires low energy but the molar absorptivity for
this transition is low and transition energy will increase in presence of polar
solvents. The most frequently used
transition is the π−π* transition for the following reasons:
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a.
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The molar absorptivity for
the π−π* transition is high allowing sensitive determinations.
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b.
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The energy required is
moderate, far less than dissociation energy.
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c.
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In presence of the most
convenient solvent (water), the energy required for a
π−π* transition is usually
smaller.
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Therefore, best
molecules that may show absorption are those with π bonds or preferably
aromatic nature. Absorption to higher excited singlet states requires a very
short time (in the range of 10-14s).
2. Vibrational Relaxation
Absorption of
radiation will excite molecules to different vibrational levels of the excited
state. This process is usually followed by successive vibrational relaxations
(VR) as well as internal conversion to lower excited states. In cases where
transitions occur to the first excited state, vibrational relaxation to the
main excited electronic level will take place and/or an intersystem crossing
(ISC) to the triplet state can occur.
3. Fluorescence
After vibrational
relaxation to first excited electronic level takes place, a molecule can return
to the ground state by emission of a photon, called fluorescence (FL). The
fluorescence lifetime is much greater than the absorption time and occurs in
the range from 10-7-10-9
s. As the lifetime in the excited state is increased, the probability of
fluorescence will be decreased since radiationless deactivation processes may
take place. However, not all excited molecules can show fluorescence by
returning to ground state and most return to ground state by losing excitation
energy as heat or through collisions with other molecules or solvent.
4. Internal and External Conversion
Internal conversion
(IC) is a radiationless deactivation process whereby excited molecules return
to the ground state without emission of a photon. This process lacks rigid
understanding but seems to be the most efficient deactivation process in
luminescence spectroscopy, since most molecules do not show fluorescence.
However, molecules with close electronic energy levels, to the extent that
their vibrational energy levels of ground and excited states are overlapped,
are believed to cause efficient internal conversion. Internal conversion can
result in a phenomenon called predissociation (PD) where an electron relaxes
from a higher electronic state to an upper vibrational energy of a lower
electronic state. When the vibrational energy is large enough and is greater
than the bond synergy, bond rupture occurs in a process called predissociation.
Dissociation should be differentiated from predissociation where dissociation
involves absorption of high energy so that the molecule is directly promoted to
a high energy vibrational level where bond rupture directly occurs. External
conversion (EC) is a process whereby excited molecules lose their energy due to
collisions with other molecules or by transfer of their energy to solvent or
other unexcited molecules. Therefore, external conversion is influenced by temperature,
solvent viscosity, as well as solvent composition.
5. Intersystem Crossing
Electrons present at
the first excited electronic level can follow one of three choices including
emission of a photon to give fluorescence, radiationless deactivation to ground
state, or intersystem crossing (ISC). The process of intersystem crossing
involves transfer of the electron from an excited singlet to a triplet state.
This process can actually take place since the vibrational levels in the
singlet and triplet states overlap. However, crossing of the singlet state to
the triplet state involves a flip in electron spin in order to satisfy the
triplet state. Intersystem crossing is facilitated by presence of nonbonding
electrons as well as heavy atoms. The presence of paramagnetic atoms or species
also enhances intersystem crossing. An electron in the triplet state can also
cross back to the singlet state and can result in a photon as fluorescence but
at a much longer time than regular fluorescence. This process is termed delayed
fluorescence and has the same characteristics as direct fluorescence except
for the large increase in lifetime.
6. Phosphorescence
Electrons crossing
the singlet state to the triplet state with a flipped spin can also follow one
of three choices including returning to the singlet state (including a flip in
spin), relax to ground state by internal or/and external conversion, or lose
their energy as a photon (phosphorescence, Ph) and relax to ground state with a
second flip in spin to satisfy the singlet ground state. As can be rationalized
from the processes involved in collecting phosphorescence photons, this
involves an intersystem crossing and two flips in spin. This, in fact, requires
a much longer time than fluorescence (10-4s to up to few
s). Therefore, the probability of phosphorescence, and hence the intensity of
the phosphorescence spectrum, is very low due to high possibility of
radiationless deactivation.
Figure 2: Wavelength
pattern of Fluorescence and Phosphorescence
INSTRUMENTATION: Spectrophotometers
1. Light sources Light sources
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Low pressure Hg lamp low pressure Hg lamp
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254, 302, 313 nm lines 254,
302, 313 nm lines
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High pressure xenon
arc lamp high pressure xenon arc lamp
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Lasers
2. Wavelength
selectors Wavelength
selectors
Filters
Monochromators
3. Detectors
Photomultipliers
CCD cameras CCD cameras
4. Cells and
sample compartments Cells and sample compartments
Quartz cells quartz cells
Light tight compartments to
minimize stray light
Note: Optical Diagram of Molecular luminescence is same as
that of flourimetry.
SPECTROFLUOROMETRY
Spectroflouremetry is an Emission phenomenon. It is
primarily concerned with electronic and vibrational states. Generally, the
species being examined will have a ground electronic state (a low energy state) of
interest, and an excited electronic state of higher energy. In fluorescence
spectroscopy, the species is first excited, by absorbing a photon, from its ground
electronic state to one of the various vibrational states in the excited
electronic state. Collisions with other molecules cause the excited molecule to
lose vibrational energy until it reaches the lowest vibrational state of the
excited electronic state. The molecule then drops down to one of the various
vibrational levels of the ground electronic state again, emitting a photon in
the process. As molecules may drop down into any of several vibrational levels
in the ground state, the emitted photons will have different energies, and thus
frequencies causing fluorescence (emission) e.g. In a typical experiment, the
different frequencies of fluorescent light emitted by a sample are measured,
holding the excitation light at a constant wavelength. This is called an emission
spectrum. An excitation spectrum is measured
by recording a number of emission spectra using different wavelengths of
excitation light.
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Figure 3: Fluorescent wavelength
of Anthracene
Quantum efficiency = 
(Note: Independent of exciting wavelength)
Quantum efficiency = Quanta fluoresed / Quanta absorbed
At low concentration; If µ
c
Spectroflouremetry is most accurate at very low concentration.
Great spectral selectivity=two monochromators
Susceptible to pH, temperature, solvent polarity
Disadvantages
1. Quenching 2. Interference
Quenching
The quantum yield of a fluorophore is
dependent on several internal and external factors. One of the external factors
with practical implications is the presence of a quencher. A quencher molecule
decreases the quantum yield of a fluorophore by non-radiating processes. The
absorption (excitation) process of the fluorophore is not altered by the
presence of a quencher. However, the energy of the excited state is transferred
onto the quenching molecules. Two kinds of quenching processes can be
distinguished:
•
Dynamic quenching
which occurs by collision between the fluorophore in its excited state and the
quencher; and
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Static quenching
whereby the quencher forms a complex with the fluorophore. The complex has a
different electronic structure compared to the fluorophore alone and returns
from the excited state to the ground state by non-radiating processes.
INSTRUMENTATION
Figure 4: Optical Diagram of Spectrofluorimrtry
Figure 5: Optical Diagram of Spectrofluorimetry
Fluorescent radiation is emitted in all directions and the use of second
monochromator perpendicular to the emitted radiation is made of this fact to
avoid difficulties that may be caused by the transmission of the incident
radiation by the sample. By moving the detection system at right angles to the
cell only fluorescent radiation is detected. Some instruments are designed to
measure front face fluorescence i.e. the radiation that is emitted along a
light path at an angle to the incident radiation. Such fluorescence
measurements are comparable to reflectance measurements.
Despite the measurement of the emitted radiation by these means, it is
still possible for scattered or reflected incident radiation to reach the
detector. To prevent this, flourimeter requires a second monochromating system
between the sample and the detector. Many simple fluorimeters use filters as
both the primary and secondary monochromators but those instruments that use
true optical monochromators for both components are known as Spectrofluorimeters.
Other instruments incorporate a simple cut-off filter system for the emitted
radiation while remaining the optical monochromator for the excitation
radiation. Because the wavelengths of both excitation and emission are
characteristic of the molecule, it is debatable which monochromator is the most
important design of a flourimeter.
The main advantage of fluorescence techniques is their sensitivity and
measurements of nanogram (ng) quantities are often possible. The reason for the
increased sensitivity of flourimetry over that of molecular absorption spectrophotometer
lies in the fact that fluorescence measurements use a non-fluorescent blank
solution, which gives a zero or minimal signal from the detector. Absorbance of
the incident radiation results in a large response from the detector. The
sensitivity of fluoremetric measurements can be increased by using a detector
that will accurately measure very small amounts of radiation.
APPLICATIONS:
There are many and highly varied applications
for fluorescence despite the fact that relatively few compounds exhibit the
phenomenon. The effects of pH, solvent composition and the polarization of
fluorescence may all contribute to structural elucidation. Measurement of
fluorescence lifetimes can be used to assess rotation correlation coefficients
and thus particle sizes. Non-fluorescent compounds are often labeled with
fluorescent probes to enable monitoring of molecular events. This is termed extrinsic
fluorescence as distinct from intrinsic fluorescence where the native compound exhibits
the property. Some fluorescent dyes are sensitive to the presence of metal ions
and can thus be used to track changes of these ions in vitro samples, as well
as whole cells.
Intrinsic protein fluorescence
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The important nutrients such as vitamins, Vitamin
B, NADH etc. in food products can be determined by the technique as they are
intrinsic fluorescent compounds in biological system which is measured even at
very low concentration by this technique.
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Some intrinsic fluorescent compounds such as Hormones
and Drugs can be estimated by this technique. It is used in drugs metabolism as
well.
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The use of pesticides can also be estimated by
this technique.
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Proteins possess three
inty lorinsic fluorophores: tryptophan, tyrosine and phenylalanine, although
the latter has a very low quantum yield and its contribution to protein
fluorescence emission is thus negligible. Of the remaining two residues,
tyrosine has the lower quantum yield and its fluorescence emission is almost
entirely quenched when it becomes ionised, or is located near an amino or
carboxyl group, or a tryptophan residue. Intrinsic protein fluorescence is thus
usually determined by tryptophan fluorescence which can be selectively excited
at 295–305 nm. Excitation at 280 nm excites tyrosine and tryptophan fluorescence
and the resulting spectra might therefore contain contributions from both types
of residues.
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The main application
for intrinsic protein fluorescence aims at conformational monitoring. We have
already mentioned that the fluorescence properties of a fluorophore depend
significantly on environmental factors, including solvent, pH, possible quenchers,
neighbouring groups, etc. A number of empirical rules can be applied to
interpret protein fluorescence spectra:
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As a fluorophore moves
into an environment with less polarity, its emission spectrum exhibits a
hypsochromic shift (lmax moves to shorter wavelengths) and the intensity at lmax increases.
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Fluorophores in a
polar environment show a decrease in quantum yield with increasing temperature.
In a non-polar environment, there is little change.
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Tryptophan
fluorescence is quenched by neighbouring protonated acidic groups.
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When interpreting
effects observed in fluorescence experiments, one has to consider carefully all
possible molecular events. For example, a compound added to a protein solution
can cause quenching of tryptophan fluorescence. This could come about by binding
of the compound at a site close to the tryptophan (i.e. the residue is surface exposed
to a certain degree), or due to a conformational change induced by the
compound.
Extrinsic fluorescence
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Frequently, molecules
of interest for biochemical studies are non-fluorescent. In many of these
cases, an external fluorophore can be introduced into the system by chemical coupling
or non-covalent binding. Three criteria must be met by fluorophores in this context.
i.
Firstly, it must not
affect the mechanistic properties of the system under investigation.
ii.
Secondly, its
fluorescence emission needs to be sensitive to environmental conditions in
order to enable monitoring of the molecular events.
iii.
And lastly, the
fluorophore must be tightly bound at a unique location.
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A common
non-conjugating extrinsic chromophore for proteins is 1-anilino-8-naphthalene
sulphonate (ANS) which emits only weak fluorescence in polar environment, i.e.
in aqueous solution. However, in non-polar environment, e.g. when bound to hydrophobic
patches on proteins, its fluorescence emission is significantly increased and
the spectrum shows a hypsochromic shift; lmax shifts from
475 nm to 450 nm. ANS is thus a valuable tool for assessment of the degree of
non-polarity. It can also be used in competition assays to monitor binding of
ligands and prosthetic groups.
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Reagents such as
fluorescamine, o-phthalaldehyde or 6-aminoquinolyl-N-hydroxysuccinimidyl
carbamate have been very popular conjugating agents used to derivatise amino
acids for analysis. O-Phthalaldehyde, for example, is a non-fluorescent
compound that reacts with primary amines and b-mercaptoethanol to yield a highly sensitive fluorophore.
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Metal-chelating
compounds with fluorescent properties are useful tools for a variety of assays,
including monitoring of metal homeostasis in cells. Widely used probes for
calcium are the chelators Fura-2, Indo-1 and Quin-1. Since the chemistry of
such compounds is based on metal chelation, cross-reactivity of the probes with
other metal ions is possible.
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The intrinsic
fluorescence of nucleic acids is very weak and the required excitation
wavelengths are too far in the UV region to be useful for practical
applications. Numerous extrinsic fluorescent probes spontaneously bind to DNA
and display enhanced emission. While in earlier days ethidium bromide was one
of the most widely used dyes for this application, it has nowadays been
replaced by SYBRGreen, as the latter probe poses fewer hazards for health and
environment and has no teratogenic properties like ethidium bromide. These
probes bind DNA by intercalation of the planar aromatic ring systems between
the base pairs of double-helical DNA. Their fluorescence emission in water is
very weak and increases about 30-fold upon binding to DNA.
1 comments:
very interesting , good job and thanks for sharing such a good blog.
photoluminescence and fluorescence
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