Applications of Fluorescence Spectroscopy

Fluorescence Spectroscopy is an analytical method that studies the fluorescence emitted by a sample when it is excited by light (often UV or visible).

• It is often called fluorimetry or spectrofluorometry.
• In practice, light of a defined excitation wavelength is shone on sample, electrons are raised to excited states.
• Then molecules relax and emit light at longer emission wavelength (lower energy).
• A spectrofluorometer or similar instrument is used to measure excitation and emission spectra.
• The quantum yield (ratio of photons emitted / photons absorbed) is used to describe efficiency.
• Lifetimes (decay time) can be measured by time-domain or frequency-domain methods.
• Fluorescence spectroscopy complements absorption spectroscopy; often better sensitivity.
• Very high sensitivity is offered by this method, low concentrations (even trace) can be detected.
• Selectivity is allowed because excitation and emission wavelengths differ (Stokes shift), so interference is reduced.
• It is widely used in chemistry, biology, medicine to monitor molecules, reactions, binding, structural changes.
• Kinetic and dynamic information (e.g. lifetimes) can be extracted.
• Fluorescence phenomenon was observed long before understood (e.g. in the 1500’s, lignum nephriticum wood blue glow).
• In 1845, Herschel discovered that UV light can excite a quinine solution to emit blue light.
• Sir George G. Stokes coined “fluorescence” in 1852, and described the shift to longer wavelength (emission).
• In early 20th century, first fluorescence microscopes were built (1911–1913) by Otto Heimstaedt & Heinrich Lehmann.
• Later, Gregorio Weber pioneered many modern uses in biology (protein fluorescence, probes).
• Theoretical model of excited / ground state transitions via Jablonski diagram was formulated by Aleksander Jablonski (mid-20th century).

Principle of Fluorescence Spectroscopy

• Fluorescence spectroscopy based on the principle that certain molecules absorb light energy and then emit part of it as visible radiation when returning to lower energy state.
• When a photon of appropriate wavelength falls on a molecule, its electron is excited from ground state (S₀) to higher excited singlet state (S₁ or S₂).
• The excitation occur very fast, within about 10⁻¹⁵ seconds, and electrons move to higher molecular orbital having more energy.
• After excitation, part of absorbed energy is lost by vibrational relaxation and internal conversion, so the molecule goes to the lowest vibrational level of excited singlet state.
• From this state, it returns back to the ground state by emission of light known as fluorescence.
• The emitted photon always have less energy (thus longer wavelength) than absorbed light, because some energy is dissipated as heat – this difference termed as Stokes shift.
• The intensity of fluorescence emission is directly proportional to concentration of fluorophore within certain limit, beyond which quenching occurs.
• The whole process involve electronic transitions, and the spectrum obtained gives information about electronic structure and environment of molecule.
• Excitation and emission both can be recorded by spectrofluorometer, where monochromators select specific wavelengths for analysis.
• In most compounds fluorescence emission occur in nanosecond range, while delayed emissions (phosphorescence) happen in microsecond to second range.
• The efficiency of fluorescence process expressed as quantum yield (Φf), which represent ratio of emitted photons to absorbed photons.
• Fluorescence process is mostly shown by molecules having rigid and planar structures with conjugated π-system like anthracene, fluorescein, rhodamine, etc.
• In general, the mechanism involve 3 main stages – (i) Excitation by light absorption, (ii) Relaxation by non-radiative losses, and (iii) Emission of fluorescent light.
• Fluorescence spectroscopy therefore depends on energy transition between singlet states, and is widely used for molecular identification and microenvironmental analysis.

Applications of Fluorescence Spectroscopy in inorganic chemistry

• Fluorescence spectroscopy is used widely for studying molecular structure and conformational change of organic compounds which have aromatic or conjugated systems.
• It is applied for identification of functional groups that are responsible for emission behavior in organic molecules, especially in aromatic hydrocarbons.
• The purity of organic compounds can checked by fluorescence intensity variation, since even small impurity can quench or enhance emission strongly.
• In photochemical reactions, fluorescence spectroscopy is used to monitor intermediate formation and decay rate which happen during light-induced transformations.
• The method is useful for measuring quantum yield, fluorescence lifetime and excited state energy which help to understand reaction mechanism.
• In polymer chemistry, it is used to investigate polymerization process, molecular aggregation and excimer/exciplex formation in organic materials.
• The electronic transitions within conjugated π-systems are analyzed by fluorescence spectra which shows shifts depending on solvent polarity or substitution pattern.
• It has been applied for detection of organic pollutants, like polycyclic aromatic hydrocarbons (PAH) in environmental samples.
• In biochemical field, many organic fluorophores (like fluorescein, rhodamine, dansyl chloride) are used as probes for tracing organic reactions or biological molecules.
• The energy transfer between donor–acceptor pairs in organic compounds are studied using fluorescence resonance energy transfer (FRET) technique.
• Fluorescence spectroscopy is employed for studying reaction kinetics, particularly in reactions involving excited state or radical intermediates.
• It is also applied in designing organic light-emitting diodes (OLEDs), where fluorescence efficiency of organic materials are examined.
• Many organic reactions which involve photo-oxidation or reduction are understood by comparing fluorescence quenching or enhancement behavior under different atmosphere.
• The method provides a non-destructive, sensitive, and accurate approach for analyzing organic compounds in solution / solid state etc.

Special fluorimetric applications

• Fluorescence Quenching Studies – are used to determine molecular interaction between fluorophore and quencher molecule which may be static or dynamic in nature.
• The quenching mechanism can analyze by observing change in fluorescence intensity or lifetime that occurs when quencher interacts with excited molecule.
• Fluorescence Lifetime Measurement is applied for studying excited state decay processes, which give valuable info about environment around fluorophore molecule.
• The lifetime values often depend by solvent polarity, temperature or binding of analyte, which indicate microenvironmental changes.
• Fluorescence Polarization (FP) is used for determining rotational diffusion and molecular size in solution, larger molecules show higher polarization values.
• It is applied in binding studies where changes in polarization indicate formation of complex between small and large molecules.
• Fluorescence Resonance Energy Transfer (FRET) technique is used for measuring distance between two chromophores (donor–acceptor), usually in 10–100 Å range.
• FRET is highly sensitive to spatial changes, so it’s applied to study protein folding / conformational transition and biological macromolecule interaction.
• Synchronous Fluorescence Spectroscopy (SFS) is used for mixture analysis where simultaneous scanning of excitation and emission wavelength improve resolution of spectra.
• It helps in determination of overlapping fluorescent species like aromatic hydrocarbons in complex samples.
• Time-Resolved Fluorescence Spectroscopy allows observation of transient excited state processes, to study reaction kinetics and photophysical behavior.
• Anisotropy Measurement (also termed as polarization anisotropy) is applied for studying rotational motion, viscosity and molecular binding in various media.
• Front-face fluorescence technique is used for solid or turbid samples where normal right-angle geometry fails due to scattering or absorption effects.
• Phase-modulation fluorimetry is employed for high frequency modulation study to determine lifetime and phase shift parameters precisely.
• In single molecule fluorescence, detection of individual fluorophores is done to analyze molecular heterogeneity and dynamic fluctuation which are averaged out in bulk.
• Quantum yield determination is an important special application for comparing efficiency of fluorescence emission among different compounds.
• Such methods are used broadly in biochemistry, material science, environmental analysis and in design of optical sensors etc.
• These fluorimetric techniques provide deeper insight into molecular interactions and microenvironment, though careful calibration and control are usually required for accurate result.