Fluorimetry, or fluorimetric analysis, is an analytical technique based on the measurement of fluorescence emitted by molecules after absorbing light. When certain compounds absorb radiation, they move to an excited electronic state and then release part of the absorbed energy as visible or near-visible light. This emitted light is called fluorescence. Because fluorescence is highly sensitive, the technique allows detection of very low concentrations of analytes, making it widely used in pharmaceutical quality testing, biochemical analysis, environmental monitoring, and clinical diagnostics.
Theory of Fluorimetry
Fluorescence occurs when molecules absorb photons and transition from the ground state to an excited singlet state. After a brief lifetime (10-8 to 10-9 seconds), the molecules return to the ground state by emitting a photon of lower energy. This emission is measured at 90° to the incident beam to minimize interference from the excitation light.
The intensity of fluorescence is proportional to the number of molecules excited, making it suitable for both qualitative and quantitative analysis.
Types of Luminescence
Luminescence refers to the emission of light from a molecule that has absorbed energy. It is divided into:
- Fluorescence: Emission from a singlet excited state; extremely fast and ceases immediately once the excitation stops.
- Phosphorescence: Emission from a triplet excited state; slower and may continue even after the light source is removed.
- Chemiluminescence: Light emitted during a chemical reaction.
- Bioluminescence: Light produced by biological organisms (e.g., fireflies).
Electronic Transitions (Singlet, Doublet, Triplet States)
Electronic transitions describe how electrons move between energy levels:
- Singlet state: All electron spins are paired. Excitation from S0 → S1 leads to fluorescence.
- Triplet state: Electrons have parallel spins. Inter-system crossing from S1 → T1 can occur, leading to phosphorescence.
- Doublet state: Characteristic of radicals with unpaired electrons.
Fluorescence originates from the singlet excited state, while phosphorescence originates from the triplet state, which has a longer lifetime.
Energy Level Diagrams for Photoluminescent Molecules
Energy diagrams (Jablonski diagrams) illustrate:
- Absorption of energy and transition to excited states (S1, S2)
- Vibrational relaxation within excited states
- Internal conversion (non-radiative transitions)
- Fluorescence emission from S1 → S0
- Phosphorescence from T1 → S0
The diagram visually explains why fluorescence occurs quickly while phosphorescence persists longer.
Deactivation Processes
Once a molecule is excited, it returns to the ground state through several pathways:
- Fluorescence emission
- Phosphorescence
- Internal conversion: Non-radiative loss of energy
- Vibrational relaxation: Energy lost as heat
- External conversion: Energy transferred to solvent
- Quenching: Loss of fluorescence due to interactions with other molecules
These processes determine fluorescence efficiency and analytical sensitivity.
Factors Affecting Fluorescence
Fluorescence intensity is influenced by many variables:
- Temperature: Higher temperatures reduce fluorescence due to increased molecular collisions.
- pH: Ionization can alter molecular structure and affect emission.
- Solvent polarity: Polar solvents may enhance or reduce fluorescence depending on the molecule.
- Presence of quenchers: Oxygen, heavy metals, and halogens often reduce fluorescence.
- Concentration: Very high concentrations lead to self-quenching.
- Viscosity: Higher viscosity reduces collisions, increasing fluorescence yield.
Structural Behavior Affecting Fluorescence Intensity
The molecular structure plays a major role in fluorescence capability. Key structural factors include:
- Aromaticity: Aromatic molecules (e.g., benzene derivatives) often fluoresce strongly.
- Rigid structures: More rigidity → higher fluorescence (e.g., fused ring systems).
- Electron-donating groups: Increase fluorescence (–OH, –NH2, –OCH3).
- Electron-withdrawing groups: Decrease fluorescence (–NO2, –CN, halogens).
- Substitution pattern: Ortho and para substitutions may enhance emission.
Compounds like quinine, fluorescein, and aromatic hydrocarbons show strong fluorescence due to their rigid, conjugated structures.
Quenching
Quenching refers to any process that reduces fluorescence intensity. Types include:
- Dynamic quenching: Caused by collisions between molecules.
- Static quenching: Due to formation of non-fluorescent complexes.
- Oxygen quenching: Molecular oxygen is a powerful quencher.
- Heavy atom effect: Halogens and heavy metals increase intersystem crossing.
Understanding quenching is crucial for accurate fluorimetric analysis.
Instrumentation of Fluorimetry
A fluorometer is an instrument designed to measure fluorescence intensity. Its main components include:
- Light source: Xenon arc lamp, mercury lamp, or lasers
- Excitation monochromator: Selects specific excitation wavelengths
- Sample cell (cuvette): Usually quartz to allow UV transmission
- Emission monochromator: Selects emitted wavelengths
- Detector: Photomultiplier tube (PMT) or photodiode
The detection is usually at a right angle (90°) to minimize interference from the excitation beam.
Working of a Fluorometer
The fluorometer operates by:
- Producing an excitation beam of appropriate wavelength.
- Directing the beam onto the sample.
- Allowing sample molecules to absorb energy and fluoresce.
- Collecting emitted fluorescence at a right angle to the excitation beam.
- Passing emitted light through an emission monochromator.
- Detecting and converting light signals into electrical signals.
- Displaying the fluorescence intensity on the output system.
The instrument can operate in fixed-wavelength mode or scanning mode for full emission spectra.
Advantages of Fluorimetry
- Extremely sensitive—detects nanogram and picogram levels
- High selectivity due to unique emission wavelengths
- Useful for trace analysis in pharmaceuticals and biological samples
- Fast and non-destructive technique
- Suitable for automation and kinetic studies
Applications of Fluorimetry
- Determination of vitamins such as riboflavin
- Analysis of antibiotics (tetracyclines, quinolones)
- Assay of polycyclic aromatic hydrocarbons
- Clinical analysis of biomolecules
- Environmental detection of pollutants and pesticides
- Study of protein–ligand interactions
- Drug stability and degradation studies
Detailed Notes:
For PDF style full-color notes, open the complete study material below: