Definition:
Fluorimetry measures fluorescence emitted by a substance after absorbing light or radiation.
It involves exciting molecules to higher energy states and detecting the light emitted as they return to lower energy states.
Principle:
A fluorophore absorbs photons, exciting its electrons to higher states.
As electrons return to the ground state, they emit lower-energy (longer wavelength) photons.
The emitted light's intensity and characteristics reveal information about the fluorophore's environment, concentration, and interactions
Key Characteristics:
High Sensitivity: Detects very low concentrations.
Selectivity: Distinguishes fluorophores by their unique excitation and emission wavelengths.
Dynamic Range: Measures a wide concentration range.
Non-Destructive: Preserves sample integrity during analysis.
Components:
Excitation Source: UV or visible light source to excite molecules.
Monochromator: Selects specific excitation wavelengths.
Sample Cell: Holds the sample for analysis.
Emission Monochromator: Filters emitted light to isolate desired wavelengths.
Detector: Measures the intensity of emitted fluorescence.
Applications in Pharmaceuticals:
Quantification: Measures concentrations of naturally fluorescent drugs.
Complex Mixtures: Fluorescent tagging makes non-fluorescent molecules detectable.
Kinetic Studies: Tracks reaction rates via fluorescence changes.
Impurity Detection: Identifies trace fluorescent impurities undetectable by other methods.
Advantages:
High Sensitivity: Detects very low concentrations.
Selectivity: Differentiates compounds by unique excitation and emission wavelengths.
Limitations of Fluorimetry:
Limited to fluorescent compounds or those that can be tagged.
Fluorescence influenced by pH, temperature, and quenching agents