Spectrofluorometry is an analytical technique that measures the fluorescence emitted by a
substance when it is excited by light of a specific wavelength.
Key idea:
• Certain molecules absorb light → get excited to a higher electronic state → then emit light of
a longer wavelength when returning to the ground state.
• By measuring this emitted light, you can detect and quantify very low concentrations of
fluorescent substances.
Basic Principle of Fluorescence
Let’s break down the steps:
Excitation:
A molecule absorbs a photon → an electron jumps from the ground state (S₀) to an excited singlet
state (S₁ or S₂).
Relaxation:
The excited electron loses some energy non-radiatively (vibrational relaxation) within the excited
state.
Emission (Fluorescence):
The electron drops back to the ground state, emitting a photon of lower energy (longer wavelength)
than the absorbed light..
What molecules fluoresce?
Molecules with rigid, planar structures and conjugated double bonds fluoresce well.
Common examples:
• Aromatic hydrocarbons (e.g., anthracene, pyrene)
• Biological molecules (e.g., tryptophan, NADH)
• Dyes (fluorescein, rhodamine)
• Many drug molecules
Instrumentation: Detailed Components
1) Light Source
• Xenon arc lamp: Most common, provides intense, continuous light over UV–visible range.
• Lasers (e.g., Argon-ion, He–Cd): Used in laser-induced fluorescence — higher intensity,
better resolution.
2) Excitation Monochromator
• A device (usually a diffraction grating) that selects the precise excitation wavelength from the
broad light source.
• Only the chosen wavelength reaches the sample.
3) Sample Cell
• Usually a quartz cuvette (glass absorbs UV light so quartz is preferred for UV).
• Path length commonly 1 cm.
4) Emission Monochromator
• Isolates a specific wavelength of the emitted fluorescence light.
• Can scan across a range to produce an emission spectrum.
5) Detector
• Photomultiplier tube (PMT): Very sensitive to low light levels — perfect for weak
fluorescence signals.
• Converts emitted light into an electrical signal for measurement.
Applications
Field Typical Use
Biochemistry & Molecular Quantify nucleic acids, proteins (e.g., tryptophan fluorescence), enzyme
Biology activity (with fluorescent probes).
Environmental Science Detect pollutants like PAHs (polycyclic aromatic hydrocarbons) in water.
Clinical & Medical
Measure biomarkers, vitamins (e.g., riboflavin, bilirubin).
Diagnostics
Assay drugs that naturally fluoresce or are derivatized with a
Pharmaceutical Analysis
fluorophore.
Food Industry Detect contaminants or monitor food quality (e.g., aflatoxins in grains).
Forensics Detect trace evidence using fluorescent dyes or labels.
Limitations
Limitation Explanation
Not all substances fluoresce Some compounds need derivatization.
Fluorescence quenching Other species can reduce fluorescence signal (e.g., oxygen, heavy ions).
Photobleaching Fluorophores can degrade if exposed to light too long.
Matrix effects Other sample components may interfere with signal.