Instrumentation in Fluorimetry is sophisticated, designed to measure fluorescence with high sensitivity and specificity.
The key components of a fluorimeter include the light source, excitation and emission monochromators or filters, sample holder, and detectors.
Components of a Fluorimeter (Instrumentation in Fluorimetry)
1) Light Source
Xenon Arc Lamp
Provides continuous spectrum from UV to visible regions.
High intensity and stability.
Mercury Vapor Lamp
Emits intense lines at specific wavelengths.
Suitable for fixed-wavelength excitation.
Lasers
Offer monochromatic and coherent light.
Ideal for high-sensitivity applications.
LEDs
Energy-efficient and long-lasting.
Available in various wavelengths.
2) Excitation Monochromator
Function: Selects the specific wavelength of light used to excite the sample.
Types: Prisms or diffraction gratings.
3) Sample Holder
Cuvettes
Made of quartz (for UV range) or glass.
Designed to minimize light scattering.
Front-Face Sample Holders
Used for solid or opaque samples.
Excitation and emission are measured from the same side.
4) Emission Monochromator
Function: Isolates the emitted fluorescence at specific wavelengths.
Considerations: High resolution and stray light rejection are important.
5) Detectors
Photomultiplier Tubes (PMTs)
Extremely sensitive to low light levels.
Fast response times.
Charge-Coupled Devices (CCDs)
Allow for simultaneous detection of multiple wavelengths.
High quantum efficiency.
Filters
Bandpass Filters
Transmit a specific wavelength range.
Used to eliminate unwanted light.
Cut-off Filters
Block wavelengths below or above a certain threshold.
Data Processing System
Computers and Software
Control instrumental parameters.
Collect and analyze data.
Provide spectral displays and quantitative results.
Working of Fluorimetry
Excitation:
A sample is illuminated with UV or visible light.
Absorption:
Molecules in the sample absorb the light and become excited to a higher energy state.
Non-Radiative Relaxation:
Some energy is lost through non-radiative processes, bringing the molecule to a lower vibrational level within the excited state.
Emission:
Excited molecules return to the ground state, emitting light (fluorescence) of a longer wavelength than the excitation light due to energy loss.
Detection:
A detector, placed at a right angle to the excitation beam, measures the intensity of the emitted fluorescence.
Spectral Analysis:
Emission intensity is plotted against emission wavelength, providing qualitative and quantitative data about the sample.
Quantification:
Fluorescence intensity is compared to calibration standards to determine the concentration of fluorescent species.
Applications of Fluorimetry
Quantitative Analysis: Determines the concentration of fluorescent compounds.
Molecular Dynamics: Studies molecular environments, interactions, and conformational changes.
DNA Analysis: Uses fluorescent dyes for sequencing and detection.
Clinical Diagnostics: Detects and quantifies biomolecules for disease diagnosis.
Environmental Monitoring: Identifies pollutants in water, air, or soil.
Drug Discovery: Examines drug interactions and properties.
Cell Biology: Visualizes cellular processes using fluorescent dyes/proteins (e.g., GFP).
Food Industry: Detects contaminants and measures compound concentrations.