Fluorimetry, which measures fluorescence from a sample, is a valuable analytical tool, especially when high sensitivity and selectivity are required.
The basic principle behind fluorimetry is that when a molecule absorbs light (usually ultraviolet or visible light), it can be elevated to an excited electronic state.
As it returns to its ground state, it can emit a photon with a longer wavelength, which is then detected as fluorescence.
Let's explore the primary components of a typical fluorimeter and their roles in the process:
1. Light Source:
Purpose:
To provide the excitation light that will be absorbed by the sample to produce fluorescence.
Common Sources:
Xenon or Mercury Arc Lamps:
These have been traditionally used because they provide a broad and intense spectrum of wavelengths.
LEDs:
Modern fluorimeters might use light-emitting diodes due to their long lifespan, stability, and the ability to produce specific wavelengths.
2. Excitation Monochromator or Filter:
Purpose:
To select a specific wavelength (or a narrow band of wavelengths) from the broad spectrum provided by the light source.
Function:
Works by diffracting light using a grating or a prism and then isolating the desired wavelength through a slit.
In some devices, specific filters are used instead of a monochromator to allow only the desired wavelength to pass.
3. Sample Holder (or Cuvette):
Purpose:
To contain the sample that is to be studied.
Material:
Typically made of quartz or other UV-transparent materials, especially when studying samples that are excited by UV light.
4. Emission Monochromator or Filter:
Purpose:
To select and isolate the specific fluorescence wavelength emitted by the sample.
This is necessary because the sample can emit light over a range of wavelengths.
Function:
Similar to the excitation monochromator, it isolates a specific emitted wavelength or a narrow band of wavelengths. Some fluorimeters use filters for this purpose.
5. Detector:
Purpose:
To detect the emitted fluorescence and convert it into an electrical signal.
Common Detectors:
Photomultiplier Tubes (PMT):
These are highly sensitive detectors that can detect low light levels.
They work by amplifying the signal from individual photons, making them especially useful for low-concentration samples.
Photodiodes:
Used in some modern fluorimeters, these are semiconductor devices that convert light into an electrical current. They can be faster than PMTs but might be less sensitive.
6. Signal Processor and Display:
Purpose:
To convert the electrical signal from the detector into a usable format (usually an intensity vs. wavelength plot) and display or record it.
Components:
Amplifier:
Boosts the signal from the detector.
Analog-to-Digital Converter (ADC):
Converts the analog signal into a digital one for processing.
Microprocessor or Computer:
Analyzes and displays data, often using specialized software.
Modern fluorimeters can also store data, fit curves to determine concentrations, and provide real-time readouts.
7. Additional Components:
Temperature Control:
Some experiments require the sample's temperature to be controlled, so a temperature-regulating system may be integrated.
Stirring Mechanism:
In cases where the sample needs to be continuously mixed.
Time-Resolved Modules:
For experiments that require measurement of fluorescence lifetimes or kinetics.