Spectrofluorometer
A spectrofluorometer, also known as a fluorescence spectrophotometer or fluorometer, is an
instrument used to measure the fluorescent properties of a sample. It works on the principle of
fluorescence spectroscopy, a type of electromagnetic spectroscopy that analyzes the light
emitted by fluorescent molecules.
Here's a breakdown of the principle and key aspects:
Principle of Operation:
1. Excitation: A sample containing fluorescent molecules (fluorophores) is illuminated
with a beam of light of a specific wavelength, typically in the ultraviolet (UV) or visible
range. This light is selected by an excitation monochromator or filter.
2. Absorption: The fluorophores absorb photons of the excitation light, causing their
electrons to jump to a higher energy level (excited state).
3. Vibrational Relaxation: The excited state is typically unstable. Before emitting light,
the molecule quickly loses some of its vibrational energy through collisions with
surrounding molecules. This results in the molecule reaching the lowest vibrational level
of the excited electronic state.
4. Emission (Fluorescence): From this lower excited state, the molecule returns to the
electronic ground state, releasing the remaining energy as a photon of light. This emitted
light is the fluorescence.
5. Stokes Shift: The emitted fluorescent light always has a longer wavelength (and thus
lower energy) than the absorbed excitation light. This difference in wavelength is called
the Stokes shift.
6. Detection: The emitted fluorescent light is then passed through an emission
monochromator or filter to select a specific wavelength or to scan a range of
wavelengths. The intensity of the emitted light at the selected wavelength(s) is measured
by a detector, such as a photomultiplier tube (PMT), photodiode, or charge-coupled
device (CCD).
Key Components of a Spectrofluorometer:
Excitation Source: Usually a high-intensity light source like a xenon arc lamp or a laser,
providing a broad range of wavelengths.
Excitation Monochromator (or Filter): Selects the specific wavelength of light used to
excite the sample.
Sample Holder: A compartment to hold the sample, often a quartz cuvette for liquid
samples or specialized holders for solid samples.
Emission Monochromator (or Filter): Selects the wavelength(s) of the emitted
fluorescent light to be detected.
Detector: Measures the intensity of the fluorescent light.
Signal Processor and Display: Amplifies the detector signal and displays the
fluorescence intensity as a function of wavelength.
Applications of Spectrofluorometry:
Spectrofluorometer
Spectrofluorometers are versatile tools used in a wide range of scientific disciplines for both
qualitative and quantitative analysis. Some key applications include:
Biochemistry and Molecular Biology: Studying the structure and function of proteins,
nucleic acids (DNA, RNA), enzymes, and other biomolecules. Monitoring biological
reactions, binding events, and conformational changes.
Pharmaceutical Analysis: Determining the concentration of drugs, studying drug
interactions, and analyzing pharmaceutical formulations.
Environmental Monitoring: Detecting pollutants in water, air, and soil, such as
petroleum contaminants, polycyclic aromatic hydrocarbons, and pesticides.
Clinical Diagnostics: Detecting and diagnosing diseases, including cancer, through the
use of fluorescent probes and dyes.
Food Science: Analyzing food components, detecting adulterants, and assessing food
quality.
Forensic Science: Analyzing inks, fibers, drugs, and biological samples.
Material Science: Characterizing the optical properties of materials, studying
photoluminescence in polymers and semiconductors.
Water Quality Analysis: Identifying organic contaminants and monitoring algal blooms.
In summary, a spectrofluorometer is a sensitive instrument that utilizes the phenomenon of
fluorescence to analyze the composition and properties of various samples across diverse
scientific fields. Its ability to selectively excite molecules and measure their characteristic
emission spectra makes it a powerful tool for both fundamental research and practical
applications.
Spectrofluorometer:
Principle: Measures the fluorescence emitted by a sample. It excites the sample with a
specific wavelength of light, causing fluorescent molecules (fluorophores) to emit light at
a longer wavelength. The instrument then analyzes the intensity of this emitted light at
different wavelengths.
What it measures: Emission spectra (intensity of emitted light versus wavelength) and
excitation spectra (efficiency of different excitation wavelengths in producing
fluorescence at a fixed emission wavelength).
Sample requirement: Requires the sample to contain fluorescent molecules, either
naturally or through the use of fluorescent probes or dyes.
Sensitivity: Generally more sensitive than spectrophotometers for fluorescent substances
because it measures emitted light against a low background.
Spectrofluorometer
Applications: Ideal for studying biomolecules (proteins, DNA), drug interactions,
environmental pollutants that fluoresce, and in applications like cell imaging and
diagnostics using fluorescent markers.
Spectrophotometer:
Principle: Measures the absorbance or transmission of light through a sample as a
function of wavelength. It passes a beam of light through the sample and compares the
intensity of the incident light with the intensity of the light that passes through
(transmitted light). The difference indicates how much light was absorbed by the sample.
What it measures: Absorption spectra (absorbance versus wavelength) and can be used
to determine the concentration of substances based on Beer-Lambert Law.
Sample requirement: Can be used with a wide variety of samples, including colored
solutions, turbid samples, and even solids with appropriate accessories. The sample
doesn't need to be fluorescent.
Sensitivity: Generally less sensitive than spectrofluorometers for detecting trace amounts
of substances, especially non-colored ones.
Applications: Widely used for determining concentrations of solutions, studying
chemical reactions, quality control in various industries (pharmaceutical, food,
environmental), and characterizing materials based on their absorption properties.
In summary, the key difference lies in what they measure:
Spectrofluorometer: Measures emitted fluorescent light.
Spectrophotometer: Measures absorbed or transmitted light.
Therefore, if you were interested in measuring the fluorescent properties of a sample, a
spectrofluorometer would be the appropriate instrument. If you were interested in measuring
how much light a sample absorbs, a spectrophotometer would be used.
