Introduction to Spectrofluorimetry

Spectrofluorimetry is an analytical technique used to measure the fluorescence emitted by a sample when it absorbs light at a specific wavelength. Fluorescence is the emission of light that occurs when a molecule absorbs light at one wavelength (usually UV or visible light) and then re-emits it at a longer wavelength (usually visible light). This technique is commonly used to study the properties of molecules, particularly those with aromatic structures, and is highly sensitive, making it useful for detecting trace quantities of substances.

  • Principle: When a molecule absorbs photons (light) of a specific energy, it undergoes a transition to an excited state. The molecule then relaxes back to the ground state, emitting a photon (fluorescence) in the process.

 

Theory of Fluorescence

Fluorescence occurs through the following steps:

  1. Excitation: The molecule absorbs photons, promoting electrons from a lower energy state (usually the ground state) to a higher energy excited state.
  2. Relaxation: After excitation, the molecule quickly relaxes to a lower excited state, emitting some of the absorbed energy as heat.
  3. Fluorescence Emission: The molecule then returns to its ground state by emitting light (fluorescence) at a longer wavelength than the excitation light. This phenomenon is a result of energy loss during the relaxation process.
  • Fluorescence Stokes Shift: The difference between the absorption and emission wavelengths is called the Stokes shift. Since some energy is lost as heat during relaxation, the emitted light is at a longer wavelength than the absorbed light.
  • Fluorescence Lifetime: The time interval between the absorption of light and the emission of fluorescence. Fluorescence typically lasts on the order of nanoseconds.

 

Factors Affecting Fluorescence

Several factors influence the intensity and properties of fluorescence:

1. Molecular Structure

  • Aromatic Compounds: Fluorescence is more pronounced in compounds with conjugated π-electron systems (e.g., aromatic hydrocarbons, fluorescein).
  • Functional Groups: The presence of certain functional groups (e.g., hydroxyl, carbonyl, or amino groups) can alter the fluorescence intensity, emission wavelength, and quantum yield.

2. Solvent Effects

  • Polarity of Solvent: Fluorescence emission can be affected by the solvent's polarity. Polar solvents can sometimes quench fluorescence by stabilizing the excited state, reducing the energy available for emission.
  • Solvent Viscosity: Higher viscosity solvents can slow down non-radiative processes like internal conversion, leading to enhanced fluorescence.

3. Temperature

  • Temperature Effects: Higher temperatures can increase non-radiative processes (e.g., vibrational relaxation), which can lead to a decrease in fluorescence intensity. Conversely, lower temperatures often reduce these processes and can enhance fluorescence.

4. pH

  • pH Sensitivity: Some fluorophores exhibit pH-dependent fluorescence. This is due to changes in the protonation state of functional groups within the molecule that alter its ability to fluoresce.

5. Concentration of the Fluorophore

  • Quenching Effects: At higher concentrations, fluorophores may undergo quenching, where the emitted fluorescence decreases due to interactions between molecules (e.g., aggregation, energy transfer).

6. Presence of Quenchers

  • Quenching: Quenching refers to the process by which the fluorescence emission of a fluorophore is reduced due to the presence of another substance. This could occur via:
    • Dynamic Quenching: Occurs when a molecule in its excited state collides with a quencher molecule, transferring energy non-radiatively.
    • Static Quenching: Occurs when the fluorophore forms a non-fluorescent complex with the quencher.

 

Quenchers in Fluorescence

Quenchers are substances that decrease the fluorescence intensity of a fluorophore. They can do this through various mechanisms:

  • Collisional Quenching: The excited fluorophore interacts with the quencher through collisions, resulting in energy dissipation without light emission.
  • Energy Transfer: The excited fluorophore transfers its energy to the quencher molecule, preventing photon emission.

Common quenchers include:

  • Oxygen: Molecular oxygen is a common quencher and can reduce fluorescence intensity by non-radiative processes.
  • Heavy Metal Ions: Ions such as Fe³⁺, Cu²⁺, or Hg²⁺ can quench fluorescence by altering the electronic properties of the fluorophore.

 

Instrumentation of Fluorescence Spectrophotometer

A fluorescence spectrophotometer is designed to measure the fluorescence emitted by a sample. It consists of several key components:

1. Light Source

  • The light source provides the excitation light. In fluorescence spectrophotometers, a xenon lamp or mercury vapor lamp is typically used for broad-spectrum excitation in the UV-visible range.

2. Monochromator/Filter

  • A monochromator or optical filters are used to select a specific wavelength of light for excitation. The monochromator typically uses a diffraction grating or prism to disperse the light, while filters are used to pass only the selected wavelength.

3. Sample Holder (Cuvette)

  • The sample is usually placed in a quartz cuvette, which is transparent to UV and visible light. The cuvette is positioned in the path of the excitation beam.

4. Emission Monochromator/Filter

  • After the sample absorbs the excitation light and re-emits fluorescence, a second monochromator or filter is used to isolate the specific wavelength of the emitted fluorescence.

5. Detector

  • The detector collects the emitted fluorescence and converts it into an electrical signal. Common detectors include:
    • Photomultiplier Tubes (PMTs): These are highly sensitive and commonly used for measuring weak fluorescence.
    • Photodiodes: For less sensitive applications.

6. Readout System

  • The readout system displays the fluorescence intensity versus the wavelength of emitted light, often in the form of a spectrum. A computer or digital display is used to present the data.

 

Applications of Fluorescence Spectrophotometry

Fluorescence spectrophotometry has a wide range of applications across various fields:

1. Analytical Chemistry

  • Quantification: Fluorescence is highly sensitive, and it can be used to detect very low concentrations of analytes. It is frequently used in the analysis of trace metals, organic compounds, and biomolecules.
  • Environmental Monitoring: Fluorescence is used to detect pollutants in air, water, and soil. It is particularly useful for measuring the concentration of organic contaminants such as polycyclic aromatic hydrocarbons (PAHs).

2. Biochemical and Biomedical Research

  • Fluorescent Labeling: Fluorescence is widely used in biological and biomedical research for tagging and tracking molecules, such as proteins, nucleic acids, and cells.
  • Immunoassays: Fluorescence is used in immunoassay techniques like enzyme-linked immunosorbent assays (ELISA) to detect specific proteins or antibodies in complex biological samples.
  • Cell Imaging and Cytometry: Fluorescent dyes are used for imaging cells, tissues, and organs to study biological processes such as gene expression, cell division, and apoptosis.

3. Medical Diagnostics

  • Fluorescence-based Diagnostic Kits: Fluorescence is used in diagnostic tests, including those for detecting infections, cancers, and genetic disorders.
  • Fluorescence Lifetime Imaging Microscopy (FLIM): FLIM is a technique that measures the fluorescence decay time, providing insights into molecular dynamics and interactions in living cells.

4. Environmental and Food Safety

  • Food Contaminants: Fluorescence spectrophotometry is used to detect harmful substances in food, such as pesticides, toxins, and preservatives.
  • Pollution Monitoring: Fluorescence is used in environmental analysis to monitor pollutants like industrial chemicals, dyes, and oils in waterways.

5. Forensic Science

  • Detection of Drugs and Explosives: Fluorescence spectrophotometry is employed in forensic investigations to identify drugs, explosives, and other illicit substances based on their fluorescent properties.