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PHDAnalytical chemistrySpectroscopic Methods


Fluorescence Spectroscopy


Fluorescence spectroscopy is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. It involves using a beam of light, usually UV light, to excite electrons in the molecules of certain compounds and cause them to emit light; this emitted light is often at a longer wavelength than the absorbed radiation.

Basic principles

The process of fluorescence can be understood by studying the Jablonski diagram:

    S2 │ excited │
       │ State │
               
    S1 │ excited │
       │ State │
         
    __  __
      /
       /
       /
       
    S0 │ Ground │
       │ State │
    

When a molecule absorbs a photon, it goes from a ground state (S0) to an excited state (S1 or S2). This transition occurs in about 10-15 seconds and is marked by the absorption of energy. After being excited, the molecule relaxes and falls to the lowest vibration level of S1. This process, called internal conversion, is usually faster than other processes.

The molecule can return to its ground state in a few ways. One possibility is by emitting a photon; this is fluorescence. Generally, the emitted light has a longer wavelength than the absorbed light because of some energy loss during non-radiative decay.

Properties of fluorescence

Fluorescence spectroscopy provides various noteworthy properties:

  • Quantum yield: The ratio of the number of photons emitted to the number of photons absorbed. A high quantum yield indicates that most of the absorbed photons result in emitted fluorescence.
  • Stokes shift: The difference in wavelength between the positions of the band maxima of the absorption and emission spectra. This is important for fluorescence detection because it gives the difference between the excitation and emission wavelengths, thereby reducing noise.

Applications of fluorescence spectroscopy

Fluorescence spectroscopy is widely used in various fields due to its high sensitivity and selectivity:

1. Biochemistry: It is used to study proteins, nucleic acids, and other biomolecules. For example, researchers can use amino acids such as tryptophan or tyrosine, which are naturally fluorescent, to study protein structure.

2. Medical diagnosis: Fluorescence techniques are widely used in medical diagnosis (e.g., flow cytometry, fluorescence microscopy) to identify and quantify biomolecules or cells.

3. Environmental studies: The presence of pollutants, toxins or other environmental parameters can be monitored efficiently using fluorescent methods, such as oil spill detection with fluorescent dyes.

4. Forensic analysis: The ability to detect small quantities of substances makes fluorescence spectroscopy invaluable in forensic investigations.

Instrumentation of fluorescence spectroscopy

A typical fluorescence spectrometer consists of several basic components:

  • Light source: The source must provide sufficient intensity. Typically, xenon or mercury lamps are used; these emit broad spectrum light. In modern spectrometers, lasers or LEDs are also used for excitation.
  • Excitation monochromator or filter: It isolates a specific excitation wavelength to avoid interference from other wavelengths.
  • Cuvette: The sample is contained here. Cuvettes are usually made of glass, quartz, or plastic and must have a flat surface and optical-quality surface.
  • Emission monochromator or filter: This isolates the fluorescence light emitted from the sample and removes any scattered excitatory light.
  • Detectors: Typically, photomultiplier tubes (PMTs) are used to detect and measure the light intensity. Other detectors such as photodiodes or CCD cameras are also used.

Visual representation of fluorescence spectroscopy

Below is a simplified visual representation of fluorescence spectroscopy in action:

    │ light │ __ __ │ │ __ ____ │ emitter │__ __
    │ source │/ /  │ stimulation │/ / │ / / 
    └────────────┘ │ mono- │ └──────────┘
                             │ Chromatator/ │
                             │ Filter │
                             
                 
                 │Sample (cuvette)│ │
                 │ < stimulation │ emission > │
                 
                             
                             │ Emission │
                             │ mono- │
                             │ Chromatator/ │
                             │ Filter │
                             
                             │Detector │                             
    

Advantages of fluorescence spectroscopy

1. Sensitivity: This technique is extremely sensitive and can detect substances at very low concentrations, often as low as parts per billion (ppb).

2. Selectivity: Different excitation and emission wavelengths make it possible to selectively analyze specific compounds, even in complex mixtures.

3. Non-destructive: Samples can often be analyzed without any consumption or destruction.

Limitations of fluorescence spectroscopy

1. Quenching: Certain conditions or compounds can interfere with the fluorescence process, reducing the intensity - this is called quenching. Typical quenchers include oxygen, heavy metals, and other chemicals.

2. Interference: Scattering from the sample or container can introduce noise and complicate the measurement.

3. Photobleaching: Prolonged exposure to excitation light can degrade the sample, permanently reducing fluorescence.

Future trends

Advances in technology continue to enhance the applications of fluorescence spectroscopy. For example, the integration of fluorescence spectroscopy with electronic and optical technologies allows the development of miniaturized, portable instruments with rapid, on-site testing capabilities.

Applications in high-throughput screening and in-vitro diagnostics are expanding, with innovations such as single-molecule fluorescence microscopy achieving unprecedented levels of detail and understanding in chemical and biochemical systems.

Overall, fluorescence spectroscopy remains an invaluable tool in modern scientific research, with vast potential for future discovery and technology development.


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