Fluorescence Spectroscopy

BY- K. Sai Manogna (MSIWM014)

It is a type of electromagnetic spectroscopy that analyses fluorescence from a sample. It is also known as fluorometry or spectrofluorometry. It requires the use of a light ray, usually, ultraviolet light, which excites the electrons of certain compounds in molecules and causes them to emit low-energy light, typically, but not always, visible light. Absorption spectroscopy is a complementary technique. Fluorometers or fluorimeters are called instruments that measure fluorescence. 

What is Fluorescence Spectroscopy? - HORIBA

Theory of Fluorescence spectroscopy:  

Molecules have different states, referred to as levels of energy. Electronic and vibrational states are mainly concerned with fluorescence spectroscopy. In general, the species being studied would have an interest in the ground electronic state (a low energy state) and a higher energy excited electronic state. Various vibrational states are within any of these electronic states. 

1. In fluorescence spectroscopy, from its ground electronic state to one of the different vibrational states in the excited electronic state, the species is first excited by absorbing a photon. 

2. Collisions with other molecules cause vibrational energy to be lost by the excited molecule before the excited electronic state’s lowest vibrational state is reached. 

3. The molecule then drops down, releasing a photon in the process to one of the ground electronic state’s different vibrational levels again. 

4. The emitted photons have different energies, and therefore frequencies, as molecules can drop down into any of many vibrational levels in the ground state. 

5. Therefore, the structure of the various vibrational levels can be calculated by studying the different wavelengths of light emitted in fluorescent spectroscopy and their relative intensity. 

Principles and Theory of Fluorescence Spectroscopy - HORIBA

The various fluorescent light frequencies produced by a sample are calculated in a standard experiment, maintaining the excitation light at a constant wavelength.  It is called the continuum of pollution. An excitation spectrum is measured using different wavelengths of excitation light by recording several emission spectra. 

Instrumentation

There are two general types of instrument

a. In order to separate incident light and fluorescent light, filter fluorometers use filters. 

b. In order to insulate the incident light and fluorescent light, spectrofluorometers use diffraction grating monochromators. 

Both types use the following system: 

1. The light passes through a monochromator or filter from an excitation source and strikes the sample. 

2. The sample absorbs a proportion of the incident light, and some of the molecules fluoresce in the sample. 

3. In all directions, fluorescent light is released. 

4. To minimize the chance of emitted or reflected incident light hitting the detector, some fluorescent light passes through a monochromator or second filter and enters a detector usually positioned at 90° to the incident light beam. 

5. Various light sources, including lasers, photodiodes, and lamps, can be used as excitation sources, xenon arcs, and mercury vapor lamps in particular. 

6. At a very narrow wavelength interval, usually below 0.01 nm, a laser only emits high-irradiance light, making an excitation monochromator or filter unnecessary. 

7. The drawback of this approach is, it is impossible to adjust a laser’s wavelength by much. 

8. A mercury-vapor lamp is a line lamp, which means that it emits light near peak wavelengths. 

9. The xenon arc, on the other hand, has a continuous emission spectrum of almost constant intensity in the 300-800 nm range and ample irradiance for measurements down to just over 200 nm. 

10. Fluorimeter filters or monochromators can be used. With an adjustable tolerance, a monochromator transmits light at an adjustable wavelength. 

11. A diffraction grating is the most common type of monochromator, i.e., collimated light illuminates a grating and exits depending on the wavelength at a different angle. 

12. To choose which wavelengths to transmit, the monochromator can then be modified. 

13. The addition of two polarization filters is required to enable anisotropic measurements: one before the emission monochromator or filter and one after the excitation monochromator or filter.

Schematic representation of a fluorescence spectrophotometer. The light...  | Download Scientific Diagram

As stated before, the fluorescence relative to the excitation light is most commonly measured at a 90 ° angle. Instead of positioning the sensor at the excitation light line at an angle of 180 ° to prevent interference with the excitation light transmitted, this geometry is used. No monochromator is perfect and some stray light, that is, light with wavelengths other than the target, will be transmitted. An ideal monochromator can transmit only light in the specified range and have a high wavelength-independent transmission. Only the light scattered by the sample induces stray light when measured at a 90° angle and results in a higher signal-to-noise ratio relative to the 180 ° geometry, reducing the detection limit by about a factor of 10000. Besides, fluorescence from the front may also be measured, often done for turbid or opaque samples. 

Detectors

1. The detector can be either single- or multi-channeled. 

2. The single-channel detector can only detect one wavelength’s intensity at a time, while the multi-channel detector simultaneously detects the intensity at all wavelengths, rendering the monochromator or filter of the emission unnecessary. 

3. The most flexible fluorimetry can record both an excitation spectrum and a fluorescence spectrum with dual monochromators and a continuous excitation light source. 

4. When measuring fluorescence spectra, the excitation light wavelength is kept constant, ideally at a high absorption wavelength, and the emission monochromator scans the spectrum. 

5. The wavelength going through the emission filter or monochromator is kept constant for calculating excitation spectra, and the excitation monochromator is scanned. 

6. As the fluorescence intensity is equal to the absorption, the excitation spectrum is usually similar to the absorption spectrum. 

Data Analysis

The fluorescence intensity usually is proportional to the concentration of the fluorophore at low concentrations. 

1. To achieve ‘real,’ i.e., machine-independent spectra, multiple variables influence and distort the spectra, and corrections are required. 

2. Here, the various forms of distortions will be categorized as either instrument or sample-related. 

3. Firstly, it addresses the distortion that occurs from the instrument. During each experiment and between each experiment, the intensity of the light source and wavelength characteristics differ over time. 

4. Besides, at all wavelengths, no lamp has constant power. 

5. To correct this, following the excitation monochromator or filter, a beam splitter may be added to direct a portion of the light to the reference detector. 

6. Furthermore, attention must be given to the transmission efficiency of monochromators and filters. These can alter over time as well. 

7. Depending on the wavelength, the propagation efficacy of the monochromator often varies. This is the reason why the excitation monochromator or filter should be put after an optional reference detector. 

8. The percentage of fluorescence that the detector collects depends on the device as well. 

9. Besides, the detector’s quantum efficiency, that is, the percentage of photons detected, differs between different detectors, as the detector eventually deteriorates with wavelength and with time. 

10. A tedious method is the adjustment of all these instrumental variables to achieve a ‘normal’ continuum, which is only implemented in practice when it is strictly necessary. 

11. This is when the quantum yield is measured. For example, when the wavelength with the highest emission intensity is detected. 

As noted earlier, distortions also emerge from the sample. Any elements of the sample must be taken into account as well. Firstly, over time, photodecomposition can decrease the fluorescence intensity. The dispersion of light must also be noticed. Rayleigh and Raman’s scattering is the primary form of scattering in this context. The light dispersed by Rayleigh dispersion has the same wavelength as the incident light, while the dispersed light typically shifts wavelengths to longer wavelengths in Raman dispersion. The dispersion of Raman is the result of a simulated electronic state caused by the light of excitation. The molecules can relax from this virtual state back to a vibrational level other than the vibrational ground state. It is often seen in fluorescence spectra at a constant wavenumber difference compared to the excitation wavenumber, e.g., the peak appears lower than the excitation light in the water at a wavenumber of 3600 cm-1. 

The inner filter effects are other things to remember. Reabsorption involves these. Reabsorption occurs because at the wavelengths at which the fluorophore releases radiation, another molecule or part of a macromolecule absorbs it. Any or all of the photons released by the fluorophore may be absorbed again if this is the case. Due to large concentrations of absorbing molecules, including fluorophores, another inner filter effect occurs. The consequence is that in the solution, the excitation light’s strength is not constant, by which a small amount of the excitation light, which is apparent to the detection system, enters the fluorophores. Both the intensity and spectrum of the emitted light are modified by the internal filter effects and must be considered when examining the fluorescent light emission spectrum.

Tryptophan Fluorescence

1. Tryptophan is an effective intrinsic fluorescent (amino acid) probe that can be used to estimate the tryptophan microenvironment’s existence. 

2. The microenvironment of tryptophan growth changes when conducting experiments with denaturants, surfactants, or other amphiphilic molecules. 

3. E.g., if a protein containing a single tryptophan is denatured at a growing temperature in its ‘hydrophobic’ center, a red-shift emission spectrum will appear. 

4. Compared to a hydrophobic protein interior, this is due to the proximity of the tryptophan to an aqueous environment. 

5. In comparison, if the tryptophan is incorporated in the surfactant vesicle or micelle, the addition of a surfactant to a protein containing a tryptophan exposed to the aqueous solvent can produce a blue-shifted emission spectrum. 

6. A fluorophore can be bound to proteins that lack tryptophan. 

7. The tryptophan emission spectrum at 295 nm is dominant over tyrosine and phenylalanine’s weaker fluorescence. 

Applications of fluorescence spectroscopy

For the study of organic compounds, fluorescence spectroscopy is used in biochemical, medical, and chemical research fields, among others. 

It is used in differentiating malignant, bashful skin tumors from benign tumors has also been documented. 

It can also be used to redirect photons.

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