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. 


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. 


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.

Types Of Spectroscopy

BY- K. Sai Manogna (MSIWM014)


Absorption spectroscopy:

Absorption spectroscopy is a technique that compares the power of a beam of light determined before and after a sample contact. It is also referred to as Tunable Diode Laser Absorption Spectroscopy (TDLAS) when done with a tunable diode laser. To decrease the device’s noise, it is most often paired with a modulation technique, most often wavelength modulation spectrometry (WMS) and sometimes frequency modulation spectrometry (FMS). 

Fluorescence spectroscopy

To excite a sample, fluorescence spectroscopy uses higher-energy photons, which will then release lower energy photons. This method is known for its biochemical and medical applications and can be used for confocal microscopy, energy transfer of fluorescence resonance, and lifetime imaging of fluorescence. 

X-ray spectroscopy

When X-rays with appropriate frequency interact with a material, the atom’s inner shell electrons are excited into empty outer orbitals, or they can be entirely expelled, ionizing the atom. Then electrons from the outer orbitals would fill the inner shell “hole.” In this de-excitation process, the energy available is released as radiation (fluorescence), or other less-bound electrons are extracted from the atom (known as Auger effect). The frequencies (energies) of absorption or emission are characteristic of the individual atom. Moreover, there are minor frequency variations for a single atom that is typical of chemical bonding. These specific X-ray frequencies or Auger electron energies can be determined with an appropriate instrument. In chemistry and material sciences, X-ray absorption and emission spectroscopy are used for determining the elemental composition and chemical bonding. X-ray crystallography is a method of scattering; X-rays are dispersed at well-defined angles by crystalline materials. If the incident X-ray wavelength is known, the distances between the atoms’ planes inside the crystal can be measured. The scattered X-ray intensities provide information about the atomic positions and measure the atoms’ arrangement within the crystal structure.


Samples of liquid solution are aspirated into a combination of a burner or nebulizer/burner, dissolved, atomized, and often excited to a higher electronic state of energy. During analysis, the use of a flame includes fuel and oxidant, usually in gases. Gases such as acetylene (ethyne) or hydrogen are used as typical fuel gases. Oxygen, air, or nitrous oxide are common oxidant gases used. These methods can also analyze metallic element analytes in the concentration ranges of part per million, billion, or probably lower. In order to identify light with the analysis data coming from the flame, light detectors are required. 

Atomic Emission Spectroscopy: This technique uses the flame’s excitation; atoms are excited to emit light from the flame’s heat. The total consumption burner with a round burning outlet is usually used in this technique. A more significant temperature flame is usually used to induce analyte atoms’ excitation than atomic absorption spectroscopy (AA). Since the flame’s heat excites the analyte atoms, no particular elemental lamps must shine into the flame. A high-resolution polychromator can be used to generate an emission intensity vs. wavelength spectrum over a range of wavelengths exhibiting multiple-element excitation lines, meaning multiple elements can be detected in one run. Alternatively, a single wavelength monochromator may be set to focus on studying a single element at a specific emission line. A more advanced variant of this process is plasma emission spectroscopy. 

Atomic absorption spectroscopy (often referred to as AA) – A pre-burner nebulizer (or nebulizing chamber) is widely used to produce a sample mist and a slot-shaped burner that gives a longer flame pathlength. The flame temperature is low enough that sample atoms are not excited from their ground state by the flame itself. The nebulizer and flame are used to dissolve and atomize the sample, but for each type of analyte, the analyte atoms’ excitation is achieved by using lamps that glow through the flame at different wavelengths. The amount of light absorbed after passing through the flame defines the analyte quantity in the sample in AA. For greater sensitivity, a graphite furnace is typically used to heat the sample for desolvation and atomization. The graphite furnace process can also analyze any substantial or slurry samples. It is still a widely used analysis method for some trace elements in aqueous (and other liquid samples, due to its strong sensitivity and selectivity. 

Atomic Fluorescence Spectroscopy: A burner with a circular burning outlet is widely used in this technique. To solve and atomize the sample, the flame is used. However, a lamp shines a light into the flame at a particular wavelength to excite its analyte atoms. Then the atoms of some components will fluoresce, emitting light in another direction. For quantifying the amount of analyte component in the sample, this fluorescent light’s strength is used. A graphite furnace is also used for atomic fluorescence spectroscopy. This technique is not as widely used as spectroscopy of atomic absorption or plasma emission. 

Plasma Emission Spectroscopy:

It has virtually replaced in several respects similar to flame atomic emission spectroscopy. 

  1. Direct-current plasma (DCP) An electrical discharge between two electrodes creates a direct-current plasma (DCP). It needs a plasma support gas, and Ar is standard. Samples could be deposited on one of the electrodes, or one electrode can be built up by conducting them. 
  2. Glow discharge-spectrometry of optical pollutants (GD-OES) 
  3. Plasma-atomic emission spectrometry, inductively coupled (ICP-AES) 
  4. Laser-Induced Breakdown Spectroscopy (LIBS) (LIBS), also called plasma spectrometry induced by laser (LIPS) 
  5. Plasma caused by microwave (MIP) 

Spark or arc/emission spectroscopy – used in solid samples for the study of metallic elements. In order to make it conductive, a sample is ground with graphite powder for non-conductive materials. A sample of the solid was usually ground up and damaged during research in conventional arc spectroscopy methods. The spark or electric arc is passed through the sample to excite the atoms, heating the sample to a high temperature. The excited analyte atoms glow at different wavelengths, producing light that can be detected by standard spectroscopic methods. Since the conditions generating the arc emission are usually not quantitatively regulated, the study is qualitative for the components. Nowadays, under an argon atmosphere, spark sources with controlled discharges allow this method to be considered eminently quantitative, and its use is widely applied worldwide through the production control laboratories of foundries and steel mills. 

Visible Spectroscopy:

Many atoms emit visible light or absorb it. In order to achieve a continuum of fine lines, the atoms must be in the gas phase. It suggests the material has to be vaporized. In absorption or emission, the spectrum is studied. In UV/Vis spectroscopy, visible absorption spectroscopy is mostly paired with UV absorption spectroscopy. While this type may be unusual as a similar indicator is a human eye, it still helps identify colors. 

Ultraviolet light Spectroscopy

In the Ultraviolet (UV) field, all atoms are absorbed because these photons are energetic enough to excite outer electrons. Photoionization takes place if the frequency is high enough. In quantifying protein and DNA concentration and protein ratio to DNA concentration in a solution, UV spectroscopy is also used. Several amino acids, such as tryptophan, usually present in proteins, absorb light in the range of 280 nm, and DNA absorbs light in the 260 nm range. For this reason, in terms of these two macromolecules, the 260/280 nm absorption ratio is a good general measure of the relative purity of a solution. It is also possible to make fair estimates of protein or DNA concentration using Beer’s law. 

Infrared Spectroscopy:

The IR absorption spectrum analysis shows what kind of bonds are present in the sample, especially in organic chemistry. The study of polymers and components such as fillers, pigments, and plasticizers is also necessary. 

Raman Spectroscopy:

To study the vibrational and rotational modes of molecules, Raman spectroscopy uses the inelastic scattering of light. An interpretation help is the resulting ‘fingerprints.’ 

Coherent anti-Stokes Raman spectroscopy (CARS) is a recent technique for in vivo spectroscopy and imaging with high sensitivity and robust applications. 

Nuclear Magnetic Resonance Spectroscopy (NMR): 

To determine the various electronic local environments of hydrogen, carbon, or other atoms in an organic compound or other compounds, nuclear magnetic resonance spectroscopy analyses such as atomic nuclei’s magnetic properties. This is used to assist in assessing the compound structure. 



Mossbauer spectroscopy modes of transmission or conversion-electron (CEMS) probe individual isotope nuclei’s properties in various atomic environments by studying the resonant absorption of characteristic gamma-ray energy as the Mossbauer effect.

In the next chapter we will discuss in detail about each spectroscopic methods.