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The UV-Vis and Fluorescence methods are both measured in the same range of wavelengths i.e., 190-800nm. Although, they use the same wavelengths, different phenomena are used. UV-Vis measures the absorption of light whereas fluorescence measures the emission of light that is previously absorbed.
UV-Visible spectroscopy is an analytical technique used for the determination of different compounds by measuring the absorbance of the sample through which a monochromatic light source is passed through. This method involves the measurement of absorbance of light in the UV-Visible range of electromagnetic spectrum.
The molecules pass from a lower energy state (ground state) to a higher energy state (excited state). In other words, the molecule passes from the Highest Occupied molecular Orbital (HOMO) to the Lowest Unoccupied Molecular Orbital (LUMO).
The most transition is the π to π* transition. The smaller the energy difference (HOMO-LUMO gap) between the orbitals, the less energy is needed for excitation. The lesser the energy, the longer is the wavelength absorbed.
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The HOMO-LUMO gap is based on the conjugated system i.e., bigger the molecule, smaller is the gap. Functional groups attached to the conjugated double bonds of a molecule also affect the gap between ground and excited state (Introduction to spectroscopy; Donald l. Pavia ... et al., 2015) (Pharmacopoeia.com, 2019). The wavelength at which the most absorption takes place is called as the λmax. this is measured by the UV spectrophotometer. The peaks obtained in the UV are always broad and absorption occurs over a range of wavelengths thereby causing simultaneous vibrational and electronic excitation.
Advantages of UV-Vis Spectroscopy:
Disadvantages of UV-Vis Spectroscopy:
Fluorescence Spectroscopy is a method of electromagnetic spectroscopy which utilizes light to analyze the fluorescence present in the sample. Fluorescence is a complimentary technique that occurs at the same wavelength as UV. The difference between UV and fluorescence is that fluorescence results from the excited higher energy state emitting a photon to a ground lower energy state. The orbitals involved are same i.e., HOMO and LUMO where HOMO refers to electronic ground state and LUMO refers to first excited electronic state. Apart from the electronic energy levels, there are also the presence of vibrational levels termed as vibrational sublevels. The vibrational sublevels present above the excited state are known as “the excited state, vibrationally excited.”
When the molecule absorbs a photon from UV light, an electron moves from HOMO to LUMO to create excited state in ground vibrational level or excited state vibrationally excited. This means that, the electron that gets emitted is relatively lower in energy than when it is initially absorbed. The diagram used to describe this process is called Jablonski diagram which is shown in Figure 3. In the diagram, absorption takes place first, followed by vibrational relaxation (curved lines) and then fluorescence (downward arrows). Not all molecules that are electronically excited fluoresce. Some of them gets decayed to ground state along with heat emission. Adding a fluorescence label to the non-fluorescent molecules can make them fluoresce (Chromedia.org, 2019) (Ludwig Brand and Johnson, 2008).
Advantages of Fluorescence Spectroscopy:
Disadvantages of Fluorescence spectrum:
Collection of a Fluorescence spectrum:
Fluorescence spectrum can be obtained by using a Spectrophotometer. The difference between UV and fluorescence spectrum collection is that instead of passing white light directly into the detector through sample (in case of UV), the sample is illuminated from the rough side of the cuvette. The rough side of the cuvette helps in proper scattering of the light all-over the sample. This is done because direct transmission of light is not desirable for fluorescence. The analyte/sample concentration is in direct proportion to the intensity of the emission. The detector is paced at 90˚. During the performance of a fluorescence spectrum, calibration of the equipment is not required. This is because the light used for fluorescence will not drift over time unlike in the UV spectrum (Orgchemboulder.com, 2019).
The sensitivity of Fluorescence detection is nearly 1000 times greater than the sensitivity of absorption spectroscopy i.e., UV-Vis spectroscopy. It results in the higher limits of detection while using less amounts of sample. Hence it is of great use when working with precious or limited quantity compounds. The reason for high sensitivity of the Fluorescence Spectroscopy is that the emission signal is measured against a zero-background level (Chemistry Stack Exchange, 2019).
This means that the photons can be measured against a dark background leading to high sensitivity. The sample is directly measured without any comparison to reference values. In case of UV spectroscopy, the absorbance is measured with a bright reference beam by using the difference between 2 intensities i.e., the intensity of light before sample and intensity of light after passing through sample (I and I0) (Lakowicz, 2010). Fluorescence is temperature sensitive and occurs at low temperatures. This is because the solvent relaxation is slow at low temperatures. Also, molecular vibration is less at these temperatures thereby leading to increase in the chances of fluorescence and its lifetime. At higher temperatures, quenching of fluorescence is observed. Quenching refers to the process of decreasing the intensity of fluorescence (Giri, 1992).
The above figure refers to the quenching of quinine dissolved in water. Normally, quinine fluoresces blue color. This is visible in the right sample. But the left sample does not show any blue color. This is because of the presence of chloride ions which quenches the fluorescence of quinine.
The violet color is due to scattered laser light. The viscosity of the sample also effects the fluorescence observed. When the sample is more viscous, the intramolecular rotation is slowed down. This intramolecular rotation rate controls the excited states. So, when the sample is more viscous, it leads to increase in the fluorescence and vice-versa. It should also be noted that viscosity is dependent on temperature (Vyšniauskas et al., 2015).
Scattering of light is an important aspect in fluorescence. The incident light is scattered in all directions either from molecules themselves or scattering by small particles in a fine suspension. The scattering of light by molecules themselves is called Rayleigh scattering and the scattering by small particles is called Tyndall scattering. Proper care should be taken to separate the scattered light from fluorescence as both are collected together. Rayleigh and Tyndall scattering causes decreased sensitivity and inaccurate results (Introduction to fluorescence spectroscopy, 2000) (Clarke and Oprysa, 2004). Solvent interaction leads to change in fluorescence. Any process that stabilizes the excited state electrons before emission results in the decrease of fluorescence. In polar solvents like water and ethanol, a bathochromic or red shift is observed for fluorophores. This is due to a phenomenon called ‘solvent sheath’.
This means that the molecules of the polar solvent rearrange themselves around the dissolved molecules thereby reducing the interaction energy. This leads to solvent relaxation and decrease in the fluorescence observed (Akers, Haidekker and Supervisor, 2005). To summarize, although UV spectroscopy is a widely used method for measurement of absorbance, the sensitivity is still low when compared to the Fluorescence spectroscopy. The fluorescence method of spectroscopy detects the emission signal of the molecule using very low quantities. But for the fluorescence spectroscopy to measure the molecule, presence of a fluorophore in necessary. This can be overcome by attaching a fluorescent label to the molecule so that it can emit fluorescence. Fluorescence is susceptible to change with changes in temperature, viscosity and solvent interactions. Fluorescent spectroscopy is highly sensitive and selective thereby resulting in its wide usage.
UV-Vis vs. Fluorescence Spectroscopy: Wavelength Overlap, Principles, and Sensitivity. (2024, Feb 20). Retrieved from https://studymoose.com/document/uv-vis-vs-fluorescence-spectroscopy-wavelength-overlap-principles-and-sensitivity
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