UV-Vis Spectroscopy Principle, Instrumentation, Applications, Advantages, and Limitation

UV spectroscopy, also known as UV-visible spectrum (UV-Vis also known as UV/Vis) refers to absorption or reflectance spectroscopy within a portion of...

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UV-Vis Spectroscopy Principle, Instrumentation, Applications, Advantages and Limitation
UV-Vis Spectroscopy Principle, Instrumentation, Applications, Advantages and Limitation

UV spectroscopy, also known as UV-visible spectrum (UV-Vis also known as UV/Vis) refers to absorption or reflectance spectroscopy within a portion of the ultraviolet spectrum and in the complete, adjacent visible areas in the spectrum of electromagnetic radiation. This is because it utilizes light from the visible and adjacent areas. Reflectance or absorption in the visible spectrum directly influences the perception of color of the compounds involved. In this area that is visible, atoms as well as molecules undergo electronic transformations. Absorption spectroscopy is a complement to fluorescence spectroscopy in that it deals with the electrons’ transitions between the excited and ground states while absorption studies changes between the ground state and that of the excited state.

Principle of  UV-Vis spectroscopy

In essence, spectroscopy is linked to the interaction between light and matter. When light is absorbed by matter and reflected back, it results in an increased amount of energy in molecules or atoms. When ultraviolet radiations are absorbed this causes the excitation of electrons in the ground state to the higher energy state. Molecules that contain p-electrons, or non-bonding electrons (n-electrons) are able to absorb energy from ultraviolet light that stimulates electrons into higher non-bonding orbitals. The more easily stimulated the electrons are, the greater the wavelength of light that it is able to absorb. There are four types of transitions (p-p*, N-P*, s -s* and even n-s*) and are arranged in the following order the following order: s-s* > n-s* > p-p* > n-p*. The absorption of radiation by chemical substance creates an identifiable spectrum that aids in identifying the substance.

How does a UV-Vis spectrophotometer work? / Instrumentation of UV Spectroscopy

There are a variety of variations to the UV-Vis spectrophotometer to get more understanding of how it works we will look at the primary components, as shown below in the figure below.

Instrumentation of UV Spectroscopy
A simplified schematic of the main components in a UV-Vis spectrophotometer. Credit: Dr. Justin Tom.

1. Light source

For a light-based method using a steady source to produce light over many wavelengths is vital. One xenon light source can be employed as a high-intensity light source that can be used in both visible and ultraviolet ranges. Xenon lamps arehowever priced higher and less reliable when compared to tungsten or Halogen lamps.

When instruments use two lamps such as halogen or tungsten, a lamp is typically used to provide visible light. A deuterium lamp can be the standard lamp that emits UV light. Because two sources of light are required to cover both UV light wavelength and the visible, the source of light inside the instrument needs to change when measuring. In the real world the most common time for this switchover is during the scanning between 300 and 350 nm . In this case, it is possible to see light emissions comparable to both sources and the transition is improved by smoother transitions.

2. Wavelength selection

The next step is to select specific wavelengths of light suitable to the particular type of sample as well as the analyte used for detection should be chosen for examination of the sample by examining the broad wavelengths of light emitted by the source of light. Methods for this are:

a. Monochromators

Monochromators separate light into a small band of wavelengths. It’s typically constructed using diffraction gratings, which can be rotated to select angles for reflection and incoming to choose the length of the light. The diffraction grating’s groove speed is typically measured in terms of the amount of grooves per mm. A higher groove frequency offers greater optical resolution however, it has a shorter acceptable wavelength. 

A lower frequency groove provides more usable wavelengths however, it has a lower optical resolution. 300-2000 grooves per millimeter are acceptable to use for UV Vis spectroscopy purposes but at least 1200 grooves per millimeter is the norm. The quality of measurements of the spectroscopic spectrum is affected by physical imperfections that occur in the diffraction grating as well as in the setup for optical. Therefore that diffraction gratings with rules tend to be more prone to defects than blazed Holographic difffraction gratings. The diffraction gratings that are blazed tend to offer significantly higher quality measurements.

b. Absorption filters

Absorption filters are usually constructed from colored glass or plastic that is designed to absorb certain frequencies of light.

c. Interference filters

Also known as dichroic filters they consist of a variety of different layers of dielectric materials, where interfering occurs between the thin layers of material. They can be utilized to block unwanted wavelengths through damaging interference, acting as a wavelength-selector.

d. Cutoff filters

Cutoff filters permit light above (shortpass) or over (longpass) the specified wavelength to traverse. They are usually implemented using interference filters.

e. Bandpass filters

Bandpass filters enable a range of wavelengths to be passed through. They can be used by combining longpass and shortpass filters.

Monochromators are the most popular choice for this purpose because of their flexibility. However, filters are typically employed in conjunction with monochromators to limit the light wavelengths further to make more precise measurements as well as to boost the signal-to noise ratio.

3. Sample analysis

The wavelength selector that is utilized for the spectrophotometer light will then pass through the sample. In all analyses the measurement of a reference sample which is often called”the “blank sample”, such as a cuvette containing similar solvents that was used for the preparation of the specimen is vital. In the event that an aqueous-buffered sample that contains the sample is being used to conduct measurements and analysis, then the aqueous buffered sample without the substance of importance is used as a reference. For examining bacteria using sterile media, this will be used as the reference. The signal from the reference sample can then be used by the instrument in order to determine the actual absorbance that the analytes absorb.

It is essential to know material and the conditions used for UV-Vis spectroscopy research. For instance most plastic cuvettes are not suitable for studies on UV absorption because they absorb the UV light. Glass is filtering, usually taking in all UVC (100-280 nm)^2 as well as UVB (280-315 nm)^2 however permitting certain UVA (315-400 nm)^2 in the range of 315 to 400 nm to go through. So glass sample holders made of quartz are essential to conduct UV analysis since quartz is translucent towards the bulk of UV light. It is also considered to be an obstructor since the wavelengths of light that are shorter than 200 nanometers are absorption by molecular oxygen within the air. A specific and expensive setup is required to measure with wavelengths that are less than 200 nanometers, typically with an optical system that is filled with pure argon gas. Cuvette-free systems also exist that allow for the analysis of extremely small samples, such as in DNA or RNA studies.

4. Amplifier

The alternating current that is generated by the photocells gets transferred into the amp. The amplifier is connected to a tiny servometer. The majority of the current generated by photocells is low intensity. The main function of the amplifiers can be used to amplify the signal multiple times in order to obtain clear and reliable signals.

5. Detection

Once the light has passed through the sample and a detector is employed to transform the light into a electronically readable signal. Typically, detectors are based on semiconductors or photoelectric coatings.

A photoelectric coating releases negatively charged electrons upon exposure to light. As electrons are released the current of electricity proportional to light intensity is created. The photomultiplier tube (PMT) is among the most popular detectors utilized to study UV-Vis light spectroscopy. The principle behind a PMT is built on the principle of photoelectricity to release electrons at first exposure to light. It is then and then subsequently multiply the ejected electrons in order to generate more current of electricity. PMT detectors are particularly effective to detect very small amounts of light.

If semiconductors are subjected light, an electric charge proportional to the intensity of light can flow through. Particularly, photodiodes and Charge-coupled device (CCDs) comprise two popular detectors built in semiconductors.

When the electric current has been generated by the detector employed, the signal is then detected and sent to a screen or computer. The following two illustrations provide simplified examples of Schematic diagrams for UV-Vis spectrophotometer arrangement.

Schematic diagram of a cuvette-based UV-Vis spectroscopy system.
Schematic diagram of a cuvette-based UV-Vis spectroscopy system. | Source:
Schematic diagram of a cuvette-free UV-Vis spectroscopy system.
Schematic diagram of a cuvette-free UV-Vis spectroscopy system. | Source:

6. Recording devices

The majority of the time amplifiers are connected to a pen recording device that is linked to a computer. Computers store all the data produced and generates an array of compound.

Advantages of UV-Vis spectroscopy

Each technique is not perfect, and UV-Vis spectroscopy makes no exception. It does however possess a few major advantages that are listed below to make it a popular.

  • The process is not destructive and allows the material to be reused or to undergo further analysis or processing.
  • It is possible to make measurements quickly, which allows for easy integration into protocols for experiments.
  • Instruments are simple to use and require no training before using.
  • Data analysis typically requires little processing, meaning that no user training is required.
  • The instrument is typically affordable to purchase and use which makes it affordable for numerous laboratories.

limitations of UV-Vis spectroscopy

While the benefits of this method seem to be to be overwhelming, they are certain flaws:

  • Stray light:  In true instrument the wavelength selectors aren’t 100% accurate and a tiny amount of light from a large range of wavelengths may be transmitted by the light source, leading to serious measurement errors. Stray light can also be emitted from the outside or the loosely-fitted compartment within the instrument.
  • Light scattering: Light scattering can be due to suspended solids in liquid samples which can cause significant measurement mistakes. Bubbles present in the cuvette or sample may scatter light, which can lead to unprovable results.
  • Interference from multiple absorbing species: A sample might include, for instance, several types of blue-green pigment called chlorophyll. The various chlorophylls show overlapping spectra when studied in the same sample. To conduct a thorough qualitative analysis every chemical species must be removed by the test sample, and studied in isolation.
  • Geometrical considerations: The misaligned placement of any of the instruments’ components, particularly the cuvette that holds the sample, can result in unreproducible results and may be inaccurate. This is why it is crucial to ensure that each part of an instrument are aligned to the same direction and set in the same place to take every measurement. Basic user education is generally suggested to prevent misuse.

Applications of  UV-Vis spectroscopy

UV-Vis has been adapted to numerous situations and purposes such as but not limited:

1. DNA and RNA analysis

Rapidly determining the purity and amount of DNA and RNA is one of the most popular applications. The spectrum of wavelengths that are used for their analysis as well as what they represent is given in Table 1. When you prepare the DNA and RNA sample for instance, for downstream applications like sequencing, it’s crucial to ensure that there isn’t contamination from one with one or the other, or with proteins or other chemicals that are a result of the process of isolation.

The 260 nm/280nm absorbance (260/280) ratio can be important for determining contamination in nucleic acids samples, which is summarized in Table 2. Pure DNA generally has an 260/280 ratio of 1.8 and the ratio of pure RNA typically 2.0. Pure DNA is less of a ratio of 260/280 than RNA, due to the fact that thymine which is replaced by uracil DNA and DNA, has a lower ratio of 260/280 than the uracil. Proteins in the samples will lower the ratio of 260/280 due to a higher absorption rate at around 280 nm.

The 260 nm/230 nm absorption (260/230) ratio can also be helpful in determining the quality of DNA samples and RNA. It can reveal chemical or protein contamination. Proteins are able to absorb light at 300 nm, which can lower the 260/230 ratio , and thus indicating the presence of protein in RNA and DNA samples. Guanidinium thiocyanate as well as guanidinium iso two commonly used compounds in purifying nucleic acids, absorb light at 230 nm, that will reduce the ratio of absorbance 260/230.

2. Pharmaceutical analysis

The most popular applications of UV-Vis spectroscopy can be found within the pharmaceutical industry. Particularly processing UV-Vis spectrums using mathematical derivatives permits overlapping absorbance peaks of the spectrum to be separated to reveal individual pharmaceutical compounds. For example, benzocaine an local anesthetic, as well as chlortetracycline which is an antibiotic can be detected simultaneously in commercial powders for veterinary use by applying the mathematical derivative to absorbance spectrum. The simultaneous measurement of both compounds was possible in a microgram per milliliter concentration by creating an appropriate calibrator function that was specific to each chemical.

3. Bacterial culture

UV-Vis spectroscopy is commonly employed in the culturing of bacterial. OD measurements are regularly and quickly recorded using the 600 nm wavelength to measure the concentration of cells and to monitor the growth. 600 nm is the most commonly used wavelength and is preferred due to their optical qualities of the bacterial culture mediums that they grow in and also to prevent damage to cells in situations where they are needed for ongoing testing.

4. Beverage analysis

The detection of specific alcohol-related compounds is another typical use that makes use of UV Vis spectroscopy. The caffeine content of a drink has to be within a certain limit set by the law and UV light is able to aid in quantification. Certain types that contain colored compounds, like anthocyanin, which is found in blueberries blackberries, raspberries and cherries, can be identified by comparing their peak absorbance wavelengths in wines to determine quality by measuring UV Vis absorption.

5. Other applications

This method can be utilized in other industries. For instance the color index is helpful for checking the oil in transformers as a precautionary measure to ensure that electric power is delivered safely. The measurement of the absorbance of hemoglobin to determine the concentration of hemoglobin could be employed in research into cancer. When treating wastewater UV-Vis spectroscopy may be employed in monitoring and kinetic studies to verify that specific dyes and dye-byproducts have been eliminated correctly using spectrograms of their respective spectra across the course of.

UV-Vis spectroscopy can also be beneficial in qualitative aspects of specific research. The ability to track changes in wavelength that corresponds to the peak absorbance is helpful for analyzing specific structural changes in proteins and also in determining the composition of batteries. Changes in the peak absorbance wavelength are also useful for more advanced applications such as the study of tiny nanoparticles. This technique’s applications are diverse and seem to be infinite.

Brief Applications of UV Spectroscopy

  • The detection of Impurities: The method is among the most effective methods to detect impurities within organic molecules. Other peaks may be seen due to impurities within the sample. Then, it can be compared to that of the standard raw material. In addition, by measuring the absorbance at a specific wavelength, impurities are identified.
  • Elucidation of organic structures: It can be useful in the analysis of the structure of organic molecules, for instance in determining in the absence or presence of unsaturation or presence of heteroatoms.
  • UV absorption spectroscopy is utilized to quantify the presence of substances which absorb ultraviolet radiation.
  • UV absorption spectroscopy is a method to identify the types of substances that absorb UV radiation, and is being used to determine the qualitative nature of compounds. The identification is made by using the absorption spectrum to compare with the spectra of the known compounds.
  • This method is employed to determine any presence, or lack of a functional groups in the compound. The absence of a band at a specific wavelengths is thought to be indication that there is no group.
  • The kinetics of reactions can be examined using UV spectroscopy. The UV radiation passes through the cell that is reacting and variations in the absorbance can be seen.
  • A lot of drugs can be found made from raw materials or in forms of formulation. They can be tested by making an appropriate solution of substance in a solvent, and then testing the absorbance at a particular wavelengths.
  • Molecular weights of compounds can be measured spectrophotometrically by preparing the suitable derivatives of these compounds.
  • UV spectrophotometers can be used to detect for HPLC.
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