JOURNAL OF PHARMA INSIGHTS AND RESEARCH

REVIEW ARTICLE

A Review on UV-visible spectroscopy

Alekhya Mandru 1*, Jyothi Mane 2, Ramya Mandapati 3
1
Student, B.Pharmacy, Sri Vasavi Institute of Pharmaceutical Sciences, Pedatadepalli, Andhra Pradesh, India
2
Student, B.Pharmacy, Sri Vasavi Institute of Pharmaceutical Sciences, Pedatadepalli, Andhra Pradesh, India
3
Student, B.Pharmacy, Sri Vasavi Institute of Pharmaceutical Sciences, Pedatadepalli, Andhra Pradesh, India

Publication history: Received on 21st October; Revised on 18th November; Accepted on 22nd
November

Article DOI: 10.5281/zenodo.10232708

Abstract: UV spectroscopy, as one of the earliest instrumental techniques for analysis, has proven to be a versatile and
indispensable tool in various scientific domains. Its capability to distinguish different types of materials makes it a valuable asset
in analytical chemistry. This method is commonly employed to ascertain the identity, strength, quality, and purity of diverse
samples. The fundamental principle of UV spectroscopy lies in the absorption of specific wavelengths of light by samples, and
it provides valuable insights into the responses of substances to this absorption. The application of Beer's law, a universal principle
in UV spectroscopy, elucidates the absorption of radiant energy by samples. Notably, this method is characterized by its accuracy,
simplicity, and a broad spectrum of applications, including drug discovery, structural elucidation of organic molecules, molecular
weight determination, and the detection of impurities. Both quantitative and qualitative analyses can be conducted effectively
using UV spectroscopy. The equipment operates within a wavelength range of 200nm to 800nm, allowing the analysis of both
colorless and colored compounds in both the UV and visible regions. In summary, UV spectroscopy stands as a robust analytical
technique with wide-ranging applications, making it a cornerstone in scientific research and analysis.
Keywords: UV Spectroscopy; Beer's Law; Analytical Chemistry; Molecular Characterization; Quantitative Analysis

1. Introduction
Spectroscopy involves the study of interaction between light or electromagnetic radiation and matter. This interaction manifests a
crucial phenomenon where 'energy is either absorbed or emitted by the matter in discrete amounts termed quanta.' Across the
electromagnetic spectrum, spanning from gamma rays to radio waves, absorption and emission processes unfold, each offering
unique insights into the nature of the studied materials [1, 2].

Among the various spectroscopic techniques, UV spectroscopy emerges as a powerful analytical tool, utilizing light within the UV
or visible region with wavelengths ranging from 200 to 800 nm. This technique stands versatile, capable of analyzing both colorless
compounds in the UV range (400-200 nm) and colored compounds in the visible range (800-400 nm). In essence, UV spectroscopy
quantifies the discrete wavelengths of UV or visible light absorbed or transmitted by a sample in comparison to a reference or blank.
This property, intricately linked to the sample's composition, holds the potential to unveil details about the sample's constituents
and their concentrations. The outcomes of UV spectroscopy are graphically represented as spectra, providing a visual and
quantitative depiction of the spectral data. This review aims to comprehensively explore the principles, applications, and
advancements in UV spectroscopy, shedding light on its significance in diverse scientific domains. Through a systematic examination
of the literature, we intend to elucidate the pivotal role of UV spectroscopy in analytical chemistry, molecular characterization, and
quantitative analysis, offering a nuanced understanding of its contributions to contemporary scientific endeavors [3,4].

2. UV Spectroscopy

2.1. Principle
UV absorption spectra arises from the transition of electrons within a molecule or an ion from a lower to a higher energy level and
the UV emission spectra arise from the reverse type of transition. The UV radiation has sufficient energy to promote or excite the
valence electrons in a molecule or an ion from a ground state orbital to a higher energy level, excited state orbital or anti bonding
orbital which can be detected as absorption [5].

Chromophores: Many organic molecules absorb ultraviolet/visible radiation and this is usually because of the presence of a particular
functional group. The groups that actually absorb the radiation are called chromophores. Some electronic transitions are statistically


Corresponding author: Alekhya Mandru
Copyright © 2023 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0.
 Journal of Pharma Insights and Research, 2023, 01(02), 091–096

probable and strong. Other transitions have a probability of zero and are said to be forbidden but some particularly useful forbidden
transitions are- d→d absorptions of transition metals; the n→π * absorption of carbonyl groups at ca 280 nm; and the π→π*
absorption of aromatic compounds at ca 230–330 nm, depending on the substituents on the benzene ring [6].

*Auxochromes: The Colour of a molecule may be intensified by groups called auxochromes which generally do not absorb
significantly in the 200-800nm region, but will affect the spectrum of the chromophore to which it is attached. The most important
auxochromic groups are OH, NH2, CH3 and NO2 and their properties are acidic (phenolic) or basic. The actual effect of an
auxochrome on a chromophore depends on the polarity of the Auxochromes, e.g. groups like CH3-. In general, it should be possible
to predict the effect of non-polar or weakly polar auxochromes, but the effect of strongly polar auxochromes is difficult to predict.

*Solvents: The effect on the absorption spectrum of a compound when diluted in a solvent, will vary depending on the chemical
structures involved. Generally, non-polar solvents and non-polar molecules show the least effect. However, polar molecules exhibit
quite dramatic differences when interacted with a polar solvent. The interaction between solute and solvent leads to absorption
band broadening and a consequent reduction in structural resolution and ε max. Thus care must be taken to avoid an interaction
between the solute and the solvent. Water and 0.1N solutions of HCl and NaOH are commonly used solvents for absorption
spectrometry. Methodology requires buffering, solutions have to be non-absorbing and generally both the composition and pH will
be specified. For reactions in the 4.2 to 8.8 pH region, mixtures of 0.1N dihydrogen sodium phosphate and 0.1N hydrogen disodium
phosphate are generally used [7]

Potentially, there are three ground state orbitals involved. They are: σ (bonding) molecular orbital, π (bonding) molecular orbital and
n ( non bonding) atomic orbital Similarly, the anti-bonding orbitals involved are: σ* orbital and π* orbital. There is no such thing as
an n* anti-bonding orbital as the n electrons do not form bonds [8].

Then, the possible electronic transitions in uv and visible regions are as follows:

1. σ to σ*

2. n to σ*

3. n to π*

4. π to π*

Figure 1 Electronic transitions

2.1.1. Beer Lambert’s Law

Different molecules absorb different amounts of energy and this can be represented as a graph of absorbance on the y-axis and
wavelength on x-axis. This graph is typically referred to as an absorption spectrum. We can determine the λmax (wavelength at
which the sample is showing maximum absorbance) of the sample and thereby the concentration It is mainly based on the principle
of Beer-Lambert's Law which states that ‘the absorbance of a sample is directly proportional to the concentration of the absorbing
species and the path length [9].

A= εcl

Where, A – Absorbance, ε - molar extinction coefficient, c - concentration of the absorbing species, l - path length

Alekhya Mandru et al 91
 Journal of Pharma Insights and Research, 2023, 01(02), 091–096

For a fixed path length, it can be used to determine the concentration of an absorber in a sample. Thus, it is necessary to know how
rapidly the absorbance changes with the concentration

2.2. Instrumentation
There are two types of absorbance instruments to collect uv absorption spectra. They are:

• Single beam UV spectrophotometer (Figure 2)

• Double beam UV spectrophotometer (Figure 3)

The single beam instrument has a filter or a monochromator between the source and the sample to analyze one wavelength at a
time. The double beam instrument has a single source and a monochromator and then there is a splitter and a series of mirrors to
get the beam to a reference sample and the sample to be analysed, this allows for more accurate analysis. The simultaneous
instrument, double beam UV spectrophotometer is usually much faster and more efficient [10].

Figure 2. Single Beam UV spectrophotometer

Figure 3. Double Beam UV spectrophotometer

The basic instrumentation (as shown in Figure 4) of a UV spectrophotometer includes:

2.2.1. Light source

Sources of UV radiation: It is important that the power of the radiation source does not change abruptly over its wavelength range.
The electrical excitation of deuterium or hydrogen at low pressure produces a continuous UV spectrum. The mechanism for this
involves the formation of an excited molecular species, which breaks up to give two atomic species and an ultraviolet photon.

Both deuterium and hydrogen lamps emit radiation in the range 160 - 375 nm. Quartz windows must be used in these lamps, and
quartz cuvettes must be used because glass absorbs radiation of wavelengths less than 350 nm.

Sources of visible radiation: The tungsten filament lamp is commonly employed as a source of visible light. This type of lamp is
used in the wavelength range of 350 - 2500 nm. The energy emitted by a tungsten filament lamp is proportional to the fourth power
of the operating voltage. This means that for the energy output to be stable, the voltage to the lamp must be very stable indeed.
Electronic voltage regulators or constant-voltage transformers are used to ensure this stability [11].

Alekhya Mandru et al 92
 Journal of Pharma Insights and Research, 2023, 01(02), 091–096

Figure 4. Instrumentation of UV Spectrophotometer

2.2.2. Monochromator (Wavelength selector)

All Monochromators contain the following components;

• An entrance slit

• A collimating lens

• A dispersing device (usually a prism or a grating)

• A focusing lens

• An exit slit

Polychromatic radiation (radiation of more than one wavelength) enters the monochromator through the entrance slit. The beam
is collimated and then strikes the dispersing element at an angle. The beam is split into its component wavelengths by the grating or
prism. By moving the dispersing element or the exit slit, radiation of only particular wavelength leaves the Monochromator through
the exit slit [12]

Figure 5. Turner Grating Monochromator

2.2.3. Sample cell

The containers for the sample and reference solution must be transparent to the radiation which will pass through them. Quartz or
fused silica cuvettes are required for spectroscopy in the UV region. These cells are also transparent in the visible region. Silicate
glasses can be used for the manufacture of cuvettes for use between 350 and 2000 nm. Glass cuvettes should not be used in uv
region as they may absorb uv radiation but can be used in visible region.

Alekhya Mandru et al 93
 Journal of Pharma Insights and Research, 2023, 01(02), 091–096

2.2.4. Detector
A detector converts a light signal into an electrical signal. It should give a linear response over a wide range of low noise and high
sensitivity.

1. Photomultiplier tube detector: The photomultiplier tube (Figure 6) is a commonly used detector in UV-Vis spectroscopy. It
consists of a photoemissive cathode (a cathode which emits electrons when struck by photons of radiation), Anodes (which emit
several electrons for each electron striking them). A photon of radiation entering the tube strikes the cathode, causing the emission
of several electrons. These electrons are accelerated towards the first Anode (which is 90V more positive than the cathode). The
electrons strike the first anode, causing the emission of several electrons for each incident electron. These electrons are then
accelerated towards the second anode, to produce more electrons which are accelerated towards the anode. The electrons are
collected at the anode. By this time, each original photon has produced 106-107 electrons. The resulting current is amplified and
measured. Photomultipliers are very sensitive to UV - visible radiation. They have fast response times [13].

Figure 6: Photo multiplier tube

2. Photo diode detector: The Photodiode detector (Figure 7) is an example of a multichannel photon detector. These detectors are
capable of measuring all elements of a beam of dispersed radiation simultaneously. A linear photodiode array comprises of many
small silicon photodiodes formed on a single silicon chip. There can be between 64 to 4096 sensor elements on a chip, the most
common being 1024 photodiodes. For each diode, there is also a storage capacitor and a switch. The individual diode-capacitor
circuits can be sequentially scanned. In use, the photodiode array is positioned at the focal plane of the monochromator (after the
dispersing element) such that the spectrum falls on the diode array. They are useful for recording UV-Vis absorption spectra of
samples that are rapidly passing through a sample flow cell, such as in an HPLC detector [14].

Figure 7: Photodiode array detector

Alekhya Mandru et al 94
 Journal of Pharma Insights and Research, 2023, 01(02), 091–096

3. Applications

3.1. Structural Elucidation of Organic Compounds

UV spectroscopy serves as a valuable tool for unraveling the structural details of organic compounds through the analysis of their
UV absorption patterns [15].

3.2. Determination of Molecular Weight

The technique is employed for the precise determination of molecular weights, aiding in the characterization of molecules based on
their UV absorption characteristics.

3.3. Detection of Impurities

UV spectroscopy is instrumental in detecting impurities within substances, ensuring the purity and quality of compounds.

3.4. Dissociation Constant of Acids and Bases

It is utilized to determine the dissociation constants of acids and bases, providing insights into their chemical properties.

3.5. DNA and RNA Analysis

UV spectroscopy plays a crucial role in the analysis of DNA and RNA, facilitating the study of nucleic acid structures and
concentrations.

3.6. Bacterial Culture

The technique finds application in the analysis of bacterial cultures, offering insights into microbial growth and metabolism.

3.7. Quantitative Analysis of Pharmaceutical Compounds

UV spectroscopy is widely employed for the quantitative analysis of pharmaceutical compounds, ensuring accurate concentration
measurements [16].

3.8. Qualitative and Quantitative Analysis

It is used for qualitative analysis, allowing the identification of substances based on their unique UV absorption patterns. UV
spectroscopy contributes to the assay of medicinal substances, ensuring the efficacy and quality of pharmaceutical formulations.

3.9. Detection of Functional Groups

The technique is applied to detect specific functional groups within molecules, aiding in the identification of chemical moieties.

3.10. Used in Chemical Kinetics

UV spectroscopy finds utility in chemical kinetics studies, providing real-time insights into reaction mechanisms.

3.11. As HPLC Detector

UV spectroscopy serves as a detector in High-Performance Liquid Chromatography (HPLC), enhancing its capabilities in compound
separation and analysis.

3.12. Beverage Analysis

UV spectroscopy is applied in the analysis of beverages, ensuring quality control and adherence to standards.

3.13. Evaluation of Raw Materials

It contributes to the evaluation of raw materials in various industries, ensuring the quality and integrity of starting materials.

3.14. In Drug Discovery

UV spectroscopy plays a vital role in drug discovery, aiding in the screening and characterization of potential therapeutic compounds.

Alekhya Mandru et al 95
 Journal of Pharma Insights and Research, 2023, 01(02), 091–096

4. Conclusion
In conclusion, UV spectroscopy stands as a pivotal and indispensable characterization technique, offering profound insights into
the properties of diverse samples through the analysis of their interaction with electromagnetic radiation. This method, characterized
by its ease of use, simplicity, accuracy, validity, and cost-effectiveness, emerges as a valuable analytical tool, particularly in
determining the concentration of absorbing species when applied to pure compounds with appropriate standard curves. Its
widespread application spans across fundamental scientific disciplines such as chemistry, pharmaceuticals, physics, and material
science. Significantly, UV spectroscopy serves as a fundamental tool, enabling the exploration and analysis of the composition,
physical structure, and electronic structure of matter at the atomic and molecular levels. In essence, its contributions resonate across
various fields, solidifying its position as a cornerstone in contemporary scientific research and analysis.

References
[1] Guo Y, Liu C, Ye R, Duan Q. Advances on water quality detection by uv-vis spectroscopy. Applied Sciences.
2020;10(19):6874.
[2] Li P, Hur J. Utilization of UV-Vis spectroscopy and related data analyses for dissolved organic matter (DOM) studies: A
review. Critical Reviews in Environmental Science and Technology. 2017 Feb 1;47(3):131–54.
[3] Demchenko AP. Ultraviolet spectroscopy of proteins [Internet]. Springer Science & Business Media; 2013 [cited 2023 Nov
30]. Available from:
https://books.google.com/books?hl=en&lr=&id=H3b1CAAAQBAJ&oi=fnd&pg=PR11&dq=UV+spectroscopy&ots=
9qbnjOxw39&sig=LCAfTbDJRmfTudGhmX_L2_sNk8c
[4] Antonov L, Stoyanov S. Analysis of the overlapping bands in UV-vis absorption spectroscopy. Applied spectroscopy.
1993;47(7):1030–5.
[5] Aleixandre-Tudo JL, Du Toit W. The role of UV-visible spectroscopy for phenolic compounds quantification in
winemaking. Frontiers and new trends in the science of fermented food and beverages. 2018;200–4.
[6] Rocha FS, Gomes AJ, Lunardi CN, Kaliaguine S, Patience GS. Experimental methods in chemical engineering: Ultraviolet
visible spectroscopy—UV‐Vis. Can J Chem Eng. 2018 Dec;96(12):2512–7.
[7] Mäntele W, Deniz E. UV–VIS absorption spectroscopy: Lambert-Beer reloaded [Internet]. Vol. 173, Spectrochimica Acta
Part A: Molecular and Biomolecular Spectroscopy. Elsevier; 2017 [cited 2023 Nov 30]. p. 965–8. Available from:
https://www.sciencedirect.com/science/article/pii/S1386142516305558
[8] Lichtenthaler HK, Buschmann C. Chlorophylls and carotenoids: Measurement and characterization by UV-VIS
spectroscopy. Current protocols in food analytical chemistry. 2001;1(1):F4-3.
[9] Mangam VT, Sarella PN, Siddhantapu S, Sudhabattula S, Surampudi VA. Novel colorimetric approach for amikacin
estimation in pure powder and its pharmaceutical formulations
[10] Domeizel M, Khalil A, Prudent P. UV spectroscopy: a tool for monitoring humification and for proposing an index of the
maturity of compost. Bioresource Technology. 2004;94(2):177–84.
[11] Picollo M, Aceto M, Vitorino T. UV-Vis spectroscopy. Physical Sciences Reviews. 2019 Mar 26;4(4):20180008.
[12] Perkampus HH. UV-VIS Spectroscopy and its Applications [Internet]. Springer Science & Business Media; 2013 [cited 2023
Nov 30]. Available from:
https://books.google.com/books?hl=en&lr=&id=6ejwCAAAQBAJ&oi=fnd&pg=PA1&dq=UV+spectroscopy&ots=b
QIUsu0UZS&sig=4R2Z2k0KHJkws7nlF52FoftQQH8
[13] Macheroux P. UV-Visible Spectroscopy as a Tool to Study Flavoproteins. In: Flavoprotein Protocols [Internet]. New Jersey:
Humana Press; 1999 [cited 2023 Nov 30]. p. 1–8. Available from: http://link.springer.com/10.1385/1-59259-266-X:1
[14] Li C, Korshin GV, Benjamin MM. Monitoring DBP formation with differential UV spectroscopy. Journal AWWA. 1998
Aug;90(8):88–100.
[15] Korshin GV, Li CW, Benjamin MM. Monitoring the properties of natural organic matter through UV spectroscopy: a
consistent theory. Water Research. 1997;31(7):1787–95.
[16] Förster H. UV/vis spectroscopy. Characterization I: -/-. 2004;337–426..

Alekhya Mandru et al 96