Forensic Physics
UV-Visible Spectroscopy: Principle, Working and Applications

UV-Visible Spectroscopy: Principle, Working and Applications

UV-Visible spectroscopy is an analytical technique that identifies a component that shows absorption or reflectance of ultraviolet(UV) and visible electromagnetic radiations under the range from 200-800 nanometers (UV range 200-400nm and Visible range 400-800nm).

The absorption of UV-Visible radiations results in the transfer of energy from the radiations to the electrons of a molecule. When a molecule is at rest, the electrons are at their ground energy level. On absorbing the energy from the radiations, the electrons show changes in three basic energy levels- electronic energy, rotational energy, and vibrational energy levels.

Electronic energy facilitates the transition of electrons from ground level to higher energy levels. Rotational energy allows the electrons to rotate about various axes while vibrational energy allows the atoms to vibrate relative to each other.

The energy of UV-Visible radiations is enough to affect the changes in all three types of energies.

This absorption spectroscopy is possible due to the activities of three types of electrons in a molecule. They are the sigma electrons, pie-electrons, and non-bonding electrons. 

Working Principle of UV-Visible Spectroscopy

UV-Visible spectroscopy generally involves the fact that when a beam of light falls on a solution or homogeneous media, a portion of the light is reflected from the surface of the media, and a portion of the light is absorbed within the medium while the remaining is transmitted through the medium. These phenomena are based on the principles of Beer-Lambert Law. 

The Lambert law states that “When monochromatic light passes through an absorbing media at right angles to the plane of the surface of the medium or solution, the rate of decrease in intensity of light is directly proportional to the thickness of the medium.” It is represented as:

-dI/db = kI


k= proportionality constant
I= intensity of light of a particular wavelength
Iₒ= intensity of incident light
Iₜ= intensity of transmitted light
b= thickness of the medium

Beer-Lambert law was proposed by Pierre Bouger in 1729, but it was Johann Heinrich Lambert, who in 1760 cited Bouger’s work and modified it into today’s acceptable form.

In 1852, Bernard and August Beer independently attenuated another factor responsible for the transmittance of light by the solution. The law then came to be known as Beer’s Law which states that “the intensity of incident light decreases exponentially with the arithmetic increase in the concentration of the solution.” This can be expressed as:

Iₒ= intensity of incident light
Iₜ= intensity of transmitted light
c= concentration of the solution

In UV-Visible spectroscopy, both principles are combined which forms the Beer-Lambert Law, which is expressed by:

Iₒ= intensity of incident light
Iₜ= intensity of transmitted light
b= thickness of the medium
c= concentration of the solution
𝜀= molar absorptivity

Molar absorptivity is a constant that depends on the wavelength of the incident radiation and the nature of absorbing material. 

UV-Visible Spectroscopy Instrument

UV-Visible spectroscopy is a simple instrument whose functioning is straightforward. It is composed of the following components:

1. Radiation Source

A radiation source is responsible for illuminating the incident light on the sample. The radiation source used in the instrument should be stable, free from fluctuations, emit a continuous spectrum of high and uniform intensity, and should not show fatigue on continued use. The most common radiation sources used in UV-Visible spectroscopy are:

  • Hydrogen or Deuterium LampThis lamp consists of a pair of electrodes enclosed in a glass tube provided with a silica or quartz window and is filled with hydrogen or deuterium gas at low pressure. Electric current is passed through the electrodes which excite the hydrogen or deuterium gas electrons to a high energy state. When these electrons return to their ground state, they release radiation in the region of 180-350 nm. Therefore these lamps are suitable for producing UV radiation. Deuterium lamps produce high-intensity radiation as compared to hydrogen lamps. 
  • Tungsten Filament lampThis incandescent lamp is capable of producing visible as well as infrared radiation. The tungsten filament is heated by a stabilized power supply or storage battery. It is the most common and inexpensive source of visible radiation.
  • Mercury Discharge LampIt is a lamp that can produce both UV as well as Visible radiations. It is usually a mercury lamp kept in a glass tube for the visible region while in a fused silica envelope for the UV-Visible region. 

2. Collimating System

The emitted radiations from the source are made parallel (collimated) by using a collimator which is usually a combination of lenses, mirrors, prisms, and slits. For visible radiations, silicate glass is used for the lenses, whereas fused silica or quartz is suitable for UV radiations.

Similarly, glass prisms are used in visible radiations and quartz prisms are used in UV radiations. The mirrors used in the collimating system are aluminized to minimize the light losses from the front surface. Slits are important in resolving polychromatic radiation into its individual wavelength or monochromatic radiation. The slit width is a key factor in the resolution of polychromatic radiation. 

3. Monochromator

Monochromators in a spectrophotometer are employed to narrow down polychromatic radiation into monochromatic radiation. It is usually done by adding filters or prisms or gratings after the collimating system. 

  • FiltersThey allow the transmission of only limited wavelength regions while absorbing most of the radiations of undesired wavelengths. The most common types of filters used are glass filters, gelatin filters, and interference filters. Glass filters are usually pieces of colored glass that transmit limited wavelengths. The metal oxides of vanadium, chromium, nickel, cobalt, iron, manganese, copper, etc. are used to give colors to the glass filters. For example- cobalt gives blue color, manganese gives purple, iron gives green, etc. the glass filters are effective for the bandwidth of 20-50nm. 
  1. Gelatin filters are thin gelatin sheets colored with dyes. The sheet is sandwiched between a pair of glasses to obtain the filter. They transmit a 10-30nm band of wavelength superior to glass filters.
  2. Interference filters are two parallel glass plates that are silvered internally and separated by a thin film of the transparent dielectric spacer of low refractive index. Magnesium fluoride is usually used as the dielectric spacer. These filters have a bandwidth of 10-15 nm.
  • PrismsThey are made up of quartz and are used in UV radiations, while glass is used as the prism material in visible radiations. The phenomena involved here is the dispersion of light which helps isolate the desired wavelengths. The effective separation of wavelengths depends on the prism material’s dispersive power and the prism’s apical angle. There are two types of prism mountings involved in the instrument.
  1. One is the Cornu-type prism in which the apical angle is 60 degrees and it is adjusted in such a way that on rotation the emerging light is allowed to fall on the exit slit.
  2. The other type of prism is the Littrow type which has an apical angle of 30 degrees and its surface is aluminized which reflects light back to pass through the prism and to emerge on the same side of the light source.
  • GratingsThese are also known as diffraction gratings, which consist of a large number of parallel lines and grooves about 15000 to 30,000 per inch ruled on a highly polished surface of aluminum. These lines and grooves act as the scattering centers for light beams falling on them. Gratings provide a narrow wavelength of light. 
Different types of Prisms

4. Sample Holder

The sample used should always be in a liquid state which is filled in the cuvettes or test tubes. To study in the UV region the cuvettes are prepared from fused while for the visible region, glass is used for preparing cuvettes. The cuvettes are usually regular-shaped tubes with an internal diameter of 1 cm to 4 cm. Generally the cuvettes are lid-less but for volatile samples, cuvettes with lids are used. 

5. Detector

Detectors measure the amount of transmitted radiation from the sample, in the form of electrical signals. An effective detector should be sensitive and have a low noise level, it should have a short response time and generate sufficient signals that can be measured easily. The common detectors used in UV-Visible spectroscopy are as follows:

  • Barrier Layer CellIt is also known as a photovoltaic cell which is the most simple and sturdy detector used in this spectroscopy. It is constituted of a base plate of copper or iron on which a thin layer of selenium is deposited. Above this is a thin transparent layer of silver. And in between the selenium and silver layer, there is a hypothetical barrier layer. Finally over the silver layer collecting rings are present which collect the electrons. When the radiation falls on the surface of a cell, it passes through the silver layer followed by the hypothetical layer, and finally impinges on the selenium layer. The radiation excites the electrons of the selenium layer and becomes positively charged. The excited electrons are collected by the electron rings and become negatively charged. Thus it generates its own electromotive force. 
  • Photo Tubes– It is also known as a photo-emissive tube. It is a spherically shaped vacuum bulb containing a photoemissive cathode and an anode. The inner surface of the semi-cylindrical cathode mounted inside the bulb is coated with photo-sensitive material like cesium oxide and a metal wire nearby is an anode. When radiant energy falls on the surface of a photosensitive cathode, there is an emission of electrons that are attracted to the anode causing electric flow. 
  • Photomultiplier TubesIt is a modified photo-tube detector. In these detectors inside a vacuum tube, a primary cathode is fixed which receives radiation from the sample. Eight to ten dynodes are fixed each with an increasing potential of about 90V. Near the last dynode is fixed an anode or electron collector electrode. The light received by the cathode releases electrons which through a series of dynodes produce more electrons. These electrons from the last dynodes are collected by the last anode and a photo-current is produced. 

6. Amplifier and Recorder

Usually, the signals produced by the detectors are very low in intensity. Therefore amplifiers are employed which amplify the signals and are collected by the recorders that show them in the form of graphs between absorbance and wavelength on a computer screen.

Absorption Spectra of UV-Visible Spectroscopy

As discussed earlier there is a transition of electrons between the energy levels of certain types of groups present in the molecule. For UV-Visible spectroscopy, the groups which are responsible for absorption are called chromophores. These chromophores are attached to a molecule that shows absorbance in the UV-Visible region at a specified wavelength. 

The common chromophores are usually unsaturated groups such as alkenes, amines, nitro compounds, etc. These groups although show absorption in the spectra but the maximum absorption is shown by the sample molecules.

There are some other saturated groups attached to the sample molecules which do not absorb radiation at wavelengths more than 200 nm. They are the auxochromes that have non-bonding valence electrons and examples include- (-OH), (-Cl), etc. These groups do modify and shift the absorption bands. 

There are situations when a shift in absorption bands is seen. This is observed when an auxochrome is attached to a chromophore. When the autochrome shifts the chromophore absorption band to longer wavelengths, it is called the redshift or bathochromic shift and the effect is called the hypsochromic effect. If the bands are shifted to shorter wavelengths, then it is called a hypsochromic shift or blue shift and the effect is a hypochromic shift.

Limitations of UV-Visible Spectroscopy

  • Regular calibration is required.
  • Maintenance is required.
  • If the sample holder is not thoroughly cleaned then there will be interference in the results.
  • Deviations from Beer’s law are a disadvantage.
  • It is only effective for homogenous solution samples.

Forensic Applications of UV-Visible Spectroscopy

  • UV-Visible spectroscopy is useful in the examination of drugs, poisons, and toxins.
  • It is helpful for ink analysis in questioned document examinations.
  • It is used to examine biological samples like blood, urine, saliva, hair, fiber, etc.
  • Examination of chemicals, dyes, metals, and petroleum compounds is also carried out by this instrument.
  • Alcohol examination is also possible using UV-Visible spectroscopy.
  • Paint analysis is carried out by this technique.
  • The examination of smokeless powder in firearm analysis is possible with this technique.


UV-Visible spectroscopy is a qualitative and quantitative analytical technique widely used in various disciplines of science. One of the important features of this method is that it is a colorimetric technique. It involves two different radiations namely ultraviolet and visible radiations that range from 200-800 nm in wavelength. It is a very useful and easy technique employed in forensic science. 

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