UV-Vis spectrophotometry is a laboratory technique used in the measurement of absorbance of light across ultraviolet and visible regions. When the incident light strikes the sample, it is usually reflected, transmitted, or absorbed. The absorbance of light causes the transition of molecules from the ground state to the excited state. UV-Vis spectrophotometer, an analytical instrument, serves this principle and measures the intensity of light absorbed by the molecule.

To obtain high precision and a more dynamic range, it is necessary to understand the design of a UV-Vis spectrophotometer. Further, to disperse the wavelengths of light and detect the absorbed wavelength in the sample, it is essential to know about the working of the UV-Vis spectrophotometer. This article details the way of designing a cheaper and efficient UV-Vis spectrophotometer and the way of working a UV-Vis spectrophotometer.

Design of UV-Vis Spectrophotometer

The design of the UV-Vis spectrophotometer includes both the parts of the spectrometer and photometer. The spectrometer part contains a light source, sample cell, monochromator, and slits. The microcontroller controls grating in the monochromator, whereas the photometer part consists of a photodiode, preamplifier, and data acquisition system.

  • The light source can be a deuterium lamp. Spherical and concave mirrors are present in the monochromator. The sample cell is a quartz cuvette. A detector is a silicon photodiode. An amplifier is a chopper-stabilized operational amplifier. A computer is used as a display.
  • The light source is focused on the entrance slit of the monochromator. Then it passes into an ellipsoidal mirror, which collects light from the entrance slit and refocusses into the concave mirror and to the grating.  The Ellipsoidal mirror enhances throughout the monochromator. It refocusses light to the large spherical concave mirror. The concave mirror lets the grating be fully illuminated and collects all light. 
  • The sample cell is required to meet the challenge of a low quantity of samples. The spectral range of the monochromator is calculated by the efficiency of grating and the maximum angle of rotation of grating. Mirrors free from aberration should be used. 
  • A Stepper motor is used to rotate the holographic grating to scan all the wavelengths coming from the concave mirror.
  • Photodiode contains an effective photosensitive area, which receives light from the optical system. The wavelength range of photodiodes covers from UV to visible region. To detect a small amount of light, a larger area photodiode is selected. The power of the light source increases the current from the photodiode.
  • Input signal from the photodiode is passed to the amplifier. For displaying data on the computer, the interface between amplifier and computer is established using a microcontroller and cables. Along with the microcontroller, external capacitors, transmitters, and receivers are also used in this connection.
  • For the light source, 1W LED can be used along with a collimator lens, which allows radiation patterns to be concentrated and reflected in a small portion of the diffraction grating. LED provides an economical alternative for radiation sources with lower energy consumption. 
  • For monochromators, low-cost diffraction grating and a small slit are used for the selection of wavelengths. A diffraction grating is cheaper and shows better performance when compared to prisms and gratings. 
  • The concentration of a chemical substance is directly proportional to the amount of light it absorbs when exposed to monochromatic radiation. The position covered by the monochromator corresponds to the level of voltage in the detector. The voltage in the detector is directly proportional to the light transmitted through the reference and sample under study. 
  • The microcontroller is used to implement the control algorithms needed for the spectrophotometer operation. The stepper motor sweeps all the wavelengths of the spectrum using micro stepping. The voltage produced by the detector is obtained in each step and the resulting monochromatic light passed through the sample has been measured. In a microcontroller, an analog-to-digital converter reads the voltage, whereas a simple average digital filter filters the voltage. After this step, the value of absorbance is computed and stored in a table. 
  • Detectors are in a spot of light insulation, which reduces the interference in the experiments and guarantees zero voltage while the radiation source is switched off.

Microcontroller Algorithm

Working of UV-Vis Spectrophotometer

There are many parts in the instrumentation of the UV-Vis spectroscopy system, which are indispensable in the functioning of the UV-Vis spectrophotometer. Ultraviolet-visible spectrophotometer system focuses electromagnetic radiation from the light source to the sample. Depending on the configuration set in the system, light is transmitted through the sample or reflected off it. Then, the light is collected from the sample through reading.

Initially, light is focussed into the entrance slit of the monochromator from the source. Monochromator uses dispersing elements, namely optical grating to separate the light by wavelength. The light is passed into a charged coupled device (CCD), which is made up of individual tiny detectors, hence the intensity of light at each wavelength will be measured. CCD is read-off to a computer and the result obtained is a spectrum, which shows the intensity of each wavelength of light. Spectrophotometers are able to measure the electromagnetic radiation from ultraviolet to infrared. Spectrum will show the intensity of light versus the wavelength. 

Types of UV-Vis Spectrophotometer

There are 3 classifications of UV-Vis spectrophotometer. They are as follows:

  • Single beam spectrophotometer
  • double beam spectrophotometer 
  • Split beam spectrophotometer 

Single Beam Spectrophotometer

In a single-beam spectrophotometer, light from the monochromator alternatively passes through reference and sample solution. As it is affected by power supply fluctuations, this type is not suitable for high-demanding pharmaceutical and quality inspection experiments. 

Double Beam Spectrophotometer

In a double-beam spectrophotometer, there are two wavelengths of monochromatic light, which alternatively irradiate samples at regular intervals. It performs better for turbid samples, eliminating the light source instability and detector sensitivity changes.

Split Beam Spectrophotometer

In a split beam spectrophotometer, the light beam from the monochromator is split into two beams. One beam passes through the detector directly and the other one passes through the sample and reaches the detector. It is able to monitor errors in the light source, but can not eliminate the effects in the reference cell. 

Working of UV-Vis Spectrophotometer


Design of a spectrophotometer should be done with a low cost, good quality and low power consuming materials. The materials used should be sensitive to minute changes and accurate, ensuring good reproducibility. In addition, deviation should be minimal between already established equipment and newly designed instruments.It should be made sure that the designed equipment will be compatible to all types of samples.

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