UV-visible spectroscopy is a quantitative optical technique to determine the chemical composition of liquids and solidsbased on their optical properties (transmittance, reflectance, and absorbance). It measures the amount of discrete wavelengths of UV or visible light that are absorbed or transmitted through a sample in contrast to a reference or blank sample. ... Read More
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Ultraviolet (UV)-Visible Spectroscopy
UV-visible spectroscopy is a quantitative optical technique to determine the chemical composition of liquids and solids based on their optical properties (transmittance, reflectance, and absorbance). It is used to classify substances during research, production, and quality control in various industries. UV-vis spectroscopy is used to gain qualitative and quantitative information about a sample. It is routinely used to characterize a range of samples, including semiconductor materials, dyes, pigments, biological materials, transition metals, organic compounds, and many other manufacturing materials. Spectrophotometers that can perform micro-volume measurements are available for testing of small volume samples.
When a light beam comes in contact with a solid sample, it may be reflected, transmitted, or absorbed. The electrons in the liquid sample get excited after absorbing some of the light from the optical source. The intensity of light absorbed is directly related to the concentration of a molecule in the liquid sample. The absorbance spectrum of a sample can be compared to the available standards to identify and quantify a compound.
A UV/Vis spectrophotometer is the tool used in ultraviolet-visible spectroscopy. It determines the light’s intensity after passing through a sample (I), and compares it with the light’s intensity before passing through the sample (Io). The ratio of I and Io is called transmittance (T) which is expressed as a percentage. The Absorbance (A) is calculated as below:
This methodology is frequently employed in a variety of practical and theoretical applications since it is reasonably affordable and simple to execute. The sample must only be a chromophore and absorb in the UV-Visible range. Aside from the measurement wavelength, variables of importance include absorbance (A), transmittance (%T), and reflectance (%R), as well as how they change over time.
Video 01: Working of a UV-Visible Spectrophotometer
Ultraviolet (UV)-Visible Spectroscopy Common Uses
Quantification of the bacterial cells in a cell culture
Study of conformational changes (such as partial unfolding) in proteins
Characterization of solid nano-bio materials that are homogeneously dispersed in a solvent
Calculation of reaction kinetics by measuring the variation of the concentration of the compounds throughout the reaction
Determination of the iron(III) concentration in wastewater
Elemental analysis of astronomical objects
Easy to use and maintain
A cost-effective technique to analyze the sample
High sampling speed
A non-invasive technique that keeps the integrity of the sample
Light sources in the deep UV range (100–200 nm) are expensive, so most spectrophotometers do not operate in this range
Results can be influenced by sample conditions like pH, temperature, impurities, and contaminants
It doesn’t work well for the samples in which solid particles are suspended in the liquid because such samples scatter light more than they absorb
The material of the sample holder should have no absorption at the measurement wavelength
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