UV-Vis Spectroscopy

UV-Vis spectrum of Paracetamols

Ultraviolet Visible spectroscopy (UV-Vis spectroscopy) is an analytical technique, which measures the amount of discrete wavelengths of ultraviolet-visible light absorbed or transmitted through a sample in comparison to a blank or reference. It relies on the use of light. Absorbance and transmittance properties are influenced by composition of sample, providing information on what is in the sample and in what concentration.

Some strengths of UV-Vis spectroscopy make it so popular for many applications. They are as follows:

  • Technique is non-destructive, which allows the sample to be reused for further processing or analysis
  • Measurements can be performed quickly
  • Little user training is need as instrument is easier to use and handle
  • Only minimal processing is required for data analysis.
  • The instrument is not costly as it is easy to acquire and access in laboratories

Having introduced you to the technique and its advantages, this article elaborates on different applications of UV-Vis spectroscopy in different fields.

Applications of UV-Vis Spectroscopy Technique

The applications of UV-Vis spectroscopy are enormous. The following are the main fields in which UV-Vis spectroscopy is used: 

  • DNA & RNA analysis
  • Pharmaceutical analysis
  • Bacterial culture
  • Beverage analysis
  • Other applications

DNA & RNA analysis

Uv-Vis spectroscopy deals with the purity of nucleic acids.

  • Quick verification of concentration and purity of DNA and RNA
  • This is essential before preparation of DNA and RNA in downstream applications like sequencing
  • The 260 nm/280 nm absorbance ratio is essential to display contamination in nucleic acids by proteins as the ratio in pure DNA is 1.8 and that in pure RNA is 2.

Pharmaceutical analysis

UV-Vis spectroscopy is an indispensable equipment in production of pharmaceutics.

  • Overlap of absorbance peaks in uv spectra can be used to find out the pharmaceutical compounds using mathematical derivatives
  • Chlortetracycline (antibiotic) and benzocaine (anaesthetic) are identified simultaneously in veterinary powder formulation using first mathematical derivative
  • By calibration function of each compound, simultaneous quantification was done on micrograms per milliliter concentration.

UV-Vis Spectra of 4-nitrophenol and 4-nitrophenolate

Bacterial culture

UV-Vis spectroscopy is essential in the biomass growth curve studies.

  • Routine OD measurements are taken at 600 nm for estimation of cell concentration and growth tracking. 
  • 600 nm is chosen due to the optical properties of media in which bacteria is growing and to avoid damage to the cells when there is need for continuous experimentation.

Beverage analysis

Uv-Vis spectroscopy is also used in FMCG industries.

  • Identification of particular components in drinks
  • Quantification of caffeine content as they should be within legal limit
  • Identification of anthocyanin in blueberries, blackberries, raspberries and cherries for quality control in wine

Other applications

Besides, there are extensive applications of UV-Vis spectroscopy in other fields also. Below are the other applications of UV-Vis spectroscopy:

  • Characterization of smaller nanoparticles.
  • Examination of structural protein changes by tracking changes in peak wavelength absorbance.
  • Determination of battery composition
  • Kinetic and monitoring studies of dyes and dye byproducts
  • Absorbance of hemoglobin for the determination of concentration of hemoglobin in cancer research
  • Measuring color index to monitor transformer oil
  • Determination of concentration of proteins through direct measurement or colorimetric assays
  • Study of enzymatic reactions
  • Monitoring growth curve of bacterial cell suspensions
  •  Can be used as a detector for HPLC
  • Through preparation of suitable derivatives of compounds, molecular weights of compounds can be measured. Example is molecular weight determination of amine through preparation of amine picrate, having absorption peak at 380 nm.
  • Impurities in organic compounds by measuring absorbance of a compound at specific wavelength through a presence of an additional peak.
  • Structural elucidation of organic compounds like presence of hetero atoms, presenc of unsaturation via the location and combination of peaks.
  • Quantitative analysis is performed on the basis of Beer’s law. Quantitative analysis are done by any of the following methods:
    • calibration curve method
    • simultaneous multicomponent method
    • difference spectrophotometric method 
    • derivative spectrophotometric method
  • Qualitative analysis performed for the unknown compound by comparing the obtained spectra with that of known compounds
  • Kinetics of a reaction
  • Detection of functional groups due to absence of functional group, which has absorbance at particular wavelength
  • Examination of polynuclear hydrocarbons as benzene and polynuclear hydrocarbons possess characteristic spectra in visible and ultraviolet regions. Polynuclear hydrocarbons are a type of hydrocarbon molecules with two or more closed rings. Example is naphthalene C10H8 with two benzene rings present side by side
  • Examination of solvent purity.
  • Peaks’ position in UV spectra reveal the molecular structure of the sample. Example is carbon-carbon double bond absorbs at specific wavelengths.
  • Calculation of extinction coefficient of the sample
  • Calculation of melting point of nucleic acids and proteins through measurement of UV spectra at different temperatures
  • Determination of rate of reaction by monitoring UV spectra as a function of time.
  • Position and profile of the peaks in UV spectra give information regarding the microscopic environment. For example, due to impurities, the peak becomes broadened.
  • Differentiating saturated and unsaturated fatty acids in olive oil and monitoring its quality

Kinetic Studies of formation of NADPH

Application Fields

In addition to the above applications, there are still some fields where UV-Vis spectroscopy is widely used.

  • Quality control
  • Cosmetic industry
  • Petrochemistry
  • Pharmaceutical research
  • Optical components
  • Food and agriculture
  • Life sciences
  • Traditional chemistry
  • Microbiology
  • Material Science
  • Analytical Chemistry
  • Chemical and Biotech industry


The applications of UV-Vis spectroscopy are seemingly endless. Still, there are few limitations for UV-Vis spectroscopy. Presence of stray light may cause measurement errors. Presence of suspended solids and bubbles in the cuvette can cause scattering. Also, geometrical considerations are significant to obtain reproducible results. Since, there will be interference from multiple absorbing species, each chemical species should be isolated from the sample measured individually for proper quantitative analysis.

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