Spectrophotometer: kinetic curves

November 27, 2022by Kalstein

A spectrophotometer is a piece of equipment used to measure the absorbance of a sample, as a function of the wavelength of electromagnetic radiation. There are several types of spectrophotometers. These are grouped according to the type of sample analyzed; There are atomic absorption and molecular absorption (commonly known as a UV-VIS spectrophotometer).

Spectrophotometers are used in laboratories to determine what the beam of electromagnetic radiation or light is and thus identify, qualify and quantify what its intensity is like. In the same way, it allows determining its efficiency, sensitivity, resolution and spectral range. Which will depend on the variabilities of the design and the choice of the optical components it contains.

The study at the biochemical level of any biomolecule requires the use of analytical techniques that allow its qualitative and quantitative determination, as well as its physical-chemical and biological characterization. One of the simplest, most accessible, useful and widely used methods is spectrophotometry, in general, and ultraviolet-visible, in particular. Biomolecules can be identified and quantified in solution and in biological samples, with the use of specific reagents that react with the compound to be analyzed and form a colored product that allows it to be detected in complex samples.

What is the foundation of a spectrophotometer?

The foundation of spectrophotometry is due to the ability of molecules to absorb radiation, including radiation within the UV-visible spectrum. The wavelengths of radiation that a molecule can absorb and the efficiency with which they are absorbed depend on the atomic structure and the conditions of the environment (pH, temperature, ionic strength, dielectric constant), so this technique constitutes a valuable tool for the determination and characterization of biomolecules.

What is a kinetic curve?

Enzyme kinetics studies the speed of chemical reactions that are catalyzed by enzymes. The study of the kinetics and chemical dynamics of an enzyme allows us to explain the details of its catalytic mechanism, its role in metabolism, how its activity is controlled in the cell and how its activity can be inhibited by drugs or poisons or enhanced by other types of molecules.

An enzyme assay is a procedure, carried out in a laboratory, by which the rate of an enzyme reaction can be measured. As enzymes are not consumed in the reaction that they catalyze, enzymatic assays usually measure the changes experienced either in the substrate concentration (which is decreasing), or in the product concentration (which is increasing). There are various methods to perform these measurements. Spectrophotometry allows detecting changes in the absorbance of light by the substrate or the product (depending on their concentration) and radiometry involves incorporation or release of radioactivity to measure the amount of product obtained per time. Spectrophotometric tests are the most widely used, since they allow the reaction rate to be measured continuously.

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