A spectrophotometer is commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases. Although many biochemicals are colored, as in, they absorb visible light and therefore can be measured by colorimetric procedures, even colorless biochemicals can often be converted to colored compounds suitable for chromogenic color-forming reactions to yield compounds suitable for colorimetric analysis. However they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 200 nm - 2500 nm using different controls and calibrations. Within these ranges of light, calibrations are needed on the machine using standards that vary in type depending on the wavelength of the photometric determination.
The absorption of light is due to the interaction of light with the electronic and vibrational modes of molecules. Each type of molecule has an individual set of energy levels associated with the makeup of its chemical bonds and nuclei, and thus will absorb light of specific wavelengths, or energies, resulting in unique spectral properties. This is based upon it's specific and distinct makeup.
UV-visible spectrophotometry:
The most common spectrophotometers are used in the UV and visible regions of the spectrum, and some of these instruments also operate into the near-infrared region as well.
Ultraviolet-visible (UV-vis) spectroscopy involves energy levels that excite electronic transitions. The energy of light at 400nm is ~250 kJoules/mol (~60kcal/mol), which is of the order of the energy of chemical bonds. Absorption of UV-vis light excites electrons that are in ground-state orbitals to their excited-state molecular orbitals[1]
Visible region 400–700 nm spectrophotometry is used extensively in colorimetry science.
- ^ P., Ballou, David; Marilee., Benore, (2010). Fundamental laboratory approaches for biochemistry and biotechnology. John Wiley. ISBN 9780470087664. OCLC 420027217.
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