Summary
Highlights
UV visible spectroscopy is a widely used technique in chemical and life science labs, primarily for determining the concentration of various substances and molecules in a solution, and sometimes for cell concentration in microbiology. While it can offer some insight into chemical groups, it's not highly reliable for that purpose due to similar data for different groups.
UV visible spectroscopy utilizes UV or visible light. Understanding the electromagnetic spectrum is crucial, especially the relationship between wavelength, frequency, and energy. Higher frequency means higher energy and shorter wavelength. The operative range for UV-Vis spectroscopy is typically 200-800 nanometers, with 200-400 nm for UV and 400-800 nm for visible light.
The basic setup includes a light source, a monochromator (which selects a single wavelength from the broad spectrum), a beam separator, two chambers (one for control, one for the sample within a cuvette), and a detector. The monochromator uses a prism to separate different wavelengths, allowing only a specific one to pass through.
When light of a specific wavelength hits a molecule, the molecule's electrons get excited, jumping from a ground state to a higher energy state. This process absorbs some of the light energy. The change in light intensity before (i0) and after (I) passing through the sample is measured. The more molecules present, the more light is absorbed.
Transmittance (T) is defined as I/i0. Absorbance (A) is the negative logarithm of transmittance (log(i0/I)). Absorbance and transmittance are inversely related. The wavelength at which maximum absorbance occurs is called Lambda Max (λmax). The Beer-Lambert Law states that absorbance is directly proportional to concentration (C) and path length (L), A = εCL, where ε is the molar absorption coefficient. This relationship allows for the accurate determination of unknown sample concentrations by plotting a linear graph of absorbance versus known concentrations.
At the molecular level, electrons reside in different energy states (e.g., sigma, pi, non-bonding orbitals) and can jump to higher anti-bonding states (sigma star, pi star) upon absorbing specific light energy. The type of bond and available energy dictates which transition occurs. Different chemical groups absorb at different wavelengths, but this technique is mainly used for concentration analysis due to variations.