However, a common misconception is that the wavelength of peak absorption (λ max) for a compound is the color it appears. The appearance of broad bands or shoulders on the UV-Vis structure is due to the numerous vibrational and rotational states of a molecule, which lead to separate energy band gaps of slightly different energies.įor molecules with absorption in the visible region, the compounds will often appear colored. Sigma-σ* transitions, associated with single bonds, are higher energy and fall in the deep UV, so they are less useful for routine use. Pi orbitals adjacent to each other that are connected, called conjugation, typically increases absorption. Thus, the best UV-Vis absorption is by molecules that contain double bonds. Pi orbitals arise due to double bonds, and n orbitals are for non-bonding electrons. The most common transitions that fall in the UV-Vis range are π-π* and n- π*. Thus, molecules with different chemical structures have different energy band gaps and different absorption spectra. The energy of the photon must exactly match the band gap for the photon to be absorbed. Typically, these orbitals are called bonding and anti-bonding. The energy difference between the HOMO and the LUMO is called the band gap. UV-visible light has enough energy to promote electrons to a higher electronic state, from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). When a photon hits a molecule and is absorbed, the molecule is promoted into a more excited energetic state. Typical UV-Vis spectrometers use a deuterium lamp for the UV that produces light from 170–375 nm and a tungsten filament lamp for visible, which produces light from 350–2,500 nm. Light sources are more difficult to find for this range, so it is not routinely used for UV-Vis measurements. The 100–200 nm range is called the deep UV. The UV extends from 100–400 nm and the visible spectrum from 400–700 nm. UV-Vis is often called a general technique because most molecules will absorb in the UV-Vis wavelength range. UV-Vis has a similar sensitivity to other absorbance measurements, such as infrared spectroscopy. Other spectroscopy techniques such as fluorescence have higher sensitivity, but they are not as generally applicable, as most molecules are not fluorescent. Typically, UV-Vis is not the most sensitive spectroscopy technique, because not a lot of light is absorbed over a short path length. UV-Vis is also a very popular detector for other analytical techniques, such as chromatography, because it can detect many compounds. UV-Vis measurements are generally taken with a spectrophotometer. Kinetics of chemical reactions are also measured with UV-Vis spectroscopy by taking repeated UV-Vis measurements over time. UV-Vis spectroscopy is used to quantify the amount of DNA or protein in a sample, for water analysis, and as a detector for many types of chromatography. It can also be used in a quantitative manner, as concentration of the analyte is related to the absorbance using Beer's Law. UV-Vis can be used in a qualitative manner, to identify functional groups or confirm the identity of a compound by matching the absorbance spectrum. The amount of absorbance at any wavelength is due to the chemical structure of the molecule. An absorbance spectrum is obtained that shows the absorbance of a compound at different wavelengths. From the transmittance (T), the absorbance can be calculated as A=-log (T). With UV-Vis spectroscopy, the UV-Vis light is passed through a sample and the transmittance of light by a sample is measured. Ultraviolet-visible (UV-Vis) spectroscopy is one of the most popular analytical techniques because it is very versatile and able to detect nearly every molecule.
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