Question

Why are we able to use a UV aborption wavelength of 280 nm to quantitatively measure the amount of protein in a sample?

Answer 1

UV-Visible (UV-Vis) is often called a general technique because most molecules will absorb in the UV-Vis wavelength range. The UV extends from 100-400 nm and the visible spectrum from 400–700 nm. The 100-200 nm range is called the deep UV. Light sources are more difficult to find for this range, so it is not routinely used for UV-Vis measurements. 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.

When a photon hits a molecule and is absorbed, the molecule is promoted into a more excited energetic state. 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). The energy difference between the HOMO and the LUMO is called the band gap. Typically, these orbitals are called bonding and anti-bonding. The energy of the photon must exactly match the band gap for the photon to be absorbed. Thus, molecules with different chemical structures have different energy band gaps and different absorption spectra. The most common transitions that fall in the UV-Vis range are $\pi$ - $\pi$ * and n- $\pi$ *. $\pi$ -orbitals arise due to double bonds, and n-orbitals are for non-bonding electrons. $\pi$ * is anti-bonding $\pi$ -orbitals. Thus, the best UV-Vis absorption is by molecules that contain double bonds. $\pi$ -orbitals adjacent to each other that are connected, called conjugation, typically increases absorption. $\sigma$ - $\sigma$ * transitions, associated with single bonds, are higher energy and fall in the deep UV, so they are less useful for routine use. 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.

Hence, one will be able to use a UV absorption wavelength of 280 nm to quantitatively measure the amount of protein in a sample.