UV-vis |
UV-visible spectrum analysis
Ultra violet (180-400 nm) – visible spectrum (400-700 nm) spectroscopy is a method based on absorbance (or transmission) of a conjugated compound. This can be in gaseous, liquid, aqueous or solid phase. Light, electromagnetic radiation, has an energy $$E=h \cdot f$$ where \(h\) is Planck’s constant, \(f\) the frequency of light. \(f\) can be found in the relation \(c=f \cdot \lambda\) (c: speed of light, \( \lambda \) : wavelength in nanometre). Combining these formulas gives rise to: \begin{equation} E=h\times\frac{c}{\lambda} \end{equation} Here, there is a clear correlation between the energy and associated wavelength. Lowering the wavelength results in a higher energetic transition occurring. Aromatic amino acid residues present in proteins such as tryptophan, tyrosine and phenylalanine are conjugate systems with molecular orbitals. The principle behind UV-vis is the molecular electronic shift from highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). These can be indicated as, for example, aromatic \(\pi->\pi *\) transitions. Only transitions between similar types of bonds can take place. The spectrophotometer is able to identify compounds based on their maximum absorption at specific wavelengths. Using the Beer-Lambert law: \begin{equation} A= \varepsilon \times c \times l \end{equation} where \(A\): absorbance (arbitrary), \(\varepsilon\) : molar absorption coefficient, \(M\)-1 \(cm\)-1, \(c\) : concentration, g/L (or mg/ml), \(l\): path length (in M or cm), accurate measurements can be made depending on purity of the samples. Protein absorbance is normally measured at 280nm.
Sample volume of approx 2-3 ml is pipetted into a UV-vis polystyrene cuvette. Spectrum analysis depends on the type of sample which is either liquid or powder. The spectrophotometer (Lambda 650S, PerkinElmer, USA) is setup to record the spectrum. Data are output in arbitrary units of absorbance.