What is UV absorbance method?
Detection and quantification of protein by measuring the UV absorbency is perhaps the simplest method among various methods of protein pharmaceutical detection. UV absorption is a process where a molecule absorbs ultraviolet light that excites electrons (makes them high energy). This energy causes an electronic transition from a ground state to an excited state. UV absorption can be detected from proteins depending on their specific amino acid residues and thus make it is possible to quantify protein concentrations.
What is the principle of UV absorbance method?
This method is based on protein absorbing UV light at the wavelength of 280 nm due to the presence of aromatic amino acids. Protein determination at 280 nm is mainly a function of tyrosine and tryptophan residues. The UV absorbance values (A280) of sample solution is proportional to the protein concentration. This approach is popular for protein determination as it is fast, non-destructive and easy to perform. However, this method is relatively insensitive according to two reasons. The first one is that an error will be obtained if protein content of tyrosine and tryptophan vary to significant extent. The other one is that if the sample contains an UV absorbance substance, such as nucleic acids, the results may be interfered.
What is UV absorbance method applied to?
Due to the disadvantage of the technique, this method is rarely used to determine the protein concentration of the final product. However, it is routinely used during downstream processing to detect protein elution form column chromatography.[1] The test solution should have a protein concentration of 0.2 to 2 mg/ml.
[1] Pharmaceutical Biotechnology: Concepts and Applications, Gary Walsh, 2007.
