Re: What wavelength of light is used to detect protein via HPLC and why?

hi Ritvik,

The wavelength used in a HPLC detector depends to some degree on the make of the machine. However, the range of wavelengths used is limited to the ultraviolet end of the spectrum: usually between 200-300 nm, with 280 nm commonly used. So the more interesting question - why is that?

Chemical bonds in molecules absorb electromagnetic radiation with a characteristic wavelength. In proteins, the peptide bond between amino acid residues absorbs at around 200 nm. The aromatic ring of tryptophan and tyrosine absorbs at about 280 nm.

In principle, you can use UV absorbance to estimate the concentration of a protein in solution using the Beer Lambert Law. It helps if you have a pure preparation of the protein of interest, because then you can determine the concentration using an independent method, measure your absorbance at 280 nm and calculate the molar extinction coefficient for your protein, for future use in spectrophotometric measurements. However, there are numerous factors that can lead to inaccuracy when measuring protein concentration using only absorbance at 280 nm. They include the presence of contaminants (such as nucleic acids which also absorb close to 280 nm), variations in pH or ionic strength and the fact that some proteins exhibit a low UV absorbance due to their structure or a low content of aromatic amino acids.

Most times though, in an HPLC machine, you just want an indication that your protein is eluting from the column and are not concerned with accurate concentration measurement, so absorbance at 280 nm is commonly used for that purpose.