I have a peptide dissolved in DMSO and MES buffer, and I aim to determine its absorbance using UV-Vis spectroscopy. However, I'm encountering issues with obtaining meaningful results at 220 nm, the specified detection wavelength for peptides. I suspect the problem is related to the absorbance of my blank sample, which is in the range of 2 or 3, potentially due to matrix interference.
Could you please guide how to address and resolve this issue?
Spectrophotometers have limits to how high an absorbance they can measure accurately. Depending on the instrument, this may be as low as 1 or as high as 3. If you are struggling to measure the absorbance of your peptide because of high background absorbance, one solution is to use a shorter pathlength. Remember that absorbance is directly proportional to the pathlength of light through the sample. If you are using a standard 1-cm-pathlength cuvette and get an absorbance of 2, if you switch to a 0.2-cm-pathlength cuvette, the absorbance will be 0.4, well within the useful range.
Certainly, that's accurate. However, if I lack access to this specific cuvette, what alternative solution could be considered?
You could change the measurement wavelength to one at which the solvent has less absorbance but the analyte still has a decent absorbance. Of course, you would need to determine the extinction coefficient at the new wavelength.
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