posted by Mak on .
Given a colored end product is produced, the presence of protein could be detected by spectrophotometry. Outline how you would go about determining the appropriate wavelength to use and the amount of protein in a sample.
For the first part, is that like a simple procedure on how to use a spectrophotmeter? The last part stumped me. How do you know the amount of protein in a sample? I don't think you have an actual number but, the more light absorbed, the higher the wavelength. So the higher the wavelength the higher the amount of protein used? Is that it? Or am I totally off?
Yes. You can do this with measuring absorbency or wavelength shift. The amount of protein affects the absorbency. You are off on your thinking about absorbency vs higher wavelength. The absorbency varies with wavelength depending on what the protein is. Short wavelength (200-280nm) are great.
See this classic:
To determine the appropriate wavelength you would need to run the spectrum of the protein. As this is coloured you would expect a peak or peaks in the visible region. I am not clear on the reason for the use of the spectrometry to detect the protein. If this is to be done in the presence of the starting materials and/or reagents then it might also be prudent to run these. Or at least the starting mixture. Sometimes there are overlaps in the spectra so the maximum in the protein's spectrum may not be the most useful.
To determine the amount of protein in the sample the usual way is to generate a Beer-Lambert calibration with a sample of the protein, at the chosen wavelength. The measured absorbance of the end product would then allow you to determine how much protein is present.
Analyze the spectrum of the protein you are looking for, using available references on spectrophotometry of proteins. Pick a wavelength where there is a strong absorption maximum where the possiblilty of strong contribution by other species that may be present is low.
You should pick a wavelength where the absorption lines overlap (continuous spectrum), otherwise Beer's law for exponential absorption will not be valid. For complex polyatomic species like proteins, the lines will probably overlap.
Determine the molar absorption coefficient of the species you are looking for, from a literature search or (preferably) a reference measurement on your own spectrophotometer, using a standard sample. Do the calibration at several concentration levels.
For an unknown sample and a specific spectral band, spectrophotometer transmission T will obey
T = exp(-k C L)
where k is an experimentally determined spectral absorption coefficient, L is the cell path length and C is the concentration