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:
http://books.google.com/books?id=6HU7q5XKdQgC&pg=PA17&lpg=PA17&dq=protein+detection+wavelength&source=web&ots=7jbAihaOxb&sig=LdjlYXyWmyJ2L3B0oXQpza4TC4U&hl=en&sa=X&oi=book_result&resnum=5&ct=result
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
Determining the appropriate wavelength to use in spectrophotometry and quantifying the amount of protein in a sample involve separate steps. Let's break down the process:
1. Selecting the appropriate wavelength:
To determine the appropriate wavelength, you need to identify the absorbance spectrum of the compound that provides the color in your end product. In this case, you are looking for the protein's absorbance spectrum.
To do this, you would typically perform a UV-Vis spectroscopy scan over a range of wavelengths (e.g., 200-800 nm) and record the absorbance at each point. The wavelength at which the protein absorbs the most light/has the highest absorbance is the appropriate wavelength to use for further analysis.
2. Quantifying the amount of protein in a sample:
Once you have determined the appropriate wavelength, you can proceed to quantify the amount of protein in your sample. To do this, you will need a standard curve.
Here's an outline of the steps involved:
- Prepare a series of protein standards with known concentrations. These standards are typically created by diluting a known concentration of a pure protein solution.
- Measure the absorbance of each standard at the appropriate wavelength determined previously. Record the absorbance values.
- Plot a standard curve by graphing the known concentrations of the standards on the x-axis against their respective absorbance values on the y-axis.
- Using the equation of the line (generated from the standard curve) or another appropriate mathematical relationship, determine the amount of protein in your sample by measuring its absorbance at the same wavelength and interpolating from the standard curve.
Please note that this process assumes a linear relationship between protein concentration and absorbance. If your sample contains interfering substances, you may need to account for those as well.
While the more light absorbed does correlate with a higher concentration of protein, it is important to use a standard curve to accurately quantify the amount of protein in your sample. The correlation between absorbance and concentration is not simply based on the wavelength alone. The absorbance at a specific wavelength depends on the molar absorptivity of the compound, which is specific to each substance.