Youâre trying to find the protein-concentration in an unknown sample using a spectrophotometer. The absorbance measurements for the standard curve gave a linear curve in the range 0.100 - 0.700. The measured absorbance for the unknown sample was 1.800. This means that the sample will have to be diluted again; how much and why? Can the mixture be diluted directly, or do you need a new sample?
Youâre trying to find the protein-concentration in an unknown sample using a spectrophotometer. The absorbance measurements for the standard curve gave a linear curve in the range 0.100 - 0.700. The measured absorbance for the unknown sample was 1.800. This means that the sample will have to be diluted again; how much and why? Can the mixture be diluted directly, or do you need a new sample?
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BIOCHEM LAB HELP:
here are our data :
concentration | absorbance |
0.02mL | 0.071A |
0.04mL | 0.090A |
0.06mL | 0.115A |
0.1mL | 0.156A |
0.2mL | 0.282A |
0.3mL | 0.357A |
0.4mL | 0.423A |
0.6mL | 0.626A |
0.8mL | 0.811A |
1.0mL | 1.040A |
UMKNOWN | 0.546 |
please answer all questions
Spectrophotometric Analysis of Protein
1. Provide the standard curve for your experiment and the calculation of the unknown concentration from absorbance.
2. Is the concentration of the unknown you calculated from your standard curve the same as the concentration of the unknown stock solution? Why or why not?
3. Explain the process through which the Biuret reagent complexes with proteins to produce the characteristic color.
4. Why is it important to condition your cuvette prior to each reading?
5. Why is it important to blank your spectrophotometer, prior to use?
6. The lab manual discusses how proteins have characteristic absorption at 275-280 nm due to the presence of tyrosine and tryptophan (to some extent phenylalanine as well) in addition to the absorbance that occurs at 190 nm. Why not simply measure the absorbance of our protein sample at this wavelength and forego the Biuret reagent entirely? Discuss this question in light of the information provided in your lab manual, your knowledge of protein structure, and limitations inherent to a teaching laboratory.
7. According to Beerâs law, the relationship between concentration and absorbance should be perfectly linear, as would be indicated by a linear regression (R^2) value of 1. However, often the regression value is less than one, indicating an imperfect correlation. Considering the possible limitations of the Beer-Lambert law, and give a detailed explanation as to why this result may have been observed.