c. For each peptide draw the predominant form at the pH at which you plan to con
ID: 202946 • Letter: C
Question
c. For each peptide draw the predominant form at the pH at which you plan to conduct the separation, make sure to indicate the pH at which you have drawn the peptide. (6 pts) d. You use UV absorption at 280 to monitor your purification process. On your first column you only see two peaks elute. What is the most obvious explanation for this phenomenon? (2 pts). e. How could you verify which fractions your peptides are contained in following the purification process? (2 pts) f. Aside from UV spectroscopy, what is another way you might measure protein concentration? (2 pts)Explanation / Answer
d) Maximum absorbance of protein solution at 280nm, aromatic acid in amino acid are the primary reason for the peak formation at 280nm.
f) The method used to determine the concentration of protein in addition yo UV absorbance method.
1) Amino acid analysis: Amino acid's mass analysis through HPLC after acid hydrolysis would able to provide you to find a protein concentration. This needs specialized instrumentation, which takes so much time and is not accurately used for protein determination.
2) Kjeldahl nitrogen determination: This method also needs specialized instrument and more amounts of ammonium sulfate. The acidified ammonium solution and the concentration of released ammonia is calculated by a titration. This is not regularly used in biochemistry.
3) Biuret reaction: In alkaline solutions, complex of cupric ion with the protein's peptide bonds and peptides to produce a complex of purple charge (max = 540 nm). The color intensity is proportional to the concentration of protein. This reaction takes place with the peptide bond only and not with the side chain of amino acid. Because the peptide bonds number per unit weight is roughly equal for all proteins, this method is regularly valid and reasonably precise, irrespective of the protein mixture composition. Other advantage is that the time in color development is comparatively short and the intensity of color stay constant for a rational amount of time. A major drawback of this method is lack of its sensitivity, 2mg protein being the lower limit. Larger sensitivity can be determined by measuring the protein –cupric ion complex absorbance at 310 nm instead of 540 nm.
10) chromatographic method use to separate peptides:
size exclusion chromatography (SEC) separation method based on the diameter of the molecule.
ion – exchange chromatography (IEC) uses molecules electric charge for the separation.
reversed phase chromatography (RPC) depends on the hydrophobic domain presence.for separation
affinity chromatography (AC) uses biospecific interaction of peptides to separate it.
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