Question

You have a solution containing human myoglobin (16.7 KD, pi7.0), hemoglobin (64.5 KD; pi7.1). and serum albumin (66,5 KD: pl 4,9). You want to separate these proteins and you run one portion of the solution through anion exchange chromatography at pH 8 and a second portion of the solution through gel filtration. You measure the protein concentration in fractions collected from both columns and you get the following elution profiles Anion Exchange filtration Protein Concentration Eurim Volume Elution Volume Based upon MW and pl, predict which proteins are in the peaks shown above. 1a. Peak 1 Peak 2 Anion Exchange Gel Filtration 1b. To test your prediction, you also measure the iron concentration in the solution eluted from both columns. Use a dotted line in the profiles above to indicate the iron concentration in column fractions (assuming your answer to part 1a is correct). 10. Neither technique alone successfully separates the three proteins. But you realize you can separate the three proteins by using the two techniques, one after the other. So, you take the material in Peak 1 from anion exchange, run it through gel filtration, and generate the following elution profile. Identify the proteins in these two peaks. Protein Concertatim Elution Volume

Answer 1

anion exchange is a method of separating analysts based on charge.proteins are separated based on net charge ,which is dependent on pH.The first stage is equillibrium in which the ion exchanger is brought to starting state in terms of ph and ionic strength hich allows the bibding of the desired solute molecules..separation is obtained since different substances have different degrees of interaction with their ion exchange due to difference in charges ,charge densities .the proteins are mostly eluting according to their isoelectric point.(PI).

peak 1 haemoglobin

peak 2 myoglobin

gel filtration separates molecules according to differences in size as they pass through a gel filtration medium packed in a coloumin gel filtration molecules do not bind to the chromatography medium so buffer composition does not directly affect the solution.gel filtration is suited for biomolecules that may be sensitive to changes in PH ,concentration of metal ions, harsh environments

peak 1 serum albumin

peak 2 hemoglobin