Question

	MW(Da)	pl
Fibronectin	440000	5,8
Hemoglobin	64500	7,1
Lysozyme	14300	11,0
Egg albumin	45000	4,6
Serum albumin	68500	4,9

The protein mixture is seperated by using a DEAE (anion exchanger) chromatographic column

The pH of sample is set to 8.5 and the column is also equilibrated with the same buffer solution. What is the expected order of elution by applying a pH gradient? Explain.

Answer 1

It is an anion exchanger i.e peptide carrying negative charge will be retained on the matrix.

If pH > pI then peptide is negatively charged and vice versa so here the order representing negative to positive is

Egg albumin > serum albumin > fibronectin > Haemoglobin > lysozyme.

So , order of elution will be reverse i.e peptide carrying positive charge will be eluted first and peptide carrying negative charge will be eluted at last.

Here is order of elution : lysozyme > Hb > Fn > serum albumin > egg albumin.