Question

You have a mixture of the proteins listed below.

Protein	pI	Molecular Weight (kDa)
A	3.1	265
B	6.9	93
C	10.3	96
D	7.1	43
E	8.6	189

1) You load the protein mix onto a cation exchange column at pH 5. Next, you apply a "washing" step by passing through buffer at pH 5. Finally, for your elution step, you apply a pH gradient starting from pH 2.0 to pH 13.0. (A gradient buffer system allows you to gradually and continuously change the pH of your mobile phase starting from pH 2 up to pH 13).

Indicate the order in which proteins will exit the column during the elution step. Explain.

2) You load this same mixture onto a size exclusion column. Please indicate the order of elution. Explain.

3) If your protein of interest is protein B, would using anion exchange or size exclusion be completely successful at separating it from the other proteins? Yes or No? Explain.

Answer 1

1) Ion exchange chromatography is a chromatographic technique to separate components based on their charge. Proteins can have many ionizable groups. These include the end amino and acid groups as well. To separate the proteins we should know its charge at pH 7. The isoelectric point of proteins the value of pH at which the protein does not have a charge. So from the pI values we will get the charge of the protein. The stationary phase using in ion exchange chromatography are charged resins. If we are using positively charged resin then during the process of separation, negatively charged proteins will bind to the stationary phase. Thus positively charged proteins will be eluted first and negatively charged last. If we are using negatively charged resin then during the process of separation, positively charged proteins will bind to the stationary phase. Thus negatively charged proteins will be eluted first and positively charged last.

If pH > pI the protein has a negative charge, if pH = pI the protein has no charge and pH < pI the protein has a positive charge. From the given data the order of charge of protein from negative to positive is A, B, D, E and C. A is negtaivly charged and C is positively charged. The ion exchange resin using in this case is cation exchange resin. Cation exchange resin is negatively charged. Thus negativley charged proteins will be eluted first and positively charged one last.

So the order of elution will be 1. A, 2. B, 3. D, 4. E and 5. C.

A will be eluted first and C at last.

2) Exclusion chromatography or Gel filtration chromatography is a separation technique base on the size of the components in the mixture. The stationary phase in this chromatographic technique is porous beads of well defined size. During the process of separation the proteins can enter into these pores. Only the proteins which are small enough to enter into these pores can only stay in stationary phase. The other big sized proteins will be excluded. So in this chromatography big sized will be eluted to first (will come out of the column first) and the smallest ones last.

Based on the size of the given proteins the elution order will be 1. A, 2. E, 3. C, 4. B and 5. D.

A will be eluted first and D at last.

3) I will choose exclusion chromatography if my protein of interest is B. because its pI is very near to pH 7. That means the protein is only slightly negative charged. So the separation will not be good if use ion exchange technique.