7 of 12 Duplication of this document in conjunction with une of accompanyling re

Question

7 of 12 Duplication of this document in conjunction with une of accompanyling reagones is permitsed for casroom/laboratory use orly This documen, or ary part muy not be reproduced or dstrbuted for other purpose what the wraen consere of EDVOTEK.be. Copyryk 1995. 1997·1999, 1999-2000.2007. 2011. 2012 EDOTEK. inc-all ngto reserved EVT 201202 24AM The Biotechnology Education Company 81-800-EDVOTEK www.edvotek.com 10 EDVO-Kit # 243 Ion Exchange Chromatography Study Questions Answer the following study questions in your laboratory notebook or on a separate worksheet. 1. What is the basis for the separation of different compounds by ion ex- change? 2. How can molecules with the same charge at varying amounts be sepa- rated by chromatography? 3. Why are celluloses often used as supports to separate large biologically active proteins? What do you think would happen if 0.5M K- acetate were used first to elute the sample? Why? 4. S. Why is it important to prepare a standard curve for each spectropho- tometer?

Explanation / Answer

1. Basis of ion exchange chromatography:-

  Protein separation based on NET charge; much like affinity chromatography, but is less specific .

Method:---Stationary phases (resins) have a ligand with different charges at a defined pH - positive or negative

Cation-exchange chromatography:
2) negatively charged resin (negative charges on the surface); therefore, there is an exchange of cations
- generally bound to Na+ or K+

Anion-exchange chromatography:
3) positively charged resin (positive charges on the surface); therefore, there is an exchange of anions
- generally bound to Cl-

4) Exchange resin is bound to counterions
5) Mixture of proteins is loaded on the column and allowed to flow through
6) Proteins that have a net charge opposite to that of the exchanger stick to the column, exchanging places w/ the bound counterions
7) Proteins that have no net charge or same charge as the exchanger elute
8) After unwanted proteins are eluted, eluent is changed either to a buffer that has a pH that removes the charge on the bound proteins or to one w/ a higher salt concentration
9) Higher salt concentration will outcompete the bound proteins for the limited binding space on the column
10) Desired molecules are eluted.

2. Answer:-  Increasing or decreasing the pH which is in effect causing more molecules of a compound to be deprotonated or protonated will cause molecules to separate.

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