Of course as always you must also prepare your procedure.

Reading
Good website about ion exchange -http://www.proteinchemist.com/tutorial/iec.html
For those of you with a deeper interest (warning big PDF ahead) go here
Robyt and White-Introduction to chromatography Chapter 4, pay special attention to  4.12.3

Questions (completed in notebook prior to class):
1.  What type of ion exchanger is CM Sephadex?  What is the structure of the ion exchanging moiety?  What is its approximate pKa?   Based on its structure and pKa explain how it serves as an ion exchanger.
2.   Predict the order of elution of the three analytes based on a gel filtration mechanism of purification.
3.  Predict the order of elution of the three analytes based on an ion exchange mechanism.
4.  Why are two different concentrations of buffer being employed in this experiment.
5.  Explain the statement (i.e. why?):  Anion exchange columns are usually eluted with a decreasing pH gradient and cation exchange columns are often developed using an increasing pH gradient.
6.  What is band broadening?  What factors can be controlled to minimize band broadening?

Introduction:

In this experiment three compounds with varying molecular weights will be separated by a combination of two different mechanisms.  Blue dextran, cytochrome C, and DNP glycine have molecular weights of  >500,000, ~12,400, and 241, respectively.  Blue dextran, as the name implies, is blue; it is nonionic.  Cytochrome C is red and has pI of 10.7; below this pH it is a net cation.  DNP glycine is yellow and above pH 3 is an anion.  Since the sizes (based upon molar mass) of these molecules differ greatly, gel filtration (size exclusion) chromatography can be utilized to separate them.  In addition, the varying ionic behavior provides a mechanism for purification by ion exchange.  In this experiment you will separate the compounds under varying eluting conditions.  CM Sephadex G-50 will be used as a stationery phase to separate the compounds at pH 6, based upon both  their differing sizes and charges.  Two columns will loaded with the mixtures and each eluted with a different buffer  (0.1 M KOAc, pH 6, 1.0 M KOAC, pH 6 ) and the results compared.  From the order of elution, the predominant mechanism of separation can be determined.  Since the compounds are colored, the separation is easy to follow.

Chromatographic Mechanisms

Gel filtration - Gel filtration is also called size exclusion.  Gel filtration is probably the easiest to understand.  The gel contains pores of differing sizes.  Depending on the size, and to a certain extent the shape, of the analyte it will be retained or excluded from the pores to varying extents.  Inclusion of the analyte in the pore leads to retention on the column.  Therefore, large molecules that interact poorly with the resin elute quickly while smaller molecules are retained.  Size exclusion resins are often sold with a particular molecular weight cut off.  This means that the resin is capable of excluding molecules larger than this value.  Gel filtration is usually done with an isocratic mobile phase.

Ion exchange - Ion exchange chromatography separates analytes based upon electrostatic interactions.  A cation exchange resin will bind cations, displacing the positively charged counter ion associated with the resin (provided by the buffer salt).  A cation exchanger is therefore, negatively charged.  Ion exchange can be accomplished by exploitation of the different charges of the analytes at varying pHs.  Changing the pH of the resin, or alternatively, the ionic strength of the resin, will alter the electrostatic interaction between the resin and the analyte.  For example, suppose an analyte contained a carboxyl group with pKa of 4.2.  Above pH 4.2, the carboxyl group will be a carboxylate, negatively charged.  This analyte will be attracted to an anion exchange resin at pH values above 4.2.  Below 4.2, the compound will be eluted.  A mixture containing this compound could be effectively loaded (bound to column) at pH 6.  Rinsing with the pH 6 buffer will remove nonionic or cationic compounds, while the carboxylate remains bound to the resin.  Changing the buffer to pH 3 will then protonate the carboxylate, producing the carboxyl group which is neutral.  The neutral compound will then had no affinity for the resin and be washed away and can be collected.  This is an example of using a pH gradient to elute and ion exchange column.  Anion exchange columns are usually eluted with a decreasing pH gradient and cation exchange columns are often developed using an increasing pH gradient.  However, use of a pH gradient, although theoretically simple, can often be problematic.  the absorbed molecules on the resin will affect the local and overall pH of the mobile phase.  In addition, the various eluents will alter the buffer pH.  For example, a decrease in pH of 0.5 in the bulk solvent introduced by buffer change may correspond to as much as a decrease in pH of 1.5 units in the micro environment of the analyte.  Remember this is a log scale so a single pH unit is a factor of 10 in concentration.  Most often, changes in ionic strength are employed rather than changes in pH for ion exchange chromatography.  As the concentration of counter ion in the mobile phase increases the counter ions interact and compete with the analyte molecules bound to the resin.  The higher the concentration of salt, the weaker binding of the analyte to the resin because of this competition.  If the concentration of the salt in the mobile phase is continually increased, the last band (last eluted = strongly bound) of analytes moving down the column will always experience a higher salt concentration than the first band (first eluted = weakly bound).  This effect provides compact bands or zones of eluting analytes, minimizing local effects which cause the poor resolution often seen with pH gradients.

1. Label two columns appropriately (to distinguish the mobile phases)
2. Prepare two columns.  Mark each (sharpie) to give a resin volume of approximately 5-6 cm³.  Column sizes will vary by group.  However, it is essential that you utilize two equal sized columns/volumes resin for your comparison.
3. Add the resin in the buffer slurry.  Resin is in high salt buffer   The column should be no shorter than 8 cm.   Allow the resin to settle to the appropriate volume.  Elute excess buffer but do not allow the column to run dry.   Add appropriate buffer as necessary to change the ionic strength.
4. Load each column with 0.2 mL of the analyte solution.  Be careful not to disturb the resin bed.  Adsorb the analyte to the resin.
5. Elute each column with the appropriate buffer.  Be careful not to disturb the bed or allow the resin to run dry.  Use an adequate volume of buffer to elute each analyte and collect the separate fractions.  Once the first colored analyte has eluted from the low salt column, change the buffer to the high salt column to finish the elution.
6. Compare the results from the two columns.

Questions:
1.  Explain the degree of migration of each colored component in each column
2.  Explain the order of elution in terms of physical properties of each analyte and the nature of the resin.
3.  Predict and justify the results you would obtain had you used the same column equilibrated with 0.1 M HCl.
4.  Why was the buffer changed from low salt to high salt in the low salt column?
5.  If you have an aqueous solution containing alanine, fructose, glycogen, ribose-5-phosphate and tRNA, how would you separate them?
6. Discuss the differences in separation that would result from use of a linear vs. stepwise gradient.

Report:  See the syllabus for the report type.
Address the following:
The degree of migration of each colored component in each column
The predominant mechanism of separation for each column.
The order of elution in terms of physical properties of each analyte and the nature of the resin.