Chapter 4:

Section 4.4
Ion Exchange Chromatography

Background

The term “chromatography” applies to a wide range of separation techniques that are based on the differential interaction of molecules between a moving phase and a stationary phase. The interaction of molecules can be based on any of the protein properties, charge, hydrophobicity, affinity, solubility or molecular weight (size). As the name indicates, column chromatography is performed with the stationary phase packed in a cylindrical container, the column. The mobile phase passes through the column driven by gravity or by a pump. Fractions of the mobile phase are collected as they leave the column. Each fraction can then be assayed to determine the location of the target protein.

Column chromatography is a commonly used technique for protein purification. Column chromatographic methods are generally more complicated to set up than batch methods such as ammonium sulfate precipitation; however, they are capable of high resolution separation and are usually necessary for a successful purification. Like other separation techniques, different column chromatography methods exploit different properties of the protein. Ion exchange chromatography separates molecules based on charge. Gel filtration chromatography separates according to size. Hydrophobic Interaction chromatography separates based on hydrophobic properties of proteins. Affinity chromatography separates based on biological affinity for specific molecules, such as antigen-antibody interaction, metal binding capability, hormone receptor interactions, etc. Although the focus of this chapter is on ion exchange chromatography since that is used in the current purification, other types of chromatography will be discussed in lecture.

Figure 4.5 Elution of a sample mixture from a chromatographic column a) A sample containing 3 different molecules is loaded onto a column. As the mobile phase carries the sample components through the column, their rate of movement is affected by the relative tendency to adsorb to the stationary phase, slowing their elution. B. The three molecules are graphed according to their elution times.

Ion Exchange Chromatography: Ion exchange chromatography (IEC) is a high resolution technique for separating proteins according to their charge. It is the most commonly used chromatographic method of protein separation due to its ease of use and scale up capabilities. Large volumes of protein solution can be applied to ion exchange columns, often much greater than the volume of the column itself. Therefore, IEC is both a high resolution and a high capacity method.

In IEC, the packing material (resin) of the column has many charged molecules that are securely bound to it by covalent bonds. Manufacturers make and sell many different types of packing materials. Some have negatively charged groups and some have positively charged groups. When the packing material is suspended in buffer, the charged groups become loosely associated with ions of the opposite charge, since the buffer contains NaCl that dissociates into Na+ and Cl-. The loosely bound ions are called mobile counter-ions. As you can see in Figure XY, positively charged beads associate with, and therefore “exchange” with negatively charged counter ions. Since negatively charged molecules are also called “anions”, this type of column resin is called anion exchange. Negatively charged beads associate with and exchange positive counter-ions (cations) and these types are called cation exchangers.

Since proteins are charged molecules, proteins in the mixture will interact with the column resin depending on the distribution of charged molecules on the surface of the protein, displacing mobile counter ions that are bound to the resin. The way that a protein interacts with the packing material depends on its overall charge and on the distribution of that charge over the protein surface. The net charge on a given protein will depend on the composition of amino acids in the protein and on the pH of the buffering solution. The charge distribution will depend on how the charges are distributed on the folded protein.

Proteins carry both positive and negative charges and the overall, net charge is dependent on the pH. The isoelectric point ( pI) is the pH at which a particular protein is overall electrically neutral -- in other words, the number of positive charges is equal to the number of negative charges. Below its pI, a protein has more positively charged amino acids and therefore an overall positive charge. Conversely, above its pI, a protein has more negatively charged amino acids and an overall negative charge. Above its pI, the protein will bind to anion exchangers; below its pI it will bind to cation exchangers. At its pI, a protein will not bind to either a cationic or an anionic exchanger. In principal, one could use either a cation or an anion exchanger to bind to the target protein by selecting the appropriate pH. For example, the pI of ß-galactosidase is 4.6. At a pH below 4.6, ß-galactosidase carries a net positive charge and would bind to a cation exchanger (Figure 2.5) However, we know that the pH stability range for ß-galactosidase is pH 6-8. Therefore, it would not be prudent to use a buffer at pH 4.6. For this purification strategy, an anion exchange resin, DEAE Sepharose, will be used with a buffer of pH 7.2

The above explanation makes it sound like protein behavior can be completely predicted based on pI; in practice, separation conditions are often determined by trial and error. It is also wise to allow a 1 pH point “margin of error” on either side of the pI when determining whether to use anion or cation exchange.

Equilibration: The first step in the process is to “equilibrate” the column. Since the packing material of the column contains charged molecules, these will interact with the ions in the buffer. Just as the pH of a solution changes when you add charged molecules to it, the pH of the column-buffer environment will change as buffer is added to the column. This process should be stabilized before your protein mixture is applied to the column. When the pH of the buffer coming out of the column is the same as the pH going into the column, the column is said to be equilibrated.

In our procedure, we will first apply our sample protein mixture to the IEC column using buffer conditions that allow ß-galactosidase to bind to the packing material. After washing the column thoroughly to remove any unbound molecules, we will change the incoming buffer by connecting a gradient maker that will slowly and progressively increase the ionic strength of the buffer, adding back the mobile counter-ion in the buffer at increasingly higher concentrations (gradient from 0.2 to 0.5 M NaCl). As we do this, ions in the buffer begin to exchange with and displace proteins that had adsorbed to the beads. The displaced proteins then flow out of the bottom of the column. At a certain point, the concentration of ions in the buffer is such that ß-galactosidase is no longer adsorbed and it elutes from the column.

As the liquid exits the column, the A₂₈₀ is read by a UV monitor and these readings give an indication of where protein is found in the eluent. All the material that elutes from the column is collected in a numbered series of test tubes (1 ml each). These 1 ml aliquots are called “fractions”. Certain fractions will contain our ß-galactosidase but most of the fractions will not. After assays, we can combine the fractions containing ß-galactosidase. By removing those fractions that do not contain our target protein, we will significantly improve the purity of our ß-galactosidase preparation.

Chromatography is often performed using instrumentation including a pump to force liquid through the column, a UV detector with chart recorder to automatically measure and record the absorbance at 280 , and a fraction collector to automatically move the tubes after a volume of liquid has been collected. A typical configuration is shown in Figure XY. Although convenient and expedient, such instrumentation is not required and the procedures can be performed adequately (but slower) by manually collecting fractions, reading their absorbance on a UV spectrophotometer and plotting the results on graph paper.

Procedure 4.4 Ion Exchange Chromatography

Summary of IEC Procedure: It is helpful to break the column chromatography phase into several steps:

• Set up: Assemble the column, ring stand and any accessory equipment (pumps, chart recorders, fraction collectors, etc.) and perform necessary calibration steps.
• Equilibration: Prepare column resin, pour and equilibrate the column.
• Loading sample
• Washing unbound
• Elution (Gradient)
• Regeneration of column (Optional)

Description of the Overall Process: The column must first be equilibrated with the buffer that will be used to run the column. Equilibration of the column ensures that the column environment is stable at the correct pH. After the column is completely equilibrated, the protein sample is loaded on to the column. Next, the column is washed thoroughly to remove all unbound proteins. SAVE all column flow through until you verify that there is no enzyme activity present. Finally, after the column has been washed extensively with 0.2 M NTM to remove unbound protein (and the A₂₈₀ of the flow-through is less than 0.1), the gradient maker is connected to start the elution process. At this point, slow the flow rate and collect 1 ml fractions. Store the fractions on ice as soon as they are collected.

Procedure 4.4A: Equipment Set up:

• Mount the column vertically on a suitable ring stand and determine the volume of your column. If you are planning to use 10 ml of ion exchange resin, you can fill your column with 10 ml of water and mark it at the 10 ml level. Empty the column.
• If you are using an automated column chromatography set up you will need to connect all the equipment in sequence according to the diagram.
• Calibrate the pump according to the procedure in the manual.
• Calibrate the fraction collector – How many drops are in one ml?
• Test that the UV monitor and the chart recorder are communicating.
• You will need to zero the UV monitor on 0.2M NTM buffer.
• The chart recorder is a relative scale; how will you know what it means?
• Attach a short piece of Tygon tubing a few inches long to the outlet of the column. Replace the air in the tubing with buffer by filling the bottom of the column with buffer and running a few ml of 0.2 M NTM buffer through the tubing. Close the column outlet after all the air is removed from the tubing.

Check list:

Some settings: Pump speed should be no more than 1 ml a minute with the column hooked up. If you are flushing the tubing with the column disconnected, higher speeds may be used.

Chart recorder speed should be set at 5 mm/minute. (Arbitrary, but it helps if students are consistant.)

UV RANGE should be set to 1.0 AUFS

Chart recorder input should be set at 1 V

Note: While running the columns, it is important to mark fraction numbers on the chart recorder so you can later associate the chart peaks with specific fractions. Use the event marker to mark when one fraction starts and then stops.

Procedure 4.4B: Equilibration

• In preparation for ion exchange chromatography (IEC), calculate the amount of ion exchange resin you need, based on the amount of protein you have. The resin we will be using is DEAE Sepharose CL-6B. Check the product literature for the binding capacity of this resin. Binding capacity is reported in milli-equilavents of charge and in mg of protein for a specific protein like bovine serum albumin (BSA) or hemoglobin. Since the number of charged groups varies from protein to protein, the amount of protein that the column resin can “hold” varies depending on the charges present on the proteins in your sample.
  
  Although it is possible to determine the amount of ß-galactosidase that would bind experimentally by loading more and more on the column until ß-galactosidase activity is present in the flow through, we will adopt a best guess approach. Since you don’t know exactly what percentage of the proteins in your sample will bind to the resin, assume that you need resin capacity for l/3 to l/2 of all the protein in your sample in milligrams.
• DEAE Sepharose CL-6B comes pre-swollen in an ethanol/water mixture that must be removed and replaced with 0.2 M NTM buffer. An aliquot of resin has been placed in a tube for you, so the resin will have settled, leaving the ethanol at the top. Pour off excess ethanol and add some 0.2 NTM buffer with 10X Tris. (We have found that the addition of 10X TRIS speeds up the equilibration process.) The resin should be about 75% of the total volume of the final slurry. Check the pH with pH paper.
• The final slurry of ion exchanger and buffer should be fairly thick but it should not so thick as to retain air bubbles. Usually about 75% settled gel is suitable. Optional: Put the slurry into a flask and degas it.
• With the column outlet turned off, pour the slurry into the column in an even, continuous motion. A glass rod can be used to prevent splashes. Let the slurry settle.
• STOP POINT: When all the suspension has settled, you may store the column at 4°C.
• Open the column outlet and allow the slurry to pack (settle) as the buffer drains out of the column. Do NOT let the column run dry.
• Begin equilibrating the column with 0.2 M NTM with 10X TRIS. If the suspension does not fill the column, buffer should be carefully added until the level is 1 cm from the top of the column before connecting the headpiece. Connect the tubing attached to the headpiece to a beaker containing 0.2 M NTM with 10X TRIS. (If you are using a pump, connect the tubing to the buffer reservoir and turn the pump on at 1 ml/min.)
• Let equilibrating buffer flow through the column at a rate of approximately 1 ml/min. Washing the column will go faster or slower depending on the hydrostatic pressure, which can be regulated by raising or lowering the height of the beaker containing the buffer. (If a pump is available, this step is unnecessary.) Some matrices are more resistant to pressure than others. Check the product literature for recommended flow rates.
• After washing the column for about 30-60 minutes, check to see if the pH of the buffer going into the column is the same as the pH coming out of the column, by pipetting a drop of solution onto pH paper. If the two pH readings are identical, change the column buffer to 0.2M NTM with 1X Tris (NOT 10x Tris). This is very important. If you have any doubt about whether or not your column has equilibrated completely, it is better to keep running buffer through it. The column is ready when all the 10X Tris has been washed out using 2-3 column volumes of 1x Tris. You can do other tasks while it is equilibrating such as making a detailed flow chart for column loading and sample elution, measuring the flow rate under different conditions and deciding on the methods you want to use to collect fractions, etc. When we are equilibrating the column, the counter ion for the DEAE resin will be one of the ions found in the buffer (What ion is that?) (What would happen if you apply your protein before the column is fully equilibrated?)
  
  STOP POINT: After the column is equilibrated, it should be stored at 4°C unless you are planning to apply your protein sample right away.

Procedure 4.4C: Loading the column:

Note: If at all possible, Procedure 4.4c, 4.4d and 4.4e should be done in the same day, without stop points. You should allow at least 3 hours (four is better).

OVERVIEW: In this procedure, you are applying your sample to the column and therefore allowing any proteins that can bind to the column to do so. Then, you are washing the column thoroughly with buffer to remove any unbound proteins BEFORE YOU START THE GRADIENT. After you have verified that all the unbound protein has been removed from the column, then you may begin the elution process by connecting the gradient.

• Remember to add dithiothreitol (DTT) to your buffers before starting. Apply the sample to the column with a flow rate no higher than 1ml per minute. In the interests of time (or if you are using a manual set-up without the pump), it is helpful to close off the column outlet, remove excess buffer from the top of the column and pipette the sample into the column. Then connect the buffer reservoir to the top of the column and open up the column outlet. If you are using a pump, you simply need to stop the pump and then start it again.

Procedure 4.4D: Washing the column:

• After the sample has been loaded on the column, connect the buffer reservoir containing 0.2 M NTM buffer and start running the column, again at a flow rate no greater than 1 ml/minute. Collect the effluent (flow-through) in a beaker or test tube and save it. Check the A₂₈₀ regularly. Continue to run buffer through the column until the A₂₈₀ is less than 0.1 with 0.2M NTM as the blank. (Check this on the DU 64 spec, not just the chart recorder.) Save this effluent. It contains any protein that did not stick to the column and will need to be checked for enzyme activity.
• Stop the column when the A₂₈₀ is less than 0.1 and prepare to connect the gradient.

Procedure 4.4E: Elution Phase

Note: Elution speed should be 0.5-0.8 ml/minute. Your resolution will be much better, i.e., you will get tighter peaks, if you maintain a slow flow rate during the elution phase. Monitor the flow rate and adjust it by carefully raising or lowering your gradient apparatus, or changing the pump speed.

• Prepare your gradient maker. If a commercial gradient maker is available, check it to make sure it is working and close off the outlet between the two cells. After verification, add 30 mls of 0.2 M NTM to one side and 30 mls of 0.5M NTM to the other side. Add a small stir bar to the side with 0.2M NTM. The reservoir that is directly hooked up to the column is the reservoir that has the 0.2M NTM buffer.

Home made gradient makers: If a commercial gradient maker is not available, a home-made gradient maker can be made with two small beakers and a glass U tube. The U tube is a piece of glass tubing a few inches long that has been bent into the shape of a U. Place 30 ml of 0.2 M NTM in one beaker and 30 ml of 0.5 M NTM in the other. Fill the U tube with 0.2 M NTM buffer. Then carefully invert the filled U-tube so that it straddles the two beakers, being very careful not to introduce any air bubbles into the tube. The tube should reach close to the bottom of each of the two beakers. Place a small stir bar in the 0.2 M NTM beaker and place the beaker setup on top of a stir plate. The gradient maker set-up is depicted below in Figure 4.2.

Figure 4.6 A gradient maker made with a U-tube.

• Assemble and label enough small test tubes to collect 1 ml fractions from the column(80+ tubes). (If you are collecting them manually, it is helpful to mark the 1 ml mark with a Sharpie.) Your fractions will need to be kept on ice once they are collected.
• To start running your column, place the tubing connected to the column headpiece into the gradient maker by securing the open end of the tubing in the bottom of the beaker containing the stir bar. Use a syringe to get the buffer flowing if needed and connect the tubing to the column. Make sure there is enough buffer on top of the resin bed so that the drops coming from the gradient maker do not disturb the resin. To ensure a smooth gradient make sure the stirrer is turning fast enough to mix the solution but not so fast as to make bubbles or be unstable. When you open the column outlet, the column will start running. The gradient will form automatically in the beaker containing 0.2 M NTM. As the buffer flows out of the low salt beaker, buffer from the high salt beaker will be siphoned into the low salt beaker through the U-tube, increasing ionic strength in a linear fashion.
• Store all fractions at 4° C until time to assay. Make sure all tubes are properly labeled
  

Procedure 4.4F:

Regeneration: When the gradient has been run completely, if the column is to be reused, you need to clean the column with 2-3 column volumes of 1 M NTM buffer until the A₂₈₀ of the effluent drops below 0.05. Then run 2-3 column volumes of 0.2M NTM buffer to re-equilibrate the column. Store the column at 4° C.

Contact Us:

Lisa Seidman
lseidman@matcmadison.edu
(608) 246-6204

Jeanette Mowery
jmowery@matcmadison.edu
(608) 243-4307