Column Chromatography

Column chromatography is one of many forms of chromatography. Others include paper, thin-layer, gas, and HPLC. Most forms of chromatography use a 2-phase system to separate substances on the basis of some physical-chemical property. One phase is usually a stationary phase. The second phase is usually a mobile phase (often a buffer in biochemistry) that carries the sample components along at different rates of mobility. The separation is based on how well the stationary phase retards the components versus how quickly the mobile phase moves them along. Substances with different properties will thus elute (exit) from the column at different times. Some common types of column chromatography used in biochemistry are gel filtration, ion exchange, and affinity. You will have the opportunity to use one or more of these during your projects. In this exercise, you will use gel filtration chromatography.

1. Gel Filtration (permeation) Chromatography. Gel filtration uses a gel matrix as the stationary phase. The matrix consists of very small porous beads. The large molecules of a sample solution do not get “caught” in the pores of the gel and will travel through the column more rapidly because they can go around the beads. They are said to be “excluded” from the matrix. Smaller molecules that can enter the gel pores must go through the beads, thus taking more time to reach the bottom of the column. Medium-size molecules can enter larger pores, but not small ones. This form is also referred to as “molecular sieve” chromatography, because the components of a sample are separated according to their molecular size (and to a certain extent, molecular shape). The gel matrices are commonly made of crosslinked polysaccharides or polyacrylamide, both of which can be made with varying pore sizes. The information supplied by the manufacturer will state the size of the beads, the approximate size of molecules that will be excluded, and the range of molecular weight range that can be separated. By using gels of different sizes and porosities, one can separate samples that have a large variety of components.
   
   A few useful definitions: Bed volume (V_t) is the total volume inside the column.
   
   Void volume (V₀) is the volume of solution not trapped in the beads.
   
   Internal volume (V_i) is the volume of solution trapped in the beads.
   
   Volume of the gel matrix (V_g):
   V_t = V₀ + V_i + V_g.
   Elution volume (Ve) is the volume necessary to elute a substance from the column


2. Ion Exchange Chromatography. In this type of chromatography, the matrix is covalently linked to anions or cations. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. There are 2 basic matrix types; anion exchangers bind anions in solution and cation exchangers bind cations. As the sample components go through the column, those with the appropriate charge bind and the others are eluted. Proteins have many ionizable groups with different pK values, thus, the charge on the protein will depend on the pH of the buffer used. Thus, one must carefully choose the exchanger and pH of the buffer used for the mobile phase. Once all unbound substances have passed through the column, the bound molecules can be eluted by changing the buffer. One way is to increase the ionic strength (either gradually using a gradient or all at once depending on whether you wish to fractionate the bound components elute them all at once respectively). The anions or cations in the salt will compete with the bound molecules and cause them to dissociate from the matrix. The higher the charge density on the bound molecules, the higher salt concentration will be required to effectively remove them. Another option is to change the pH, and thus the charge, of the proteins. Problem: You use ion exchange chromatography with DEAE cellulose (an anion exchanger) to separate proteins with the following pI values: 3.5, 5.2, 7.1,and 8.5. The proteins are loaded onto the column in a low ionic strength buffer, pH = 7.0. The column is then washed and eluted with a gradient of 0.05–0.50 M NaCl in the same buffer. What is the order of elution of the proteins?


3. Affinity Chromatography. Affinity chromatography utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. For example, the immobilized molecule may be an antibody to some specific protein. When solute containing a mixture of proteins is passed by this molecule, only the specific protein reacts to this antibody, binding it to the stationary phase. This protein is later eluted by changing the ionic strength or pH. Alternatively, an excess of the molecule immobilized on the stationary phase may be used. For example, if the molecule you wish to purify binds glucose, it can be separated from molecules that don’t by using a glucose affinity column (the matrix contains immobilized glucose molecules). Only glucose-binding molecules will bind to this matrix. The bound molecules can be eluted by adding glucose to the elution buffer. This will compete with the matrix-bound glucose for the binding sites on the protein and the proteins (now bound to free glucose) will dissociate from the matrix and elute from the column. This method is gentler, but can only be used in some cases. This elution method is only feasible when the immobilized molecule is small, readily available, and cheap, as is the case with glucose.

Exercise for Gel Filtration Chromatography
Determine the “bed volume” of the glass column by filing the column with water and measuring with a graduate cylinder.

Preparation of the Gel

1. You will use Sephadex G-100 for this experiment. The gel has a fractionation range for proteins of 4000–150,000 daltons. Sephadex is supplied as a dry powder and must be hydrated before use. The amount of water absorbed and the time required depends on the type of gel. Sephadex G-100 takes 3 days at room temperature or 3 hours in a boiling water bath. One gram of dry powder will make about 15–20 mL of gel. Weigh out the powder and add a large amount of water. Gentle stirring may be used, but vigorous stirring will break the beads.


2. When the gel is ready, decant the water. Some of the very fine particles will also be decanted. This is not a problem. In fact, it is good to remove the “fines” as they will pass through bottom support screen of the column or clog the column and slow the flow. Replace the water with phosphate buffered saline (PBS) and stir to equilibrate the gel with the buffer. Allow to settle and decant again.


3. Degas with a gentle vacuum just before use.

Packing the Column

4. Close the outlet of the column. Stir the gel to create a slurry and carefully fill the column without creating areas of different densities. The most even packing will be achieved if you pour all the necessary slurry into the column at once. If necessary, stir the settling gel to prevent layers of gel from forming. Open the outlet and add buffer as the gel packs. Do not let the buffer drop below the top of the gel bed! If it is necessary to add more Sephadex, stir the top of the gel bed before adding more slurry.


5. If layers or air bubbles are still present in the column, invert the column and allow it resettle, doing this as many times as is necessary to obtain a well-packed column.


6. Connect the column to the peristaltic pump and equilibrate the column by eluting 1 bed volume of PBS buffer at a flow rate of 1 mL/min. Collect the eluent in a graduated cylinder.


7. Determine the void volume and check the packing. Blue Dextran is a large polysaccharide (average molar mass is about 2 million daltons). It is excluded from the beads and will be eluted in the void volume. Add Blue Dextran solution to the top of the column and let it run into the gel. Immediately start collecting the eluent in a graduated cylinder. Gently put more buffer over the gel and run the peristaltic pump. Measure the amount of PBS eluted during the time it takes the Blue Dextran fraction to run the length of the column. This volume is the void volume. If your column was evenly packed, the Blue Dextran should run as a horizontal well-defined band through the column.


8. Prepare your protein mixture to 1 mg/mL concentration and add it carefully to the top of the column like you did for the Blue Dextran. For the best resolution, the sample volume should not exceed 1%–2% of the column volume. You will run the following substances: hemoglobin, myoglobin, cytochrome c, and vitamin B₁₂. Vitamin B₁₂ has a molar mass of 1355 D and should be completely included in the Sephadex beads. All of these substances are colored various shades of red or brown, so you should see them as they make their way down the column and in the collected fractions. Run the column at a rate of 0.5 mL/minute. Rates that are too fast will decrease resolution and compress the gel. Start collecting 1-mL fractions and start the chart recorder as soon as you add the sample. The eluent passes through an absorbance detector (280 nm) and will detect the proteins as they elute.


9. Note the elution volume of each substance. A plot of log molar mass versus elution volume should be linear over the useful fractionation range (for roughly spherical proteins).