### Gel Filtration Theory Gel filtration is a simple and reliable chromatographic method for separat — Organic Chemistry Chemistry Question

### Gel Filtration Theory

Gel filtration is a simple and reliable chromatographic method for separating molecules according to their size. Within a fractionation range chosen, molecules are eluted in a decreasing order of their size. Versatility of the method makes it applicable for purification and characterization of biological substances of all classes, including macromolecules not readily fractionated by other techniques.

Some gel forming organic polymers with a 3D network structure (usually referred to as gel filtration media, GFM) possess properties of molecular sieves and can separate molecules according to their size and shape. A chromatography column should be filled with swollen gel and equilibrated with corresponding buffer solution. The separation mechanism is non-adsorptive and independent of the eluent system used, thus being fairly gentle. Liquid inside porous gel beads of GFM is the stationary phase, whereas eluent solution outside the beads is the mobile one.

In a column, all sample molecules can be present in the liquid between the beads. The total volume of such “outside” liquid is referred to as the void volume in gel filtration and is equal to about 30% of the column volume. Sample molecules are partitioned between the eluent (the mobile phase) and the accessible part of bead pores (the stationary phase). This partitioning acts to establish a dynamic equilibrium of sample molecules between the mobile and stationary phases and is driven exclusively by diffusion. The mobile phase transports the sample molecules down the column. The molecules present in the pores are "stationary" and not subjected to transportation. Migration rate of a sample zone depends on the fraction of sample molecules present in the mobile phase. Separation of individual macromolecules can only be achieved in the case of their partial access to the pores of the GFM. Applicable sample volume is restricted to 0.5 – 5% of that of the column, since no concentration effect is active in gel filtration. Flow rate is kept low to avoid peak broadening due to incomplete mass transfer, whereas columns used are long to allow optimum resolution.

### Materials
* Blue dextran (molecular mass, MW = 2 MDa), 4 mg
* Proteins:
- Ovalbumin (MW = 43 kDa), 1.5 mg
- Cytochrome C (MW = 13 kDa), 0.4 mg
- Bovine serum albumin (BSA) (MW = 67 kDa), 2.2 mg
- Chymotrypsinogen (MW = 25 kDa), 1 mg
- Hemoglobin (MW = 64.5 kDa), 1.5 mg
* HCl, 230 cm₃ of aqueous solution, (c = 0.1 mol dm^-3)
* KCl, solid, 22.35 g
* Buffer: Tris (2-Amino^-2-(hydroxymethyl)propane^-1,3-diol; 6.05 g
* GFM: Toyopearl HW^-50 (or HW^-55), fine, 70 cm₃

### Apparatus
70 cm₃ chromatography column; packing reservoir; stand; peristaltic pump; UV-cord connected to plotter; Eppendorf centrifuge; analytical balances; water-jet pump; one 1000 cm₃ measuring cylinder; one 250 cm₃ volumetric flask; one big Buchner funnel with glass filter; one 1000 cm₃ Bunsen flask; one 1000 cm₃ round-bottom flask; one 100 µdm₃ micropipette with tips; one 1000 µdm₃ micropipette with tips; one 2 cm₃ syringe connected to 20 cm tubing; four Eppendorf tubes; one 100 cm₃ measuring cylinder; one 200 cm₃ flask; one 100 cm₃ beaker; big steel spatula; small spatula; glass rod; filter paper.

### Procedure

Step 1. Preparation of buffer solution
To prepare Tris buffer solution (c = 0.2 mol dm–3), dissolve 6.05 g of Tris in 250 cm₃ of distilled water in the 250 cm₃ volumetric flask. Mix 125 cm₃ of the Tris solution and 230 cm₃ of HCl solution (0.1 mol dm–3) in the 1000 cm₃ measuring cylinder. Add distilled water to 800 cm₃. Add 22.35 g of KCl to the Tris^-HCl solution and stir thoroughly until the salt completely dissolves. Add water to 1000 cm₃ (the final concentration of KCl is 0.3 mol dm–3)

Step 2: Preparation of a chromatographic column
Packaging the column is one of the most important stages in chromatography, as it determines the separation quality to a great extent. The column should be packed uniformly, and the upper and lower gel surfaces should be strictly horizontal.
1. Equilibrate gel material to room temperature.
2. Gently shake the bottle to make an even slurry.
3. Pour 70 cm₃ of gel slurry into a beaker and dilute with buffer to 100 cm₃.
4. Stir with a glass rod to make a homogeneous suspension free from aggregates.
5. Add eluent buffer solution to the column to check for leaks, wet the walls of the column and remove air from the bed support. (It is better to fill the column bottom-up using the water-jet pump). Drain buffer leaving about 1 cm above the gel surface. For columns with bottom glass porous filter, a filter paper circle with a diameter equal to the inner column diameter should be placed on the glass filter to prevent from gel leakage from the column.
6. Mount the column vertically and attach the addition packing reservoir firmly to the column. It should be twice shorter than the column.
7. Wash the gel with three portions (of about 100 – 120 cm₃) of Tris-buffer solution on Buchner funnel with glass filter attached to 1000 cm₃ Bunsen flask using water-jet pump. Try not to dry Toyopearl. After each washing disconnect the water-jet pump when the upper gel surface just starts turning dry. Then add next portion of buffer, stir with big steel spatula to make a homogeneous suspension, and subject to suction.
8. Transfer the gel from the funnel into 1000 cm₃ round – bottom flask, add 50 cm₃ of buffer solution and connect the flask to water-jet pump using a connector. Vacuum degassing should proceed for at least 5 min.
9. Re-suspend and pour the gel slurry into the column in one continuous motion. Pouring down a glass rod held against the wall of the column prevents from air bubbles [VISUAL]. Try gel slurry to flow along the column wall.
10. Carefully fill the reservoir to the top with buffer solution, disturbing the gel as little as possible. Connect the reservoir with the peristaltic pump, which should in turn be joined to buffer stock in the 200 cm₃ flask. Turn on the pump and open the column outlet.
11. Buffer solution should be pumped through the column until the gel stops settling. After two bed volumes remove the gel reservoir and insert flow adaptor.

Step 3: Preparation of solutions
Weigh blue dextran and proteins using balance and small spatula. Prepare solution of Blue dextran by dissolving it in 1 cm₃ of Tris-buffer solution in an Eppendorf tube. Prepare two solutions of standard proteins in Eppendorf tubes. The first solution contains Ovalbumin, Cytochrome C, 0.07 cm₃ of blue dextran solution and 0.93 cm₃ of Tris-buffer solution. The second solution contains Bovine serum albumin, Chymotrypsinogen, 0.07 cm₃ of blue dextran solution and 0.93 cm₃ of Tris-buffer solution. Prepare solution of Hemoglobin (unknown protein) in 1 cm₃ of Tris-buffer solution. Centrifuge two solutions with standard proteins and the solution of unknown protein for 5 min.

Step 4: Application of samples
1. Apply sample solutions carefully, trying not to disturb the gel. To make it easier, filter paper circle could be placed at the top of gel (still take into account possible protein absorption on the paper). Remove flow adaptor, disconnect the peristaltic pump and open the column outlet. Let the buffer soak into the gel (the gel surface should be free of buffer but not dry) and close the column outlet. Add sample solution slowly using pipette with wide tip or 2 cm₃ syringe connected to 20 cm tubing, open the column outlet and allow the solution flow inside the gel. Close the column outlet and add buffer solution (about 1 cm₃) slowly and carefully (as during the sample application). Open the column outlet and let the buffer soak in the gel. Repeat the procedure. This allows the sample solution flowing deeper inside the gel and prevents from backward diffusion. Close the column outlet and carefully make a buffer layer with height of about 2 cm over the gel.
2. Connect the peristaltic pump to the column inlet and the UV-cord to the column outlet (the tube length should be as short as possible) and start elution.

Step 5: Column chromatography
1. Carry out calibration of the column in two steps:
A) Apply the first solution of standard proteins containing Blue dextran, Ovalbumin and Cytochrome C to the column. Start elution with the rate of about 1 – 2 cm3/min, collecting the eluate into 100 cm₃ measuring cylinder. The elution process is monitored by following the eluate absorbance at 280 nm, which is registered by the UV-cord. Measure Elution volumes for Blue dextran and proteins using cylinder (record the volumes corresponding to maxima of the eluate absorbance).
Note: in the case of using a spectrophotometer and test-tubes, the procedure should be modified as follows. Collect the eluate in a measuring cylinder up to 25% of the column volume. Then continue collecting the eluate in test-tubes in portions of 1 mL. Determine the eluate absorbance at 280 nm in each test-tube by using a spectrophotometer and record the total volumes corresponding to maxima of the eluate absorbance).
After the three peaks are registered, the column should be washed with the buffer solution until the total elution volume becomes equal to that of the column.
B) Apply the second solution of standard proteins and proceed as described above.
2. Apply the solution of unknown protein. After the peak is registered, stop the peristaltic pump, close column outlet and turn off the UV-cord.