CHROMATOGRAPHY Lab 3 INTRODUCTION

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1 CHROMATOGRAPHY Lab 3 contributed by Mike Brundage CAUTIONS AND PITFALLS Your column is packed with Sephedex beads and is very fragile. Do not move or bump the column while running the experiment.. Be sure that the column does not run dry or the column bed will collapse. Wipe cuvettes clean of fingerprints and smudges before placing them into the colorimeter. OBJECTIVES After completing this lab you should be able to: 1. Separate small molecules from large molecules using size exclusion chromatography. 2. Determine the order of molecular separation by using spectrophotometry on the samples collected. 3. Explain the theory and usefulness of spectrophotometry and chromatography in molecular biology. INTRODUCTION Chromatographic techniques are among the most useful methods available for the separation or isolation of organic compounds from a mixture. The most widely used chromatographic techniques include column chromatography, paper chromatography, and thin-layer chromatography(tlc). These techniques are frequently used to isolate proteins, enzymes, lipids, hormones, plant growth substances, pigments, and other naturally occurring organic materials. More sophisticated chromatography, involving molecular separation by instrumentation, is available in the form of gas liquid chromatography(glc) and high performance liquid chromatography(hplc). Using a chromatography column to separate specific molecules is relatively easy but does require that you understand the theory behind the technique. All forms of chromatography employ the same general principle: a mixture of solutes in a moving phase pass over a selectively absorbing matrix, the stationary phase. Separation occurs because the solutes have different affinities for the stationary and moving phases. Solutes that have greater affinity for the moving phase will spend more time in the moving phase and therefore will move along faster than solutes that spend more time in the stationary phase. In this experiment we will employ size exclusion chromatography (sometimes referred to as molecular filtration or gel permeation chromatography). This chromatographic technique separates molecules

2 according to size. Unlike other methods of chromatography, charge and solubility have no effect on the separation. In biochemical laboratories this technique is commonly used during the purification of high molecular weight molecules such as proteins, polysaccharides and nucleic acids from low molecular weight molecules. Flow Direction Faster Migration Rate Slower Migration Rate Time Figure 3.1 Separation by differences in migration rate. The stationary phase, or matrix inside the column, is composed of sephadex beads. Each tiny bead contains hundreds of microscopic pores and channels. Filling the column with the matrix is referred to as "packing". The packed matrix is called the "bed". It is very important not to allow the bed to run dry during any part of the experiment because pockets of air in the bed will cause an uneven flow of molecules through the column. In a clean, well-packed column, size excludes large molecules from entering the microscopic pores in the sephadex beads. Thus molecules with higher molecular weight move more rapidly through the matrix. Smaller molecules tend to spend more time flowing into and out of the beads thereby taking a less direct route through the column bed. As the mixture passes through the column, the smaller molecules get "detoured" inside the porous beads and filter down more slowly. The elution buffer, or mobile phase, flows through the matrix and out the column carrying the dissolved solutes with it. In today's experiment you will be collecting eluted samples in 2 ml fractions. You will then analyze each sample using a spectrophotometer to detect the presence of protein molecules, and an indicator to detect the smaller inorganic compound.

3 This laboratory activity demonstrates the separation of hemoglobin (M.W. 64,500) and ammonium sulfate (NH 4 ) 2 SO 4 using size exclusion chromatography. The separation occurs as a mixture of the molecules is applied to the top of the column and is washed down the column by the elution buffer. The compounds in the sample move down the column at different rates, depending on their size and how much time they spend "detoured" inside the beads. See Pre-lab Explanation Figure 3.1 Example of a Column Chromatography Apparatus

4 Calculate the molecular weight for ammonium sulfate and predict which molecule will pass more quickly through the matrix or gel bed. Explain your reasoning MATERIALS Hb/ammonium sulfate solution (10 mg Hb/ml) Tris buffer (25 mm, ph 8.0) column (25cm x 10mm) plastic syringe or funnel rubber tubing 2 pinch clamps Sephadex (G-25 course grade) dropper bottle of barium chloride (BaCl 2 ) ml beakers ml beaker for Sephadex 1 50 ml beaker for Hb solution Spectrophotometer (Spec 20) 12 cuvettes test tube rack 2 1-ml pipette + pipette pump 1 10-ml pipette + pipette pump stirring rod nonwater soluble marker 100 ml distilled water PROCEDURE Read through the procedures and develop a flow diagram for the experiment to insure that you understand the procedures. PART 1. SETTING UP THE COLUMN (Part 1 may be completed for you.) 1. Filling the column bed. Remove the syringe and tubing and open the top of the column. Be sure the frit in the bottom of the column is clean. 2. Stir the sephadex and slowly fill the column. Allow the liquid to run through the column by opening the bottom pinch clamp. 3. Continue stirring and adding the sephadex beads as you fill the column to within 2 cm from the top. Be sure to keep the matrix covered with liquid at all times. 4. Reattach the syringe and close the column. Be sure that the syringe is attached to the highest point on the ring stand, that the tubing runs in a straight line (no bends) and that all connections are tight.

5 5. Fill the syringe to the brim and test the flow rate by opening both pinch clamps. If the buffer flow is slow, tap the column near the valve to release air bubbles from the frit. Once you are satisfied that your column is packed and flowing properly, fill the entire syringe with fluid to wash the column bed one final time. Close the top and bottom pinch clamps when the buffer has almost reached the bottom of the syringe. Caution: no buffer should be leaking from the bottom tube - if so the column will run dry. 6. Turn on the Spec 20 and allow it to warm up before beginning Part 2. Set the wavelength to 450 nm. This setting is the maximum absorbance value for hemoglobin. We will use the Spec 20 to determine the presence of Hb. PART 2. SEPARATING THE MOLECULES 1. Before you actually load your samples confirm that there is no residual hemoglobin or ammonium sulfate left in the column. Collect a sample of elution buffer and test it as described in Part 3. If you detect the presence of hemoglobin or ammonium sulfate, wash your column with about 20 ml of buffer and test another sample. Repeat this procedure until the column is clean. 2. Prepare to draw off twenty 2.0 ml samples. Use a one ml pipette to transfer 2.0 ml of distilled water into one cuvette and mark the surface level with a marker. Discard the water and place a small mark on all remaining tubes at the same height. You are now ready to separate the molecules. 3. Allow the elution buffer to run out of the column until it is near the top of the bed.. Caution: Do not let the buffer run below the top edge of the matrix. 4. Carefully add 2.0 to 3.0 ml of the Hb/ammonium sulfate solution to the surface of the column bed. Add the mixture by placing your pipette against the wall of the column so as not to disturb the bed.. 5. Allow the Hb/Ammonium sulfate solution to enter the bed by opening the bottom clamp to remove a 1.5 to 2.0 ml fraction from the column. This loads the samples in a tight band in the column for better separation. Remember, at no point during the separation should you allow the fluid level to fall below the column bed. 6. Gently add buffer to fill the column and attach the upper tubing. Again fill the funnel with buffer. You are now ready to collect samples. 7. Open the clamps and allow the buffer to flow slowly through the column. The buffer solution will wash the Hb/ammonium sulfate solution fairly quickly through the column. 8. Immediately begin to collect a total of twenty 2.0 ml fractions or samples. Continue adding elution buffer to the top of the column to avoid allowing the bed running dry. PART 3. ANALYZING THE SAMPLES 1. Remove the level mark from all test tubes using an alcohol swab and a Kimwipe. Smudges and marks interfere with light transmittance. 2. Add 1.0 ml of distilled water to each tube so that the fluid level is above the beam of light produced by the colorimeter. 3. Calibrate the Spec 20 colorimeter using a cuvette filled with 2.0 ml of buffer and 1.0 ml of distilled water. 4. Insert each of the ten sample tubes in sequence into the Spec 20 and record percent transmittance in Table 3.1. Be sure to follow proper spectrophotometer protocol by using a tissue to remove

6 smudges or fingerprints before inserting each cuvette into the chamber. 5. Once you have detected the presence of hemoglobin using the Spec 20 you are ready to search for the presence of ammonium sulfate using barium chloride as an indicator. Add 5 drops of 200 mm BaCl 2 and roll each tube between the palms of your hands. 6. Record the amount of precipitate formed in Table 3.1 using the following symbols: no ppt.(0), small amount ppt. (+), large amount ppt. (+++). PART 4. WASHING THE COLUMN 1. Run 20 ml of elution buffer to wash out all residual ammonium sulfate. 2. Collect 2.0 ml near the end of the wash and test for the presence of Hb and ammonium sulfate.

7 Table 3.1 Analysis of Column Chromatography Samples Sample %T ppt. Sample %T ppt. HINTS FOR WRITING THE LAB REPORT. INTRODUCTION: Discuss the theory behind separation by chromatography How is size exclusion chromatography different from other chromatographic techniques? What is the molecular weight of the two molecules being separated? State your hypothesis and prediction. MATERIALS AND METHODS: Flow diagram RESULTS: Table 3.1 Describe trends Which type of molecule moved more rapidly through the matrix? DISCUSSION: Did your results support your prediction? Discuss evidence to support this conclusion? Explain how pore size influences the separation of molecules in size exclusion chromatography. What would happen if the pores in each bead were larger? What would happen if the pores in each bead were smaller?