Size-Exclusion Chromatography Separating Large Molecules from Small Daniel J. McLoughlin Xavier University, Cincinnati, OH 45207 Most discussions on separating molecules by chromatomaohic orocedures revolve around the attraction of the koiecules for two separate phases. The first of these phases is commonly called the stationary phase or matrix, and the second is called the mobile phase or solvent. Renews concerning the classroom instruction of these techniques generally have focused on partition chromatography or ion exchange chromatography These techniques rely on polar interactions between the two phases and the molecules to be separated. They generally use paper or thin layer surfaces ( I ) . Separation by Size Chromatographic techniques that separate molecules by size are rarely discussed. Exceptions are procedures that are difiicult to carrv out in the classroom due to several factors: the size of the columns, the specialized chemicals involved. and the leneth of time reauired for the ex~eriment (214). I n biochemistry, methods that allow the separation of molecules by size provide valuable information about the molecular weights and shapes of biopolymers and about their interactions with smaller molecules (5).Chromatographic methods that separate species by size have several names, including gel filtration chromatography, gel permeation chromatoeraohv. molecular sieve chromatoeraohv The mecha&m o"f and size-exclus~on'c~omatography. seoaration is discussed in most introductorv biochemistrv
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Theory of the Technique In size-exclusion chmmatomaohv the stationarv *ohase consists of a column of porous beads whose pores sizes are, on an average. cornoarable to the size of the molecules to be separatei. ' ~ a r &molecules fit into fewer pores than small molecules. Thus, the larger molecules spend less time on the stationary phase (within the pores on the gel) and more time in the mobile phase (the solution moving past the pores). A very large molecule may not fit into any pore of the stationary phase, and thus will emerge from the column rapidly Molecules of intermediate size will spend a small amount of time in the pores, and they will emerge later than the larger molecules. Small molecules will spend a relatively large amount of time in the pores, and they will be retained on the column for a considerably greater period of time. Thus, a mixture of molecules of various sizes will be separated on the column, with the largest molecules emerging first and the smallest molecules coming last. This paper will address a simple method of demonstrating size-exclusion chromatography in the classroom. A simple prelaboratory demonstration model is included. The experimental procedure demonstrates -
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the differences in size among a set of molecules the formation of a complex between a macromolede and a small molecule the separation of this mmplex on a chromatographic column
Demonstration Materials Hardware wire screen: 1 ft x 3 ft; U2-in. square openings 'Piece of stiff cardboard: 1 ft x 3 ft 'Styrofoam balls, four each with the following diameters: 1-112in., 1 in., U4 in. Procedure Simple filtration is easy to demonstrate as shown in Figure 1 by placing the wire screen on a desk top and then placing the Styrofoam balls on top ofthe screen. Lifting the screen will separate the balls of the largest two sizes from the smallest balls, which will pass through the screen. Size-exclusion (or gel filtration) chromatography can be demonstrated by first placing a piece of cardboard on the desk, and then placing the screen on top of the cardboard. The balls are then placed on the screen along the length of one of the shortest sides, as shown in Figure 2a. Slowly lift the end of the screen and cardboard backing together so that the balls roll over the screen as they move toward the table as shown in Figure 2b. As the unit is lifted, the largest balls roll down first, followed by the medium-size balls, then by the smallest balls. The hallswill separate by size with the largest balls spending less time within the openings of the screen, while the smallest balls spend more time and thus are retained longer. (You may wish to bend upward the last 3 in. of the wire screen to catch the balls as they roll down the screen.) Experimental Materials (for 10 student groups) 40 disposable glass Pasteur pipets (Fisher X13-676-6A) - 2 0 mL of hydrated Sephadex G-50 80-100 mesh beads (Sigma G50-80)
'Soluble starch solution Iodine solution 1can of red soda or foe3 colon 60 test tubes
Figure 1. Set-upfor demonstration of simple titration Volume 69 Number 12 December 1992
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of wire. This filter paper will serve as a bed for the column packing. Using a second dropper, add a slurry of the hydrated Sephadex beads to the prepared chromatography column until the column is 112 to 213 full of packed Sephadex beads. If too full, remove some of the packing with a pipet or dropper. The column should now be placed in a test tube or small glass so that it remains vertical during the course of the experiment. Safety and Disposal .Caution: Iodine solutions stain some materials and may temporarily stain the skin. Students should be told to use
caution while handling this material. The Se~hadex can be reused. Save and store in saturated NaCl solntion to inhibit bacterial growth. All excess'chemicals in this ex~erimentcan be dis~osedbv flushine down the sink. Experimental Procedure Examination of the Iodine Solution
Fig~re2. Above: The set-~p for dernonslrarlng size-excl~sionchromatography. Below. Tne dernonslrallon n progress, showmg part c es lravelmg o!nerently according to s ze. Sephadex Gel
Prepare a slurry of hydrated Sephadex gel by adding about 5 g of the Sephadex to approximately 100 mL of water and allow to swell for 1 h. This will make enough packing for 50-100 individual columns. Other gel sizes and types may also be used. (Sephadex G-75 or G-100 may be substituted, as well as the polyacylamide Bio-Gel P-30 or P-60 Gels (Bio-Rad Laboratories).) The chromatographic gels can also be reused. Store them in saturated NaCl solution to inhibit bacterial growth. Starch Solution
Prepare a saturated starch solution by suspending several mams of soluble starch in 100 mL of water. h u t the suspension to boiling. Stir and allow the suspension to cool to room temperature. By decanting, remove the upper clear solution for use in the experiment. Store the solution in a dropper bottle.
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Iodine Solution
Tincture of iodine solution from the local pharmacy may be used. A more saturated solution will give better visual results. I t can be prepared by adding approximately 250 mg of solid iodine to 100 mL of 50% alcohol aqueous solution. Store the solution in a dropper bottle. Column Preparation
The chromatography column is prepared in a disposable glass dropper pipet, commonly called a Pasteur Pipet. A description ofthe use of this type of pipet for column chromatography has appeared previously in this Journal (8). The column should be prepared by loosely blocking the bottom opening of the disposable dropper with a small piece of filter paper. The paper should be loosely lodged in the opening by pushing it into the pipet with a small piece 994
Journal of Chemical Education
Add 1 drop of iodine solution to the column, and constantly wash the column by adding drops of water to the top. At no time should more than a few drops of solution remain on the top of the column. Collect the drops into a small test tube that contains several drops of starch solution. The student should count the number of drops that fall from the column. Start counting with the first drop after adding the iodine solution to the top of the column. S t o ~ counting when the color of the sol;tion in the collection test tube c h a m s . The color of the band as it moves down the column should also be noted, as well a s the color change when the iodine band leaves the end of the column. Examination of the Starch Solution
Add 1 drop of starch solution to the column, and wnstantly wash the column by adding drops of water to the top. At no time should more than a few drops of solution remain on the top of the column. Collect the drops into a small test tube containing several drops of iodine solution. Count the number of drops that fall from the column. Start counting with the first drop after adding the starch solution to the top of the wlumn. Stop counting when the color of the solution in the collection test tube changes. The color (or lack of color) of the band as it moves down the column should be noted, as well as the color change when the starch band leaves the end of the column. Adding Iodine Solution, Followed by the Starch Solution
Add 1 drop of iodine solution to the column, and constantly wash the column by adding drops of water to the top. At no time should more than a few drops of solution remain on the top of the column. Collect the drops into an empty test tube. When the yellow-brown iodine band is about half way down the column, add 1drop of starch solution to the top of the column. Continue to add water to the top of the column. The color (or lack of color) of the band as it moves down the column should be noted by the student. Separation of Starch-Iodine Solution
In a small test tube, mix together two drops of starch solution with two drops of iodine solution. Add 1 drop of this starch-iodine solution to the column, and constantly wash the column by adding drops of water to the top. At no time should more than a few drops of solution remain on the top of the column. .,
Collect the drops into an empty test tube and count the number of d r o ~ that s fall from the column. Start counting with the first &op after adding the starch-iodine solution to the top ofthe column. Stop counting when color develops in the empty collection test tube. The wlor (or lackof color) of the band a s it moves down the column should be noted by the student. Discussion These procedures offer several advantages for the instruction of chromatographic techniques. T h e experiment is inexpensive
It is easily performed in less than 1h. (This time can be cut in half when only the iodine solution followed by the starch solution and the separation of the starch-iodine solution is attempted. T h e materials ore nontoxic n n d easy to acqwre. -3lodifirnrmns in the ~ n c ~ . d uilreensil~ re mndr to o h t a n the separation of hlwd protein (a collection of large macmmolecules) from small molecules, such as those found in food colors or colored soda. The procedure gives the beginning stuilent a n appreciation for column chromatography, a s opposed to the thin layer or paper chromatography techniques most commonly used in the classroom. The ~roceduresallow the instructor to acquaint the student with two basic facts:
that molecules have a range of sizes that techniques are available to separate them by size This procedure also demonstrates the interaction of a small molecule (iodine) with a large macromolecule (starch), and it separates the complex that is formed between these molecules. Interested teachers can pursue questions concerning equilibria between these molecules and analogies between the starch-iodine complex and protein-substrate interactions in biochemistry. Acknowledgment This demonstration and procedure were developed while working on a project funded with grants from the Ohio Chemical Council, The Cincinnati Section of the American Chemical Society, The Ohio State Board of Regents, The National Science Foundation, and The U.S. Department of Education. Their assistance and the assistance of the K-12 teachers in the Miami University (Ohio) "Partners for Terrific Science" program, who tested this procedure in their classrooms, is gratefully acknowledged. Literature Cited 1. Mickey, C. D.J Chem. Ed-. 1881,58,997-1003. 2. McGuinness, E.T. J C h e m . E d u . ls?S,50,826830. 3. Melins, P J C k m . E d u . 1971,48,76&766 4. Hurlburt, J.A;Schonbezk,N. D. J Chem. Edue. 1984,61,1021-1022. 5. Stelhagen, E. In Methaia in E ~ y m o l o g y Denkcher, ; M., Ed.; 1890,182,317328. 6. Stryer. L.Blochamistry 3 r d d . ; W. H. Freeman:NY, 1988:4748. 7. Boyer, R. F Modern E r p ~ n m n f o BlochemUfry:Addiaon-Wwwley; l p 87-96. 8. Mewaldt, W : Rodolph, D.: Sady, M. J. Chem. Educ 1985,62,53&.531.
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