In the Laboratory
Size Exclusion Chromatography: An Experiment for High School and Community College Chemistry and Biotechnology Laboratory Programs Linda S. Brunauer* Department of Chemistry, Santa Clara University, Santa Clara, CA 95053; *
[email protected] Kathryn K. Davis William C. Overfelt High School, San Jose, CA 95122
Chromatography refers to the separation of chemical substances by differential partitioning into two phases. One phase, the mobile phase, is generally a liquid or gas while the other phase, the stationary phase, is most commonly a solid. The various components in a mixture are separated by differences in their tendency to associate with the two phases. The technique may be used preparatively or analytically and is an exceedingly powerful tool in the chemist’s toolkit. Students in high school science programs and college introductory natural science courses often gain exposure to chromatographic separations in the form of experiments employing thin-layer or paper chromatography (1–2). While extremely useful, these techniques are generally not appropriate for the separation of macromolecules such as proteins. In this article we describe a simple experiment that utilizes a common column chromatographic technique, gel filtration, to allow students to explore the nature of variations in the size of biological molecules. Background Gel filtration (also known as gel permeation or size exclusion chromatography) was first introduced in 1959 (3) as a technique for desalting and separating macromolecules under mild conditions. The technique separates molecules on the basis of molecular size or, perhaps more properly, their Stokes radius. The solid stationary phase consists of a relatively inert resin prepared from cross-linked polymers, most commonly dextrans (e.g., Sephadex), polyacrylamide (e.g., Bio-Gel), or a combination (e.g., Sephacryl). Gel filtration resin particles are porous, hollow beads and are available in a wide range of pore sizes. Columns packed with resin may be roughly divided into two volumes: the volume outside of the beads, known as the void space or void volume (Vo), and the volume inside of the beads, known as the included volume (Vi). Samples are applied to the top of the resin in a narrow band and are then eluted through the column with an appropriate solvent. Large molecules that are unable to enter the beads are excluded from the interior of the beads and elute from the column first at a volume equal to Vo. Small molecules that are able to equilibrate in the total volume of liquid elute at a volume equal to the sum of Vo and Vi. Molecules of intermediate size elute between these two extremes and may be considered to be partially included. Thus the elution position of different molecules (Ve) will be dependent on their molecular sizes. Selection of an appropriate bead pore size is therefore often a critical element in designing an effective separation. Other parameters,
such as the dimensions of the column and the flow rate, may also directly affect the quality of the separation. In general, long and narrow columns are more effective than short and wide columns; slow flow rates usually give results that are superior to those obtained with fast elution rates (4). In the current experiment, students prepare a short gel filtration column and use it to separate a standard mixture of three highly colored molecules of vastly different molar mass: blue dextran (blue; approximately 2,000,000 g∙mol), bovine hemoglobin (reddish-brown; approximately 64,000 g∙mol), and vitamin B12 (pink; approximately 1400 g∙mol). Students subsequently apply and elute a sample of the enzyme wheat germ acid phosphatase (WGAP) on the same column, using a quick and simple microscale well plate enzyme assay to determine the elution position of the enzyme in column eluant. The relative molar mass of the enzyme is estimated by comparison of its elution volume to the elution profile of the molecules in the standard mixture. The use of gel filtration to estimate the molar mass of native proteins by comparison to the elution volume of proteins of known molar mass has been widely used in biochemical analysis and has been featured in some college biochemistry laboratory manuals (5–7). The current study provides a protocol by which instructors may perform successful gel filtration analyses in the high school and community college environments where access to standard biochemical laboratory equipment may be limited and where student laboratory time may be restricted to periods of as little as one hour. Because the experiment features the use of visibly colored molar mass markers and enzyme reaction products, analysis of elution position may be done via direct visual examination rather than quantitative spectrophotometric determination (8). The experiment provides students with experience in column chromatography, enzyme assay, microscale manipulations, and graphical analysis of data. Materials and Methods Materials: Sephacryl S-300-HR, bovine hemoglobin, vitamin B12, blue dextran, wheat germ acid phosphatase (WGAP), tris(hydroxymethyl)aminomethane hydrochloride (Trizma HCl), Triton X-100, p-nitrophenyl phosphate (PNPP), potassium hydroxide, and sodium acetate were purchased from Sigma Chemical Company (St. Louis, MO). All procedures were carried out at ambient temperature. Preparation of the gel filtration column: Sephacryl S-300HR resin was equilibrated in Tris buffer [50.0 mM Trizma HCl,
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 85 No. 5 May 2008 • Journal of Chemical Education
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In the Laboratory 3.0
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Fraction Number Figure 1. Gel filtration analysis of colored molar mass standards. Dark symbols: absorbance at 618 nm (blue dextran); light symbols: absorbance at 549 nm (hemoglobin, vitamin B12). The order of elution is blue dextran (fractions 7–10), hemoglobin (fractions 11–15), and vitamin B12 (fractions 17–21).
Figure 2. Gel filtration analysis of WGAP. Aliquots of each column fraction were incubated with PNPP substrate at pH 5.2 as described in the text. After a 10 minute incubation at ambient temperature the reaction was terminated by the addition of KOH and the relative enzyme activity determined by the absorbance of the p-nitrophenolate product at 405 nm.
0.01% (v∙v) Triton X-100, pH 7.0] and suspended in buffer to yield a slurry that was approximately 75% settled resin. A 10 mL portion of the suspension was poured into a 0.7 × 20 cm chromatography column fitted with a two-way stopcock and approximately 35 cm of fine-bore flexible tubing. The resin was allowed to settle and was used the next day. Preparation of the colored molar mass standards: Stock solutions of blue dextran (25 mg∙mL), bovine hemoglobin (50 mg∙mL), and vitamin B12 (10 mg∙mL) were prepared in Tris buffer. Each sample was centrifuged for several minutes in a microcentrifuge at 5,000 × g to remove any undissolved material; alternatively, filtration through a small quantity of glass wool in a glass Pasteur pipet may be used to remove undissolved material if a microcentrifuge is not available. A mixture of these components was made by combining 75.0 μL each of blue dextran and hemoglobin plus 37.5 μL of vitamin B12. Separation of the colored molar mass standards: Excess buffer was carefully removed from the top of the gel filtration column. A 175 μL sample of the standard mixture was applied to the top of the gel filtration resin using a micropipet. Care was taken not to disturb the resin bed or to allow the resin top to become dry. The stopcock was opened and the sample allowed to enter the resin. Once all of the sample had entered the column, the stopcock was closed, and a 150–200 μL sample of buffer was applied to the top of the column. The stopcock was opened and the liquid allowed to enter the resin. Once all of the buffer had entered the resin, the stopcock was closed; this step served to ensure that residual sample clinging to the glass wall of the column entered the resin prior to the addition of the bulk of the eluting buffer. Buffer was next added atop the resin bed until the reservoir was about ¾ full. The stopcock was opened and fractions of approximately 0.50 mL were collected until all traces of color were gone from the column and the tubing; the buffer in the reservoir was replenished as needed to maintain an
adequate supply of buffer. Approximately 12–13 mL of buffer were required to completely elute all of the colored solutes from the column. The presence of color (the particular color as well as the relative color intensity) was evaluated for each fraction either by visual examination or by measurement in a microplate reader at suitable wavelengths (549 nm for hemoglobin and vitamin B12; 618 nm for blue dextran). Gel filtration of WGAP: A 175 μL sample of WGAP (10 mg∙mL in Tris buffer) was carefully applied to the gel filtration column and eluted as described for the colored molecule standard mixture. Enzyme assay: The relative activity of WGAP in eluant fractions was determined by measuring the extent to which column fractions could dephosphorylate the substrate p-nitrophenyl phosphate (PNPP). A 50 μL aliquot of each column fraction was transferred to separate wells in a 96-well microplate. At 20 second intervals, 200 μL of enzyme substrate (5.00 mM PNPP dissolved in 0.100 M CH3COONa buffer, pH 5.2) was added to each well. After a 10 minute incubation the reaction was terminated by the addition of 50 μL of 1.00 M KOH. This serves to both denature the enzyme, thus halting the reaction, and to increase the pH of the reaction mixture, converting the product p-nitrophenol to the highly colored p-nitrophenolate anion. The presence of yellow color was evaluated for each fraction either by visual examination or by measurement in a microplate reader at 405 nm (9).
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Hazards Appropriate personal protective equipment (e.g., chemical splash goggles, gloves) should be worn throughout the experiment. KOH is a strong base and highly caustic. Sephacryl S-300-HR, Trizma HCl, PNPP, and Triton X-100 are irritants. Vitamin B12 is toxic.
Journal of Chemical Education • Vol. 85 No. 5 May 2008 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
Results and Discussion
Acknowledgments
The three colored molar mass standards separate effectively; each component elutes as a separate peak that is easily determined by visual examination or by microplate analysis (Figure 1). The use of standards that are readily detectable by visual examination avoids the necessity of generating colored compounds by reaction with labeling agents such as fluorescein isothiocyanate (10). Previous articles have reported chromatographic separation of a variety of colored compounds with varying resolution (11–13). The resolving power of the resin chosen for the current study, Sephacryl S-300-HR, is sufficient to separate blue dextran (representing the void volume), vitamin B12 (representing the included volume), and proteins of intermediate molar mass such as hemoglobin (64,000 g∙mol). Analysis of WGAP enzyme activity indicates that the enzyme (55,000 g∙mol; ref 14) has an elution volume similar to that of hemoglobin (Figure 2) as predicted by the similarities in their molar masses. The data presented in Figures 1 and 2 were collected using a Molecular Devices SpectroMax microplate reader (Sunnyvale, CA) however qualitatively similar results were also obtained by simple visual examination of column fractions and reaction products (data not shown). The potential use of this procedure is not limited to separation of molecules that possess significant visible color (11–13). Enzymes that may not be detected by direct visual examination may instead be detected in column eluant by use of an appropriate enzyme assay that generates a visibly colored product. For example, PNPP or similar compounds (such as p-nitrophenyl acetate) may be used to detect a variety of hydrolases; such techniques allow students to gain experience in performing timed microscale colorimetric assays. Alternatively, detection of protein may be made directly using a suitable microscale protein assay such as the biruet assay (8) or a dye-binding assay (15). The protocol was incorporated into the regular curriculum of an advanced placement biology class (14 students) and required four hours of laboratory time, spread across several days, to complete. Upon completing the experiment the class was informally surveyed to evaluate the experiment. High school students who traveled to a near-by university to quantify their data using a microplate reader indicated that they enjoyed the opportunity to gain exposure to the college environment. In general, students indicated a high satisfaction level associated with the experiment; several reported that they especially enjoyed the visual appeal of watching the standards elute from the column. The primary complaint was the quantity of time required to elute the compounds. This may be partially avoided by developing the column at a greater flow rate, sacrificing a measure of the quality of the separation to reduce the tedium of monitoring the columns for a prolonged period of time.
The authors would like to thank Terry O. Tran for assistance in testing the gel filtration protocol and George Doeltz for preparation of figures used in the student handout. We also wish to thank James Chen and Ram Subramaniam for their helpful comments on the article. KKD was supported by a summer IISME (Industry Initiatives for Science and Math Education) fellowship from Santa Clara University. Literature Cited 1. Quach, Hao T.; Steeper, Robert L.; Griffin, G. William. J. Chem. Educ. 2004, 81, 385–387. 2. JCE Editorial Staff. J. Chem. Educ. 2000, 77, 176A–176B. 3. Porath, J.; Flodin, P. Nature 1959, 183, 1657–1659. 4. Smith, Charles A.; Villaescusa, F. Warren. J. Chem. Educ. 2003, 80, 1023–1025. 5. Farrell, Shawn O.; Ranallo, Ryan T. Experiments in Biochemistry: A Hands-On Approach, 2nd ed.; Thomson Brooks/Cole: Belmont, CA, 2006; pp 181–209. 6. Dryer, Robert L.; Lata, Gene F. Experimental Biochemistry, 1st ed.; Oxford University Press: New York, 1989; pp 364–367. 7. Ninfa, Alexander J.; Ballou, David P. Fundamental Laboratory Approaches for Biochemistry and Biotechnology, 1st ed.; Fitzgerald Science Press: Bethesda, MD, 1998; p 117. 8. Rowe, H. Alan. J. Chem. Educ. 1993, 70, 415–416. 9. Minch, Michael. J. Experiments in Biochemistry: Projects and Procedures; Prentice Hall: Englewood Cliffs, NJ, 1989; pp 169–176. 10. Chakravarthy, Manu; Snyder, Laura; Vanyo, Timothy; Holbrook, Jill; Jakubowski, Henry V. J. Chem. Educ. 1996, 73, 268–272. 11. Hurlbut, Jeffrey A.; Schonbeck, Niels D. J. Chem. Educ. 1984, 61, 1021–1022. 12. Blumenfeld, Fred; Gardner, James. J. Chem. Educ. 1985, 62, 715–717. 13. Gorga, Frank R. J. Chem. Educ. 2000, 77, 264–265. 14. Verjee, Z. Eur. J. Biochem. 1969, 9, 439–444. 15. Bradford, M. M. Anal. Biochem. 1976, 72, 248–254.
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2008/May/abs683.html Abstract and keywords Full text (PDF) with links to cited URLs and JCE articles Supplement
Student handouts
Instructor notes including detailed information on reagent and equipment preparations, CAS and Sigma Chemical Company catalog numbers, an approximate cost analysis for reagents and equipment, and suggestions for variations on the experiment
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 85 No. 5 May 2008 • Journal of Chemical Education
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