In the Laboratory
Survey of Biochemical Separation Techniques
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Melanie R. Nilsson Department of Chemistry, McDaniel College, Westminster, MD 21157;
[email protected] Several different separation methods are used in a modern biochemistry setting but many undergraduate laboratory courses only have time to explore a few of these techniques. This simple laboratory exercise exposes students to a wide variety of separation techniques in one laboratory period and provides a nice complement to a project-oriented program (1, 2) or to courses that also explore specific separation techniques in greater detail. The separation of a mixture of myoglobin and blue dextran is explored using syringe filtration, Centricon, dialysis, gel filtration, and solid-phase extraction. The mixture is green and separation is detected by the visual observation of the red–orange myoglobin and the blue dextran. Visual observation of colored compounds has previously been used for detection (3) because it eliminates the need to synthesize protein derivatives or employ spectroscopic methods (4). Both molecules are non-hazardous, relatively inexpensive, and the large difference in molecular weight (myoglobin 16,951 Da; blue dextran 2,000,000 Da) makes the mixture easy to separate even with less-than-optimal laboratory technique. Experiment Prepare a 1:1 mixture of myoglobin (Sigma, 2 mg兾mL) and blue dextran (Sigma, 2 mg兾mL) in distilled and deionized (DDI) water. Groups of two to four students are provided with 3 mL of the myoglobin兾blue dextran mixture. This will be a sufficient quantity of material for all of the techniques assuming the following volumes per technique: 100 µL each for syringe filtration and solid-phase extraction, 200 µL for Centricon, 1–2 mL for dialysis, and 500 µL for gel filtration. Five workstations are set up in the laboratory with one technique per station. Each station provides a variety of choices so students have to carefully consider how each technique works in selecting an appropriate option. This encourages students to think critically about how separation is achieved and to appreciate differences among the techniques. For most of the techniques, separation is based on molecular size and is achieved by passing the sample through a membrane with a pore size that will retain one of the sample components but allow the smaller molecular weight (MW) component to pass through. The only technique in this exercise that does not rely on MW differences for separation is solid-phase extraction (SPE), which separates materials based on charge, hydrophobicity, or other properties depending on the choice of column packing material. Brief descriptions along with the required materials for each technique are provided below. Additional technique information on gel filtration and dialysis can be found in many textbooks (e.g., ref 5 ).
Syringe Filtration Filtration using a syringe filter is frequently used to remove particulate matter from samples prior to HPLC analy112
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sis, eliminate bacteria from solutions, and to remove excess solid from saturated solutions. This technique is cheap, easy, and the materials are disposable. Unfortunately, only a limited number of filtration pore sizes are available (the smallest pore size is about 20 nm) and the material retained on the filter is not easily recoverable. In this exercise, the sample was forced through a 0.45-µm Millex-HV syringe filter using a 1-mL disposable syringe.
Centricon Centricon centrifugal filtration units (Millipore) are disposable, easy to use, and exceptionally reliable. The sample is placed in the upper chamber of the Centricon unit and forced through the membrane into the lower chamber by centrifugation. Although Centricon units are frequently used to concentrate or desalt protein samples, they can also be used to separate biomolecules of different sizes. Centricons of various molecular weight cut-off (MWCO) sizes were provided; most students selected the 50 kDa or 100 kDa units. The sample, 200–500 µL, was placed in the top chamber and centrifuged at 10,000 rpm for 15 minutes. Note that this will require access to a centrifuge but Microcon units can also be used that only require a microcentrifuge. This experiment could also be extended to include a demonstration of an Amicon stirred cell system (Millipore). Dialysis Dialysis is a common protein separation and desalting technique in which the sample is placed into tubing with a specific pore size, sealed, and then placed into a beaker of solvent. The smaller MW component diffuses out of the tubing into the solvent thereby achieving separation of the two sample components. It is relatively inexpensive, easy, disposable, and an appropriate choice of buffer can prevent protein denaturation during separation. Unfortunately, this method takes a long time (hours to days), generally has to be performed at 4 ⬚C to prevent bacterial growth, and the low MW sample component cannot be easily recovered. Students were provided with an assortment of dialysis tubing from different companies, but the most commonly used tubing was a Spectra兾Por Biotech 10-mm RC membrane with a MWCO of 60,000 Da. The tubing was presoaked for 30 minutes in a 4-L beaker of DDI water, approximately 1 mL of sample was added to the tubing, and the ends of the tubing clamped with Spectra兾Por Universal nylon closures. The tubing was placed into a 4-L beaker of DDI water (with stirring) for the remainder of the laboratory period. Multiple student samples were placed into a single beaker and identified by different colored nylon closures. Gel Filtration Gel filtration is commonly taught in undergraduate laboratories because it is relatively inexpensive and is applicable to a wide range of molecular weight materials (6–8). Gel fil-
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In the Laboratory
tration packing material of different excluded volume sizes (e.g., 15, 25, and 100 kDa) and different phases (solid or premade slurry) was provided in addition to phosphate buffer, pH 7–8 (most buffers will work), and disposable columns of different diameters and widths (e.g., 15 × 80 mm, 7 × 100 mm). Most students selected the Bio-gel P-100 (Biorad), Sephacryl S-100 (Amersham Pharmacia), or Sephadex G-100 (Sigma) gel filtration packing material. The students prepared a slurry, poured the column, applied 100–500 µL of sample, eluted the column with phosphate buffer, and observed for separation. Extensions of this part of the exercise could include determining the gel filtration void volume or the experimental determination of the MW of myoglobin (9).
Solid-Phase Extraction Solid-phase extraction (SPE) has applications in biochemistry in addition to environmental, organic, and medicinal chemistry. Typically this technique is used as a rapid pre-purification step and, in biochemistry, a common application is to desalt protein samples prior to mass spectrometry. This method utilizes a small column on which the sample is adsorbed and components of interest are eluted. Columns are available with a variety of packing materials (e.g., normal phase, reversed phase, ion exchange). This technique is easy to use and molecules can be separated based on a variety of characteristics. This method is designed for small volumes of sample and is relatively expensive compared to other techniques. A variety of 1-mL column types (e.g., LC- 8, LC18, LC-Si) were obtained from Supelco (now a division of Sigma-Aldrich). Regardless of the packing material, the column was prewashed with solution A (10 mM HCl), 100 µL of sample was loaded onto the column followed by elution with 500 µL of solvent A and then 500 µL of solvent B (10 mM HCl, 20% DDI water, 80% acetonitrile). Solvents were simply chosen to be the same as the reversed phase HPLC solvents that had been previously encountered in the course; an extension exercise could involve solvent optimization for each column type. The color of the eluents was noted in addition to the final color of the column packing material. Hazards The solutions for eluting the SPE columns contain hydrochloric acid (toxic, corrosive) and acetonitrile (toxic, flammable). Results and Discussion The laboratory report for this exercise required students to specify for each technique which option they chose, whether or not the technique should have worked in theory, their results, and possible explanations if separation was not achieved. Representative quotes from student laboratory reports are provided below.
Filtration “The separation ability of this apparatus is difficult to predict because it is hard to relate a MW to a metric length measurement. If the filter can separate particles smaller than 2,000,000 Da [then] the myoglobin should filter through and the blue dextran remain in the filter. This did not work bewww.JCE.DivCHED.org
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cause the solution that came out of the filter was all green. This didn’t work because both myoglobin and blue dextran must be smaller than 0.45 µm. This caused them to both pass through the holes on the filter and no separation was achieved.”
Centricon “This should work in theory. The filter is large enough to allow myoglobin to pass through it, and it is small enough to keep the blue dextran from doing the same. Myoglobin should separate into the bottom chamber while blue dextran should remain in the top. The results showed red going through the filter and blue staying above the filter. In order to enhance the level of separation, one could add some solution and centrifuge the apparatus again.” Dialysis “In theory this should work because the MW of myoglobin is 16.9 kDa and the MW of [blue] dextran is 2000 kDa. The myoglobin should diffuse out of the bag, leaving the [blue] dextran. The solution observed inside the dialysis tube was blue in color, changed from the green of the original solution.” [Note: generally, some separation was observed but the results varied depending on the length of time the sample was allowed to dialyze.] “I think that if it was allowed to be in the water longer it would separate to a greater degree.” Gel Filtration “This should work due to the large molecular weight difference. The blue dextran should come off the column with the void volume because it is too large to enter the beads. The myoglobin is able to enter the beads and will thus come off the gel column after the blue dextran. This method ran as predicted with the blue dextran coming off before the myoglobin.” [Note: quality resolution was obtained by all students using the Bio-gel P-100 and Sephacryl S-100, but the Sephadex G-100 did not yield reliable separation.] “The problem was more than likely due to a poorly created slurry mixture.” Solid-Phase Extraction “It is hard to predict whether these different columns will work or not. They separate on MW and hydrophobicity. It is not readily apparent which factors will be the most important, how they will balance out, etc. The LC-Si SepPak did work with the blue dextran coming off first and the myoglobin coming off second.” [Note: in general, some degree of separation was observed with most column types (except LC-NH2).] “In the cases in which it didn’t work, the two materials must not have had a large difference in the property being used to separate them. Also, perhaps the solvent was not optimal for separation, or was not ideal for that particular column type.” Summary This laboratory exercise provides exposure to a broad range of modern biochemical separation techniques in one laboratory period. Students learn the basic principles of five different separation methodologies and get hands-on prac-
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In the Laboratory
tice with each technique. This exercise has been performed for the past three years with comparable results each year. It provides breadth in an otherwise project-oriented program and reinforces separation concepts learned earlier in the semester. Acknowledgments I would like to thank all the students in Biochemistry 3321, fall 2004 who provided permission to use quotes from their laboratory reports in this manuscript. W
Supplemental Material
Instructions for the students and notes for the instructor are available in this issue of JCE Online.
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Literature Cited 1. Stahelin, Robert V.; Forslund, Raymond E.; Wink, Donald J.; Cho, Wonhwa. Biochem. Educ. 2003, 31, 106–112. 2. Craig, Paul A. J. Chem. Educ. 1999, 76, 1130–1135. 3. Gorga, Frank R. J. Chem. Educ. 2000, 77, 264–265. 4. Chakravarthy, Manu; Snyder, Laura; Vanyo, Timothy; Holbrook, Jill; Jakubowski, Henry V. J. Chem. Educ. 1996, 73, 268–272. 5. Boyer, R. Modern Experimental Biochemistry, 3rd ed; Benjamin/ Cummings: New York, 2000. 6. Blumenfeld, Fred; Gardner, James. J. Chem. Educ. 1985, 62, 715–717. 7. Hurlbut, Jeffrey A.; Schonbeck, Niels D. J. Chem. Educ. 1984, 61, 1021–1022. 8. Russo, Salvatore F.; Radcliffe, Angie. J. Chem. Educ. 1991, 68, 168–169. 9. Rowe, H. Alan. J. Chem. Educ. 1993, 70, 415–416.
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