In the Laboratory edited by
Harold H. Harris
Design for a Simple and Inexpensive Cylinder-within-a-Cylinder Gradient Maker for the Undergraduate Biochemistry Laboratory
University of Missouri- St. Louis St. Louis, Missouri 63121, United States University of Missouri-St. Louis St. Louis, MO 63121
Paul A. Sims,* Gary B. O'Mealey, Nabeel A. Khan, and Chelsea M. Larabee Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, United States *
[email protected] The principles and practices of ion-exchange chromatography are important topics in the undergraduate biochemistry laboratory. Part of the theory typically includes a discussion of the use of gradients of gradually increasing salt concentration to elute a compound of interest from an ion-exchange column. In practice, one often uses a gradient maker to generate the gradient of increasing salt concentration, and for many undergraduate labs, the cost of several gradient makers (as required in relatively large labs) may be prohibitive. A previous article in this Journal (1) described the construction of an inexpensive gradient maker that consisted of two graduated cylinders (or plastic vials) placed side-by-side and connected by flexible tubing.1 Although this design works well, we recognized that equipment or space limitations might preclude some labs from adopting it. For example, if one uses the side-by-side design with a small magnetic stirrer, one has to center one of the cylinders on the stirrer and build a support for the other cylinder that is the same height as the stirrer. Such an arrangement may be difficult to attain for some labs, and a more compact design might be more feasible. Thus, the design we chose and describe uses a cylinder-within-a-cylinder arrangement, which is similar to that found in the commercially available Pace gradient maker.2 To minimize the cost of construction, we used readily available and relatively inexpensive materials such as plastic bottles, glass tubes, epoxy resin, an opensided tubing clamp, cork stoppers, and vinyl tubing. Construction of the Gradient Maker We constructed the gradient maker in the following manner. First, we used heavy-duty scissors to cut the tops off two plastic bottles of differing diameters (Figure 1A). Next, we drilled a small hole (∼0.6 cm diameter) near the bottom of the smaller bottle, and then we epoxied the smaller bottle to the bottom of the larger bottle (Figure 1B). Specifically, we used a 500 mL bottle (Nalgene, mfr. no. 2002 0016) for the inner chamber and a 1000 mL bottle (Vitlab, mfr. no. 100689) for the outer chamber. The diameters of these bottles nearly satisfied the equation pffiffiffi ð1Þ do di 2 where do is the diameter of the outer chamber and di is the diameter of the inner chamber. We used this equation as a guide in selecting bottles because the cylinder-within-a-cylinder design requires that the inner and outer chambers of the gradient maker hold similar volumes at the same height. Although eq 1 neglects the thickness of the inner bottle and the resulting 508
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Figure 1. Design scheme for the cylinder-within-a-cylinder gradient maker: (A) Cutting the tops off the two plastic bottles. (B) Creating a hole toward the bottom of the inner chamber and epoxying this chamber to the inside bottom of the outer chamber. (C) Assembling the components to complete the design.
height difference when the inner bottle is epoxied to the inside of the larger bottle, we found that the above two chambers came reasonably close to satisfying the volume and height requirements. For example, when we stoppered the hole at the bottom of the inner chamber and filled the inner and outer chambers each with 400 mL of water, the level of water in the inner chamber was higher than that of the outer chamber. To establish equal fluid levels, we removed 50 mL of water from the inner chamber. This difference in volume did not affect the linearity of the gradient. We connected the inner chamber to the environment (e.g., ion-exchange column) by using a length of vinyl tubing attached to two glass tubes (Figure 1C). We also used a shorter length of vinyl tubing to cover the end of the glass tube that contacted the bottom of the inner chamber (Figure 1C). This vinyl “boot” protected the glass tube in the inner chamber from the spinning magnetic stir bar (Figure 1C). Finally, we used a small piece of tape to secure the outer glass tube to the outside of the outer chamber (not shown).
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Vol. 88 No. 4 April 2011 pubs.acs.org/jchemeduc r 2011 American Chemical Society and Division of Chemical Education, Inc. 10.1021/ed1008427 Published on Web 01/24/2011
In the Laboratory
Operation of the Gradient Maker We operated the gradient maker in the following manner. First, we stoppered the hole near the bottom of the inner chamber with a size 00 cork stopper. Then, we filled the inner and outer chambers (in that order) to the same height with the two different elution buffers. The buffer that filled the inner chamber (buffer A) had the lower concentration of salt whereas the buffer that filled the outer chamber (buffer B) had the higher concentration of salt. After we filled the two chambers, we used tongs to remove the cork stopper that connected the inner and outer chambers.3 Next, we placed the apparatus on a magnetic stirrer such that the outlet was below the gradient maker, and then we used a syringe to create negative pressure at the outlet and thus establish flow from the inner chamber to the environment. Finally, we used a tubing clamp (Figure 1C) to regulate flow from the gradient maker to the environment. As buffer A flowed out of the inner chamber, buffer B flowed into the inner chamber where the slowly spinning stir bar gently mixed the two buffers. Thus, the fluid in the inner chamber gradually became more concentrated, which conductivity tests on the eluent (collected as fractions) confirmed (r2 > 0.99). Some buffer (∼40 mL) remained in the outer chamber at the end of each run because the bottom of the inner chamber rests slightly above the bottom of the outer chamber. This observation likely explains why the gradient remains linear despite the initial difference in volume between the two chambers. Summary The design of the gradient maker is economical, compact, and easy to construct and to operate. We estimate that the cost of one such unit is approximately $15; thus, 15-20 units can be built for the price of one commercial unit. These potential
r 2011 American Chemical Society and Division of Chemical Education, Inc.
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savings may help make the inclusion and use of gradient makers in the undergraduate laboratory feasible. Acknowledgment The authors gratefully acknowledge Jim Cornell for suggesting the use of the vinyl boots and Katie Branscum and Paul Cook for helpful comments. The authors also are grateful to Rachel Mason for allowing us to use her conductivity meter and to Allison Fleshman and Matt Petroswsky for help in setting up and using the conductivity meter. Notes 1. An additional report (2) described the construction of a simple and inexpensive gradient maker for use in density gradient centrifugation; this particular design would not work for ionexchange chromatography but may be of interest to people who use density gradient centrifugation. 2. At present, the primary vendors for the Pace gradient maker appear to be bioWORLD (3) and Laboratory Essentials (4). 3. We also tried an alternative design that used a length of vinyl tubing and two glass tubes to connect the outer and inner chambers. Although this design produced linear gradients, it proved more difficult to establish flow between the outer and inner chambers without getting an air bubble in the connector. The air bubble restricted flow between the two chambers and allowed the inner chamber to “outrun” the outer chamber such that the two chambers did not empty at the same rate.
Literature Cited 1. Flurkey, W. F. J. Chem. Educ. 2000, 77, 1041. 2. Tessier, H. Sep. Sci. 1972, 7, 303–305. 3. bioWorld Home Page. http://www.bio-world.com/index.php (accessed Dec 2010). 4. Laboratory Essentials Home Page. http://www.laboratoryessentials. com/index.php (accessed Dec 2010).
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Vol. 88 No. 4 April 2011
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