In the Laboratory edited by
Cost-Effective Teacher
Harold H. Harris University of Missouri—St. Louis St. Louis, MO 63121
An Inexpensive Gradient Maker for the Biochemistry Laboratory William H. Flurkey Department of Chemistry, Indiana State University, Terre Haute, IN 47809;
[email protected] Some biochemistry experiments use ion exchange chromatography to demonstrate protein purification techniques. Proteins bound to ion exchangers can be eluted with increasing salt concentrations or a change in the pH. We have used step gradients and linear salt gradients to elute proteins from mini-DEAE ion exchange columns (1–2 mL). Linear salt gradients can be generated from homemade gradient makers. These gradient makers were made from two 25-mL plastic graduated cylinders or from two plastic vials (see diagram). A small-diameter metal rod was heated in a flame and used to “melt” a hole through the plastic near the base of each cylinder at the 1-mL graduated mark. A 2–3-in. piece of flexible tubing (polyethylene, polypropylene, or silicone) slightly larger than the hole diameter was pushed forcibly through the holes to connect the vials or cylinders so that a clamp could be used to prevent solution flow between the two chambers. If the hole is symmetrical, the tubing expands and there is no need for sealing the hole with glue or epoxy. One of the cylinders had another hole melted through it and a longer piece of silicon tubing was forced through this hole. This tubing was connected to the ion-exchange column. For more stability, cylinders or plastic vials can be glued or taped to stiff cardboard or glued to a small rectangle of plastic to facilitate placing a magnetic stirrer under the gradient maker. With practice, gradient makers can be made with no leaks, but if leaks occur they can be sealed with glue. Gradient makers can also be made of chambers with different diameters to create concave or convex gradients. A test of gradient makers made with identical cylinders showed a linear increase in salt
concentrations as measured by changes in conductivity. The total cost for each gradient maker is less than $20 for plastic graduated cylinders and probably a few dollars or less for plastic vials. These gradient makers provide a very inexpensive alternative to commercial gradient makers. To use this apparatus for generating a linear gradient, clamp shut the tubing connecting the two chambers. Pour a predetermined volume of the more concentrated solution into the chamber containing only one tubing outlet (usually the chamber farthest away from the column). This is the “heavy” side. Clamp shut the tubing from the other chamber going to the column. This chamber is the “light” side. Open the clamp between the two chambers and let a little of the more concentrated solution flow into the light-side chamber. As soon as the solution enters the chamber, clamp the connection shut and remove any solution entering the chamber. Then fill the light side chamber with the less concentrated solution and add a small stir bar. Open the clamp on the tubing going to the column and let the less concentrated solution fill this tubing completely. Clamp the tubing and connect it to the column. Remove the less concentrated solution from the light-side chamber until the volumes in the two chambers are at equal levels. Place the chamber on a magnetic stirrer and start the stirrer. Start the flow through the column and then open the connection in the tubing from the light side to the column. Finally, open the connection between the two chambers to start flow from the heavy side to the light side. Collect fractions and monitor the salt concentration using a conductivity meter or by refractive index measurements.
Figure 1. Examples of gradient makers constructed from (left) plastic graduated cylinders and (right) plastic vials. Cylinders are connected by pliable tubing and are shown glued to a solid plastic support.
JChemEd.chem.wisc.edu • Vol. 77 No. 8 August 2000 • Journal of Chemical Education
1041
