I intended this exercise to lead into an analysis of hydrolysis products of starch by amylases but I've never gotten that far. Students at least saw the separation of acetone from blue dextran on the basis of their different molecular weights.

The GFC matrix they used was Sepharose CL-6B, but Sephacryl S-300, with similar separation properties, would work as well; both are nearly infinitely reusable. In fact, almost any gel filtration media would give clean separation of the blue dextran and acetone used as samples. Students poured a 75% slurry of CL-4B into inexpensive 1 x 20 cm columns with plastic end fittings (glass tubes plugged with glass wool work nearly as well) and equilibrated the columns with 25 mL of 0.1 M NaCl. When the bed was drained, they pipetted 0.5 mL of a solution of 0.4 mg/mL blue dextran and 1% acetone onto the top and let it drain in. Then they eluted with thirty 0.5 mL portions of 0.1 M NaCl, collecting each separately. Finally, they determined the absorbance at 280 nm of each fraction and plotted the results. From the absorbance peaks, they estimated the void and included volumes.

Since I had them on hand, students used Eppendorf pipets to add the 0.5 mL portions to the column. Burets would have been more accurate, if a little unwieldy. This would have been easier if I had fraction collectors and pumps. Even though they didn't get to use these columns for anything else, they still saw the principle of GFC.

Revised 6/14/07