Question

Why is it important to avoid air bubbles when you pack columns for column chromatography? If you accidently introduce air bubbles into the columns, what are the strategies you could take to remove the air bubbles? When running the column, is it okay to run the liquid below the column level? Why?

Answer 1

Solution:

Column chromatography purification method often use gravity-flow to process sample solutions through a resin bed that is packed into a column. In column chromatography the efficient separation of different components in a mixture depends on efficient and uniform contact of solution with the solid support or resin bed. If air bubbles form in the resin bed, they limit contact with the resin and impede flow through the column. Therefore, it is very important to pack the column as evenly as possible to avoid the formation of air bubbles, cracks etc. because cracks, air bubbles, and channels would lead to a poor separation.

If bubbles do form in a column resin bed by improper resin packing or handling, the following methods can be used to correct the problem.

(A) Removing Small Bubbles from Below the Bottom Filter Disc

1. Always remove the column’s top cap before the bottom cap and replace the bottom cap before the top cap.

2. By placing the thumb over the sealed column top and invert the column until the bubble is in the exit tip.

3. Applying gentle pressure with the thumb to the diaphragm film on the column top until the trapped air is expelled from the tip.

4. Return the column to an upright position and remove the diaphragm film.

(B) Stirring Method for Removing Air Bubbles from Column Resin Bed

1. Capping the column bottom and by adding water or solvent so that the resin bed is covered by a height of 1-2 cm of solution.

2. By gently stirring the resin matrix with a clean weighing spatula or pasteur pipette, until all portions of the resin are loosely suspended in the solution.

3. With the bottom cap still in place, allow the column to stand for several minutes until the resin mostly settles (packs) itself back into a bed.

(C) Centrifugation Method for Removing Air Bubbles from Columns

1. First, cap the column bottom and add water or solvent so that the resin bed is covered by a height of 1-2 cm of solution.

2. Place the entire bottom-capped column in a centrifuge tube and centrifuge for 10 minutes at 1,000 × g.

When running the column, the liquid should always be above the column level to avoid formation of air bubbles inside the column bed.