Why should the bottom cap of the chromatography column be removed before the chromatography process?

The reason the bottom cap of the chromatography column should be removed before the chromatography process primarily relates to the need for the liquid buffer to flow freely through the column. By removing the cap, it's possible to allow the buffer to drain out of the column while also ensuring that the flow path is clear for both the buffer and the sample application.

During chromatography, the absence of any obstruction at the column's bottom ensures that the buffer can properly equilibrate the packed media, facilitating effective interaction between the sample and the stationary phase. This step is crucial for achieving optimal results in terms of separation and purification of components, such as proteins like GFP, within the chromatography medium.

While there are other considerations, such as managing pressure and ensuring beads are well suspended, the primary function of removing the bottom cap directly concerns the drainage and flowing requirements of the buffer setup necessary for successful chromatography.