Adsorption Chromatography - Preparing the Column

[00:00:02]now that you've prepared the media the next step is to prepare the column all column components must be in perfect condition inspect the glass components paying particular attention to the ends columns with chipped ends should be replaced the column and Reservoir should be rinsed in 20% ethanol to remove all traces of Grease inspect the Nets or filters discard any that have holes or otherwise not intact make sure to use a filter or net with a mesh size that is compatible with your media the tubing must be securely fastened inside the endpiece and adapter this important point is commonly overlooked remove all traces of air in the bottom end piece by forcing 20% ethanol up through the net using a syringe connected to the tubing air bubbles will disrupted flow ethanol is better than pure water it breaks surface tension and therefore does not leave a hydrophobic film to entrap air leave a meniscus of ethanol over the net attach the stop plug insert the end piece into the column and tighten securely next add distilled water to a height of about 2 cm in the bottom of the column attach the packing connector to the column making sure it fits snugly attach the reservoir to the packing connector again tightening securely a reservoir is essential to make a well-packed column Mount the assembly to a ring stand level the column if not level the packed column May produce broad or crooked bands