Biochemical Techniques and Instrumentation

2.2.4. Technique

1. Gel preparation

Packing gel materials are swollen by soaking them in suitable buffer for a predetermined time prior to use. Swelling time varies with the type of gel materials (Table 2.2.4). Space between the polymer chains increases during swelling. Swelling time can be reduced by heating the gel in boiling water bath for 1-5 h.

2. Column packing

Swollen gel is degassed by applying vacuum and then packed into the column. Three volumes of running buffer is allowed to flow through the column.

3. Separation technique

Conventional simple glass column are run under gravitational pressures in descending mode and it is time consuming. Desalting technique are performed under gravitational pressure. Modern technique use pumps to control the flow rate and achieve more reliable and reproducible separation of the sample components. Commercial columns with flow adapters are required to perform this separation.