Preparative Chromatography - Packing Preparative Columns > Axial Compression > Page 38

 

Because any density gradients caused by the compression are longitudinal rather than radial they will not affect the flow profile along the bed. Thus, the bands remain undistorted and very high efficiencies can be obtained with this technology. In addition, there is no restriction on column diameter, and HPLC columns of this type with diameters as large as 80 cm have been made. A diagram of the axial compression packing system is shown in figure 18.

 

The apparatus consists of a column (capped at the top with a stainless steel frit) having a precisely controlled internal diameter and finish. The column contains a close fitting piston. The piston, which is mounted on a constant-pressure hydraulic jack, can be moved throughout the entire length of the column. It contains a porous frit at the top and a channel for passage for the mobile phase through its center. The channel is connected to the detector and thence to the fraction collector. To pack the column, the piston is withdrawn to bottom of the column and the column filled with the measured mass of packing. The amount of packing used will determine the effective length of the column. The hydraulic jack is activated and the packing compressed to the maximum pressure which is determined by the mechanical strength of the packing. If the packing is crushed by the use of too great a pressure, many fines will be produced, which cause the permeability to be extremely low and seriously reduce the column efficiency. The pressure to the column is maintained continuously and during each separation. In addition to stabilizing the bed when packed, the system allows the rapid packing and unpacking of media which can range from gel packings such as cross–linked agaroses to 8 mm silica.

At the extreme, these techniques can allow the scale of preparative chromatography to be much increased and can permit intrinsically expensive materials (such as antibiotics and other biotechnology products) to be processed in kilogram quantities and more. There are, however, other methods of increasing the scale of chromatographic separations