Preparative Chromatography - Packing Preparative Columns > LC Columns > Page 34

When slurry packing preparative LC columns, care must be taken not to operate at pressures in excess of the bursting strength of the tube used for the column. As the column diameter increases, the maximum permissible pressure rapidly falls unless extremely thick walled tubing is employed. The safe maximum pressure for any tube can usually be obtained from the tube or column supplier. A slurry is made of the packing (110% of that needed to fill the column) and placed in the packing reservoir. The reservoir is rapidly connected to the pump (which must have both an adequate delivery rate and an adequate operating pressure - the delivery rate will depend on the column diameter and the applied pressure on the wall thickness and the column length). The pump is started and the column exit valve opened and the flow continued until it has been reduced to a constant value. The flow is then arrested and some packing will remain in the reservoir which ensures that the top of the column is tightly packed. The column is disconnected, the packing secured with a suitable frit, and then connected to the chromatograph.

The major difference in equipment used for larger scale chromatography lies in the technology required to pack and maintain high-performance LC columns of large diameter. Another difference is that industrial-scale equipment is often dedicated to just one specific separation.

Once the diameter of an HPLC column approaches 5 cm, additional difficulties arise in its preparation and operation. It is possible to pack a column 5 cm in diameter by conventional high-pressure slurry packing techniques but this may not be easy to do, particularly in a laboratory environment, and it becomes even more difficult and expensive for columns of wider diameter. There are other problems that need addressing, once packed, the practical lifetime of a column is also uncertain. The changes in performance of a preparative HPLC column that occurs with time depends upon the stability of the packed bed. Frequently, the bed settles after operation for even a short time and the top of the column needs to be repacked. Sometimes channels are formed in the bed, in which case the entire column has to be repacked. The rate of settling again depends upon the diameter of the column. This bed instability arises because there is a significant change in wall support as the column diameter increases. In analytical columns the walls are relatively close to the center of the column and 'bridges' of packing particles can be formed across the bed, as shown in Figure 16. These bridges allow the longitudinal forces acting on the packing within the column to be dissipated to the walls. When a column is packed, it is never in its optimal configuration and there are always areas where the packing is not as tightly packed as could be desired. The presence of the bridges protects the bed from the full pressure of the system, stabilizing the more poorly packed areas. When wider columns are used, the supporting bridges can no longer form, and the metastable packing areas are no longer conserved by bridges and are free to rearrange. This is a function of both column and particle diameters and also the frictional forces between particles and the wall.