Practical Procedures with Columns and Samples

The useful lifetime of hplc columns is shortened by the appearance in the column packing of cracks or voids, especially at the top of the 

A void at the top of the column, (ii), produces additional dispersion. Uneven packing as in (iii) adds to the dispersion and may cause a solute to appear as two or more peaks. 

Although hplc columns are packed under high pressure, the column bed can be disturbed by sudden pressure variations, or mechanical 

For use with 25 cm x 4.6 mm analytical columns, guard columns and scavenger columns are often 4.6 mm internal diameter and 3-10 cm in length. They can be packed with microparticulate stationary phases or with porous layer beads. Porous layer beads are cheaper than microparticulates and are easier to pack, but they have lower capacities and will require changing more often. It is usually difficult to know how long a pre-column will last before it requires changing. In routine work, precolumns are usually repacked or replaced to a fixed schedule. 

If the performance of a column is no longer satisfactory it can sometimes be reconditioned by washing with a suitable solvent, or series of solvents. Some bonded phase columns, C-18 for instance, tend to collect non-polar impurities, which can sometimes be removed by washing the column with a non-polar solvent, eg heptane. Assuming the mobile phase normally used with the column is CH3OH/H2O 50 50, we cannot wash directly with heptane because of miscibility problems, we have to get to heptane via a miscible solvent or series of solvents. 

Because scavenger columns are located upstream of the injection valve, they do not add to the dispersion of the chromatogram, and their size is not critical in this respect. Guard columns, on the other hand, do cause a slight loss of efficiency, and so need to have a relatively small volume. Reducing the volume, of course, reduces the life of the guard column. 

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