HPLC analysis of biological samples requires complicated sample preparation, depending on the component of interest. This sometimes results in very small amounts of sample being available for injection. If the injection mass is extremely small on a typical analytical column with internal diameter of 4.6mm, the component of interest might not be detected due to diffusion of substance in the column. A semi-micro column with a small internal diameter can be helpful in detecting substances in such small amounts. Such columns are also useful for saving eluent or applications involving LC/MS since lower flow rates can be selected in analytical operations.
Although the use of semi-micro columns can be useful as mentioned above, care must be taken during operations as described below. Factors causing poor column performance include dead volume of the tubing system and that of the flow cells of the detector.
The figures below show how internal diameters of tubes and internal volumes of flow cells can affect chromatograms. Starting with a tubing system compatible with analytical columns of standard sizes, column performance (theoretical plate or peak symmetry) is reduced as a result of sample diffusion outside the column. Using semi-micro columns requires suppression of this sample diffusion by the tubing system. However, it should also be noted that a small flow cell volume means a shorter path length, which in turn results in a lower peak height. It is important to optimise the system for both tubing diameter and flow cell volume, keeping in mind the considerations discussed above, in order to obtain the most appropriate column performance and detection sensitivity for the separation.