HPLC analysis of bio samples requires complicated sample preparation depending on the component of interest and sometimes calls for very small amounts of sample for injection. If the injection mass is extremely small in a column in general use with internal diameter of 4.6mm, the component of interest might not be detected as a peak due to diffusion of substance in the column. A semi-micro column with a small internal diameter will be helpful in detecting substances in such small amounts. It is also helpful for eluent saving or application to LC/MS because lower flow rates can be selected in analytical operations.
Although the semi-micro column is helpful as mentioned above, care must be taken during operations as described below. Factors causing poor column performance include dead volumes of tubing system including flow cells of the detector.
The figures below depict how internal diameters of tubes and internal volumes of flow cells affect chromatograms. Given the tubing system compatible to analytical columns with standard sizes, column performance (theoretical plate or peak symmetry) degradation occurs due to sample diffusion outside the column. Using semi-micro columns requires suppression of sample diffusion by the tubing system. On the other hand, it should be noted that a small flow cell volume forces a path length to shorten resulting in a lower peak height. It is important to arrange the system environment keeping in mind the considerations discussed above to obtain the column performance and detection sensitivity appropriate to separation of interest.