Post-column derivatization involves the modification of the chromatographic system to allow the reaction to take place prior to entering the detector by inserting a post column reactor between the column and the detector. Such a reactor can affect the resolution obtained from the column and the reactor system must be designed with some care. The post-column reactor is required to fulfill the following functions:
1/ Provide a source of reagent and a means of mixing it efficiently with the column eluent.
2/ Ensure the reaction is complete before the derivatized product enters the detector.
3/ Minimize the dispersion that takes place in the reactor so that the integrity of the separation achieved by the column is maintained.
A diagram of a post-column reactor system is shown in figure 24.
Figure 24. Chromatograph with Post-Column Reactor
The post-column reactor consists of a reagent reservoir, a pump, a mixing T and a reaction chamber. The pump should provide a pulse-free flow of reagent as most detectors are flow sensitive to a greater or lesser extent. Either a syringe pump should be employed or, if a reciprocating pump is used, it should be fitted with an efficient pulse dampener. The former is preferable, but syringe pumps can be clumsy to refill. The design of the mixing T is critical (different kinds of mixing systems have been studied by Scholten et al. ). The mixing T must ensure that the column eluent is intimately mixed with the reagent to assure efficient reaction Furthermore, the mixing T must not cause any peak dispersion and impair the separation achieved by the column. The reaction chamber provides a hold-up volume to give time for the slower derivatizing reactions to proceed to completion.
This volume must be designed to minimize any peak dispersion. Low dispersion tubing (e.g. serpentine tubing can be used) , or if a relatively large reaction time is necessary then a short length of column packed with glass beads might be suitable. The reagents used in post-column derivatization will be similar to those used in pre-column derivatization. Derivatization, particularly post-column derivatization, is used as a last resort to provide sensitivity or selectivity, which is impossible to obtain other means. Post-column reactors complicate the instrument and render it more difficult to operate.