Post column, derivatization reactor

Post-column derivatization does not merely require the selection of the most appropriate reagent to react with the solute to render it detectable, but also involves the modification of the chromatographic system to allow the reaction to take place prior to entering the detector. This necessitates the insertion of a post column reactor between the column and the detector. Such a reactor can easily interfere with the resolution obtained from the column and consequently the reactor system must be designed with some care to... 

The reagents used in post-column derivatization will be specific for the solute of interest and will often be the same, or similar to those used in pre-column derivatization. Reagents that produce derivatives that absorb in the visible light range are popular, as relatively simple detectors can be employed. In general, derivatization, particularly post-column derivatization, is only used as a last resort to provide sensitivity or selectivity, which is difficult or impossible to obtain by any other means. Post-column reactors complicate an already complex instrument, render it more difficult to operate and make it more expensive. In addition, however well the post-column reactor is designed it will inevitably impair, to some extent, the separation achieved by the column. 

Nelson et al. used the oxidation of morphine to pseudomorphine 1n a post-column reactor. For the post-column derivatizing reagent, a solution of 50 mg potassium ferric cyanide in 250 ml of 4 M ammonia was used. Methanol - 0.1 M aqueous potassium bromide (12.5 87.5) was used as mobile phase for the separation of the alkaloids on a octadecyl stationary phase. The fluorescence was measured at 432 nm after excitation at 323 nm. 

Uihlein, M. Schwab, E. A novel reactor for photochemical post-column derivatization in HPLC. Chromatographia 1982, 15, 140. 

Marko-Varga, G. and Gorton, L. (1990) Post-column derivatization in liquid chromatography using immobilized enzyme reactors and amperomet-ric detection. Analytica Chimica Acta 234, 13-29. 

In post-column derivatization (see also Chapter 15), the HPLC mobile phase must be miscible and compatible with the reaction mixture in the reactor so that it dose not cause precipitation or interfere with the derivatization reaction. The reagent itself must have significant differences in spectral properties from the products. Post-column derivatization reactions are usually rapid, and fluorescent rather than UV-absorbing labels are preferred. 

Post-column reaction is a common feature of many special types of analyses, the most well-known being the amino acid analyzer that uses ninhydrin with a post-column reactor to detect the separated amino acids. In general, derivatization and post-column reactor systems are techniques of last resort. In some applications they are unavoidable, but if possible, every effort should made to find a suitable detector for the actual sample materials before resorting to derivatization procedures. 

Several precolumn derivatization techniques are available for those who wish to trade extra sample preparation time for the expense and maintenance of post column pumps and reactors. The more popular derivatives are dansyl-(32), OPA-(33), PTH-(34) and PITC-(35) amino acids. There are problems and limitations with some of these systems, however, analysis time is only 15-25 min. compared to 90-240 min. of the ion-exchange post column systems. 

FIGURE9.5 (a) Schematic diagram of the sheath-flow post-column reactor, (b) Separation of protein standards derivatized with NDA//S-mercaptoethanol at a height difference of (A) 0.5 cm, (B) 3 cm, and (C) 6 cm between the derivatization solution reservoir and waste reservoir. (Adapted from Ye, M. L., J. Chmmatogr. A, 1022,201, 2004. With permission.)... 

Oldenburg, K. E., Xi, X., and Sweedler, J. V., Simple sheath flow reactor for post-column fluorescence derivatization in capillary electrophoresis, AnoZysf, 122,1581, 1997. 

Ye, M. L., Hu, S., Quigley, W. W. C., and Dovichi, N. J., Post-column fluorescence derivatization of proteins and peptides in capillary electrophoresis with a sheath flow reactor and 488 nm argon ion laser excitation, J. Chromatogr. A, 1022, 201, 2004. 

Sample is dissolved in water and injected into LC system. The eluate is derivatized on-Une with acetylacetone by means of a post-column reactor... 

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