Precolumn versus Postcolumn Arrangements

The derivatization can be carried out in the pre-col-umn or post-column mode, i.e. before or after the separation has taken place. In the post-column approach the derivatization reaction does not have to yield a single, stable product, provided that the reactions are reproducible. There are several serious disadvantages associated with this technique: (1) excess

Table 2 Derivatization placement in LC

Mode

Reaction sequence

Pre-column, offline

Pre-column, online

Post-column, offline

Post-column, online

Derivatization away from LC-injection-separation-detection Derivatization on the LC-injection-separation-detection Injection-separation-derivatization away from LC-detection Injection-separation-derivatization on the LC-detection derivatization reagent must not interfere in the detection process; (2) reaction kinetics need to be rapid to allow real-time detection; (3) additional pumps are needed for a nonpulsating supply of derivatization reagent; (4) reaction solvents must be miscible with the separation mobile phase; and (5) an efficient mixing of derivatization reagent with the column effluent is required.

Pre-column derivatization is an alternative approach to post-column derivatization. One of its advantages is that derivatization is independent of the mobile phase and the reaction kinetics are not limited. Apart from an increase in detectability, pre-col-umn derivatization can also improve the selectivity and chromatographic resolution of the overall method. Excess reagent present in the reaction mixture must be chromatographically resolved from the analyte derivative peaks, and/or be physically or chemically removed from the sample solution prior to injection. If several analytes yield the same derivative^), then these would not be separable, and it would be impossible to determine which analyte was originally present in the sample. For example, the use of a substrate that could react with several enzymes, pre-column, would then lead to exactly the same product(s), preventing absolute identification of the enzyme actually present in the sample reaction mixture. More derivatizations have been performed online, post-column, as opposed to online, pre-column, or even offline, pre-column, for the above reasons, at least in LC areas. It is also possible to perform de-rivatizations in situ, or within the mobile phase. In this case derivatization reagent is placed in the solvents used for the LC separation. After separation has occurred, the eluent from the LC column can be heated to cause the reaction to occur, prior to the final detection stage. Unlike the online, post-column mode of operation, this does not require the addition of a mixing tee, heating coil, reagent pump, or ancillary tubing after the LC column.

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Solar Panel Basics

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