Introduction

Derivatization involves changing in some way the basic chemical or physical structure of a compound, usually to a single product, which may be more useful for the analysis of the original analyte in liquid chromatography (LC). Derivatization can be used for analytical or preparative scale LC. In the analytical mode, it can be used to improve the identification and quantitation of the analyte of interest. It may also be used to improve throughput and recovery in preparative scale LC purifications of large amounts of material. Changes in the basic structure of the analyte can also lead to improved peak shape, elution times, peak symmetry, efficiency, plate count, and other indicators of chromatographic performance. That is, elu-tion times and retention factors, as well as resolution, separation factors, reduced plate heights, and other LC parameters of performance, can all be varied and improved by suitable, selective derivatization of the starting analyte.

The most general type of derivatization involves modifying the chemical structure of the starting compound by tagging or adding another reagent to it via a suitable functional group alteration (Figure 1). Thus, most simple derivatizations involve a derivatiz-

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ing reagent, the substrate or analyte of interest, the desired derivative of the analyte, remaining excess reagent, and undesirable by-products coming from the excess derivatizing reagent reacting with solvent, water or thermally degrading (Figure 1). Ideally, only the desired derivative would remain at the end of the reaction period, without any remaining starting analyte, derivatizing reagent or by-products. However, this idealized situation is rarely observed and it is often necessary to separate prior to or during the LC analysis the desired derivative from all other possible compounds coming from the derivatization reaction and/or sample components and their possible derivatization products.

Though most derivatizations usually occur in a homogeneous solution between the analyte of interest and the reagent itself, it is possible to perform derivat-izations on the analyte in solution with an immobilized or solid-phase reagent. Figure 2 illustrates a typical immobilized or solid-phase reagent that has been described in the literature for use with LC. It is also feasible to first immobilize the analyte on a solid support, such as silica gel, Immobilon™ membrane, poly(styrene-divinylbenzene), C18 packing material, and others, and then perform the derivatization reaction on the now-immobilized analyte. Once the reaction is completed, the excess reagent is simply washed from the solid support still containing the derivative. The desired derivative is then eluted with a stronger solvent from the solid support, often in a disposable plastic tube (solid-phase extraction cartridge or Sep-PakTM), without any residual, unreacted starting

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