General Approaches to Derivatization in Liquid Chromatography

Chemical derivatization in LC requires the optimization of several reaction or separation parameters. These include temperature, pH, solvent, time, ratio of o

6-AQ tagged polymeric reagent

6-AQ tagged polymeric reagent

Derivative of amines

Figure 2 Typical immobilized or solid-phase reagent. The 6-AQ-tagged polymeric reagent reacts with amines (70°C, 10min), producing a derivative free in solution, now 6-AQ derived. (6-AQ, 6-aminoquinoline; PSt, polystyrene.)

Derivative of amines

Figure 2 Typical immobilized or solid-phase reagent. The 6-AQ-tagged polymeric reagent reacts with amines (70°C, 10min), producing a derivative free in solution, now 6-AQ derived. (6-AQ, 6-aminoquinoline; PSt, polystyrene.)

Table 1 Summary of common derivatization approaches, other than chemical reactions, used in LC

1. Photochemical conversions, photohydrolysis reactions, photocleavage or photoextrusion reactions, photobleaching, etc.

2. Electrochemical conversions (amperometric or coulometric), oxidative or reductive reactions to convert an electrochemi-cally inactive analyte into an electrochemically active derivative.

3. Enzymatic conversions, enzyme-substrate reaction detection, used to detect enzymes post-column via their reaction with substrate and formation of the conversion product, which may then be UV, FL, EC and/or MS active. This is detection of enzymes first separated by LC.

4. Microwave digestion reactions, post-column, used to digest proteins/peptides, nucleic acids and other biopolymers, leading to monomeric species that are more easily detected (EC) and/or derivatized before final detection (e.g. proteins p amino acids # OPA p tagged amino acids; UV, FL, or EC active).

5. Immunodetection (ID), post-column in LC, used to tag an antigenic species with a FL or enzyme-tagged antibody, leading to indirect detection of the untagged antigen via its complex formation in a sandwich format. A primary antibody must be immobilized on the solid ID support to initially capture the antigen after separation by LC.

6. Enzymatic conversion of a substrate, post-column, to form the turnover product with improved UV, FL or EC detection properties. This is detection of enzyme substrates, first separated by LC, then detected post-column by addition of enzyme in solution or via an immobilized enzyme reactor column, pre-detection.

reagent to substrate, separation of derivative from sample components and reaction by-products, detector optimization for derivatives, chromatographic optimization of derivative peak shape, generation of standard derivative and structure confirmation, production of derivative calibration plot for quantita-tion, etc. The purity of the derivative peak in a sample must also be demonstrated by online photodiode array (PDA) or mass spectrometric (MS) methods. The derivatization reagent must be well characterized with regard to structure and its purity demonstrated. The reaction conditions need to be optimized to minimize reagent consumption, maximize derivative yield, and eliminate the formation or presence of reaction and reagent by-products that might interfere in the final separation and detection steps. It may even be necessary or desirable initially (pre-LC) to separate the excess reagent from the derivative and then introduce just the sample and the now-formed derivative into the analytical LC column.

Sometimes the reagents used have different detector properties from the final derivatives. The excess reagent at the end of the derivatization reaction may then be transparent under the optimized detection conditions for the derivative. It may even coelute together with the derivative peak, yet not be observed under such detection conditions. This reduces the need for initial separation of excess, unreacted reagent from the derivative and sample, and/or optimization of LC conditions so that the derivative peak appears completely resolved from all the other peaks.

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