HPLC Pre-column and Post-column Derivatization Questions

1) Why is derivatization performed? The purpose of derivatization is to convert analytes that cannot be directly detected into detectable substances by the detector. It allows the analytes to selectively react with derivatizing reagents, altering their elution order in the chromatographic column and achieving better separation.

2) What are the classifications of derivatization? Based on the timing of derivatization, chemical derivatization can be classified into pre-column derivatization and post-column derivatization. Pre-column derivatization involves converting the sample into derivative compounds before injection, followed by separation on the chromatographic column. Post-column derivatization, on the other hand, involves modifying the physical or chemical properties of analytes after the chromatographic column to make them detectable.

3) What types of compounds are suitable for derivatization? Derivatization is suitable for various compounds, including organic compounds, natural products, biopolymers (such as proteins, nucleic acids), environmental samples, food components, drugs, and drug metabolites, among others.

4) What are the requirements for derivatization? Requirements for derivatization include selecting appropriate derivatizing reagents and reaction conditions to ensure selectivity and efficiency of the derivatization reaction. Additionally, it is important to avoid the formation of multiple derivatization products that could complicate chromatographic separation.

5) What are the drawbacks of pre-column and post-column derivatization? The drawback of pre-column derivatization is the potential formation of multiple derivatization products, which can pose challenges in separation. Furthermore, pre-column derivatization allows longer reaction times and various forms of reactors, but it requires additional processing steps and can be time-consuming. Post-column derivatization, on the other hand, requires the development of specific post-column derivatization systems, involving criteria such as performance, price, and form. Additionally, post-column derivatization may increase the complexity of the instrument and require adaptation of the chromatographic detector before and after the derivatization process.