Hello, everyone! Have you ever encountered situations in your daily work where it’s challenging to analyze certain compounds using conventional experimental methods? What should we do in such cases? Derivatization is a great option to consider. Today, join me to explore the application of derivative techniques in liquid chromatography. Let’s see how they help us overcome these challenges.

Chemical derivatization is a method that involves the use of chemical reactions to attach a specific functional group to the target analyte, thereby enhancing its detection sensitivity and separation efficiency. This approach aims to modify the properties of the compound through chemical derivatization reactions, making it more suitable for specific analytical processes. It finds extensive application in instrumental analysis, including chromatography techniques (such as gas chromatography, liquid chromatography, supercritical fluid chromatography, thin-layer chromatography, electrophoresis, and various hyphenated techniques), mass spectrometry, nuclear magnetic resonance spectroscopy, UV-visible spectroscopy, fluorescence spectroscopy, and electrochemical analysis, among others.

Differences Between Gas Chromatography and Liquid Chromatography Derivatization

In gas chromatography (GC), the application of chemical derivatization reactions is primarily aimed at increasing the volatility of samples or enhancing detection sensitivity. In contrast, chemical derivatization in high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) refers to a process wherein specific reagents, commonly known as chemical derivatization reagents or labeling agents, are employed under certain conditions to react with sample components. The resulting reaction products facilitate chromatographic detection or separation.

Classification of Liquid Chromatography Derivatization Techniques

Derivatization techniques in liquid chromatography serve as common auxiliary tools in chromatographic analysis. These techniques address issues related to poor retention, instability, or limited sensitivity of target compounds when conventional detection methods cannot directly detect them. The derivatization process in liquid chromatography involves the reaction of the analyte with a specific reagent, commonly referred to as a derivatization reagent, to generate compounds that are more amenable to detection. Derivatization reactions can be categorized into two types based on the formation of covalent bonds: labeling and non-labeling reactions. Labeling reactions involve the formation of covalent bonds between the analyte and the labeling reagent during the reaction process, while all other reaction types, such as ion pairing, photolysis, redox reactions, and electrochemical reactions, fall under non-labeling reactions.

Another way to categorize derivatization reactions is based on the location of the derivatization process, which includes pre-column derivatization, on-column derivatization, and post-column derivatization.

From the perspective of instrument integration, derivatization can be classified into three categories: online, offline, and at-line (automation).

Using the Italian HT4000A automatic sample handling workstation, complex and multi-step automated derivatization operations can be performed, as illustrated in the diagram below. Different liquid chromatography systems, when combined with the HT4000A automatic sample handling workstation, can achieve offline, online, or at-line derivatization.

Common Reactions in Liquid-Phase Derivatization Techniques

Currently, in HPLC, pre-column derivatization (referred to as pre-column derivatization) and post-column derivatization (referred to as post-column derivatization) are predominantly used. Common reactions in at-line derivatization include esterification, acylation, alkylation, silylation, boronation, cyclization, ionization, and photochemical reactions, among others.

HT4000A Derivatization – Vortex Mixing

Common Derivatization Methods in Liquid Chromatography

Let’s explore different derivatization methods:

Pre-column Derivatization

The first approach involves transforming the analyte into a detectable derivative before separation through the chromatographic column. This can be achieved in various ways:

  • Online pre-column derivatization involves separately pumping the analyte and derivatization reagent through different fluidic systems into a reactor for immediate reaction. The mixture then enters the chromatographic column.
  • Offline pre-column derivatization involves pre-reacting the analyte with the derivatization reagent and then introducing the derivatized product as the sample for chromatographic analysis.
  • Some literature also mentions the possibility of introducing the derivatization reagent into the mobile phase, allowing the analyte to undergo derivatization directly with the mobile phase after injection, making it an online derivatization method.

Post-column Derivatization

Post-column derivatization refers to the separation of the analyte first, followed by online mixing of the eluate from the chromatographic column with a derivatization reagent to generate detectable derivatives. The resulting derivatives are then directed into the detector. Depending on the type of derivatives generated, this method can include UV-Visible derivatization, fluorescence derivatization, Raman derivatization, electrochemical derivatization, and photochemical derivatization, among others.

Amino Acid Post-Column/Pre-Column Derivatization Schematic Diagram

  1. UV-Visible Derivatization:
    • UV derivatization involves reacting organic compounds with weak or no UV absorption with derivatizing reagents containing UV-absorbing functional groups, thereby generating compounds that can be detected using UV absorption. For example, amine compounds readily react with halogenated hydrocarbons, carbonyl, or acyl-based derivatizing reagents.
    • Visible light derivatization has two primary applications: firstly, it is used for the detection of transition metal ions. This involves reacting transition metal ions with chromogenic reagents to produce colored complexes, chelates, or ion-association compounds that can be detected using visible light. Secondly, it is used for the detection of organic ions. In this case, the counterions of the analyte ions are introduced into the mobile phase, forming colored ion-pair compounds that can be separated and detected.
  2. Fluorescence Derivatization:
    • Fluorescence derivatization involves reacting the analyte with a fluorescence derivatization reagent to produce substances that exhibit fluorescence, which can then be detected. Some fluorescence derivatization reagents themselves do not possess fluorescence, but their derivatives exhibit strong fluorescence. Derivatization techniques not only complicate the liquid chromatography system but also consume time and increase analytical costs. Some derivatization reactions also require strict control of reaction conditions. Therefore, derivatization techniques are considered only when convenient and sensitive detection methods are not available or when there is a need to enhance the selectivity of separation and detection.
  3. Photochemical Derivatization:
    • Photochemical derivatization (PCD) is a class of analytical methods based on photochemical reactions. It combines unique derivatization techniques with traditional detection methods such as fluorescence, chemiluminescence, UV-visible, and electrochemical detection. PCD enhances the sensitivity and selectivity of conventional methods and significantly expands the application scope of traditional detection methods. It finds widespread use in pharmaceuticals, complex biological samples, environmental sample analysis, and more.
      Column post-chemical derivatization reactions are primarily associated with fluorescence analysis, although there are also methods employing electrochemical detection. The main categories of photochemical reactions include intramolecular energy transfer, collisional energy transfer, quenching, photoionization, isomerization, direct reactions, and intermolecular dissociation.

Application Example of Liquid-Phase Derivatization Techniques

Detection of Aflatoxins (Photochemical Derivatization):

In the detection of aflatoxins (AFT), mycotoxins produced by certain molds, liquid-phase derivatization, particularly photochemical derivatization (PCD), is employed.

After undergoing column post-photochemical derivatization, AFT emits characteristic fluorescence. This fluorescence is captured by a fluorescence detector (FLD) and subsequently processed using a chromatography workstation. Photochemical derivatization coupled with FLD detection proves highly effective in detecting low concentrations of aflatoxins, offering significantly greater sensitivity than other detection methods.

Schematic Diagram of Photochemical Derivatization Coupled with HPLC

Amino Acid Analysis in Feed (Pre-Column Chemical Derivatization)

Chromatography Column: Welch Amino Acid Analysis Column, 5μm, 4.6×250mm.

1 Aspartic acid2 glutamic acid3 serine
4 glycine5 histidine6 arginine
7 threonine8 alanine9 proline
10 ammonium chloride11 tyrosine12 valine
13 methionine14 cystine15 Isoleucine
16 leucine17 norleucine18phenylalanine
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