Classical Column Chromatography

Classical column chromatography, also known as liquid-solid chromatography, is a technique for separating solutes based on the difference in distribution between a stationary phase (usually a solid adsorbent) and a mobile phase (solvent). The basic principle can be summarized as follows: with the help of the fixed phase, the solute of the solution is adsorbed, and then the components of the mixture are further separated into discrete components by using the different distribution coefficients between the fixed phase and the mobile phase.

In column chromatography, the stationary phase is usually composed of porous particulate matter (such as silica gel, alumina, activated carbon, etc.), which has a large specific surface area and adsorption center, and can form hydrogen bonds with solute molecules, van der Waals forces and other interactions, resulting in adsorption. The mobile phase is responsible for elution of solute molecules from the stationary phase, and its selection should be determined according to the polarity of the solute and the adsorption characteristics of the adsorbent.

In the separation process, the mixture solution is introduced into the column containing the stationary phase. With the flow of the mobile phase, the components move down the column at different speeds due to the different adsorption capacity of the stationary phase. The more polar components are usually easy to be adsorbed by the stationary phase, so they move slowly. However, the component with weak polarity is easily carried away by the mobile phase, and the moving speed is faster. In this way, each component forms a number of color bands on the column to achieve separation.

► Purpose of derivative treatment
The main purpose of derivatization is to change the physicochemical properties of analytes so that they are more suitable for separation and detection by the column chromatography. Specifically, derivative processing can achieve the following goals:

1) Improved detection performance: By introducing functional groups that are easy to detect, the analyte generates a stronger signal on the detector, improving the sensitivity and accuracy of the detection.
2) Change molecular structure or polarity: Adjust the polarity of the analyte to improve its separation on the column and reduce peak trailing and overlap.
3) Increased volatility: For some non-volatile analytes, derivatization can improve their volatility, making them easier to separate and detect in gas chromatography.
4) Stable analyte: Sensitive functional groups in the analyte can be protected by derivatization to prevent degradation or change during separation and detection.

► The type of derivative treatment
Derivatization is mainly divided into two types: pre-column derivatization and post-column derivatization:

1) Pre-column derivation:
Before the analyte enters the column, it chemically reacts with the derivates to produce the derivates.
The derivatives are then separated and detected on a column.
Precolumn derivatization has the advantages of free reaction conditions and easy multi-step reaction, but it may also introduce impurities or lose samples.
2) Post column derivation:
The analytes are first separated on the chromatographic column and then chemically react with the derivants in the derivatives cell.
The resulting derivatives are then entered into the detector for detection.

Post-column derivation has the advantages of good reproducibility and less influencing factors, but it requires additional instruments and equipment.
► Application examples of derivative processing
In the extraction and separation of natural pharmaceutical chemical components, it combined with derivative processing technology can achieve the following applications:

1) Improved detection sensitivity: For example, in amino acid analysis, the introduction of easily detectable functional groups (such as fluorophores) through pre-column derivatization can increase the signal strength of amino acids on the detector, thereby improving the sensitivity of the detection.
2) Improve the separation effect: For some compounds with similar polarity or similar structure, the polarity can be adjusted by derivatization treatment, so that the separation effect on the chromatographic column can be improved. For example, in the analysis of flavonoids, different functional groups can be introduced through derivatization treatment, thus adjusting their polarity and improving the separation effect.
3) Protection of sensitive functional groups: For analytes containing sensitive functional groups (such as phenolic hydroxyl, amino, etc.), derivatization can protect these functional groups from degradation or change during separation and detection. For example, in carbohydrate analysis, hydroxyl functional groups can be protected by acetylation derivatization to prevent their degradation during separation.