How To Choose The Right Stationary Phase For FLASH Column

 Flash purification column is the heart of Flash purification system, just like the engine in a car. Choosing the correct Flash purification column is the most critical part of the entire purification.  Today HAWACH will talk about the connotation of the purification column-stationary phase.

The stationary phase is also commonly known as the packing, which is the core part of the chromatography. To determine the stationary phase packed in the Flash purification column, three aspects of the stationary phase must be determined in sequence: matrix type, surface modification/bonding phase, and matrix parameters.

1. Matrix
The matrix is the basic material composed of the stationary phase, and the commonly used matrix materials are divided into three categories: inorganic, organic, and composite materials. Inorganic materials include silica gel, alumina, etc.; organic materials are mainly polymer materials such as gels; composite materials usually refer to composite materials that combine inorganic and organic materials through hybridization, and coating.

Silica gel is the most widely used matrix in Flash purification. Silica gel for chromatography has a porous structure, high mechanical strength, and thermal stability, excellent pore structure, and specific surface area, and its surface contains a large number of active hydroxyl groups. It is used in normal-phase purification systems. It plays a very important role in the separation and purification of compounds.

At the same time, the surface of silica gel is easily modified to form other bonded stationary phases. There are many kinds of stationary phases modified with a silica gel matrix, and the separation and purification effect is much better than other matrixes. However, the stability of the silica gel matrix is relatively poor under alkaline conditions.

At the same time, the presence of its hydroxyl groups can easily cause irreversible adsorption of some substances, especially alkaline substances. In addition, when applied to the separation and purification of some biomolecule samples, it will cause non-specific adsorption of samples, or denature the sample, resulting in the deterioration of the peak shape of the sample elution in the chromatogram and the decrease of the sample recovery rate.

The alumina matrix has high mechanical strength, good chemical stability, unique selectivity to some compounds, and good complementarity to the silica matrix. However, it is difficult to modify the surface of alumina, so it is still used in normal phase purification systems at most and a small amount in ion exchange systems.

The organic matrix is mostly polymer gel material (such as resin, polystyrene divinyl copolymer, etc.), which has better chemical stability than silica gel (applicable pH value can range from 1 to 12). The sample basically does not undergo denaturation reaction, nor is it prone to irreversible adsorption.

After modification, it has a wide range of applications in the separation and purification of biomolecules such as proteins and carbohydrates in ion exchange chromatography, size exclusion chromatography, and reversed-phase hydrophobic chromatography. However, compared with inorganic substrates such as silica gel, the separation mechanical strength that the organic matrix stationary phase can withstand is lower, and the separation and purification effect is poor, which limits its scope of application in purification.

The composite matrix is compatible with and neutralizes the advantages and disadvantages of the inorganic matrix and the organic matrix, but the corresponding stationary phase of the composite matrix is generally expensive, so it is less used in preparation and purification.

2. Surface modification/bonding phase
By modifying the matrix or bonding different groups, different separation types of stationary phases are formed, which usually include: normal phase, reverse phase, ion exchange, size exclusion, chiral, and other stationary phases.

The chromatographic mode in which the polarity of the modified groups on the matrix is greater than the polarity of the mobile phase is called normal phase chromatography, which relies on the different polarity of the sample to distribute between the stationary phase and the mobile phase to achieve separation. In this separation mode, the sample is eluted from the purification column in order from weak to strong polarity. Commonly used normal-phase stationary phases include unmodified silica gel; diol-based, amino, cyano-based, and other silica-based stationary phases; alumina, etc.

The normal phase silica gel stationary phase is mainly used in the preliminary purification of synthetic non-polar or medium-polar intermediates. The conventional flow system is n-hexane/ethyl acetate, dichloromethane/methanol, etc. Its advantages are simple purification and simple sample post-processing; its disadvantages are low separation performance (compared with reversed-phase immobilization), poor reproducibility, and strong adsorption and even irreversible adsorption of certain substances.

In addition, some polar groups (diol groups, amino groups, cyano groups, etc.) bonded normal phase stationary phases are often used under reversed-phase conditions. For polar compounds that are not strongly retained in reversed-phase stationary phases such as C18, good separation and purification effect will be achieved.

3. Matrix parameters
Matrix parameters include the shape, particle size, and pore size of the matrix. These parameters should be selected in combination with specific requirements for purification.

Taking the most widely used silica gel matrix as an example, the silica matrix used in preparation and purification is usually divided into two types: spherical and amorphous. Amorphous silica gel is cheap and crude.

Compared with amorphous silica gel, spherical silica gel has obvious advantages: good particle uniformity, stable column bed after packing and not easy to collapse, low multipath diffusion effect, high column efficiency, and good resolution. But its price is a bit more expensive, generally used in the purification stage.

The size of the matrix particle size directly affects the column efficiency and separation effect. The choice of particle size depends on the separation status of the sample. The smaller the particle size of the stationary phase is, the more expensive it is. In order to achieve the column efficiency, the smaller the particle size is, the larger the linear flow rate is selected. Therefore, the higher the backpressure, the higher the instrument requirements.

Summary
In summary, flash purification preparation of samples should be based on the actual preparation requirements, combined with the nature of the sample, the requirements for product purity and yield, and the actual situation of the instrument configuration, from the matrix, surface modification/bonded phase, and matrix parameters. Considering three aspects, the most suitable stationary phase for purification and preparation is finally determined. Still cannot decide, welcome to contact the HAWACH team for a more professional solution.

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