Matrix Parameters of Flash Purification Column Stationary Phase

The stationary phase, commonly known as the fillers, is the most central part of chromatography. People need to determine three aspects of stationary phase information in turn before determining how to select the kind of stationary phase of the flash purification column. They are matrix type, surface modification/bonding phase, and matrix parameters.

Here we talk about the matrix parameters. Matrix parameters include matrix shape, particle size, and pore size. These parameters should be selected according to the specific requirements of 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 the first choice for preliminary roughness. Compared with amorphous silica gel, spherical silica gel has obvious advantages. It has the following 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 smaller the particle size, the higher the flash column efficiency can be achieved. The choice of the particle size depends on the separation of the sample. After all, the preparation should consider the cost issue, the smaller the particle size. The more expensive the stationary phase, in order to achieve the best column efficiency, usually the smaller the particle size, the larger the linear flow rate, so the higher the back pressure, the higher the instrument requirements.

Generally, large-particle stationary phases are often selected for crude and pure, but small-particle stationary phases are mostly selected when the sample has high added value and requires high purity and high yield. Therefore, the choice of stationary phase particle size should be considered in terms of cost, sample, and purification equipment parameters.

The choice of the pore size of the porous stationary phase matrix is ​​mainly based on the molecular volume of the sample. The pore size of the stationary phase is usually greater than three times the sample molecular volume. In order to make the sample molecules effectively enter the stationary phase pores for sufficient contact to achieve the separation effect.

However, for polypeptide sample molecules, the pore size of the stationary phase does not have to meet the above rules because of the long-chain structure of the molecule. At the same time, the pore size is also related to the specific surface area. If other parameters are the same, the smaller the pore size, the larger the specific surface area and the specific surface area affects the separation effect and sample loading to a certain extent. Generally, a larger specific surface area means more surface modification groups. Then the separation effect is better and the sample load is larger.

In summary, when performing Flash purification and preparation of samples, it should be based on actual preparation requirements, combined with sample properties, product purity and yield requirements, instrument configuration, and other actual conditions, from the matrix, modification/bonding. The three aspects of phase and matrix parameters are comprehensively considered to finally determine the stationary phase required for purification.

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