What is the principle of HPLC


6.0 HPLC columns / HPLC separation columns

The HPLC columns usually consist of a stainless steel tube and screw caps at both ends with a connection for a capillary on one side as an inlet and on the other as an outlet. The HPLC columns are filled with the stationary phase, a very fine-grained, porous powder (3, 5, 8 or 10 ┬Ám grain size) under mechanical pressure, without empty space. The smaller the grain size, the higher the separation performance. The column material is always selected according to the cutting task to be processed. Probably the most frequently used HPLC column is a "C18 column". In this case, the stationary phase is a chemically modified silica gel with C18 chains built up on its surface. To ensure good reproducibility of the results, the HPLC column should be operated at a constant temperature. In the HPLC column, the sample substances are adsorbed on the stationary phase and separated by adsorption / desorption processes.

[Fig. 11]

The analytical separation column often has an inside diameter of 2 to 5 mm and a length of up to 250 mm, it is designed for a maximum flow of approx. 2 ml / min.

The semi-preparative separation column with an inside diameter of 10 - 50 mm and a length of 250 mm is suitable for a maximum flow of approx. 100 ml / min.

The preparative separation column with an internal diameter from 75 mm and a length of up to 1000 mm are used with a flow from 100 ml / min.

The Guard column is mounted directly in front of the separation column to prevent it from becoming dirty. The guard column is smaller and almost always has the same column material as the separation column.

6.1 The Pillar packing stand "Pack the columns yourself"

If you want to be flexible with the column material with the column material, you use a column packing station or also called a column packing station.

Thanks to static or dynamic axial compression, all common column materials can be packed reproducibly in the bed length required by the user.

Due to the simple packing process, the HPLC column can be quickly adapted to the needs and there is no need for a selection of expensive ready-made columns. The carrier material can be packed with high pressures through a frit that is firmly connected to the movable piston. This ensures an even distribution of the substance over the entire cross-section of the column and an optimal shape of the peaks.

[Fig. 12]

Compression of the column bed can be achieved in two ways. Either with space-saving manual hydraulics or with pneumatically driven hydraulics for static and dynamic axial compression. With static compression, the HPLC column can be removed from the packing stand. This means that several HPLC columns can be produced with one packing stand. On the other hand, with dynamic axial compression, the HPLC column remains in the packing stand and the column bed is automatically kept under pressure so that there is no free space at the column inlet that could affect the separation performance. If the separation efficiency of the removed HPLC column declines due to the resulting free space, re-pressing is sufficient to achieve the best separation results again. In addition, a column packing station can be used to create different bed lengths with a column tube and, as an option, temperature control over the entire length of the bed is possible with a column tube with thermal jacket.

The packing process:

The bottom flange is mounted on the column tube. The slurry (emulsion of carrier material and suitable solvent such as isopropanol) is poured into the column tube. The slurry (column material) is pressed hydraulically, the solvent in the slurry is displaced from the column tube, so that a homogeneous solid packing is created from the packing material. The piston is fixed (static compression) or automatically pressed (dynamic axial compression).

[Fig. 13]

6.2 Rotating column

This is the SCPC with a rotor (Fig. 6) in which the constituents of a sample are separated in over 1000 chambers connected in series (Fig. 7) and fractionated after they have passed through. Partition chromatography, also known as liquid-liquid extraction, centrifugal partition chromatography (CPC) or counter current chromatography (CCC), is used as the separation method. The separation process takes place in two, immiscible liquid phases, the ingredients of the sample being distributed in these two liquid phases according to their polarity.

This method enables the separation of samples of various kinds on an analytical, preparative and production scale. The SCPC systems are always structured in the same way regardless of size and throughput. The heart of every system is the rotating column, the rotor. It is thus the separation column, surrounded by peripheral devices such as the HPLC pump, the feed system for samples, the detector, a fraction collector and the computer with HPLC software for controlling the system and evaluating the chromatograms. Compared to HPLC systems, the SCPC systems are easier, simpler and cheaper, especially when raw extracts of different matrices have to be separated. The SCPC systems are not in direct competition with HPLC, but also accelerate the entire chromatographic process through to the pure product in combination with it.