Common Problems in Use of HPLC Chromatographic Column
For a commonly used HPLC chromatographic column, how should I activate and maintain it when I get a new HPLC chromatographic column? Why do you want to do this?
Activation of a new column is actually an equilibrium process. In addition to balancing with the mobile phase, it is sometimes necessary to equilibrate the new column with the sample to be tested, especially for the determination of peptides with relatively high molecular weight.
Because of the high molecular weight substance molecules, the diffusion speed is slow, and the time required for equilibrium is correspondingly longer. The specific balance method is also very simple. Inject samples several times until the peak area and retention time are stable, and then perform the formal sample injection measurement.
If you want to speed up the equilibration time, increase the concentration of the injection sample used for equilibration, or continuously inject multiple injections without waiting for the elution to complete. Equilibrate the new column with the analyte in order to saturate the adsorption capacity of non-specific adsorption sites on the surface of the silica matrix filler.
What are the HPLC chromatographic column technologies? For example, tail sealing, etc., where are these technologies reflected in the application?
HPLC chromatographic column technology includes packing technology and packing technology. It goes without saying that the packing technology has a decisive influence on the separation performance and selectivity of the HPLC chromatographic column. The packing technology is not as simple as imagined.
Different stationary phases, different particle diameters, different inner diameters, and lengths of the column tube, the packing process is different, it is necessary to pack a compact, stable, and uniform column bed. Art requires accumulation of experience.
Where is the gap in HPLC chromatographic column technology?
Answer: There are actually certain skills and procedures for liquid HPLC chromatography column packing, and there may be some luck. Generally, use the high-pressure homogenization method for filling. That is, the filler can be uniformly suspended in the solvent. Then use instant high-pressure compaction, which actually uses different proportions of homogenized liquid and appropriate pressure.
If the pressure is too high, the particles will be broken, the pressure will be too small, and the number of plates will be small. At the same time, the pressure needs to be stable, otherwise, the distribution will be uneven and the tailing will be serious. At the same time, the level of head flatness. Put on the sleeve and it’s ready to use.
Under what circumstances can the column be cleaned? Originally discussed this issue, I also removed it and cleaned it, but I saw that the front part of the HPLC chromatographic column was more contaminated, so I scraped it with a blade, and then installed the cleaned sieve. The problem is solved, but will the service life be reduced?
If the HPLC chromatographic column head is contaminated, remove the contaminated one, and install some packing. Because you scraped some fillers, the number of micro-plates is reduced. You don’t shave too much, only the surface maybe some dirt, so the problem is solved. But in the future, there will be the same problem. If you hang it again, it will be scratched accidentally, which will affect the column efficiency. It is recommended to install a pre-column.