Installation and Preparation Before Use HPLC Column
Installation of HPLC column
(1) First open the package to check the type, size, and storage solvent of the HPLC column. Also, please check the manufacture date.
(2) Unscrew the sealing plugs of the joints at both ends of the HPLC column and put them back into the packing box for spare.
(3) According to the mobile phase flow direction marked on the HPLC column tube, connect the inlet end of the chromatographic column to the outlet of the injection valve through a connecting tube (for example, if conditions permit, it is recommended to use a HPLC guard column in front of the column); the outlet of the HPLC column and the detector connection.
The connecting pipe is a stainless steel pipe with an outer diameter of 1.57 mm and an inner diameter of 0.1 to 0.3 mm. The connecting pipe’s both ends are equipped with hollow screws and pressure rings for sealing. Insert the connecting pipe as hard as possible after it passes through the hollow screw and the pressure ring.
Preparation before use
In addition to the structure and installation, we all should know the preparation before use well. Because of the complexity of biology, the higher the degree of sample purification, the longer the service life of the HPLC chromatographic column, so sample pretreatment is very important, for example, solvent selection, sample filtration, etc.
We can choose pure water for chromatography as the solvent, and use the solvent inlet filter to filter impurities, particles, etc., which can be more “pure” and will greatly help extend the service life of the HPLC chromatographic column. Before using a chromatographic column, other influencing factors should be considered.
For example, certain properties of the analyte can also affect the service life of the HPLC chromatographic column. Strong acids, strong alkaline substances, and protein-based biological macromolecules can interact with stationary phase fillers or form an irreversible adsorption layer, change the surface characteristics of the fillers, change the performance of the HPLC chromatographic column, and ultimately lead to separation failure.
Operating Procedures and Standards of HPLC Column
When the HPLC column is preserved, the plug should be tightened to avoid volatilization of the solvent in the HPLC column. Avoid violent shocks. Before the HPLC column is used, understand the preservation solvent after the previous use, and replace the transition with the appropriate solvent if necessary.
HPLC column tolerance pH range of silica gel matrix is generally 2-8. If the mobile phase needs to add acid or alkali, the pH value must be measured before the experiment, so as not to damage the HPLC column. When the sugar column is used, the flow rate is lower and the HPLC column temperature is higher.
In the experiment, the flow rate was corresponding to pass through the HPLC column at a small flow rate and gradually increased to the specified speed with the full expansion of the filler. Furthermore, the HPLC column should be stored in a solvent with a high organic phase ratio.
