How to use chromatographic column better
New31. After getting the column, read the instructions first to understand the specific performance of the column: temperature tolerance, pH tolerance, etc. And the washing and preservation conditions of chromatographic column. The chromatographic column can be used normally only after the column calibration test confirms that there is no problem with the chromatographic column itself.
2. The sample shall be fully pretreated to ensure the cleanness of the sample; the protective column or on-line filter shall be used, and the protective column core or filter piece shall be replaced according to the actual situation.
3. Avoid sharp changes in pressure and temperature and any mechanical vibration.
4. The composition of the solvent should be changed gradually, especially if a buffer salt is used in the mobile phase in reversed phase chromatography to avoid salting out.
5. The suitable mobile phase (especially pH) was selected to avoid the destruction of the stationary phase, the fracture of covalent bond with low pH, the hydrolysis of bond and phase, and the dissolution of silica gel with high pH.
6. During the use of chromatographic column, if the pressure rises or pollution occurs, choose appropriate conditions to wash or recoil.
Applicable to C18, C8, C4, C1, phenyl, PFP, AQ, etc
Use the polar mailing solvent (such as acetonitrile) containing ≥ 30% of the column volume at least 20 times as the mobile phase to remove the buffer salt in the column; use the polar mailing solvent (such as acetonitrile) containing 95% of the column volume at least 20 times as the column volume to wash the chromatographic column.
When the column calibration did not recover obviously after the removal of pollutants, 50% isopropanol / acetonitrile was used to clean the column 20 times of its volume at a low flow rate to remove pollutants. (the above operations can be determined according to the laboratory's own SOP, which is only the reference for saifen needle to wash its own brand of RP-HPLC column.)
Applicable to amino column and cyano column
Routine cleaning: when acetonitrile: water = 60:40, wash the chromatographic column. It is not recommended that the proportion of water phase exceed 40%. Excessive use of water phase is easy to cause hydrolysis of bond and phase.
Column regeneration: when the column is used for a long time and the column calibration is reduced, the column can be regenerated. Acetonitrile: water (add 0.05% ammonia water in the water) = 60:40, wash 30 times the column volume. (the above operations can be determined according to the laboratory's own SOP, which is only the reference for saifen needle to wash its own brand chromatographic column.)
Applicable to the washing method of SCX and sax of ion exchange column
Daily flushing: wash 30 columns with 100% water for demineralization, and then wash 20 columns with 20% methanol or acetonitrile for preservation in 20% methanol or acetonitrile.
Long term storage: wash 30 columns with 100% water to remove salt, and then use 50% acetonitrile; wash 20 columns with 50% water to store in 50% acetonitrile: 50% water.
Regeneration: first use high concentration buffer salt (no more than 1m), then wash with 100% water, 100% acetonitrile or methanol, and then store with 20% acetonitrile or methanol. (the above operations can be determined according to the laboratory's own SOP, which is only the reference for saifen needle to wash its own brand chromatographic column.)
