Conventional reversed phase chromatographic column

Recommended steps for real sample analysis with conventional reversed phase chromatographic column:
1. Make sure that the mobile phase is clean, new water phase mobile phase should be prepared every 24-48 hours, and the mobile phase solvent bottle should be replaced or carefully cleaned to avoid bacteria growth in the mobile phase. Ensure that the pure water system in the laboratory works normally.
2. Ensure that the sample does not contain particulate impurities. If the sample is too dirty or there are particles, a suitable sample preparation method should be considered. When using the sample filter, make sure that the filter membrane is 0.22 μ m and fully compatible with the sample solvent (completely soluble). High speed centrifugation can also be considered to remove particulate impurities. For example, after centrifuging for 20 minutes above 8000 rpm, the sample supernatant is removed for sampling.
3. Ensure that the sample solution is compatible with the mobile phase conditions, and there will be no precipitation or solvent immiscibility after injection. The mobile phase or the solution with weaker elution intensity than the mobile phase (i.e. lower proportion of organic solvent) is usually used to dissolve or dilute the sample, so as to obtain the best peak shape and detection sensitivity.
4. If the system generates mobile phase by on-line mixing, check the compatibility of each component of mobile phase in advance. For example, salting out may occur when phosphate buffer is mixed with a high concentration of acetonitrile (e.g. ≥ 70%).
5. Understand the operation limit of chromatographic column. If the conditions of mobile phase, column temperature and sample solution are not conducive to the life of chromatographic column, please use protective column to prolong the column life.
6. Before using the mobile phase for column equilibrium, if the mobile phase contains buffer salts which may precipitate, the organic solvent water solution with 5 times volume of column volume should be used for the transition. For example, before using the 40 / 60 methanol / phosphate buffer mobile phase, wash the column with a 40 / 60 methanol / pure water solution of 5 times the volume of the column.
7. In the routine analysis process, the pollutants that may accumulate in the column are cleaned with each needle or several probes at intervals. After the analysis, clean the column thoroughly to remove buffer salts and contaminants.
8. During the use of chromatographic column, the column efficiency test shall be carried out regularly to record the number of plates and pressure, so as to track and monitor the performance change of chromatographic column. The column efficiency test method should be fixed.
9. The reversed phase chromatographic column should be kept in a suitable high proportion or 100% organic phase (such as acetonitrile or methanol). If the chromatographic column is used in high temperature or extreme pH conditions, or the chromatographic column is stopped for more than 72 hours, it is recommended to keep the chromatographic column with 100% acetonitrile.
10. Use the original column plug and make sure the plug is tight to avoid solvent volatilization in the column. Avoid bumping and dropping when taking and placing chromatographic column.
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