In our liquid chromatography detection, the column pressure is a very important instrument parameter. We need to pay attention to the column pressure at frequently. In case of abnormality, we can analyze it through the following points.
1. New High column pressure
Reason: The loss of the chromatographic column preservation solution causes the packing at both ends to dry up. In this case, when the sample is detected, the column pressure will increase and the column efficiency will decrease.
Solution: The new column is activated according to the instructions before use; if it is used for a long time after purchase, it needs to be activated overnight with the low flow rate of the activation solution in the instructions before use.
Reason: The sieve plate particles fall off and block due to violent transportation.
Solution: If the column pressure is still high after the new column is activated according to the instructions, it may be caused by transportation, and the column can be sent back to the company to replace the sieve plate.
2. Mobile phase preparation or system setting error
Reason: Under the reversed-phase system, the same chromatographic column has different organic phase ratios, different column temperatures, and different pressures.
Solution: Carefully check the system configuration for errors. If there is any error, please correct it and then test. If the pressure is still abnormal, it is recommended to re-prepare the mobile phase and check it again.
3. The detection method damages the packing of chromatographic column
Reason: Some chromatographic conditions, such as reversed phase C18 chromatographic column, are detected at high pH value. High pH flow phase causes the packing to dissolve, resulting in the consolidation of the column end, causing the column pressure to increase, and the column efficiency to decrease.
Solution: Use mild detection conditions.
4. The buffer salt mobile phase transition method is incorrect
Reason: For the project of the mobile phase containing salt, before using the mobile phase, it is necessary to replace the high proportion of the organic phase in the chromatographic column with the transition mobile phase, and then replace the mobile phase. After the detection, it is also necessary to replace the salt with the transition mobile phase, and then rinse the chromatographic column with the chromatographic column preservation solution. Otherwise, the column pressure will increase due to salting out.
- Slight salting out: Use 10% methanol, slowly increase the flow rate from low to 1.0ml/min, rinse until the column pressure returns to normal, and then rinse for about 30 minutes.
- Severe salting out: Rinse overnight with 10% methanol at low flow rate
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