3 Things to Pay Attention to in C18 HPLC Columns
During the use of the C18 HPLC column, the following matters must be noted. Hawach will introduce you to three things you should pay attention to when using C18 HPLC columns (C18 Universal HPLC columns, C18 Aqueous HPLC column, and C18 Low PH HPLC columns).
1. Because the stationary phase of C18 HPLC columns is highly hydrophobic, try not to use high water phase conditions during use. If the water is too high, it will cause the stationary phase to collide, causing a rapid decline of column efficiency and even causing an irreversible negative impact; due to the high carbon loading, the performance is particularly obvious, it is recommended that the water ratio in the process of use does not exceed 90%;
2. C18 liquid chromatography column pressure rise, column performance decline: usually, the column is contaminated, the column can be regenerated to restore part of the efficiency;
3. C18 HPLC columns pressure swell: usually caused by salt precipitation or sieve plate blockage, if the salt is precipitated, rinse with a low flow rate of 10% methanol-water until the pressure is restored; if it is judged that salt is not precipitated, pure methanol or 10% Methanol water is the opposite flow column (reverse the column, not connected to the detector). If there is no effect after recoil for half an hour, the recoil is invalid, and other reasons need to be found;
4. Before using a C18 HPLC column, it must be washed with methanol or acetonitrile reagent at a low flow rate of 20 times column volume. The effect is to fully wet the stationary phase and thoroughly stretch the carbon chain to make the performance of the column better. status. The specific operation is: after rinsing 65% acetonitrile/water, connect the inlet end of the column to the chromatograph, and vent the outlet end (do not connect the detector to avoid contamination of the detector). Dry the diluted solution in the column at a low flow rate between 0.1ml/min and 0.5ml/min. After 20 minutes, connect the detector to the detector at a flow rate of 1.0ml/min for 2~8 hours;
5. After the liquid chromatograph is tested and analyzed, the c18 high-performance liquid chromatography column must be washed. Especially when the mobile phase contains acid or salt, it is necessary to rinse the acid or salt in the c18 liquid chromatography column. It is usually recommended to wash 20 times column volume with 10% methanol water;
6. If the C18 HPLC column is in a long-term idle state, the column should be thoroughly rinsed. It is recommended to rinse for 3-4 hours or more and then stored in the pure organic reagent or a high proportion of organic reagent solution.