Use and maintenance of chromatographic column
Maintenance of chromatographic column1. A pre column was used to protect the analytical column (silica gel has a certain solubility in polar / ionic mobile phase).
2. The mobile phase must be degassed and filtered before use.
3. Avoid drastic changes in composition and polarity of mobile phase.
4. The pH stability range of most RP-HPLC columns is 2-7.5, which should not exceed the pH range of the column.
5. If polar or ionic buffer solution is used as mobile phase, the column should be washed clean after the experiment and stored in acetonitrile.
6. Pressure rise is a signal that the precolumn needs to be replaced or the chromatographic column washed.
System considerations
1. When opening and closing the system, the flow rate should be buffered (1ml / min → 0.8ml/min → 0.4ml/min → 0ml / min), so as to avoid the collapse of column filling due to sudden pressure change.
2. The mobile phase is the buffer system. Do not switch to strong solvent directly. Sudden conversion to high concentration organic solvent may precipitate the buffer solution in the HPLC flow system, which will lead to more problems, such as column head blockage, pipeline blockage, pump leakage, piston damage or injection valve spindle failure. The strong solvent should be replaced after washing 5-10 column volumes with buffer free mobile phase (i.e. replacing buffer with water).
3. Do not wash the column directly with 100% organic solvent after the test. Wash the column with 90% water-10% organic solvent (if the mobile phase is buffer solution, pure water or 5% organic solvent water solution) for at least 30 min, then wash the column with organic solvent for about 30 min, and finally store the column in organic solvent.
4. Before replacing the chromatographic column, the chromatographic column must be washed and balanced, and then stored in organic solvent (preferably in acetonitrile).
5. When removing the chromatographic column, both ends should be sealed immediately.
6. Except for special cases, the pre protection column should be used in general.
The reason of equilibrium chromatographic column
The reversed-phase chromatographic column is stored in acetonitrile / water after delivery test. As the chromatographic column may dry out during storage or transportation, if it is not balanced, some salts, lipids, fatty substances, humic acid, hydrophobic protein and other biological substances easily interact with the HPLC column, so that these substances can be adsorbed on the chromatographic column, Contaminate the column.
How to balance the column?
Therefore, before using mobile phase to analyze samples, we should first use methanol or acetonitrile equilibrium chromatographic column with 10-20 times of column volume; if the mobile phase we use contains buffer salt, we should pay attention to "transition" with pure water. During the equilibration process, the flow rate is increased slowly and the column is equilibrated with mobile phase until a stable baseline is obtained (if the buffer salt or ion pair reagent level is low, it will take a long time to equilibrate).
What might happen after column contamination?
The sample components retained in the chromatographic column are accumulated to a certain extent to make them start to form a new stationary phase. Sometimes these analytes can interact with these impurities to form a certain separation mechanism. The interference of these non target products can be detected by the detector, and can form chromatographic peaks, bubbles, baseline disturbance and baseline drift, and "strange peaks" appear on the chromatogram ”The results showed that negative peak, wide peak, steamed bread peak, double headed peak, trailing peak and retention time fluctuated. These behaviors are usually found only through parallel experiments.
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