Notes on the use of liquid chromatography column

1. Instructions for the use of liquid chromatography column:
(1) Precautions before use of liquid chromatography column:
There is no special description for the storage solution of the HPLC column, and all of them are the mobile phase shown in the evaluation report. Before use, it is necessary to pay attention to whether the storage liquid of the liquid chromatography column is mutually soluble with the mobile phase of the sample to be analyzed. In reversed phase chromatography, if high concentration of salt or buffer solution is used as eluent, 10% or so of low concentration organic phase eluent should be used first, otherwise the salt in buffer solution will be easily separated out from high concentration organic phase and the chromatographic column will be blocked.
(2) Mobile phase:
The organic solvents used in the mobile phase should be as pure as possible. The water in the mobile phase should be ultrapure water or double distilled water from all glassware. If the mobile phase is 45.0 μ m, the mobile phase is better. In addition, the containers containing the mobile phase and the on-line filters in the chromatographic system should be cleaned or replaced regularly.
Generally, the pH value of the bonded phase filler based on conventional silica gel is 2.0-8.0. When it is necessary to use the chromatographic column under the boundary conditions of the applicable range of pH value, clean the column immediately after each use with a solvent suitable for the storage of the column and mutually soluble with the flow used, and completely replace the original mobile phase.
(3) Sample:
The sample should also be as clean as possible. Sample filter or sample pretreatment column (SPE) can be selected to pretreat the sample; if the sample is inconvenient to handle, the protective column should be used. In the analysis of samples by normal chromatography, all solvents and samples should be strictly dehydrated.
2. Preservation of chromatographic column
(1) Maintenance of reversed phase liquid chromatography column after daily experiment:
Buffer solution or mobile phase containing salt was used. After the experiment, the column was washed with 10% methanol / water for 30 minutes. The salt in the column was washed off, and then washed with methanol for 30 minutes. Note: do not wash the column with pure water, but add 10% methanol into the water to prevent the filler from collapsing.
(2) Long term storage column:
If the chromatographic column is to be preserved for a long time, it must be stored in a suitable solvent. The reversed phase column can be stored in pure methanol or acetonitrile, the normal phase column can be stored in pure hexane after strict dehydration, and the ion exchange column can be stored in water (containing preservative sodium azide or thiomersal), and the plug provided when purchasing a new chromatographic column is plugged. The best storage temperature is room temperature.
3. Problems and solutions in the use of chromatographic column
The most common problem in the use of chromatographic column is the increase of column pressure. If the column pressure increases slowly in the process of long-term use, it is a normal phenomenon. However, the column pressure suddenly rises in the process of use (except for system pipeline blockage and pressure sensor failure), the possible reasons are as follows:
(1) The filter plate of chromatographic column head is blocked or polluted;
(2) The packing of chromatographic column head was polluted by sample;
(3) The salt in the buffer solution of the chromatographic column will crystallize and precipitate when encountering high concentration of methanol or other organic solvents;
(4) If the pH value of mobile phase is too high or too small, the structure of stationary phase will be destroyed or dissolved.
The solutions are as follows:
(1) If it is determined that the filter sieve plate of chromatographic column head is polluted, the chromatographic column can be flushed to normal pressure with methanol in the opposite direction, or the chromatographic column head can be removed and placed in 10% dilute nitric acid for ultrasonic cleaning for 10 minutes, and then the chromatographic column can be loaded again after ultrasonic cleaning with pure water for 10 minutes.
(2) If it is determined that the packing of chromatographic column head is contaminated, remove the column head screw, dig out the contaminated filler in the front section of column, and refill with the same column filler. After careful repair, install the column head screw again.
(3) If it is determined that it is salt crystallization, wash the column with 10% methanol / water to dissolve all the salt in the column, and then change to high concentration methanol.
(4) If the pH value is not used properly, it is difficult to recover.
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