Use and protection of liquid chromatography column

Chromatography is a kind of separation and analysis method, and separation is the core. Therefore, the chromatographic column responsible for separation is the heart of the chromatographic system. The requirements of chromatographic column are high column efficiency, good selectivity and fast analysis speed. It is also a knowledge about the installation, maintenance and preservation of chromatographic columns and the chromatographic failures caused by chromatographic columns.
The proper use of high performance liquid chromatography column includes: installing protective column, filtering mobile phase and sample, purifying sample, avoiding high column pressure, paying attention to temperature and pH, washing correctly and timely.
01. Installation of protective column
The function of the protective column is to filter out the chemical "garbage" from the mobile phase and the sample, and effectively remove the insoluble matter in the mobile phase and sample. Although the current chromatograph is equipped with filters with different pore sizes of 1 and 2 μ m at the inlet of the mobile phase adsorption, the back of the injection valve and the two ends of the chromatographic column, the filter can only remove insoluble particles but not chemical pollution. Therefore, it is necessary to install a protective column, especially for the analysis of complex mixtures and biological samples such as traditional Chinese medicine and Chinese patent medicine.
The packing of protective column should be consistent with that of analysis column, and the particle size can be larger than that of analysis column. The protective column is a consumable. After a certain number of sample analysis (50-100 times), the protective column should be replaced when there is a significant increase in column pressure, deterioration of peak shape or baseline drift.
02. Filter the mobile phase and sample
In principle, all the mobile phases passing through the chromatographic column should be filtered, but the actual operation should be different according to the specific situation: when the organic solvent uses the chromatographic grade, the water uses the quartzite sub boiling water or other ultra pure water, the filtration has no more practical significance when the quality of the water and organic solvent can be ensured and there is no pollution.
When the mobile phase contains buffer salt, filtration is necessary. As mentioned above, when the buffer solution is placed for a period of time (depending on the ambient temperature, it generally does not exceed 48 h in summer and less than one week in winter), it should be re filtered before use.
The prepared sample solution should be filtered by 0.45 μ m (0.22 μ m is better) pore size filter before being injected into the flow system. Pay attention to distinguish the water system and oil system, and do not mix the two to prevent the filter membrane from dissolving and causing pollution.
The container containing mobile phase and on-line filter in chromatographic system should be cleaned and replaced regularly to avoid filter failure due to overload.
03. Clean the sample
When the sample contains chemical components harmful to the chromatographic column, such as proteins, sugars and other organic molecules and strong adsorption substances, as well as group ions that may damage the column packing. Before injection, the sample should be separated and purified as much as possible to remove the excess impurities.
04. Avoid excessive column pressure
Although the high performance liquid chromatography (HPLC) column can withstand a pressure of up to 6000 psi (pounds per square inch), high and excessive pressure changes will shorten the life of the column. The method to avoid this is to
(1) When the column pressure is too high, the flow rate should be as small as possible, and it should not exceed half of the maximum allowable pressure of the column.
(2) when the flow rate is large, the method of velocity gradient should be adopted to make the flow rate in place step by step.
(3) When using manual injection valve, switch the injection valve as quickly as possible. Otherwise, more than 20% of the pressure impact will quickly scrap the column. When multi column combination is used, column switching should be avoided under high pressure.
05. Pay attention to temperature and pH
Generally, the maximum operating temperature of silica gel based chromatographic column should not exceed 60 ℃, and should be lower than 40 ℃. Especially when the pH of mobile phase is close to the limit of use, the operating temperature should be reduced.
If the temperature is too high, it will accelerate the hydrolysis of the bonded phase and the dissolution of silica gel, which will change the packing properties, collapse the column bed, reduce the column efficiency and change the peak shape.
At present, the range of pH indicated by bonded silica gel column can reach 2-10. However, extreme pH conditions should be avoided in actual use, especially in alkaline conditions (pH ≥ 8). If it is necessary to use under extreme pH conditions, the following methods can be used to avoid column damage:
(1) a precolumn (saturating column) is added between the pump and the injection valve to saturate the mobile phase
(2) Reduce operating temperature
(3) Immediately after the test, clean thoroughly with a "mild solvent" that is miscible with the mobile phase used.
06. Correct and timely flushing
Before starting the test, the solvent in the column should be determined. For the new chromatographic column, the mobile phase used in the evaluation report unless otherwise specified. The current solvent in the column must be mutually soluble with the flow used for the analysis sample. If it is not miscible, a third solvent that can be miscible with both of them should be used for transition.
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