Maintenance and repair of C18 chromatographic column

In the daily separation and analysis work, whether the chromatographic column is used properly or not directly affects the life of the chromatographic column. The following describes the matters needing attention in the daily use of C18 column.
(1) During the loading, unloading and replacement of columns, the action should be light, and the joint should be properly tightened. Strong mechanical vibration must be prevented to avoid voids in the column bed.
(2) If the instrument is used for routine analysis, the sample types are limited, but the analysis times are large, it is advisable to configure a special column for each type of routine analysis, which will help to prolong the service life of the column.
(3) If the column temperature control device is used, it should be noted that the temperature can be raised only after the mobile phase is introduced.
(4) The mobile phase should be degassed before use, so as not to reduce the column efficiency and affect the detection. The sample solution should be properly pretreated and filtered to reduce column contamination and blockage.
(5) When changing the mobile phase, it is necessary to pay attention to the mutual solubility of solvents to prevent salting out.
(6) The actual operating pressure of the column should be lower than the maximum pressure when filling, preferably less than half of the maximum pressure. Generally, it does not exceed 20 593.965-29419.95 kPa. When used in the range of low pressure (≤ 14709.975 kPa), the column can keep high column efficiency for a long time.
(7) C18 column is a non-polar bonded column. The pH value of mobile phase should be strictly controlled between 2 and 7 to avoid damaging the column.
(8) After the separation and analysis work is completed, it is not necessary to stop the machine immediately. It is necessary to flush the chromatographic analysis system in time, generally more than 0.5 h, to remove impurities in the chromatographic column.
(9) If there are salts in the mobile phase, wash them with water first. If amines (such as trimethylamines or tetrabutylamines) are added to the mobile phase, wash them with a mixture of 50% methanol and 0.05% phosphoric acid solution, not just water.
(10) C18 generally uses 100% methanol as the preservation solvent to prevent the column from cracking and damage. Do not keep water or buffer solution in chromatographic flow path for a long time.
(11) A suitable C18 column was selected to protect the analytical column from the influence of impurity particles and irreversible adsorbed interfering substances. The particle size of packing in protection column should be consistent with that of analysis column as far as possible.
(12) The shelf life of the column should not be too long. The C18 column, which was not used for a short time, was washed with methanol for 30-60 min, and then both ends of the column were sealed. For columns that are not used for a long time, one is to wash them regularly and then seal them again; the other is to install a container filled with methanol with a certain capacity at both ends of the column after flushing (only one end can be filled), so as to supplement the evaporation of solvent in the column during a long storage period.
repair
In the daily use of chromatographic column, although the protection is strict and the sample and mobile phase are pretreated, it is still difficult to completely avoid the column from contamination, loss of stationary phase, hardening, collapse of column bed and decrease of column efficiency after a long time of use. Some columns can be restored by maintenance.
3.1 column pollution regeneration technology
After the column is polluted, it can be washed with suitable solvent to regenerate the column efficiency. The conventional regeneration washing method of C18 column is: methanol, chloroform, methanol / water 60 ml respectively pass through the chromatographic column in turn, and then use 100% methanol 60 ml to balance the chromatographic column, and then seal up, the column efficiency will return to normal. If necessary, the column should be washed with 0.05 mol / L H2SO4, 0.5 mol / L H3PO4 or 0.1 mol / L EDTA sodium salt, and then washed with water. Finally, the column was balanced with 100% methanol and then sealed. For the seriously polluted C18 column, water, methanol, chloroform and ethane can be used for washing in turn, and then the column should be reversed and then rinsed once again. The solvent used for each time is 60 ml, and the detector is not connected. Finally, a 100% methanol balanced chromatographic column is used for sealing.
3.2 remediation of column contamination
When the regeneration of column pollution is invalid or the column pollution is known to be serious, the column repair method can be used, but the column pollution depth should not exceed 5 mm. The method is to dig out the contaminated part with a special shovel, and then mix the stationary phase and the mobile phase which are the same as the column filler into the slurry, and then carefully fill the slurry like stationary phase into the excavated part (try to make the later filled fixed phase close to the original tightness) and level the end face. If the pore size of sieve plates at both ends of the column head is the same, the column can be reversed and used for normal time. The purpose is to recover the compactness of column bed with the help of mobile phase flushing.
3.3 restoration of column collapse
There are many reasons for column collapse. When the collapse is not serious, if the sieve plate aperture at both ends of the column head is the same, the column can be reversed and used for general time to recover the performance. When the collapse is serious (about 5 mm), it can be repaired by column pollution.
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