Column installation, commissioning and maintenance

1. Matching of column head type and stainless steel capillary joint
Chromatographic column is a consumable, not the original equipment of the instrument. If the depth of the joint and the column head do not match, there will be leakage or excessive dead volume. The depth of the joint is longer than that of the column head, so it is not easy to tighten and leak liquid; the depth of the joint is shorter than that of the column head, and there is a gap in the column head, resulting in the birth and death volume, broadening the spectrum band and tailing the peak.
2. Matching conversion of solvents
If the solvent does not match with the mobile phase, it should be converted before use. Especially when the mobile phase contains buffer salt, if the preservation solvent is pure organic phase or organic phase is higher, if the new column is used directly, the buffer salt will crystallize and precipitate in the column, and the new column will be permanently and irreversibly damaged. The preservation solvent of normal phase column is n-hexane. If it is to be converted to HILIC column mode, dichloromethane or ethyl acetate should be used during the conversion due to the poor mutual solubility of n-hexane and methanol acetonitrile.
3. Balance and aging of new column before use
Generally, the balance has been carried out in the factory inspection, but the time from the chromatographic column to the end user is different. It is better to rebalance the chromatographic column before the formal determination by the user. The best way to balance and age together is to run a complete analysis program (including injection) several times until the stability of peak shape, retention time and peak area is observed. The so-called "aging" is a term borrowed from the gas phase in order to achieve the adsorption saturation of analytes in the chromatographic column and the whole flow path of the liquid phase system. For some specific analytes, such as those with molecular weight greater than 1000, the aging time will be longer due to the slow diffusion speed, so the aging can be accelerated by large concentration injection or continuous injection of multiple needles in the same elution cycle.
4. PH application range
It is generally considered that the pH range of silica gel matrix column is 2-8, which is very rough. The type of silica gel, the temperature of use, the type of stationary phase bonded on the surface of silica gel, and the buffer salt have influence on this. The pH tolerance range of silica gel is larger than that of fillers with small pore volume and high bonding density. Some use high-quality silica gel, high-density bonding and double sealing, or special process can increase the pH tolerance range to 10. Phosphate buffer salt has strong permeability, and has the side effect of accelerating the dissolution of silica gel. Organic hybrid silica gel and silica gel filler coated with hybrid layer on the surface of silica gel can be used in the pH range of more than 12 due to the existence of organic matter. Read the instruction manual at the first time after getting the column to confirm the sealing solution in the column. If possible, column efficiency test can be carried out according to COA report.
5. Column preservation
For short-term storage (overnight or every weekend), it is better to use the mobile phase (without buffer salt) to minimize the balance time of the next use. It is generally recommended to use pure methanol or acetonitrile for long-term storage of reversed-phase columns. On the one hand, pure organic phase preservation can minimize the hydrolysis of bonding phase, but pure methanol (acetonitrile) will elute the bonded phase which has been hydrolyzed and temporarily adsorbed in the column, thus speeding up the loss process of the stationary phase and shortening the service life of the stationary phase. The pure organic phase has the disadvantage of easy volatilization and drying of the column bed. The use of about 80% organic phase is also a good choice, and can avoid the growth of bacteria. It is strongly recommended to label the column with the preservation solvent.
The CN based column is unstable in organic polar solvents (which may lead to the collapse of the column bed structure) and is suitable for low temperature storage in aqueous phase. The column bed should not be damaged due to freezing of stationary phase. 0.05% sodium azide solution can be added to the chromatographic column suitable for preservation in aqueous solution, such as ion exchange column and water-based SEC column to prevent bacterial growth.
It is recommended that the positive phase column be preserved in the mobile phase used, whether in the short term or in the long term.
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