[Chromatography Q & A] What solvents are used for washing and storage after using the column?
Most of the HPLC columns currently used are commercially available packed columns packed with packing agents. In this case, the solvent filling the column at the time of release needs to be replaced with the mobile phase to be used. Moreover, if the column is left with the used mobile phase in it, it may be deteriorated. Thus, the used mobile phase in the column needs to be replaced with an appropriate solvent. This time, centering on the washing method after using the column, points to consider when replacing the mobile phase, preservation solvent, etc. will be explained.
As shown in Figure 1, solvents in the column generally differ before, during and after use, and it is necessary to replace the solvent each time.
Solvents in column
Figure 1
Points
①First, check the type of solvent.
②Fully replace with the mobile phase to the HPLC system, and connect the column.
③When a buffer solution is used as the mobile phase, pay attention to precipitation of salt.
④The liquid volume is 15-20 times the column volume as a guide.
⑤Solvent that can elute the sample well and does not affect the column.
⑥When a buffer solution has been used as the mobile phase, pay attention to precipitation of salt.
⑦Solvents sealing at the time of purchase of the column are generally used.
⑧Do not use a solvent containing halogen as a preservation solvent.
⑨Seal the column tightly and protect from drying.
⑩Pay attention to elevation of column pressure.
1. Replacement of Sealing Solvent at Release / Preservation Solvent with Mobile Phase
Method
The liquid volume required to equilibrate from the solvent sealing in a column into the mobile phase to be used varies depending on a packing agent, column size, detector and mobile phase etc., but please consider the approximate amount to be 15-20 times the column volume. But Ion-pair chromatography may take time to equilibrate.
Points to consider
- First, check the type of solvent sealing in the column. If the solvent and the mobile phase are not mixed with each other, replace with a solvent that is mixed with both, and then replace with the mobile phase. At this time, pay attention to elevation of column pressure.
- When a buffer solution, etc. are used as a mobile phase, an organic solvent in the column may cause precipitation or crystallization of salts, which may cause column deterioration. Thus, it is necessary to consider, for example, replacing the solvent with water in advance.
- Connect the column after fully replacing also the inside of the HPLC system with the mobile phase. The column may deteriorate due to contamination with the mobile phase used previously. Special attention should be paid to the inside of the injector sample loop, damper, mixer, etc.
2. Replacement of Mobile Phase with Preservation Solvent, and Washing of Column
Method
Wash with a solvent that can elute the sample well and does not affect the column, etc., and preserve. As a preservation solvent, solvents sealing at the time of release of the column are generally used.
Points to consider
- When replacing the used mobile phase with the washing solvent, pay attention to mutual miscibility and elevation of column pressure.
- When a buffer solution has been used, flow distilled water to remove salts from the column. Direct flow of organic solvents may cause precipitation or crystallization of salts, which may cause column deterioration.
3. Column Preservation
Method
Be sure to seal tightly so as to form no gap or crack inside to protect from drying. Even when preserving for a long time, pass the solvent through the column once every 1 or 2 months.
Especially when preserving for a long time, it is necessary to wash the inside of the column sufficiently with a solvent having an appropriate elution force to wash impurities as much as possible. Figure 2 shows regeneration methods and column washing methods when the column is contaminated due to a long-term use, etc. General solvents for long-term preservation are shown in Table 1.
Figure 2. Column washing method for each packing agent
Table 1. Solvents for long-term storage of column
| Packing agent | Preservation solvent |
|---|---|
| Reverse-phase silica gel | Methanol or acetonitrile |
| Normal-phase silica gel Silica gel column Chemically modified packing agent column |
n-Hexane Ethanol |
| Ion-exchange silica gel | Water / methanol = 95 / 5 |
Points to consider
- Do not use solvents, such as those containing halogen, in which the packing agent and stainless steel tubes are eroded.
- Frequent washing or increasing the amount of washing solvent may shorten the column life, so please avoid them as much as possible.
Centering on the column washing method, points to consider when replacing the mobile phase, preservation solvent, etc. were mentioned above. Please make maintenance after using the column to ensure stable use of the column for a long period of time.