In the process of chromatographic analysis, it is often necessary to use buffer salts to adjust the pH value of mobile phase. Improper use of buffer salts may cause column pressure increase, column efficiency decrease and the retention time of compounds change.
Column pressure rise
Reason: Improper use of buffer salt leads to buffer salt out, blocking the pores between frit and bonded particles, blocking flow mass transfer and causing column pressure rise
The retention time of the same compound changes
Reason: The salt contained in the column can change the retention time of the compound if the sample is injected without rinsing
Column efficiency decline
1) Some buffer salts will penetrate into the depth of the bonding phase, damaging the silica gel matrix, resulting in column bonding phase loss, column bed loosening, and column efficiency decline
2) Condensation on the surface of the bonded phase makes it difficult for the C18 carbon chain to stretch, and the retention ability of the material decreases, resulting in the decrease of column efficiency. Therefore, it is necessary to wash the chromatographic column after using buffer salt, and special attention should be paid to the concentration of buffer salt in water.
So how do you use buffer salt correctly?
Pre-use treatment: Flush the column with a mobile phase without buffer salt before using buffer salt as the mobile phase until the baseline is stable. In principle, the mobile phase used for flushing contains the same proportion of water (or more water) as the mobile phase used for analysis, except that the mobile phase used for flushing contains no buffer salts. Buffer salts are usually more soluble in water than in organic solvents. When the mobile phase containing buffer salts (especially when the mobile phase water is saturated buffer salt solution) is used for analysis, if the proportion of water in the mobile phase used to save the column before analysis is relatively small, do not rinse off first, and the mobile phase used for sample is large in organic solvent content, but the proportion of water contained in the buffer salt is not enough to dissolve, Buffer salts will precipitate and deposit on the column, which may cause the column damage described above.
Treatment after use: Flush the mobile phase with the same water content ratio as the analysis (the only difference with the analytical mobile phase is that the mobile phase used for flushing does not contain buffer salts) for about 30min until the baseline is stable. If the column is not to be used for an extended period of time and is to be stored for a long time, an additional step is to rinse the column with a pure organic solvent until the baseline is stable.
There are a few caveats to using buffers
- Avoid using carbonate, which has a corrosive effect on steel.
- Buffer is a good fungus culture medium, buffer is best to be ready to use.
- Do not use organic solvents directly after the experiment, organic solvents will precipitate salts, resulting in liquid path or column blockage.
- The use of buffer should be timely grasp the pH range, so that there are a few in mind.
- When cleaning liquid path and column, temperature control can be heated to 30 degrees Celsius for easy washing.
- If the buffer solution is used for a long time, it is necessary to pay attention to whether there is precipitation at the joint. If there is white salt precipitation, it can be considered to wash the liquid path with 10% nitric acid for a certain period (remove the column, walk 30mL, and then rinse with 5 times water) to avoid the blockage of the liquid path.
- Reliable reagents should be used in the selection of buffers to avoid unnecessary trouble caused by impure salts.
If the proportion of organic solvents in the mobile phase is very high can not be used to wash buffer salt, is not washed out. Usually the C18 column is washed with 5%~10% methanol first, which can wash out the buffer salt, and then use pure organic solvent to protect the column. The best method is to use the mobile phase with the same concentration as the mobile phase does not contain salt for cleaning. But it’s just slower. Water is used for quick replacement, generally within 15 minutes is the best, and the flow rate of 0.8 is better. If pure water is used, it is easy to cause the loss of bonded carbon chain, so it is better to use 5%~10% methanol solution to flush. The buffer in the mobile phase can be replaced by pure water, while the organic phase remains unchanged. It’s safer to wash the post this way.
It is necessary to use buffer salt correctly, not only to prevent buffer salt out, but also to achieve the purpose of improving the column life. We can sum up its use in one sentence: filter before use, rinse after use.