How to operate the cleaning and regeneration of HPLC column?
In general, for the commonly used reversed-phase chromatographic column, if the column efficiency is seriously reduced, it is necessary to consider cleaning and regeneration. It is recommended that the following process be used for washing:[flushing process]
20 times column volume water: acetonitrile = 95%: 5% (used to remove buffer salt in the system, and the proportion can be adjusted according to the concentration of common buffer salt)
Acetonitrile 20 times column volume
Isopropanol 5 times column volume
N-hexane with 20 times column volume
Isopropanol 5 times column volume
Acetonitrile 20 times column volume
20 times column volume water: acetonitrile = 95%: 5% (the same proportion can be maintained as step 1)
Rebalance the column with the required mobile phase ratio
As for the regeneration of chromatographic column, I believe you can see a lot of related articles. It is suggested that you can choose the regeneration conditions according to the actual needs.
Today I want to talk about the volume of the column in the conditions given above.
In fact, when the chromatographic column is washed, many laboratories are used to use the washing time to evaluate whether the desired effect is achieved. For example, a master told you to flush the pillar for half an hour.
However, the volume of washing solvent should be used to evaluate the washing effect. For example, 5 times the column volume of isopropanol, 20 times of column volume of acetonitrile, etc., what do these column volumes refer to?
In fact, the volume of the column needs to be calculated according to the inner diameter and length of the column you use.
It is easy to calculate the column volume by the formula. For example, the volume of a column with 4.6 mm inner diameter and 150 mm column length is as follows:
If you want to wash the column with 20 times the volume of acetonitrile, you need about 34 ml of acetonitrile. If your column flow rate is 1ml / min, you need to rinse with acetonitrile for 34 minutes.
When the column flow rate was 1.5 ml / min, it was necessary to rinse with acetonitrile for about 23 minutes.
I don't know. Can you understand that?
In the future, when we discuss the column washing in the wechat group of chromatography school, please don't just tell me how long it took you to wash the chromatographic column, but also the flow rate!
Finally, we should remind you that when washing the chromatographic column, do not connect the detector at the end of the column, but a container that can store the waste liquid.
