Tuesday, 18 January 2011

Help on: Equilibration of HPLC columns between gradient method injections

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Question:
“I have a question about column re-equilibration after each injection ready for the next one. Firstly, is there a way how to calculate how long the re-equilibration step should be (or is it more of experience and trial and error)? I am using a 30 x 4.6mm column (2.7 particle size), HP1100 pump, 1.7ml/min flow rate, and 2 min gradient method (as you can see I’m after a fast turn-around!). I’m just a bit worried if I’m giving the column enough time to re-equilibrate ready for the following injection. My gradient is as follows:

Time - %A - %B
0.00 - 90.0 - 10.0
0.10 - 90.0 - 10.0
0.50 - 10.0 - 90.0
1.50 - 10.0 - 90.0
1.60 - 90.0 - 10.0
2.00 - 90.0 - 10.0

I am also taking advantage of the next injection’s injection time (25 seconds) which I’m assuming can also be used as part of the re-equilibration step? I have done a series of injections (~10) and the retention times are pretty constant using this 2 min method. Though I was also wondering over how many injections could you justify the method reliable, would 10 be enough?”

Answer:
“A general rule for equilibration is to allow 10 column volumes of mobile phase to pass through the column. Column volume refers to the amount of space in a column which can be taken up by the mobile phase, or the space not taken up by stationary phase packing. A previous blog on column equilibration describes the calculations for determination of this value and the MTS HPLC calculator can convert this to a time value, you may find these helpful.

For your method 10 column volumes is equal to 2.1 minutes. Given the length of your analysis I think that this would be too much time to spend on re-equilibration. Your current equilibration time is half a minute in the gradient table and another 25 sec for the injection which equates to about 5 column volumes. I feel that this should be sufficient if the method can be demonstrated to be working reproducibly. The signs of insufficient equilibration are baseline problems and variation in retention time so if you can show that these are constant then you can justify re-equilibration using 5 column volumes.

The method conditions will determine whether 5 column volumes is enough for re-equilibration, i.e. how different the starting and end conditions of the gradient are. Although your gradient is quite steep which means it has to go back from 90% B to 10% B in the re-equilibration, the 10 injections that you have performed give a good indication that the method is working fine. It would probably be good to back this up with some results over a longer time frame. Rather than setting up an experiment to test this I would suggest monitoring the use of the method when you analyse samples.”

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