decreasing retention time problems

[aceto_81]

What kind of column are you using? A special 100% water compatible column?
Maybe flushing with some organic after a few runs could restore your retention times?

Ace

[kyle432]

its a propac wcx-10 column used for protein analysis.

this method used has been in place for many years and this issue had never occurred before i dont know if this change of introducing organic would be taken on board by my supervisor as he could see how it would work.

can i ask if during a run you introduce regular organic washes, wouldn't the system then need to be conditioned again as well as flushed with the buffers as if it was another natural start of a batch? due to time restraints we have, this again would not be a possibility.

kyle

[aceto_81]

I have no experience with the propac wcx columns, and I'm not sure if the organic wash would help or not, but it is something relative easy to try.
I don't know how much equilibration is necessairy afterwards, but again, if it helps, it's an easy fix.

Some other thoughts: how did you switch your buffers when using line 1 & 2 (the experiment you described above)? Did you wash, shutdown the pump, ... or just put the line from bottle 1 to 2?
Have you already measured the flow at the beginning of your set, and at the end? (Other indication: does the injection peak also decrease in retention time?)

Ace

[HPLCaddict]

Ahh, OK, so we're talking about ion-exchange and you're using pure buffers as eluents, right?
I think most of the contributors (including me) we're thinking about reversed-phase...stupid reflex .
Then stay away from organics.

Some further questions, you just switched to the second bottle of your buffer and retention times were restored to the correct values, right? You didn't include any column washing procedures between the switch? And using that second bottle of buffer, retention times slowly started to drift again?
How did you store that second bottle? Was it capped? Possibly in the fridge? What kind of buffer is this? Possibly something like the famous phosphate "buffer" pH 5.0?

[aceto_81]

Ahh, OK, so we're talking about ion-exchange and you're using pure buffers as eluents, right?
I think most of the contributors (including me) we're thinking about reversed-phase...stupid reflex .

It would be easier troubleshooting if such method details were included in the first post... Now only after x posts we found out we're talking about ion-exchange

...
Then stay away from organics.
...

As I said: I have no experience with those type of columns, so please ignore my previous post for that part.

Ace

[kyle432]

ah again im sorry i forgot to mention that.

inbetween sample brackets we include a blank injection using our eluent. so after the first bottle was used (from line A) for one sample bracket we then switched to the other bottle (line C) for the next bracket meaning a blank was done before the samples were injected.

the initial bracket of samples on line A showed decreasing retention times however the confusion being that when we switched to line C the samples within that bracket started again as normal with good peak times however it then also began decreasing in terms of retention time.

Line initial retention time ending retention time
A 17.71 14.22
C 17.59 14.48

the bottles were treated the same. we made a 5L amount of MES buffer with ph5.7 using the guidelines of the method that has always been used and seperated the buffer into two identically sized bottles. the other buffer used for gradient contains similar constituents.

thanks again everyone

[kyle432]

sorry about that as well ace. im still relatively new to hplc/uplc and ms and this is my first ever forum post about the matters in hand so my apologies.