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Help wrt. separation needed !
Posted: Sat Jul 02, 2011 9:21 am
by Humad
I am trying to remove these product related impurities that pops up as pre and post peaks around our product on our analytical system. The only preparative techinque that works in lab is a weak anion exchanger, but the impurites are comming out as shoulders effecting the purity.
I have tried playing with the temperature, pH and conductivity gradient. Any idea how i can have shoulders to come out as peaks ?
BR
Re: Help wrt. separation needed !
Posted: Sun Jul 03, 2011 6:54 pm
by tom jupille
Possibly:
- decrease the load
- use a smaller particle size (if you are not overloaded!)
Certainly:
- use a longer column (and a proportionally longer gradient)
Re: Help wrt. separation needed !
Posted: Sun Jul 03, 2011 7:16 pm
by unmgvar
can you provide more information on the nature of your compound?
you talk of using an analytical system, yet you say you do purification
what are the details of the analytical method you have and how different is it from the prep method you are using?
maybe you do not have all the parameters transferred correctly between the 2 columns sizes
in most cases when prep is done in an analytical system, flow rate are not used matching the linear velocity between the 2 columns leading to differences in the chromatography and purity
Re: Help wrt. separation needed !
Posted: Mon Jul 04, 2011 4:05 pm
by Humad
Possibly:
- decrease the load
- use a smaller particle size (if you are not overloaded!)
Certainly:
- use a longer column (and a proportionally longer gradient)
Load is already decresed to the absolute minimum.
We are using a very small particle size ( 15 microns )
The lenght of the column is 20 cm and any thing longer that that gives us pressure problems once we scale up !
Re: Help wrt. separation needed !
Posted: Mon Jul 04, 2011 4:12 pm
by Humad
can you provide more information on the nature of your compound?
you talk of using an analytical system, yet you say you do purification
what are the details of the analytical method you have and how different is it from the prep method you are using?
maybe you do not have all the parameters transferred correctly between the 2 columns sizes
in most cases when prep is done in an analytical system, flow rate are not used matching the linear velocity between the 2 columns leading to differences in the chromatography and purity
Sure , its not a compund but instead a protein ( 15 Kda)
We use a C18 (RP) and acetonotril in our analytical system which is something we cannot use once we scale up.
In preparative mode we have a strong anion exchanger with non organic buffers. But I am having problems getting the very close product related impurities we see on analytical system to separate on the preparative system !
Re: Help wrt. separation needed !
Posted: Mon Jul 04, 2011 6:39 pm
by unmgvar
is it an AKTA system?
what is the resin exactly you are using and mobile phase?
what are the impurities nature that made you choose the WAX?
are we talking of mono, dimer, HMW or is it something else?
moe like:
15 kda is something like lyzozyme and I have know an application for it on SCX and not WAX. maybe it would be better for you?