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Problem! Retention time increase by time in Codeine analysis
Posted: Wed Dec 12, 2012 3:07 pm
by ultima_86
Hi everyone, this is my problem. I analyze Codeine with buffer prepare as following: 1.6g Sodium butansulfonat in 780ml Water and make up to volume with CH3OH then adjust pH 2.8 with HCl 2M. My mobile phase consists of 100% buffer and it is isocratic. The flow rate is 1.5ml/min, one injection lasts 25 minutes.
The RT of Codeine is always around 5-6 minute at first but after a few injections ( 3 or four) it will increase a bit (0.2 - 0.3 minute). After more than 15 injections, the RT from my first injection and my 15th injection is extremely different ( the RT of first injection is 5 and the 15th is 5.5). That make RSD of RT isn't good whereas the area after 15 injection is the same.
I also have thought that give the column more time for equilibration so I inject 15 injection before my real analysis but the RT still inceases as the above mentioned way above.
What can I do to stop the RT increase? Please help me. Thanks!
Re: Problem! Retention time increase by time in Codeine anal
Posted: Wed Dec 12, 2012 3:20 pm
by prepcolumn
Hi everyone, this is my problem. I analyze Codeine with buffer prepare as following: 1.6g Sodium butansulfonat in 780ml Water and make up to volume with CH3OH then adjust pH 2.8 with HCl 2M. My mobile phase consists of 100% buffer and it is isocratic. The flow rate is 1.5ml/min, one injection lasts 25 minutes.
The RT of Codeine is always around 5-6 minute at first but after a few injections ( 3 or four) it will increase a bit (0.2 - 0.3 minute). After more than 15 injections, the RT from my first injection and my 15th injection is extremely different ( the RT of first injection is 5 and the 15th is 5.5). That make RSD of RT isn't good whereas the area after 15 injection is the same.
I also have thought that give the column more time for equilibration so I inject 15 injection before my real analysis but the RT still inceases as the above mentioned way above.
What can I do to stop the RT increase? Please help me. Thanks!
This is possibly because of using ion-pair agent in your mobile phase. Longer chain sulfonate salts (hexane, heptane etc) may decrease this equibration time (it also changes your RT)
Re: Problem! Retention time increase by time in Codeine anal
Posted: Wed Dec 12, 2012 4:19 pm
by Vlad Orlovsky
prepare higher concentration buffer (5 times higher) and flash your column with it, then switch to your regular method
Re: Problem! Retention time increase by time in Codeine anal
Posted: Fri Dec 14, 2012 9:49 pm
by ultima_86
Thanks for your reply. I have tried using Sodium hexan instead of Sodium Butan. The RT seem to be stable but longer and I aslo got a poor, broaden peak shape. Is there some ways to resovle these problems?
To increase the concentration of buffer I thought it is very hard to do because the IP reagent quite expensive. But thanks anyway, I will try if I have a chance.
Re: Problem! Retention time increase by time in Codeine anal
Posted: Fri Dec 14, 2012 9:55 pm
by Vlad Orlovsky
May be one day you can consider mixed-mode chromatography where ion-pairing reagent is attached to the surface:
http://www.sielc.com/Compound-Codeine.html and retention time is controlled by amount of ACN and amount of ions in the mobile phase. You don't need to equilibrate your column with ion-pairing reagent, you don't need to dedicate column for analysis with ion-pairing reagents and you life will be much easier.