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Dear All
We developed a method (LC-MS/MS) for certain lipophilic molecules by
using Shimadzu Sil HTc, in isocratic mode with 80% acetonitrile there
was no carryover at all. Whenever I changed the same method to
gradient (from 10% organic to 85 % organic linear or one step
increment) it was found a huge carryover in gradient program. All
the seals and valves of the system were working fine.
Can anyone suggest me how to avoid this carryover?
Thanks in advance
Dev
devchem4.-at-.yahoo.co.in
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Dear Dev,
I have experienced the same phenomenon.
It appeared that my gradient did not completely wash the trapped
analyte of the column. Eventually I could resolve it by changing the
pre-column (it apparently trapped some of my analyte), but in your
case it could also be your analytical column of course. It's also
possible that you have some "leakage" of your analyte in your system
somewhere that is gathered on your analytical column during
restabilisation of your column with 10% modifier. You could also
change the pH of your eluent to improve elution from your column.
Good luck,
Rob ter Heine
--
Rob ter Heine, MSc, PharmD
Department of Pharmacology, Slotervaart Hospital
Amsterdam, The Netherlands
E: rob.terheine.at.slz.nl
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Make sure your injector loop "sees" the complete gradient; this may or
may not help.
Best regards,
Frederik Pruijn
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Dear Dev,
b) Don't get confused with gradient peaks as carryover.
Depending upon the gradient program and purity of solvents you have been
using for your method you can see number of peaks in gradient
separation, which cannot be found with Isocratic separation.
With Regards,
Veeravalli Vijaya Bhaskar,
Research Associate,
Aurigene Discovery Technologies Limited,
Bioanalytical Division,
Bangalore,
Email: vijayb.at.aurigene.com
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Dev,
Check if your mobile phase got contaminated with the analyte. This will
give you a peak every time you run a gradient, with the analyte
accumulating on the column during the initial step, and eluting as a
peak at the high-organic step. It is easy to check - simply run the
gradient without injecting anything (the best thing to do is to bypass
the injector completely) and see if you still get the peak.
Hope this helps,
Andrew
Andrew Volosov, Ph.D.
Associate Director, DMPK
Preclinical Development
Sepracor, Inc.
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The following message was posted to: PharmPK
Dev,
Check if your mobile phase got contaminated with the analyte. This will
give you a peak every time you run a gradient, with the analyte
accumulating on the column during the initial step, and eluting as a
peak at the high-organic step. It is easy to check - simply run the
gradient without injecting anything (the best thing to do is to bypass
the injector completely) and see if you still get the peak.
Hope this helps,
Andrew
Andrew Volosov, Ph.D.
Associate Director, DMPK
Preclinical Development
Sepracor, Inc.
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