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huge hump in the chromatogram
Posted: Fri Jun 08, 2007 5:18 pm
by mimisandhu
I am analysing drug in capsule(gelatin capsule) and getting a huge hump before the parent peak. The method works fine for the drug substance. the hump is coming where the two impurities are supposed to be. My mobile phase A is 5/95/0.1:ACN/H2O/TFA, mobile phase B is 95/5/0/1:ACN/H2O/TFA. The Column is C18 and the gradient is
Initial---------5.6%B
10.0 Min.-----50%B
15.0 Min.-----100%B
Please advice how can I get rid of the hump. Thanks in advance.
Posted: Fri Jun 08, 2007 5:35 pm
by Noser222
Have you run a blank gradient? If not, TFA is notorious for causing baseline problems. Read a little more about the reasons in this post:
http://www.sepsci.com/chromforum/viewto ... hlight=tfa
Posted: Fri Jun 08, 2007 7:41 pm
by DR
Water check:
Let sit pumping at initial conditions for ~20 min, then run back-to-back blank injections. If the hump is a lot smaller in the second blank, you need to clean up your water, then remake the A phase.
For details, search "Empore"
Posted: Mon Jun 11, 2007 1:28 pm
by mimisandhu
When I run the drug substance only with same column, mobile phase, gradient etc. I don't see the hump at all. it is when I run the drug in capsule that I start to see the hump and it grows with each injection.
Posted: Mon Jun 11, 2007 6:06 pm
by Mark Tracy
Is there dissolved gelatin in the drug sample? Run a gelatin sample and see if that is the source of your hump. If so, you may need additional cleanup prior to analysis.
Posted: Mon Jun 11, 2007 6:32 pm
by mimisandhu
yes! I filtered the sample and also centrifuged it and by doing that I get a clear solution but It is causing that hump at 4-6min. where two of my impurities are eluting. If anyone had similar problem please let me know how to solve this problem. Thanks!
Posted: Tue Jun 12, 2007 5:27 am
by JM
What is in your capsule besides drug? if your standard run is Ok , it indicates that your sample containes some other excipients or your drug interecting with geletin . run one placebo and check.
JM
Posted: Tue Jun 12, 2007 4:48 pm
by Mark Tracy
Dissolved gelatin will pass through an ordinary filter. They are kind of expensive, but centrifugal ultrafilters with a 3000 MW cutoff will remove dissolved proteins. Grace and Millipore are two sources. Another alternative is an SPE cleanup, but that is actually more work than the ultrafilter.