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Respected all,
Sir this is vasu working as scientist in India, am working on an =20
cancer drug in API 2000 , Column is giving peak fronting , even i =20
tried new columns also. and this fonting is not appearing in old =20
column. i would like to know what could be the reason , and necessary =20
precautions to be taken
Column: Flexit 150*4.6mm C18 5=B5 size .
Mobile Phase: 0.1% Formic Acid:Methanol 20:80
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Dear Mr. Basu,
#1.After how many injection the column is not able to elute the peak
effeciently?
#2. Any prehistory of the elution of drug with any other column?
#3. The probable cause is phase collapsing of the column due to
formic acid as it is seldomly used for C-18 column.
#4. Suggestion is that it need proper washing with high content
acetonitrile with water (60:40) for longer period of time.
#5. if problem still persist then you have to remove formic acid.
Regards
Vineet Singh
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The following message was posted to: PharmPK
The key thing about columns is maintenance. Sometimes it is not easy
to do that particularly for beginners.
I know of researchers who have used a single column even for two
years without changing it
For cleaning purposes always begin from what the guidelines say i.e
use methanol: water 50:50
Acetronitrle is more expensive than methanol so even if you are using
it as the mobile phase always use methano water to clean your column.
s.o.o
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Dear Basu,
I believe your problem is due to the quality of packing of column.
Have you tried another brand of column? (I like phenomenex columns
like Gemini, luna). Using formic acid is perfectly fine, as it is
volatile acid suitable for LCMS purpose.
Vinayak Nadiger
Manager , Bioanalytical Chemistry
11 Biopolis Way, Helios #08-05
Singapore 138667
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Dear Basu,
I hope I'm not repeating somebody else with this message. In my
experience the main reason for peak fronting is column overloading. I
did not understand from your message what were the concentrations you
used. The column brand you used is also unknown to me. However, try
diluting your sample. Using LC/MS/MS (ABI 2000?) you can certainly go
down quite a bit.
Best regard,
Stefan
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The following message was posted to: PharmPK
I would agree that column overloading is a common cause of reason for
peak fronting. Another common cause of peak fronting is solvent
content in the injectate, so if you have solvent in your sample try
reducing the percentage of solvent or reducing your injection volume
so less solvent is hitting the column during the injection.
Formic acid at the concentration you are using (0.1%,correct?) should
be fine for chromatography.
Hope this helps,
Brian Faulkner
Scientist
Vitalea Science, Inc
www.vitaleascience.com
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