By Anonymous on Monday, April 26, 2004 - 11:00 pm:
Hi all,
we have a very bad developed method where the peak of interest would come out right at the solvent front peak, overlaying the negative dip right in front of the solvent front.
At times, we can get this -ve dip real small as to not interfere significantly with peak of interest.
But what is the real reason for such dip, and any way to minimise it?
mobile phase: acetonitrile-lineA
Buffer ph6.3(adjusted with KOH)-line B
please advise
thanks in advance
By Anonymous on Tuesday, April 27, 2004 - 07:24 am:
I think it is due to a difference in rafractive index/UV absorbance between your solvent used to dissolve your sample and the mobile phase. try dissolving your sample in mobile phase to minimise the interference.
John
By MG on Tuesday, April 27, 2004 - 10:35 am:
Decrease acetonitrile content until peak of interest is retained on the column.
By Mike on Wednesday, April 28, 2004 - 12:40 am:
Thanks John and MG,
I am afraid that changing the sample diluent to mobile phase would not be possible since this has already been validated and in my regulated environment, highly not possible to change being the end-user.
Yes,the difference in UV absorbance may have caused the dip.
maybe i can try to decrease the acetonitrile content slightly. :(
thank you your time.
Mike
By Anonymous on Wednesday, April 28, 2004 - 01:44 am:
Shows that regulation and 'quality' often not only doesn't contribute to scientific quality but detracts from it. Surely there must be an island somewhere where we could put all the 'quality' people and make them count the blades of grass or sand grains? therby leaving the creative people to get on with things.
John
By HW Mueller on Wednesday, April 28, 2004 - 02:27 am:
Anon, April 28,
it looks more like those who try to do honest science will be on an island (or already are).
By Mike on Wednesday, April 28, 2004 - 09:19 am:
What can we do?
they want to come out with the product fast and develop this bad method.
they are already on a holiday island counting $$$, not grass or grains.
will get back to you people once having try out the suggestion.
Mike
By Anonymous on Wednesday, April 28, 2004 - 08:35 pm:
You may need to take a closer look at what has changed since the method was "validated". Are you using the exact same vendor on reagents? Try different brands. On the quality side, from a pharma analytical manager's perspective, I would advise you that if the method is not suitable for the intended purpose (appears that way), then you have a (serious) problem if you are working in a "regulated", like by FDA, environment. If the method does not give accurate and reliable results, then someone is obligated to fix it.
By Mike on Thursday, April 29, 2004 - 10:07 am:
some group is seeing to it but that may take months and years till it is resolved.
some suggested that it could be also minimised by setting on certain HPLC. Of the 5 LCs tried, 3 of them can be minimised. 2 of them, no matter of days of cleaning and flushing could not have the dip minimise.
Could different LC contribute for such cause?
Mike
By MG on Thursday, April 29, 2004 - 10:18 am:
If a peak of interest is eluting at the solvent front, the best solution is to change your LC conditions so that your peak of interest is retained. Then your solvent dip won't matter. Otherwise, what purpose is your column serving? As an expensive filter?
By MS on Friday, April 30, 2004 - 12:38 pm:
Turn off your degasser. Inject an air peak and see if the dip gets bigger. Or, as suggested above, use column/conditions that retain the peak longer. These suggestions, I realize, are a bit too logical for a validated method.
By Chris Pohl on Friday, April 30, 2004 - 01:50 pm:
Mike,
Of the five LC's you have tested, are all five with the same equipment? Or are you dealing with different UV detector brands with the five different instruments? The refractive index sensitivity of different detectors vary significantly from one vendor to another. Since your problem is most likely due to a refractive index effect in the flowcell (as noted above), it wouldn't be surprising to find that the problem was detector specific.
By HW Mueller on Monday, May 3, 2004 - 12:51 am:
From my experience one can surmise that the closer to the tm you are the more interference (overlap) you have, useful chromatography seems to start some minutes after tm (to). Also, it seems to be an impossibility to eliminate all baseline disturbances at tm. But then there are some who deliberally do this "chrom.", riding on the fronting peak, forgot what itīs called), a proceedure I never understood, anybody out there to enlighten on this?
By B.Buglio on Monday, May 3, 2004 - 06:03 pm:
MG (4/29) speaks to the critical issue here.
Theres no chromatography going on w your present
system. This means that specificity is
unachievable and this method is not validated. You
may have some data but it wont stand up to
scientific scrutiny.
You need to "slow" down the elution with either a
different mp and/or a different column and
revalidate.