By Mattias T on Thursday, March 8, 2001 - 07:31 am:
We are setting up a method for the detection
of N-acetylaspartate by ion-pairing. The initial
tests showed promising results but the
resolution wasn´t satisfactory. We therefore
decided to change to another column, a ACT
Ace C 18 column (250 x 4,6 mm, 5um,
equipped with a guard column). On our very
first run with this new column we discoverd
periodically occuring peaks each 10 minutes
when monitoring the baseline. After
equilibrating with buffer for several hours the
peaks were still there. Now even closer to
each other (about 7 minutes). We´ve tried to
bypass the pulse damper of our pump system
(Varian 5500) and changing back to our old
column without any improvement. When
washing the column with 20 % MetOH
overnight and then equilibrating with buffer the
peak separation increased to 30 minutes but
after an hour they were back at the original
7-10 minutes.
We would be very grateful to anyone who has
a clue on how to solve this problem.
Buffer composition: 1,25 % MetOH, 2,8 mM
Tetrabutylammoniumhydroxide, 25 mM
KH2PO4.
UV-detection at 210 nm.
By bill on Thursday, March 8, 2001 - 01:18 pm:
If the peaks are coming off at at a reproducable time increment; I would be looking at your detector lamp. I have come across this before with ageing lamps.
bill
By Anonymous on Thursday, March 8, 2001 - 03:58 pm:
pH ?
By Mattias T on Friday, March 9, 2001 - 12:16 am:
pH is 7,0.
By Anonymous on Friday, March 9, 2001 - 02:45 pm:
At 210 nm, you'll see practically everything. It could be that your solvent mixer stops working periodically, if you use such a thing. Or it could be air bubbles in the pump or solvent mixer. Monitor the pressure carefully to see if there is a correlation between a change in pressure and the baseline spikes! If this is the case, you may want to prepare the mobile phase manually.
By Anonymous on Monday, March 12, 2001 - 08:54 am:
What about monitoring the baseline without column?
By Mattias T on Tuesday, March 20, 2001 - 07:34 am:
Thank you everybody for your response!
The last weeks we´ve been trying out all of
your ideas and everything else it seems :).
The results have been quite puzzling and the
peaks would disappear and reappear without
any simple explanations.
Then suddenly when we were trying a series
of runs with degassed buffers (Nitrogen),
which we had done before, the peaks
disappeared and this time for good.
Why this suddenly happened is beyond our
understanding. LC never stops to suprise
you...
Anyway, thanks for your valuable ideas!
By Anonymous on Wednesday, March 21, 2001 - 06:04 am:
Is nitrogen a good choice for degassing solvents? I didn't think it was. According to solubilities in the Merck Index, nitrogen is not "significantly" less soluble in water than oxygen. You might try degassing by vacuum or sonication with vacuum.
Posting is currently disabled in this topic. Contact your discussion moderator for more information.