Hi, All:
We are doing a normal phase method validation and getting negative peaks interfering with the analyt peak. The HPLC conditions are: HP-1100, column: (R,R) Whelk-O, 250x4.6mm, Column temp.: 40C, M.P.: 75% Hexane/25% IPA. Flow: 1mL/min, Injection: 20uL, Detection: UV@220nm. Diluent: IPA. The negative peaks are at about 3-4min (a broad dip) and 10min. At the beginning of the method developement and validation (for about one month), the negative peaks are very tiny or even couldn't be observed. Then one day those peaks just come out. The height of those peaks changes from -10mAu to <-1mAu. We suspect it's the water in the column, so we flushed the column with IPA for more than 3 hours, at the same time doing 100uL injections of IPA to rinse the injector. The next day when we try to do the injections (IPA and sample), we see the negative peak at 10min grows up to -40mAu. Then we flushed the column with Hexan, do not see much change. We switch to another system, still see those negative peaks (about -6mAU~-10mAU). We tried to make fresh mobile phase, degas for a long time, using diffent lot of IPA & Hexan,try different vials, change a new column, it doesn't help. One day we just used an old mobile phase, do not degas, suddenly the negative peak disappeared, but after we made some new mobile phase, the negative peak come back again. Now we see negative peaks in IPA, sample, but no negative peak in mobile phase injection. Does anybody know what are those negative peaks? Why sometimes they appear and sometimes just disappear??? I guess it will work if we use the mobil phase as diluent. But I still would like to know where are those negative peaks come from?
Thank you in advance for your reply.

a new comer

To get peaks you either have to inject something or cause some other disturbance. Impurities in the mobile phase change only the baseline at equilibrium. Why donīt you inject, separatly, all the solvents that may be in your sample + pure hexane and iPrOH, and tell us what happened.

Hi, HW:
Thank you for your reply. The IPA and Hexan I used are all OmniSolv, which is better than HPLC grade. I injected pure IPA (the only solvent in my sample) at 10uL, 20uL,30uL and 100uL yesterday, the dip at ~10min gets bigger and bigger with the increasing of injection volume. Also I injected mobile phase (75%Hexan:25%IPA) at 10uL,20uL and 30uL, I can see a positive peak at ~10min. The peak increases with the increasing of injection volume. But it's relatively small comparing to the dip. I don't know how to explain this. Does anybody know why and how to get rid of this dip? The reason we want to use IPA as diluent is because we only have one set of sample. And we have to do two test on them, one test is using IPA as diluent. Also, I'd like to know why IPA causes a dip there.
Thank you very much for your help.

If there is no "untold story" behind this you demonstrated that the m. p.is the culprit. It apparently absorbs more than your old one (hexane contaminated?). The full answer may come out if you inject hexane, and also water (neg. peak at 10 min?).

I am not familiar with OmniSolve, but HPLC-grade solvent have been tested for use with HPLC. At 220 nm, you see a lot of things, and it could very well be an impurity in your solvents. Based on your experiments, it could be an impurity in your hexane.

Hi, Thanks for your reply. I injected the Hexane today. Saw a negative peak in around 2 min. I guess that's because of the absence of IPA. And I did not see a negative or positive peak in around 10min. But the baseline was kind of wavy. My co-worker set up a sequence today, including an IPA sample spiked with 1% H2O. We'll see what we get tomorrow.

Hi, we got some result which I can't explain. My co-worker filtered the mobile phase by a 0.45um Nylon filter. The dip at ~10min. is smaller (about 0.4mAU). we injected the 1% H2O sample (in IPA), and saw a positive peak at ~10 min. How come? I thought the H2O may not clean, but why the positive peak just come out at the place where the negative peak comes out? Does anybody know how to explain that?
Even that dip, although it's smaller this time, who knows if it's because of the filter or just we were lucky this time. So does anybody know where does the ddip come from?
Thanks in advance.

That looks like several things are varied at once. My guess was that the 10min neg. peak was due to H20 in iso-propyl. Suggestion: Do the experiments yourself, systematically, changing one thing at a time. Check all solvents with a spectrometer if possible.