Chromatography Forum

I have changed injector, injector loop.....tryed a multitude of wash solutions....purge the injector...wet primed...basically everything i can think for weeks but we still getting carryover. If i inject nothing (Empty vial) the carryover is still present....so it must be in the system somewhere right? Waters the HPLC support suggest running the system with Nitric acid...that's the only thing i haven't done yet!....any other suggestions?
Im running a gradient method...when the gradient kicks in about the 10 minute mark thats when the carryover occurs. We are developing a Related substances....and bascially the the carryover has the same peaks(approx 12) as would if u injected a normal sample!!

What injector are you using ?? I had a problem with carry over whaen using HPLC 1200 Agilent and I wash the flush system with hot pure water during 5 min, isopropil alcoohol(IPA) for 5 min and acetonitrile (tht was part of my mobile phase) for 5 more minutes.

Did you wash the injector valve system ? I put all parts in the IPA 10 min in the shaking system. There some injector parts that should be replaced when you have carry over.

Have you changed the wash frit?
Change the sample loop? (You could try with a sufficient long piece of tubing).
Change V3?

Kind regards

Ace

What is the composition of your mobile phase? At one point we were using an ion-pairing reagent in the mobile phase (gradient method) and re-equilibrating for 2 minutes at 50% B after every sample and at this concentration of mobile phase, impurities of the ion-pair would selectively retaing (accumulate) in the column during the re-equilibration step. Not until another sample was run and the gradient reached higher concentrations of %B(MeOH) the ion-pair impurity would be eluted. At first we believed this was a carry-over issue when it really was not, the small impurity peak did not affect the analytes, the problem was addressed as the peak was identified simply as a system-method peak.
Good luck!

Column :ATLANTIS T3 C18 4.6MM 150MM 3um
Wavelenght: UV @225NM
Gradient: 91:9 Phospahe buffer:MeOH to 50:50 with a 80 minute run time
Our products are penicillin derivatives....which are pretty sticky....hope this is a help.....oh yeah when the gradient kicks in at 15 minutes (55:45)....thats when the carryover appears

My first suspicion would be your sample solvent is allowing material to precipitate on the column or guard column or even in the injector.. Is your sample completely dissolved in the initial mobile phase?. Does the the sample concentration remain completely soluble in the initial mobile phase whilst at the autosampler temperature for several hours?.

If you inject a series of larger volumes of high % MeOH/Water blanks, do your carryover peaks get larger than normal, and then reduce?. What happens if you increase column temperature by 5 - 10C.

Allow the column to rinse at 50:50 for longer at the end of the run before injection, just to confirm it is carryover that you are seeing.

I would not bother with the nitric acid clean, at this stage, because the contamination is most likely either in the injector or on the front of the colunm ( which obviously isn't cleaned with nitric acid ).

You can also prewarm a sample vial of final mobile phase, and quickly inject the largest possible volume several times to try and rinse the injector, using a short isocratic run of 50:50 mobile phase..

It possible something else in the sample is binding to the carry
over, so ensure that the problem doesn't happen with the standards.

If you dilute the sample with mobile phase, and inject a sample then a few blank injections of final mobile phase, does the carryover decrease with each injection?.

Are your wash solvents sufficiently aggressive to rinse material from the injector?. What happens if you try a new column.

Another cause could be a faulty injection system, but that should show up in other analyses.

Please keep having fun,

Bruce Hamilton

I am not sure if you are truely talking about carry-over or if you see peaks in your blank gradients all the time. Please clarify, since these two things require different courses of action.

I agree with Uwe that this does not look like traditional injector carryover. This sounds to me more like "ghost peaks," which means the peaks show up even when no sample is injected. Try this simple test.

Perform a blank injection immediately after the column is equilibrated. Then equilibrate the column again, but wait longer - approximately the same length as the delay at the start of the gradient. Then perform another blank injection. If the ghost peaks are about twice as large (because you pumped twice as much mobile phase before the gradient), then they are coming from your mobile phase, most likely the A (buffer) solvent.

Well i believe the peaks have been present in the blank since beginning of the development stage of the method. We thought it was the column too.....but we hooked up a new column and ran a blank (Our blank is deionised water) and the very first injection showed the carryover!!....or ghostpeaks!!

The peaks that are coming off in the Blank are a carbon copy of the chromatography of the sample injection. We know this because we have injected(individual related subsatnces and active peak) them at high concentrations individually. The blank peaks (carryover/ghostpeaks) are at a much smaller level (AU 0.000-0.006) but as i said before nearly a carbon copy as if injected a sample except they are alot smaller.

The sample solvent is deionised water

and also we have tryed 4 different HPLC systems and the same problem occurs with all of them

which type of alliance is it?
269x or 279x?

Alliance 2695

Just for kicks have you tried water from a different source? I've come across times when there has been peaks from my milli-Q water and not from bottled water and vise versa.