decreasing retention time with increasing times of injection

[slimshady]

I found my retention time decrease after several injections. All the method setting and system conditions were the same. How to explain this ?

if the chemicals built up on the column the retention time is supposed to be longer.

[tom jupille]

f the chemicals built up on the column the retention time is supposed to be longer.

Actually, retention usually decreases as a result of chemical contamination. The junk ties up part of the stationary phase surface and therefore changes the phase ratio. Only if your analytes interact more strongly with the junk than they do with the original stationary phase will retention increase.

[ntruong]

Why rule out the pump?

[Paulhurleyuk]

Are you running a gradient ?

Often with a gradient method the column never reaches perfect equilibrium with the starting conditions (there was a paper on this in LCGC a while ago, but dang if I can find it...), but does reach a semi equilibraitation that's the same every time, so the first few runs the equilibriation is changing from your pure starting conditions to the gradient.

Try running a few blanks before your run and see if that helps.

Paul.

[Paulhurleyuk]

sorry, duped myself !

Paul.

[slimshady]

Are you running a gradient ?

Often with a gradient method the column never reaches perfect equilibrium with the starting conditions (there was a paper on this in LCGC a while ago, but dang if I can find it...), but does reach a semi equilibraitation that's the same every time, so the first few runs the equilibriation is changing from your pure starting conditions to the gradient.

Try running a few blanks before your run and see if that helps.

Paul.

I just made several injection with normal method not with gradient one.
The retention time difference between my 1st injection and 5th injection is about 2min( quite huge). All this happen within 50min .

I suspect that there is some water in my modifier. The water absorbed on the silica gel inside the column which make the retention time shorter.

[slimshady]

Why rule out the pump?

I put some TTBB into the solution. Since the retention time for TTBB in every run is the same, so can say it is not the problem of the pump

[slimshady]

f the chemicals built up on the column the retention time is supposed to be longer.

Actually, retention usually decreases as a result of chemical contamination. The junk ties up part of the stationary phase surface and therefore changes the phase ratio. Only if your analytes interact more strongly with the junk than they do with the original stationary phase will retention increase.

if that is the case what solvent can I use to flush the column?

seems like the peaks got in the next day will shift to the original place and then to move forward again. what does that indicate?

[unmgvar]

slimshady what are you doing with your column when you have done working with it?
do you wash it some how?
it looks like you have a contaminant in there like Tom suggest and that after a while it is washed out hence the fact that your RT shifts back to it's original place.

has your RT stabilised after going forward and kept steady or is it all the time going forward?
can you give more specifics about your mobile phase composition?
what is the nature of your sample and what are the preparation steps that you are performing?

[Uwe Neue]

Slimshady, you do not provide enough information to get a decent answer...

You appear to use normal-phase chromatography, you run some kind of a mobile phase, after your runs you flush the columns with something, and then when you start again, the retention has increased.

Can you figure out what is going on if I am giving you the information above?

[slimshady]

actually i am doing supercritical fluid chromatography. Thus the mobile phase is supercritical CO2 with modifier( Methanol V:V=0.18:1). My sample is Flurbiprofen (dissolved in MeOH) The peak shifting I mentioned appeared during a series of injections.


I said the peak moved back to the original place. That happened in the next day i run the same experiments again. And during that time I did not wash my column at all.

Yesterday I washed my column (Chiralpak AD-H) following the manual's instruction. after that I waited for 1h 20min for the baseline stablizing. Then I ran my experiments. For the first 2 injections the peak still moved forward. From the 3rd injection to the 5th the peak become stablize which means the reproducibility was quite good. BUT my 6th and 7th injection began to move backward compared to the 3rd to 5th injection. Although the reproducibility of 6th and 7th injections was also quite good.


How come my peaks oscilate ? First move forward and then backward. They are all continous injections under same method.

[slimshady]

the flow rate for supercritical fluid CO2 is 1 ml/min and for MeOH is 0.18ml/min

[ntruong]

Hi Slimshade,
Have you ever tried this method on another instrument? Is the retention time shifted as well? Since your run is isocratic, do you premix your mobile phase and give it a "good" sonication? Do you also prime all channels and store with the same mobile phase? Do you use a in-line degasser? How long is your equilibration time?

[Bruce Hamilton]

actually i am doing supercritical fluid chromatography. Thus the mobile phase is supercritical CO2 with modifier( Methanol V:V=0.18:1). My sample is Flurbiprofen (dissolved in MeOH) The peak shifting I mentioned appeared during a series of injections.

How come my peaks oscilate ? First move forward and then backward. They are all continous injections under same method.

We worry that people forget to tell us details, and you thought that your system is using CO2 SFC with a modifier isn't important?. I assume the method used to work OK, but if it didn't - then then sample introduction method should be reviewed to ensure consistent dissolution.

As you are aware, the critical parameters for consistent SFC retention are temperature and pressure - assuming your modifer injection is consistent and well-mixed.

The pressure is mainly controlled by flowrate. and I would want to be assured that the system is performing consistently by testing it according to the manufacturer's specifications, including verifying that the modifier injection is consistent

You should also watch for any effects of ambient temperatures on the CO2 flows/pressures from liquid storage to column. If you system has a mass flow controller/monitor, check to ensure that there are no longer-term drifts in flows.

I would perform the performance tests over the same timeframe, temperatures, and pressures as for your analyses, and see what drift in temeprature, pressure and retention times you experience.

Once I had verified that the system was behaving correctly I would revist the analysis, however the variations you indicate would suggest to me to initially focus on the instrument consistency, not the method details.

Bruce Hamilton

[ym3142]

I have no idea at all about the supercritical chrom then curious that how the system ensures only liquid CO2 but not any gasous CO2 present.