Moving peaks

[HW Mueller]

None of the suggested causes seem to address the problem of undulating retention times. If this undulating happens without changing any operations than something must be wrong mechanically. How do the retention times vary in relation to each other?

[Bryan Evans]

As per the MSDS, 1.0M (~ 6% w/w) AcOH solution has a pH of 2.4.
So the pH of 3% solution is defintely higher than 2 (especially with MeOH).

Anyways, I'd still look at sample prep, sample solubility, guard column,
and / or alternate column.

[juddc]

I've rarely met a modern silica based RP column that couldn't handle pH around 2 for a long time. Wash all of your columns with solvent, retest them, and let us know what you see.

Also, I wouldn't think that caffeine would be well retained with ~40% meoh. You'd be out of the void, but not by an awful lot.

[gurkishan]

Hi Guys,
thanks for the brilliant replies.
Well, my pH of water plus acetic acid is 0.9. Damn too low.
Also, today, after starting the column, with nothing being changed ie, mobile phase is the same bulk container, my pressure in the column has gone up from 2000 to 2400 psi and the peaks that were being eluted in 6 min yesterday are being done in 2 minutes.
I do wash my column after 50 injections with 100 % methanol for 1 hour. I dont know whether acetaminophen is being retained still on the column after washing.
What else could i do?

[HW Mueller]

Obviously you are not telling us everything. Either your pH meter is shot or you put another acid in there. Also, did the rt slow again as you mentioned before (undulating rt)? Either your pump is shot or you are running this outside of equilibrium or all of these.

[gurkishan]

Hi
Sorry, but i am trying to bring up everything i can and i do appreciate all you guys helping me out!
I calibrated the pH meter. When i added glacial acetic acid 3mL in 69 mL water, i got a pH of 0.9 and after adding methanol, the pH was 1.8
My peaks are moving up and down evryday even after equilibrating the machine for 2-3 hours. The peaks are shifting in and out.
I have tried various columns- coated and uncoated, but have not been able to get reproducible results and they keep on shifting in retention time (altough the areas do come out to be more or less the same)

[juddc]

OK...Washing with MeOH doesn't do it...

If you've tried multiple columns and get the same issue, then the column isn't your problem.

Your pH is quite low for any HOAc solution I've ever seen.

Mechanical issues may be causing this, however they'd be easy to see.

I still think you may have a sample issue. What does the system so when you inject a simple single component standard?

Please describe your sample matrix in greater detail (if you can) and what you've done to extract and clean it up.

[Kostas Petritis]

OK...

There is no way that 3% of acetic acid can give you pH 0.9, period. As Hans mentioned, either your pH is broken or you are not adding what you are saying (or what you think) you are adding.

If your pH meter was broken and as you are not buffering your mobile phase, there would be no reason to observe any variation in your retention time over time. As a result, scenario 2 is more probable.

Your glacial acetic acid is not glacial aceitic acid. It is either a strong acid or a combination of acetic acid with a strong acid (TFA maybe or even worst HCl?). In this case, maybe you indeed destroy your column and you get all the subsequent retention time shifts.

Try to start with a new bottle of acetic acid...

Having said that, I do not eliminate the build up possibility...

[gurkishan]

Ok,
I tested my Mobile phase pH with a calibrated fisher instrument. It is 2.2 pH
The column on which i had moving retention times, but still had the result-- for some reason is now at 2400 psi and stable instead of 2000psi and the peaks are not resolving now.
I had reversed the column overnight and had 100% methanol flowing through it at 0.2mL/min
But ene after this, the column is staill showing 2400 psi and not resolving the peaks
Any better method to wash column?

[HW Mueller]

You mentioned undulating retention times again, now a loss of resolution. It seems your column as well as your pump(s) are shot.

[gurkishan]

Hi
I checked out my pumps by running a different chromatography method. It gave me accurate results.
But, yet again, with this method, i am seeing the problem--- bad resolution and moving peaks

[HW Mueller]

Different column? Different mobile phase? Different pumping/mixing method? Different plumbing?

[gurkishan]

it was an assay for caffeine, using same column but different mobile phase.
That is waht is making it so confusing!

[juddc]

This really sounds like a column fouling issue to me... either sample related or bad mobile phase solvent.

What is your sample (excipients)?

What is your preparation protocol?

If you have a fresh column and inject only a standard mixture do you get stable results from injection to injection?

How do you know your water and solvents are clean?

Do you filter your samples prior to injection?

Perhaps a simple solid phase extraction cleanup would make life much easier.

More detail is needed in order for anyone to have any hope of helping you!

[HW Mueller]

Here are some more questions: How do you mix your mobile phase, how and what did you mix in the caffeine run? Was the caffeine run just a standard run, a single injection (remember juddc asked you whether standards of the problem analytes give erratic results)? Does this "moving in and out" (undulating) of rt occur spontaneously, that is you don´t change anything while this happens? How does the resolution change in relation to this? Or did the change in resolution occur separate to the undulation, on a different day and run? Are the rt, resolution, etc., always ok after you wash the column with MeOH? Does the undulation always occur when you run real samples?
In other words a coherent explanation of what is going on is essential to understand what happens.