Chromatography Forum

Hello everyone,

I'm encountering an elution problem on my capillary column. How is that possible if I separated and eluted my compound for calibration graph (with a good coefficient 0.997 applying internal standard technique) next time when I injected it couldn't see the peak. I've tried to dilute/concentrate my sample but still no result. I injected separately an internal standard and I saw it even at 1/1 000 000 dilution in FS. I also see my reaction product but when I mix it with my standard it just doesn't apear on my chromatogram.
I simply don't get it. What is going on there? Where is it? why can't I see it?
I would realy appreciate your opinions and advices!

Wish you all the best!

How is it possible that you expect sensible answers when you do not tell us anything about what it is that you are trying to analyse, or the conditions under which you are trying to run ?

We are not psychic.

Peter

Perfectly right Peter,
I'm trying to quantitate racemic metoprolol on GC-MS/IT.
Oven program: 150 grd - 1 min, 20 grd/min, 220 hold 1 min, 10 grd/min 300 hold 1 min using 95% dimethylsiloxane 5% phenyl column. I must mention that I've done calibration on it in the same condition. Haven't change anything.

Either your sample prep delivers 0% recovery, or your analyte is reacting with something in the sample that is not in the standards.

Peter

How is it possible? about an hour ago, I've injected the same solutions on a different GC MS analyzer using the same conditions as previously mentioned, using the same column and the standard eluted perfectly.

How is it possible? about an hour ago, I've injected the same solutions on a different GC MS analyzer using the same conditions as previously mentioned, using the same column and the standard eluted perfectly.

I am really interested in hearing how you expect an answer to that question when you provide absolutely no information on the composition of the samples or on what the analytes are or on how you prepare the samples.

Peter

By solutions, I meant to say standard solutions ( metoprolol standard solubilized in toluene) a serial dilutions nothing else

Right, let me test my powers of deduction.

You ran a calibration series successfully.

Then you injected something else, either a sample according to post 1 or a standard solution according to the immediately preceding post, and got no peak. ?

Then you injected some internal standard and got a peak, and some precursor and got no peak ?

Then you injected some standard solutions on another instrument and saw peaks ?.

You may have either a blocked syringe or a huge leak on the first instrument.

Peter

Yes Peter, that's the answer to all questions, except for the syringe and leak. Can be an instrument problem if internal standard and other compounds eluted correctly, respecting their retention times and ionization patterns? Beside that, I've already checked the leak before my first post. If there would be the syringe blocked, how can I see the internal standard?

Mermeth

Yes Peter, that's the answer to all questions, except for the syringe and leak. Can be an instrument problem if internal standard and other compounds eluted correctly, respecting their retention times and ionization patterns? Beside that, I've already checked the leak before my first post. If there would be the syringe blocked, how can I see the internal standard?

Mermeth

Quoting form your first post "I injected separately an internal standard and I saw it" - the critical fact is that you ran the internal standard separately, in other words in a different run to the analyte that you did not see. Therefore that internal standard tells you nothing about whether the run with the absent analyte had a problem or not.

You are right - if everything in the run looks normal except the target analyte then an instrument or operation problem is very unlikely, but that is not how you describe your troubleshooting.

If only the target analyte is affected then the two most likely problems are human error in mixing solutions, or a specific reaction of the analyte with some component of the sample/standard.

Peter

When I injected in the other instrument, I used the same solution that gave no result regarding metoprolol elution on my instrument.
Ok, I'll run again internal standard in the presence of metoprolol standard.
I'll let you know the result.

Thank you very much Peter,

Whish you a peaceful weekend!

Mermeth

When I injected in the other instrument, I used the same solution that gave no result regarding metoprolol elution on my instrument.
Ok, I'll run again internal standard in the presence of metoprolol standard.
I'll let you know the result.

Thank you very much Peter,

Whish you a peaceful weekend!

Mermeth

So there is a paradox; there cannot be anything wrong with the solution because you see your analyte peak on one of the instruments, but there cannot be anything wrong with instrument 1 because you see the internal standard and the other components of the solution (whatever they are).

"Ok, I'll run again internal standard in the presence of metoprolol standard." Does this mean that the metropolol peak was missing in only one rum ? That you did not run another injection ?

Peter

Dear Peter, You are right.

There is a paradox but it's a reality in the same time.

I've done several runs but only with metoprolol without the internal standard. Plus I injected some other compounds because I was afraid of column deterioration and they eluted exactly as I expected.
After that I injected the internal standard alone.
After your post I injected metoprolol mixed with internal standard.
I saw the IS but not my interest analyte.
I honestly don't understand why doesn't it come out on this instrument.
I've double checked it on a GC-MS and on GC -FID: same conditions, same column, using the same standard solutions. It eluted perfectly on both instruments.


Mermeth

Is the instrument that gives you both IS and metoprolol in your sample an ion trap?
What is the relative response of the internal standard and the metoprolol?
What else is in the sample that may overload the trap?

If metoprolol is small compared to your internal standard and there is a lot of extra material in your sample that isn't in your standards then the trap may fill with ions from the sample matrix and metoprolol will be hard to see.

Hello Steve,

Yes, it is an IT.

At a dilution of 1/1 000 000 for IS relative abundance is less than 5%, but I can see the m/z values, indeed very small.
Metoprolol should be much more intense compared to IS.

I can't see metoprolol.
I've only injected metoprolol solubilized in toluene.

Can IT remain overload from another samples injections?
There is a lot of run on the instrument.

What should I do in this case?

Mermeth