New peaks appearing out of nowhere?

[bunnahabhain]

Hi everybody

There is a strange thing going on in my autosampler. Injections from the same vial produce two new peaks, while the solution in a fresh vial is stable:

Sample, freshly prepared:


Same vial after about 3 h:


Same sample, filled into a fresh vial fresh vial:


What can be going on here? Note that the peak size of the original peaks does not decrease, but something new appears *additionally*. I have run these samples back in January, and the solution was stable.
(BTW: Yes, I have seen the retention time shift, this is because the column was not completely equilibrated; I don't think that this is related to the strange observation).

Information on the sample: This is Propofol and 2,4-di-tert-butylphenol in ACN/MeOH/H2O. Eluent is acetic acid in ACN/H2O 70/30.

Any ideas greatly appreciated,
Jörg

[Consumer Products Guy]

Please clarify: if you fill two vials with fresh juice and inject one now, and the other in 3 hours, then this happens? Or only in a vial that has had an injection already?

[Klaus I.]

Interesting

Would the additional signals also occur, when you remove the septa/cap from the vial befor the second injection?

[artsjeroen]

What happens if you inject a sample followed by 2 blanks from the same vial?

[bunnahabhain]

Please clarify: if you fill two vials with fresh juice and inject one now, and the other in 3 hours, then this happens? Or only in a vial that has had an injection already?

This is from a vial that already had an injection. But during runs over night, I have also seen it in one out of two vials which did not have an injection. Another one (injected 5 hours later) was clean again.

Would the additional signals also occur, when you remove the septa/cap from the vial befor the second injection?

This is a good idea, I will try that.

What happens if you inject a sample followed by 2 blanks from the same vial?

I don't understand. With "blank" do you mean an injection with volume set to zero?

I'll keep you posted!

[artsjeroen]

No, I mean inject your solvent (ACN/MeOH/H2O) twice after a sample injection.

There were no injection in between the 3 hours that you've re-injected the same vial? Otherwise injecting the same vial 12 times might give more information that the extra peaks are "formed" or that they are introduced from a source of contamination.

[Gerhard Kratz]

Dear Jörg,

Do you have other septa available in your lab? I remember I had a similar observation what finally came from the septum. After I changed the septa the problem was gone.
Good luck
Have a nice weekend. We have still sunshine in Karlsruhe.

[bunnahabhain]

Dear Klaus

Unfortunately we don't have other septa. This came to my mind, too yesterday, so I called our supplier. They will send us a different batch to try out. I am not really sure, but I seem to remember that we had black caps back in January but now we have blue caps. Septa are the same, though (Silicone/PTFE with slit).
Greetings from across the Rhein.

What happens if you inject a sample followed by 2 blanks from the same vial?

This is running now: There is a time-dependent increase of the new peak both in the blank and the sample => degradation can be excluded.

Peak area of the new peak at 4.2 min:

Code: Select all

           Sample      Blank     Blank without cap
t= 0 min    1.744       1.955     n.d.
t=15 min    7.972       9.666     n.d.
t=30 min   10.396      16.505     n.d.

(edit: Blank without cap added)

Regards
Jörg

[Klaus I.]

Dear Jörg,

When I understand your observations correctly the origin of the additional peaks is the septa. Were the vials shaken or do you think the vapor of your solvent will extract something from the septa?

If I were in your spot, I would perform a small extraction study by adding a septum to 1mL solvent in a test tube. After some minutes I would transfer the solution to an hplc vial (without cap) and inject the solution. If the additional signals reasonable occur it should be a demonstration of the origin of the contamination.

Greetings from the beginning of the Oberrhein (Upper-Rhine?)
Klaus

[bunnahabhain]

The vials were not shaken, so it must be vapor or (more likely) the liquid film on the outside of the needle which is wiped off by the septum while the needle is withdrawn from the vial. On the next injection, this solvent spot containing dissolved stuff is pushed down into the liquid. This is an old autosampler without needle wash, unfortunately.
I just received a different batch of caps (WICOM is really fast! I called yesterday after 5 pm and got the caps today at 2 pm. Quicker than amazon prime ). I will see how these behave and report on Monday.
Jörg

[bunnahabhain]

Update:
The different batch of caps showed the same peaks, but somewhat lower peak areas over multiple injections. Running the samples without caps is not an option because the solvent (ACN/MeOH) would evaporate too quickly.
I will ask a different supplier now for a sample.

[DoryFish]

Hi, I have also experienced contamination problems with septa when injecting samples with acetonitrile. My way of testing the problem was to fill three separate vials and performed multiple injections from each vial, with time delays between each new vial. In my case the first injection from each vial was always clean but replicates showed contamination peaks.

[Klaus I.]

I’m wondering if it would be possible that these problems are caused by the slit in the septa? Assuming that the usual PTFE layer is necessary to separate the silicone from the sample solution, the slit may support extraction from the septa under certain circumstances.

In my opinion is the usage of septas with slit is often not necessary for normal operation. But I have recognized the last years, that using septas with slit is the standard recommendation of one instrument vendor for almost every autosampler problem.

[bunnahabhain]

Hi, I have also experienced contamination problems with septa when injecting samples with acetonitrile. My way of testing the problem was to fill three separate vials and performed multiple injections from each vial, with time delays between each new vial. In my case the first injection from each vial was always clean but replicates showed contamination peaks.

This is exactly my observation.

I’m wondering if it would be possible that these problems are caused by the slit in the septa? Assuming that the usual PTFE layer is necessary to separate the silicone from the sample solution, the slit may support extraction from the septa under certain circumstances.

In my opinion is the usage of septas with slit is often not necessary for normal operation. But I have recognized the last years, that using septas with slit is the standard recommendation of one instrument vendor for almost every autosampler problem.

We have a Dionex ASI-100. The needle is > 1.5 mm thick, no chance for septa without slit. I have even seen septa with slit being pushed into the vial if the cap is not brutally tightened.

Jörg