More Peak Splitting Problems!

[Rob Burgess]

This seems to be a common topic of problems for chromatographers that is often overlooked in textboxes and, in my humble experience, is often very difficult to solve. I have a problem that is proving very diffcicult to get to the bottom of.



Above is a chromatogram I am obtaining with different injection volumes that is proving very problematic. It can be seen that at 30µL we have a horrid peak shape for our main component peak, whereas at 40µL the peak shape appears fine. this is a bit of a contradiction as to what we'd usually expect. I have experimented with a 50µL also, and sometimes it is OK, and at other times not!

For everyone's info, the sample is a 2° amine with a acidic salt counterion. Concentration of sample is ca. 0.2mg/mL. The sample solvent is a 40/60 v/v MeOH/water diluent (if we use lower % strength MeOH we don't appear to get a full extraction of all our impurity peaks!).

The chromatography conditions are as follows:

Agilent 1100 High Pressure Binary System
MP A: Water + 0.1% Formic
MP B: MeOH + 0.1 % Formic
Gradient profile:

0.0 min 30% B
8.0 min 70% B
17.0 min 70% B
17.01 min 30% B
20.00 min 30% B

Dwell volume ca. = 1.2 mL
Col. Temp. 29°C
UV Det. @ 254 nm

Q/ Can anyone give any insight for the cause of this strange phenomena?

Should I be lowering my injection volume further 30µL for instance. Should I be tring to afford a more conc. solution to inject / while maintaining extraction efficiency.
Can I change the gradient conditions to achieve a more reproducible peak shape i.e. increase organic content at start; intoduce a isocratic hold etc. etc.

[Rob Burgess]

I've just been running some samples today, using the parameters listed above, and it seems to be an intermittant peak splitting problem i.e. sometimes it occurs with either a 30 or 40µL injection and sometimes it doesn't.

I'm thinking of putting a a isocratic hold for 3 mins @ 30% B at the start of the run, to see if this will solve the problem. I'll let everyone know how I get on.

PPS. Sorry I couldn't get the image to post properly. I did follow the instructions but to no avail I'm afaraid!

[Consumer Products Guy]

I'd try a significantly smaller injection volume, like 10-15 ul. I always try smaller injection volumes if peak shapes are not good, just to see if things improve, as it's so easy to do.

[Rob Burgess]

Hi Mr. Products,

I get the point what your trying to make. the problem with us Pharm. Dev. analysts is that we are often at the point where we're trying to eke out every last bit of sensitivity from the analytical method.

However, as you are saying I may be forced down that road, of settling for a lot smaller injection volume. I did actually try a 10µL injection with this method and that actually split as well... as I say the problem is intermittant and consequently extremely frustrating! Cheers for your thoughts though....

[SIELC_Tech]

Rob,

I would try to dissolve sample in the presence of formic acid to mimic ionic component of the mobile phase. Do you observe clean peak split or it is "Batman Shape", you might have toutomeric equilibrium (if your structure support this), you might also experiencing problem with injector.

[Newman768]

Hi Rob Burges,

If you're using a silica based column have you ever added a little TEA to the mobile phase (around 10 µl or so)?
(Column has to be purged with enough mob phase to gain some effects from TEA).

[tom jupille]

Rob, when the peak splits, does it always also elute later the way in does in the original post?

If the problem were solvent-strength related, I would expect the retention time of the split peak to be about the same as the unsplit (maybe even a tad early). If the problem were active-site related, I would expect the splitting to be consistent rather than intermittent (although Newman78's TEA suggestion is worth checking).

It almost looks like that one compound is precipitating somewhere and then redissolving when your organic concentration gets high enough. That would account for the late elution, and I could "hand-wave" around the intermittent appearance of the problem (might depend on the details of the mixing between injector and the column), but it's contra-indicated by the lack of correlation with injection volume. You might check the solubility of the formate salt vs. whatever the original counterion, but I'll admit this is a stretch!

[Uwe Neue]

Interesting problem. I am not sure what the specific cause of the problem is, but I am rather sure about the overall nature of the problem, i.e. a mismatch between solvent composition of the sample and the mobile phase.

My suggestion would be to do the following: take you sample, dilute it 1:1 with your mobile phase A (water with formic) and then inject double the amount that you are injecting now. All peaks should have the same size. You most likely will be enriching your sample at the column inlet due to the fact that the sample is now made up in 20% methanol. Therefore peak shape should get better. I do not know, what injection volume your injector allows. You could potentially do an even higher dilution and use a still larger injection volume.

[Chris Pohl]

Rob,

I think your problem might be "ionic strength overload". I suggest that you boost the ionic strength to see if this helps (either use 0.2% formic acid or better still add some sodium formate).

[JM]

Looking at your Chromatograms and the fact that this is intermittant, i think your proportinating valves have problem. i presume you are using low pressure gradient. This kind of difference in peak shape can only happen when your organic phase stops intermittantly.

Change the solvent channel to Cor D or see if you can replace it.

JM

[Rob Burgess]

Many thanks for all your replies and suggestions. I thought it would be apt to give an update.

I did try many of your suggestions: i.e. trying smaller injection volumes, starting the gradient with a lower starting % B, having an isocratic hold at the begining of the gradient etc. but alas all to no avail!

Then JM's suggestion pushed me in the right direction. If the problem is intermittant, then perhaps there was a a problem with the pumping system itself. Note, it is not a proportioning valave though as it is a high pressure binary dual pump system.

I tested out a 50µL injection on another two high pressure systems and so far I have had good peak shapes with no splitting on both systems. The retention time of the peak is retained more, though only very slightly (less than 1%).

Does this truly suggest I had a problem with my organic pumping head on my original system? If so what could it be? It appears that its affect is very subtle but as observed catastrophic in terms of peak shape!

PS. While I'm here, many many thanks to Tom J. for correcting the original image pasting for me. Its much appreciated... cheers!

[JM]

hi Rob!!,
it is nice feeling that it helped you. To my own experience the Agilent HPLCs can give you nightmares in gradients , if the pump seals/check valves are old . one can easily detect gradient problems by ploting pressure profile along with chromatograms and compare to find out any unusual change in the pressure graph.

The Organic phase pump head usually go bad due to obvious reasons, Change your pump seals and check valves if not working properly.

Cheers!!!

JM

[tom jupille]

Rob, you're welcome! And thanks for the update (always nice to get feedback on what did/didn't work).

The fact that the problem is intermittent is a pain in the neck (or lower), but if you can spare a half-day or so, you might be able to test pump operation by running a variation on the dwell-volume measurement:

• - replace the column with a capillary restrictor (to give about the same back pressure)
  - spike the B reservoir with 0.1% acetone (or whatever else will give about 1 AU of absorbance)
  - then run a series of gradients.

If it is a proportioning valve problem, it should show up as variations in the baseline.

[Rob Burgess]

A.N.Other update:

We had an engineer look at our problem system and it appears that the crux of the problem was due to the ceramic frit at the end of the mobile phase drawing up bubbles intermittantly on the organic solvent B line. This would obviously cause a temporary pump starvation but had a drastic effect on the resulting peak shapes when it occured.

It's a nightmare when something so trivial can cause days of frustration... oh well onwards and upwards!

[Uwe Neue]

It is good that you give us feedback on what you have learned. It helps us all... The more problems one has encountered, the better will be the answer when troubleshooting a problem.
Thanks!