XBridge shoulder peak

[adrien16]

Hello everyone !

I'm using a waters column Xbridge 150x4.6 3.5µm and we observed souldering peak after several injections. The method use a ion pairing agent at 10 mM in a phosphate buffer at 10mM pH 6.8. The column temperature is 45°C and the flow rate 1.3mL/min.
A gradient is used with 80% buffer / 20% MeOH increasing in 20min at 30% buffer / 70 %MeOH.
In the beginning of the sequence, the chromatograms are well and the a shoulder on all peaks appears.

Suggestions or ideas or something critical in the method ?

Thks

[danko]

Hi Adrien,

1. What is the position of the shoulder – in front of the peak or at the tail?
2. When you say all peaks, do you mean within a single chromatogram? If more peaks – how many are there and which retention times do you achieve for them.
3. After how many injections or hours or days does the shouldering appear.
4. Which ion pairing agent do you utilize?
5. You must be encountering a pretty high backpressure under the described conditions. What is the average pressure?

Best Regards

[Uwe Neue]

The equilibration with ion-pair reagents can be a problem in gradients. Yes, more details will help...

[Victor]

If you have not done this already (probably you have), then try injecting a simple test substance like toluene around 500 mg/l in the mobile phase. Detect at 254nm. You can do this isocratically with the mobile phase you are using if you want, with e.g 30% buffer 70% methanol. Toluene should be unaffected by the buffer or ion pair agent. If you get a nice clean peak with at least 10,000 theoretical plates (should be more) then you can start worrying about your method. If the toluene test fails, then you should start worrying about the state of your column.

[adrien16]

Hi Danko,

We have 4 peaks in our chromatogram. At the beginnig of the sequence all peaks are Ok. And then at the middle of the sequence, after 15 injections (7 hours) we observe tailing peaks.

We use Octane sulfonic acid as ion pairng agent ( 2g/L )
We record a backpressure around 3000 psi in the beginng and 3800 psi max during the gradient.

According to Waters, the method should be performed ! They don't understand...me too !

I don't understand why it works, and then it doesn't work !
Is ion-pairng agent precipitate with phosphate ?
What is the influence of concentration of Ion-pairing agent ?

Thank you for your advices !

[AdrianF]

Ion Pairing and gradients are always likely to be a problem as the system is never in equilibriium. However it might be worth trying adding the ion pair agent to the methanol.

[Dan]

Adrien,

The additional information is good, but a little more is needed.

Can you tell us about the preparation of your buffer? Which phosphate salt do you use? What is the preparation procedure?

You usually do not see precipitation problems when you use a sodium phosphate salt with ion pair reagents. However, if you use a potassium phosphate salt, then you can see precipitation in some cases. I am not sure about octane sulfonic acid if there are precipitation issues.

Increasing concentration of the ion pair reagent will give increased retention times in reversed phase HPLC.

As others noted, gradients with ion pair reagents are not easy, equilibration is a big issue here.

Regards,
Dan

[adrien16]

Dan,

I agree with you, but we did the same analysis with an acetate buffer and we saw the same issues !
We prepare our phosphate buffer with potassium phosphate dibasic (1.74g /L --> 10mM) filter through 0.2µm.
All samples and standards are filtered through nylon 0.45µm.

We haven't seen any precipitation !

Do you think the buffer may precipitate in th column when a flowrate is running ?

One more question : Do you think the concentration of ion pairing agent could have an effect on peak shape ?

Thks
bye

[Dan]

It is possible that you would not see a precipitate for two reasons:

1) the particulates are so fine that they remain suspended in solution and do not actually precipitate
2) as you guessed, the precipitation may occur on column as the amount of MeOH increases

However, since you tried the acetate buffer, precipitation may not be the issue. You still need to consider equilibration time.

Can you increase the equilibration time between injections?

Also, do you know that pH 6.8 is appropriate for your analytes/separation?

Regards,
Dan

[Uwe Neue]

Have you tried to wash the column with 90% acetonitrile and 10% water, and then gone to the manufacturers plate count test as suggested by Victor?

[adrien16]

Hi,

The pH is < pKa-2 of the analytes, so i think the pH is well adjusted for this kind of analytes.
However, don't you think that ion pairing agent could damaged the column because we've tried to rinse columns with 100% ACN and the issue still occurs !
Actually, at the moment we try to reduce the amount of the ion pairing agent for increasing the lifetime of the column but i don't know if it's a good solution.

We'll try tomorrow to test our "damaged column" according to the supplier.

Thanks

[Bruce Hamilton]

Couple of questions.

Did the method work previously?
Is the sample dissolved in the initial mobile phase?.

Please keep having fun,

Bruce Hamilton

[danko]

Hi Adrien,

I’m missing some important information. I didn’t ask of course so it’s my fault.
Anyway, after the appearance of tailing peaks, is the column dead/useless or does it work fine again next time you start a sequence?
If it is the case, then you shouldn’t worry about precipitations and rinsing – especially if you are planning to use 100 % ACN.
It would only mean that you’ll need to prolong the re- equilibration step. I don’t know how long you equilibrate before the next injection, but considering the column size and the nature of the mobile phase, I would suggest at least 10 – 12 min re- equilibration step following the gradient.

If on the other hand the column is ruined after the 15th injection, then you might consider precipitations – not necessarily of the mobile phase components, but rather sample components in combination with the mobile phase. The quick test is, to mix mobile phase and sample in a test tube and observe the mixture for changes.

Finally, I still think your working conditions generate too much backpressure which with time might cause void volume at the head of the column, which will manifest in distorted peaks etc.
The solution in this case might be changing the MetOH to ACN or mixing MetOH with ACN.
It should be mentioned here that ACN has stronger elution properties than MetOH, so it wouldn’t be a simple substitution. Possible selectivity alteration should not be counted out either.

Best Regards

[Kwet]

Hello Adrien16,

I agree to investigate longer equilibration time.

I also think sample should be prepared in the initial mobile phase (MeOH / buffer 20:80). Is that the case? For both samples and standards?

I think if buffer precipitation occurs the pressure will increase after injections. You should check if pressure is the same at first injection than after 15th injections.

For me, XBridge column will not have problem with 3800 PSI. It can support 6000 PSI and < 4000 – 5000 PSI is recommended for longer life time.

To wash your column with 100% CH3CN you should first remove buffer by flushing with MeOH / water 20:80 for example (replace buffer by water). Otherwise buffer will precipitates. I suppose you take care of this but it doesn't appear clear to me in the upper discussion.

In general, decreasing buffer concentration leads to higher tailing! Octane sulfonic acid does not precipitate with phosphate because they are both anionic. But they could precipitate with a cation (an amine function below the pKa or a quartenery ammonium for examples).

You say pKa is 8.8. If it relates to an amine function, perhaps you should work at pKa +2 (pH 10.8 with NH4HCO3 5 mM – adjust pH with NH4OH). You will have a neutral amine instead of a cationic leading to an enhanced retention, avoiding ion pairing and tailing. XBridge columns are especially designed to basic pH so perhaps you can get the advantage of this.

Best regards.

[adrien16]

Hello,

Thank you very much everyone for your advices.

Kwet,

We prepare all samples and standards in mobile phase in initial conditions. We take care of every rinses steps after using a buffer.

The precipitation of amine may be a solution according to our method because our pressure is good, but some times after several injections we observe an increase of backpressure which could happen after a precipitation. But, i was thinking about a second "way" into the column that involve shouldering peaks.

Anyway, I think i 'll change the method to remove ion pairing agent and perform two separate method for all my actives drugs.

I thank you all for your advices !

Bye