Split peaks

NGray

<p><span>Hi</span></p><p></p><p><span>I've been having trouble for some time with peak splitting with both XBridge and Acquity columns. I've been using 10 </span><span>mM ammonium bicarbonate pH 10 with ACN or MeOH as I'm working with basic compounds and initailly this gives excellent chromatography with good resolution and peak symmetry. However, after around only 100 injections my peaks start to split and I've also noticed an increase in backpressure. With the Acquity BEH C18 column I was running at 600 uL/min and 40 C. I've tried washing and backflushing the columns but the peaks are still split. We opened up one of the XBridge columns where we saw a clear void. I initially thought this was due to dissolution by the high pH buffer but no one else I know has had experienced column instability at pH 10. I've been very careful to wash with water before intorducing 100 % organic on to the column to prevent buffer precipitation, although one of my gradients did reach 95 % ACN. This has occured on different instuments (UPLC and HPLC), I'm using a guard filter and all my buffers and samples are filtered with 0.2 um filters. My samples are dissolved in mobile phase composition, and all analytes I inject show split peaks, it is not compound specific.</span></p><p></p><p><span>Since, I've been using an XBridge 2.5 um column with 5 mM ammoniun bicarbonate at pH 9.5, 30 C, 400 uL/min and have not experienced any problems with split peaks or elevated backpressure.</span></p><p></p><p><span>I've noticed a few of you on here have had problems with peak splitting as well so if anyone can offer any advice I'd be most greatful!</span></p><p></p><p><span>Thanks.</span></p>

dougm

Hello,

In case you are interested, there was a previous posting earlier this year that was almost identical to yours:

http://forums.waters.com/clearspace_community/thread/1354?tstart=0

Could you please clarify something for me? You mention that you are experiencing problems with XBridge columns BUT you are also using XBridge columns without issue. I would appreciate it if you could clear that up for me.

Thank you very much,

--Doug

NGray

Hi Doug

Thanks for your response. I have seen the problem with all the XBridge columns I have used with this buffer at pH 10; keeping it at 9.5 with methanol (which I think lowers the pH slightly) is fine. It may be conincidental but as this has happened with a few columns now (3 or 4), including a Phase II Acquity column, which makes me think in some way it must be related to the pH. Perhaps, as you say in the other case linked above, it is a conbination of factors, pH, temperature and pressure, but I am concerned about trying it again. The user in the similar case says a Van Guard column helped - how would this help?

Many thanks,

Nicola

dougm

Hi Nicola,

Tracking down such things down can be difficult. Have you confirmed that all of your split peak columns are voided? As you probably know, split peaks can be caused by a contaminated frit/column as well. We've seen this lots of times.

Assuming they are voided, this can be caused by either a mechanical void (bed shifted) or chemical void (bed dissolved) or a combination. It's tough to determine exactly what is the cause. One possible way is to run the same method except use low pH (e.g., 0.1% HCOOH). Under these conditions the packing material will surely not dissolve. If the column lasts, we know that pH is playing a (bad) role. If the column still voids, you start considering a mechanical (pressure) cause. Unfortunately, I do not know of any other way to figure this out. Even autopsying a voided column cannot distinguish between a mechanically vs. a chemically voided LC column.

To make matters more confusing, as you've read and probably know, the BEH material is quite pH resistant.

Not sure if this helps or not. If there is anything else I can do, please let me (and the Community) know.

Best regards,

--Doug