Strange peak in 100% ACN

[Onson]

I am separating two compunds with HPLC using a ACN-H2O gradient. I start of with 10% ACN and go all the way to 100% ACN. I am able to separate and collect the two compounds, but there is a strange noisy peak occurring just after 100% ACN has been reached. Its not really a peak but a small increase in baseline with a lot of noise. The same peak occurs even if I am injecting 10% AcN, so it can't be any trace chemicals from my reaction. I have tried changing the mobile phases but the peak is still there. Has anyone experienced similar problems and have any suggestions of what it might be?

Thanks!

[LCbob]

Hi
Can you post a pic? It could be a number of things, including contamination, refractive index effect or even the pump playing up with the high level of ACN.

[Onson]

Hi,

Thanks for your reply. Here are two images. First one is the chromatogram for one of my compunds, second image is a close-up of the strange peak that occurs in all of my chromatograms.




Onson

[Onson]

[LCbob]

Hi

Do you mean the peak at 28mins? This looks like a noise spike.

I will assume we are talking about the increase in baseline and noise at 34mins

I also assume the blue line is the % gradient values of A:B.

The sudden increase in baseline could be due to the increased change in gradient towards the end.

Do you know your dwell volume and flow rate, with this info you may be able to work out if the two are interrelated. Also extend you runtime, at the very end the baseline seems to be rising even further.

Can I suggest you monitor the pump ripple ( pressure change ) with 100% ACN, you may find there is an increased ripple effect at the high ACN level compared to the lower level.
Are you using HPLC grade ACN and at what wavelength?

[Onson]

Hi,

No. Not the 28min peak, the noise at 34min. My flowrate is 10ml/min. The blue line is the % ACN. Dwell time is approx 8min, so 80ml. I am using a C18 column and only HPLC grade H2O and ACN. Detection at 254 and 630nm. The 34min noise can be seen at both wavelenghts.

I could take that the sudden increase in ACN towards the end would shift baseline slightly, but I cant understand why it's so noisy.

I also monitor the pressure. I get a decrease in pressure with increased ACN.

Thanks!

[Peter Skelton]

The reduction in pressure is due to the lower viscosity of acetonitrile.

What grade of ACN are you using? Also what actual HPLC system? Given that you're running at 10ml/min I take it that this is a prep HPLC method?

[Onson]

I am using HPLC-grade water and ACN from Fisher, a Waters Prep LC 4000 system, and a C18 column (19x300mm).

[Russ]

If you let the system equilibrate for a few hours at 10 % AN, do the peaks get larger? If they do then you are likely seeing small amounts of impurities in the water or AN.
Do you degas the water or have a degasser in the system? Do you have a back-pressure regulator installed after the detector? If not, you could be seeing the effects of small gas bubbles in the detector flow cell.
A couple of simple ideas from a simple person.

[Onson]

Thanks for the help.

A He-flow is used to prevent air dissolving in the water. I equilibrate in 10% ACN for 15min. A couple of hours would consume a lot of mobile phase. I don't think there is a back-pressure regulator after the detector. I will have a look at that.

[skunked_once]

My bets are on stuff eluting from the column after reaching 100% acetonitrile. Whatever the cause, is this a serious problem for your analysis or are you just curious about the cause? The baseline shift is less than 0.01 AU as compared to an absorbance of about 0.4 AU for your sample peak. Is this a case of making a mountain out of a molehill?