ZIC-pHILIC column care

[plusik]

Hello,

We are using Sequant ZIC-pHILIC column to separate adenosine nucleotides (AMP, ADP, ATP, CoA, Acetyl-CoA..) for detection with MS.

We use 10mM ammonium carbonate buffer at high pH, similar conditions as here: http://www.sequant.com/sn/ufiles/SeQuan ... 00-03A.pdf

In the beginning we were getting very nice sharp peaks, but after 1 week of using the column, some peaks show high tailing (ADP, CoA) and overally intensity is decreased.

Now my questions are:

1) Is it really safe to use ZIC-pHILIC at pH ~9? The manual says OK, but we can see peaks in the MS background that apparently come from the column particles (structure is not stable?), while we don't see these peaks at neutral pH.

2) We use ACN:2-propanol:H2O 40:40:20 as a wash solution for our LC line. Is it safe to wash the column with this? Is it better for the column to wash it often, or rather to leave it containing the mobile phase (high pH)?

Thank you!

Tomas

[Mark Tracy]

If the measured pH of the aqueous buffer is around 9, the effective pH is closer to 10 in the mobile phase. The pKa of carbonate (and other anions) shifts toward the basic end for organic/water mixtures.

[Wen Jiang]

Hi Tomas,
The ZIC®-pHILIC column is designed to run at pH 2-10. I personally think it should not be a problem to work at your separation conditions, especially using the HILIC eluent that normally contains 70% acetonitrile. We actually have not seen this problem after several hundred columns sales. Your finding is really interesting and valuable to us.

1) Is it really safe to use ZIC-pHILIC at pH ~9? The manual says OK, but we can see peaks in the MS background that apparently come from the column particles (structure is not stable?), while we don't see these peaks at neutral pH.

Can you send all detail information, e.g. the recording of background, chromatograms, sample composition and etc., to support@sequant.com. We will carefully study your data and reply you in short.

2) We use ACN:2-propanol:H2O 40:40:20 as a wash solution for our LC line. Is it safe to wash the column with this? Is it better for the column to wash it often, or rather to leave it containing the mobile phase (high pH)?

ZIC®-pHILIC column can stand this washing solution without any damage. However, it is not a good solution for cleaning column before the column storage. The reason is highly hydrophilic compounds will still be retained on column with this solution by HILIC separation mode. You can check our columnmanual to find our recommended method to clean and store your column.

Best regards,

[Uwe Neue]

The pH of an ammonium bicarbonate buffer does not change much over the entire range of solvent composition. The reason is that the pKa of the bicarbonate goes to a higher pH (as Mark said), but the pKa of the ammonium ion shifts towards a lower pH.

Are you using ammonium bicarbonate?

I can't tell you anything about the stability of the column, since I don't know it. Wen Jiang will help you with that.

[plusik]

Thank you all for your replies.

We are using ammonium carbonate, not bicarbonate. This is the product specification:
http://www.sigmaaldrich.com/catalog/sea ... LUKA/74415

Wen, thank you for your comment. I have just sent our data to support@sequant.com.

[Uwe Neue]

As you can see from the Certificate, this is a mixture of ammonium bicarbonate and ammonium carbamate. Above, I was talking about the ammoniumbicarbonate buffer, prepared from ammonium bicarbonate and ammonia. I have never worked with the carbamate, so I do not know what complications can arise, such as potentially a slow change over time.

[HW Mueller]

plusik, there seems to be nothing mentioned about the matrix of your nucleotides. I am not familiar with the pHILIC column, but have been using ZIC HILIC and Atlantis HILIC. From those I know that they are extremely sensitive toward polar/ionic "dirt" in the sample matrix. Something to whatch.
Some time ago I did a cursory check on ammonium carbonate, this carbamate equilibrium completely disqualifies this material for use in HPLC in my view.

[Wen Jiang]

Hi Tomas,
Thank you for your data. That is really a nice separation on those compounds.
We actually find the source of your problem after a first look on your chromatographic conditions.
You are using ZIC-pHILIC separation column coupled with a ZIC-HILIC guard column. Since ZIC-HILIC is a silica based zwitterionic stationary phase, its useful pH range is 2-8 as SeQuant recommends. In your case, ZIC-HILIC material was slowly dissolved in eluent at pH 9 and resulted MS background. Those pollutants from guard column will finally distort your ZIC-pHILIC column.
Despite that, SeQuant decided to exchange your ZIC-pHILIC column and replace ZIC-HILIC guard column by ZIC-pHILIC guard. We will have further contact with you through support@sequant.com.

Kind regards,

[Uwe Neue]

So, HW, or others...

I know that the "ammonium carbonate" contains the carbamate. I would also expect that the carbamate is not terribly stable in water, but I do not know anything about this. Could somebody enlighten me?

[plusik]

We actually find the source of your problem after a first look on your chromatographic conditions.
You are using ZIC-pHILIC separation column coupled with a ZIC-HILIC guard column. Since ZIC-HILIC is a silica based zwitterionic stationary phase, its useful pH range is 2-8 as SeQuant recommends. In your case, ZIC-HILIC material was slowly dissolved in eluent at pH 9 and resulted MS background. Those pollutants from guard column will finally distort your ZIC-pHILIC column.
Despite that, SeQuant decided to exchange your ZIC-pHILIC column and replace ZIC-HILIC guard column by ZIC-pHILIC guard. We will have further contact with you through support@sequant.com.

Thank you very much for the support. We had asked our dealer to provide a suitable guard column for ZIC-pHILIC and he sent us the ZIC-HILIC guard. Therefore I didn't expect any problems here.
I will contact you further by e-mail..

If the measured pH of the aqueous buffer is around 9, the effective pH is closer to 10 in the mobile phase. The pKa of carbonate (and other anions) shifts toward the basic end for organic/water mixtures.

Our buffer is 10mM ammonium carbonate + 0.2% NH4OH.
pH is 9.3. When I mix it with ACN 50:50, the pH actually drops to 9.1

plusik, there seems to be nothing mentioned about the matrix of your nucleotides. I am not familiar with the pHILIC column, but have been using ZIC HILIC and Atlantis HILIC. From those I know that they are extremely sensitive toward polar/ionic "dirt" in the sample matrix. Something to whatch.

We were using same sample during 1 week and the conditions were still changing. The sample is dissolved in ACN/MeOH.

Some time ago I did a cursory check on ammonium carbonate, this carbamate equilibrium completely disqualifies this material for use in HPLC in my view.

We were getting pretty nice results with carbonate, until the column condition got worse. Do you recommend to switch to ammonium bicarbonate, while we're still at the method development stage?

[Uwe Neue]

I absolutely recommend to go to ammonium bicarbonate. We have used it for years in many applications without any headaches.

[HW Mueller]

What I remember is that ammonium carbonate is simply undefined. I just now checked the Merck catalog, they claim that theirs is 1:1, CH6N2O2 : CH5NO3 (H2NCO2- NH4+ : NH4+ HCO3-) in the solid. If I am not confusing something this ratio has to do with the manufacturing method, whether one gets a fast equilibrium in solution I don´t remember, but even if there is one it would be temperature, ionic strength, org modifyer dependent. Again this is a vague recollection and could be a confusion: There could be some kinetic effects so that equilibrium is not reached in reasonable times or consistently (repeatably). It´s completely clear in my mind, though, that I have classified this as not worth the bother.
Also, I remember that we discussed this in more detail maybe a year ago?