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By Robin on Sunday, May 30, 2004 - 07:45 am:
Dear All,
Has any of you any experience using ion pair agents as tetrabutylammonium bromide or tetrabutylammonium hydroxide at high pH (9-...) and its impact on LC and subsequent MS? It concerns the LCMS analysis of phosphorylated (1-4) biomolecules (mW< 1000). Is separation of such compounds still feasible at these conditions and how is the mass spectrometric detection behaving? Is there a lot of ion suppression or other problems?
Thanks in advance
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By MG on Tuesday, June 1, 2004 - 07:19 am:
I would not recommend quaternary ammonium salts with LC/MS. With positive ionization, it won't work. You will see no signal for your analytes. Take my word for it. With negative ionization, I can't recall if I've ever tried these additives, but I still see the potential for problems.
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By Einar Pontén - SeQuant AB on Tuesday, June 1, 2004 - 03:17 pm:
Robin, we would be interested in learning more about your application. Why do you need to run at elevated pH values?
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By Robin on Wednesday, June 2, 2004 - 04:09 am:
In answer to MG: We would use ES(-) mode (cf the phosphate groups). Low concentrations on TBAB are described as an ion pair reagent in a LCMS procedure (ES-), but not a high pH conditions.
In answer to Einar. I can not go into detail (yet) about the application. But I'm also interested in the ZIC-HILIC phases. (I followed the developments of Alpert some years ago...nice work).
Greetings
Robin
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By MG on Wednesday, June 2, 2004 - 01:36 pm:
Robin, if anything I would expect the high pH conditions to work better for ESI(-). I would be concerned about the volatility of the salts. But if it has been done in the literature at low pH (I was not aware of that work), I think it is worth a try at high pH.
A couple of things to keep in mind with the LC: (1) Make sure your column is of the type that can handle high pH; (2) Be aware that the column may not be usable for other projects, once exposed to the TBA. I was once given a column to use for an LC/MS analysis (positive mode), and saw nothing but constant m/z 242 signal. The column had once been used with TBA, and I was unable to wash it enough to make it usable with LC/MS.
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By A.Mouse on Friday, June 4, 2004 - 12:02 am:
And watch your interface. If you see white stuff building up, it is the TBAB.
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By Robin on Friday, June 4, 2004 - 03:17 am:
Dear All,
Thank you for your interest! I already have some experience with the use of ion pair LCMS (at pH =7 and no Tetraalkylammoniumsalts), yet not with the mentioned two conditions (TBAB or TBAOH) and the high pH. I was planning to use a polymer phase or the Xterra columns of waters.
About the inferface: the use of Z-sprays allow reasonable salt contents (I even used some phosphate buffers with them (low concentrations of course) and believe or not, the MS-sensitivity was not compromised to much (contrary to the background increment).
If anyone of you has some experience with normal ion pair LCMS; here is another topic I'm concerned about. We have problems to genereate reproducible LCMS runs under these conditions (shifting retention times). This is probably due to the many parameters which control IP mechanism. Has any of you any advice about this phenomenon?
Greetings,
Robin
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Friday, June 4, 2004 - 02:36 pm:
Real ion-pair chromatography (under isocratic condition) takes a long time until the surface is covered / saturated with the ion-pair reagent. Several hours of equilibration time are not unusual. The issue is that the molar quantities of ion-pair reagent in the mobile phase are very small, probably even smaller in your case where your detection is MS.
If you are running gradients, you are always out of equilibrium, and a consistent reequilibration procedure is important.
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By Einar Pontén - SeQuant AB on Friday, June 4, 2004 - 03:00 pm:
We have introduced a new polymeric column, ZIC®-pHILIC that is designed for HILIC at elevated pH. Most likely this column will solve your problem and then you will be free the ion-pair agent problems.
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By AllsepTech on Friday, June 4, 2004 - 05:19 pm:
For reverse phase column with immobilized ion-pairing reagent check this link:
http://allsep.com/Technology_RetentionO ... pounds.php
You do not have to use ion-pairing reagent because in our case it is bonded to silica gel. You can use veriety of LC/MS friendly conditions (ammonium formate, ammonium acetate, formic acid, TFA). Our columns have reverse phase properties so your compounds will retain by two mechanisms RP and ion-exchange. Contact us by email or phone if you need more information
Dear All,
Has any of you any experience using ion pair agents as tetrabutylammonium bromide or tetrabutylammonium hydroxide at high pH (9-...) and its impact on LC and subsequent MS? It concerns the LCMS analysis of phosphorylated (1-4) biomolecules (mW< 1000). Is separation of such compounds still feasible at these conditions and how is the mass spectrometric detection behaving? Is there a lot of ion suppression or other problems?
Thanks in advance
-------------------------------------------------------------------------------------------------------
By MG on Tuesday, June 1, 2004 - 07:19 am:
I would not recommend quaternary ammonium salts with LC/MS. With positive ionization, it won't work. You will see no signal for your analytes. Take my word for it. With negative ionization, I can't recall if I've ever tried these additives, but I still see the potential for problems.
-------------------------------------------------------------------------------------------------------
By Einar Pontén - SeQuant AB on Tuesday, June 1, 2004 - 03:17 pm:
Robin, we would be interested in learning more about your application. Why do you need to run at elevated pH values?
-------------------------------------------------------------------------------------------------------
By Robin on Wednesday, June 2, 2004 - 04:09 am:
In answer to MG: We would use ES(-) mode (cf the phosphate groups). Low concentrations on TBAB are described as an ion pair reagent in a LCMS procedure (ES-), but not a high pH conditions.
In answer to Einar. I can not go into detail (yet) about the application. But I'm also interested in the ZIC-HILIC phases. (I followed the developments of Alpert some years ago...nice work).
Greetings
Robin
-------------------------------------------------------------------------------------------------------
By MG on Wednesday, June 2, 2004 - 01:36 pm:
Robin, if anything I would expect the high pH conditions to work better for ESI(-). I would be concerned about the volatility of the salts. But if it has been done in the literature at low pH (I was not aware of that work), I think it is worth a try at high pH.
A couple of things to keep in mind with the LC: (1) Make sure your column is of the type that can handle high pH; (2) Be aware that the column may not be usable for other projects, once exposed to the TBA. I was once given a column to use for an LC/MS analysis (positive mode), and saw nothing but constant m/z 242 signal. The column had once been used with TBA, and I was unable to wash it enough to make it usable with LC/MS.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Friday, June 4, 2004 - 12:02 am:
And watch your interface. If you see white stuff building up, it is the TBAB.
-------------------------------------------------------------------------------------------------------
By Robin on Friday, June 4, 2004 - 03:17 am:
Dear All,
Thank you for your interest! I already have some experience with the use of ion pair LCMS (at pH =7 and no Tetraalkylammoniumsalts), yet not with the mentioned two conditions (TBAB or TBAOH) and the high pH. I was planning to use a polymer phase or the Xterra columns of waters.
About the inferface: the use of Z-sprays allow reasonable salt contents (I even used some phosphate buffers with them (low concentrations of course) and believe or not, the MS-sensitivity was not compromised to much (contrary to the background increment).
If anyone of you has some experience with normal ion pair LCMS; here is another topic I'm concerned about. We have problems to genereate reproducible LCMS runs under these conditions (shifting retention times). This is probably due to the many parameters which control IP mechanism. Has any of you any advice about this phenomenon?
Greetings,
Robin
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Friday, June 4, 2004 - 02:36 pm:
Real ion-pair chromatography (under isocratic condition) takes a long time until the surface is covered / saturated with the ion-pair reagent. Several hours of equilibration time are not unusual. The issue is that the molar quantities of ion-pair reagent in the mobile phase are very small, probably even smaller in your case where your detection is MS.
If you are running gradients, you are always out of equilibrium, and a consistent reequilibration procedure is important.
-------------------------------------------------------------------------------------------------------
By Einar Pontén - SeQuant AB on Friday, June 4, 2004 - 03:00 pm:
We have introduced a new polymeric column, ZIC®-pHILIC that is designed for HILIC at elevated pH. Most likely this column will solve your problem and then you will be free the ion-pair agent problems.
-------------------------------------------------------------------------------------------------------
By AllsepTech on Friday, June 4, 2004 - 05:19 pm:
For reverse phase column with immobilized ion-pairing reagent check this link:
http://allsep.com/Technology_RetentionO ... pounds.php
You do not have to use ion-pairing reagent because in our case it is bonded to silica gel. You can use veriety of LC/MS friendly conditions (ammonium formate, ammonium acetate, formic acid, TFA). Our columns have reverse phase properties so your compounds will retain by two mechanisms RP and ion-exchange. Contact us by email or phone if you need more information
