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By LYR on Wednesday, July 7, 2004 - 09:04 am:
Hi all,
I have a v difficult problem developing a LC-SRM-MS method for a v polar non-protein amino acid in natural products. The compound has 2 COOH gps, 1 NH2 gp, reported pka for this compound is 1.95, 2.95, 9.25.
I tried all possible C18 and it cannot be retained.
So, I recently got a new Waters Atlantis HILIC silica column, 50x2.1mm, 3um. This is my 1st experience using HILIC and now I’m facing a challenging problem:
It can be retained in HILIC already. But there is this serious problem of peak broadening and tailing. Peak width can go up to 3min wide! The best so far is still a broad 1.2min wide.
The tubings diameter and length are all appropriate to reduce postcolumn broadening. The concentration used is about 1ng/ul, and injection vol is 2ul. There shouldn’t be any overloading.
I have tried using various isocratic as well as various gradient conditions without any success. There is always the same peak broadening despite what I tried.
I have tried using solvent A: 0.2%formic acid, B: ACN.
I have also switched to A: 10mM Ammonium formate (pH3), B: ACN to try. An eg.: running isocratic at 60%B gave a retention at about 1.4min, but a v broad peak of about 1.5min!
I really don’t know what is happening? How to optimise it? What else can I try to solve this problem? Anyone has experience with this Atlantis HILIC silica?
Hope to receive some help and advices from this forum. Thanks!
Regards,
LYR
-------------------------------------------------------------------------------------------------------
By tom jupille on Wednesday, July 7, 2004 - 10:24 am:
Is there any chance that your amino acid can exist in two different conformations in solution? If so, you may be seeing interconversion and increasing the temperature may help.
-------------------------------------------------------------------------------------------------------
By SIELC_Tech on Wednesday, July 7, 2004 - 11:44 am:
Dear Lyr,
If you get tired of HILIC optimization, try our Primesep approach to the retention of amino acids, you can choose among several columns to retain your compound and run LC/MS comaptible method.
http://allsep.com/makeChr.php?chr=Chr_009 (analysis of underivitized aminoacids)
Peak shape should be good (just see the comparisson). This example is not similar to yours but shows comparisson of peak shape for several leading brand LC columns.
http://allsep.com/makeChr.php?chr=Chr_068
You can always contact us if you need more information.
-------------------------------------------------------------------------------------------------------
By MG on Thursday, July 8, 2004 - 11:25 am:
I have found that using higher ammonium formate in the aqueous (strong) mobile phase will reduce broadening and tailing, up to 100-200 mM (but it's a good idea to check solubility by mixing some ACN with your buffer in a beaker). Unfortunately such high buffer conc can reduce your LC/MS sensitivity.
-------------------------------------------------------------------------------------------------------
By LYR on Monday, July 12, 2004 - 02:53 am:
Thanks all for your suggestions...
i do not know if my amino acid will have different conformations in solution. but i have tried increasing temperature, it did not help.
i have continued trying with pH 5.9 buffer, changing ACN to MeOH etc, all without any success. Although i noticed changing to MeOH seems to narrow the peak v slightly. but still a fat peak....v frustrating.
MG: thanks, ok, i will try a higher concentration of formate next.
i heard that HILIC is better for cationic basic compounds. My compound has both acidic and basic groups. If the pH is reduced to below 1.95 (pka of the lowest COOH gp), so that the net charge is positive, just wondering if it will help?? but the problem is not many solvents can reduce the pH to below 2 too....except TFA which will also reduce LCMS sensitivity....
-------------------------------------------------------------------------------------------------------
By Einar Pontén - SeQuant AB on Monday, July 12, 2004 - 03:09 pm:
This problem is likely caused by the relatively unspecific interaction with the silanols on plain silica. You should also expect long equilibration times.
I agree with MG that you should try to increase the ion strength and also keep control of the pH in the mobile phase.
If you contact us we would enjoy to supply you with a ZIC®-HILIC column for comparision. That will probably solve your problem as well.
-------------------------------------------------------------------------------------------------------
By SIELC_Tech on Monday, July 12, 2004 - 03:47 pm:
LYR,
We are willing to develop a method for you if you send us a sample. While we try to develop LC/MS compatible method on our Primesep columns you can continue your attempts to develop HILIC approach. Contact us at mail@sielc.com
-------------------------------------------------------------------------------------------------------
By A.N. Onimis on Friday, July 16, 2004 - 11:59 am:
I'll be different and make an actual chromatographic suggestion in between the advertising war between SeQuant & SIELC.
What is your injection solvent? Try making up your standard in the initial mobile phase conditions (or weaker). For example, maybe 60, 70 or 80% ACN. ACN is your weak solvent just like water in RP.
A 50 x 2.1 mm column is quite small and very easy to overload if your injection solvent is too strong (too much water).
Try 0.2% formic acid as a low pH additive. Or 100 mM ammonium formate, pH 3.
Now back to the SeQuant & SIELC show.
-------------------------------------------------------------------------------------------------------
By LYR on Sunday, July 18, 2004 - 11:04 am:
Grateful to all your offers of help:)
My sample can only dissolve in water. but after preparing the stock solution, i dilute the stock at least 100x using 100%organic solvent(75:25 ACN:MeOH, as recommended in the column brochure), so the amount of water is definitely very small.
i have just tried increasing the ammonium formate concentration last week. and yes, it does help in narrowing the peak. seeing some improvements.
just tried 40% 200mM ammonium formate pH3 with 60% ACN. This high concentration of about 80mM ammonium formate pH3 ON COLUMN seems to improve the peak to about 0.5min with lesser tailing, instead of the previous 1-3min broad tailing peak.
do you all think this concentration is too high? is this method with such high conc. acceptable?
wondering how sharp should an acceptable peak preferably be? seems hard to reduce peak width to <0.2min narrow peak. but i am doing LC-SRM, so resolution problem should be less.
and for a 50x2.1mm column, is a RT of 0.8min ok or too early?
thanks again.
-------------------------------------------------------------------------------------------------------
By SIELC_Tech on Monday, July 19, 2004 - 07:56 am:
LYR,
Are glutaric and aspartic acids match your aminoacid? ASP and GLU both have two -COOH and one -NH2 groups. If yes, We will run them both on Primesep column with LC/MS compatible conditions and see if we can get a better peak shape.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Tuesday, July 20, 2004 - 07:05 pm:
Lyr:
Never mind the previous comments, they do not address your fundamental problem.
Please tell us about the solvent that you use to dissolve your sample in HILIC! HILIC is more sensitive, and it requires the opposite of reversed-phase: you need to dissolve your sample preferentially in the organic solvent, and most samples do not like that. In order to get good peak shape on your HILIC column, the sample needs to be dissolved in a mobile phase with a higher or equal organic content than the mobile phase. Most likely, you dissolved your sample in water, where its solubility is best. If you do this right, you'll do ok with the silica HILIC column.
-------------------------------------------------------------------------------------------------------
By LYR on Thursday, July 22, 2004 - 09:19 am:
SIELC: my amino acid is not glutaric nor aspartic. it is an unusual non-protein type of amino acid that is not commonly available commercially. it is even more polar than aspartic.
A.Mouse: thanks for comment. pls also refer to my same reply on 18Jul---my sample stock solution is dissolved in water. but it is already diluted at least 100x using 100%organic solvent(75:25 ACN:MeOH, as recommended in the column brochure), so the amount of water is very small and of higher organic content than the mobile phase. guess it is not this solvent problem?
-------------------------------------------------------------------------------------------------------
By SIELC_Tech on Thursday, July 22, 2004 - 11:24 am:
"Fundamental problem" here is that LYR is trying to use the wrong tool. His original message was generated on July 7, so two weeks later after following bunch of suggestions the problem still exists and he can try to solve it for another two weeks. Is this productive and cost effective? Or does it have any scientific value.
LYR:
Even if you aminoacid is even more polar than aspartic and glutaric you can retain it on a mixed mode column by ion-excahnge mechanisms. We still are willing to run your sample or can send you a column for a trial.
Hi all,
I have a v difficult problem developing a LC-SRM-MS method for a v polar non-protein amino acid in natural products. The compound has 2 COOH gps, 1 NH2 gp, reported pka for this compound is 1.95, 2.95, 9.25.
I tried all possible C18 and it cannot be retained.
So, I recently got a new Waters Atlantis HILIC silica column, 50x2.1mm, 3um. This is my 1st experience using HILIC and now I’m facing a challenging problem:
It can be retained in HILIC already. But there is this serious problem of peak broadening and tailing. Peak width can go up to 3min wide! The best so far is still a broad 1.2min wide.
The tubings diameter and length are all appropriate to reduce postcolumn broadening. The concentration used is about 1ng/ul, and injection vol is 2ul. There shouldn’t be any overloading.
I have tried using various isocratic as well as various gradient conditions without any success. There is always the same peak broadening despite what I tried.
I have tried using solvent A: 0.2%formic acid, B: ACN.
I have also switched to A: 10mM Ammonium formate (pH3), B: ACN to try. An eg.: running isocratic at 60%B gave a retention at about 1.4min, but a v broad peak of about 1.5min!
I really don’t know what is happening? How to optimise it? What else can I try to solve this problem? Anyone has experience with this Atlantis HILIC silica?
Hope to receive some help and advices from this forum. Thanks!
Regards,
LYR
-------------------------------------------------------------------------------------------------------
By tom jupille on Wednesday, July 7, 2004 - 10:24 am:
Is there any chance that your amino acid can exist in two different conformations in solution? If so, you may be seeing interconversion and increasing the temperature may help.
-------------------------------------------------------------------------------------------------------
By SIELC_Tech on Wednesday, July 7, 2004 - 11:44 am:
Dear Lyr,
If you get tired of HILIC optimization, try our Primesep approach to the retention of amino acids, you can choose among several columns to retain your compound and run LC/MS comaptible method.
http://allsep.com/makeChr.php?chr=Chr_009 (analysis of underivitized aminoacids)
Peak shape should be good (just see the comparisson). This example is not similar to yours but shows comparisson of peak shape for several leading brand LC columns.
http://allsep.com/makeChr.php?chr=Chr_068
You can always contact us if you need more information.
-------------------------------------------------------------------------------------------------------
By MG on Thursday, July 8, 2004 - 11:25 am:
I have found that using higher ammonium formate in the aqueous (strong) mobile phase will reduce broadening and tailing, up to 100-200 mM (but it's a good idea to check solubility by mixing some ACN with your buffer in a beaker). Unfortunately such high buffer conc can reduce your LC/MS sensitivity.
-------------------------------------------------------------------------------------------------------
By LYR on Monday, July 12, 2004 - 02:53 am:
Thanks all for your suggestions...
i do not know if my amino acid will have different conformations in solution. but i have tried increasing temperature, it did not help.
i have continued trying with pH 5.9 buffer, changing ACN to MeOH etc, all without any success. Although i noticed changing to MeOH seems to narrow the peak v slightly. but still a fat peak....v frustrating.
MG: thanks, ok, i will try a higher concentration of formate next.
i heard that HILIC is better for cationic basic compounds. My compound has both acidic and basic groups. If the pH is reduced to below 1.95 (pka of the lowest COOH gp), so that the net charge is positive, just wondering if it will help?? but the problem is not many solvents can reduce the pH to below 2 too....except TFA which will also reduce LCMS sensitivity....
-------------------------------------------------------------------------------------------------------
By Einar Pontén - SeQuant AB on Monday, July 12, 2004 - 03:09 pm:
This problem is likely caused by the relatively unspecific interaction with the silanols on plain silica. You should also expect long equilibration times.
I agree with MG that you should try to increase the ion strength and also keep control of the pH in the mobile phase.
If you contact us we would enjoy to supply you with a ZIC®-HILIC column for comparision. That will probably solve your problem as well.
-------------------------------------------------------------------------------------------------------
By SIELC_Tech on Monday, July 12, 2004 - 03:47 pm:
LYR,
We are willing to develop a method for you if you send us a sample. While we try to develop LC/MS compatible method on our Primesep columns you can continue your attempts to develop HILIC approach. Contact us at mail@sielc.com
-------------------------------------------------------------------------------------------------------
By A.N. Onimis on Friday, July 16, 2004 - 11:59 am:
I'll be different and make an actual chromatographic suggestion in between the advertising war between SeQuant & SIELC.
What is your injection solvent? Try making up your standard in the initial mobile phase conditions (or weaker). For example, maybe 60, 70 or 80% ACN. ACN is your weak solvent just like water in RP.
A 50 x 2.1 mm column is quite small and very easy to overload if your injection solvent is too strong (too much water).
Try 0.2% formic acid as a low pH additive. Or 100 mM ammonium formate, pH 3.
Now back to the SeQuant & SIELC show.
-------------------------------------------------------------------------------------------------------
By LYR on Sunday, July 18, 2004 - 11:04 am:
Grateful to all your offers of help:)
My sample can only dissolve in water. but after preparing the stock solution, i dilute the stock at least 100x using 100%organic solvent(75:25 ACN:MeOH, as recommended in the column brochure), so the amount of water is definitely very small.
i have just tried increasing the ammonium formate concentration last week. and yes, it does help in narrowing the peak. seeing some improvements.
just tried 40% 200mM ammonium formate pH3 with 60% ACN. This high concentration of about 80mM ammonium formate pH3 ON COLUMN seems to improve the peak to about 0.5min with lesser tailing, instead of the previous 1-3min broad tailing peak.
do you all think this concentration is too high? is this method with such high conc. acceptable?
wondering how sharp should an acceptable peak preferably be? seems hard to reduce peak width to <0.2min narrow peak. but i am doing LC-SRM, so resolution problem should be less.
and for a 50x2.1mm column, is a RT of 0.8min ok or too early?
thanks again.
-------------------------------------------------------------------------------------------------------
By SIELC_Tech on Monday, July 19, 2004 - 07:56 am:
LYR,
Are glutaric and aspartic acids match your aminoacid? ASP and GLU both have two -COOH and one -NH2 groups. If yes, We will run them both on Primesep column with LC/MS compatible conditions and see if we can get a better peak shape.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Tuesday, July 20, 2004 - 07:05 pm:
Lyr:
Never mind the previous comments, they do not address your fundamental problem.
Please tell us about the solvent that you use to dissolve your sample in HILIC! HILIC is more sensitive, and it requires the opposite of reversed-phase: you need to dissolve your sample preferentially in the organic solvent, and most samples do not like that. In order to get good peak shape on your HILIC column, the sample needs to be dissolved in a mobile phase with a higher or equal organic content than the mobile phase. Most likely, you dissolved your sample in water, where its solubility is best. If you do this right, you'll do ok with the silica HILIC column.
-------------------------------------------------------------------------------------------------------
By LYR on Thursday, July 22, 2004 - 09:19 am:
SIELC: my amino acid is not glutaric nor aspartic. it is an unusual non-protein type of amino acid that is not commonly available commercially. it is even more polar than aspartic.
A.Mouse: thanks for comment. pls also refer to my same reply on 18Jul---my sample stock solution is dissolved in water. but it is already diluted at least 100x using 100%organic solvent(75:25 ACN:MeOH, as recommended in the column brochure), so the amount of water is very small and of higher organic content than the mobile phase. guess it is not this solvent problem?
-------------------------------------------------------------------------------------------------------
By SIELC_Tech on Thursday, July 22, 2004 - 11:24 am:
"Fundamental problem" here is that LYR is trying to use the wrong tool. His original message was generated on July 7, so two weeks later after following bunch of suggestions the problem still exists and he can try to solve it for another two weeks. Is this productive and cost effective? Or does it have any scientific value.
LYR:
Even if you aminoacid is even more polar than aspartic and glutaric you can retain it on a mixed mode column by ion-excahnge mechanisms. We still are willing to run your sample or can send you a column for a trial.
