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By Raymond on Thursday, March 25, 2004 - 02:59 am:
I am currently analyzing an amino sugar. As i do not have much experience with HILIC, what conditions shld i try first to obtain seperation of these type of compounds. I have a zic HILIC column as well as a waters silica HILIC column.
Please Advice
Thank You
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By AllsepTech on Thursday, March 25, 2004 - 06:34 am:
Raymond,
After you try HILIC consider the following method specially developed for the retention of amino sugars.
http://www.allsep.com/makeChr.php?chr=Chr_012
This was performed on our Primesep 100 column. The column is a combination of reverse phase and ion-exchange columns. You can retain other compounds by any of two mechanisms (or by combination of both). If you change conditions (increase amount of acid in the mobile phase) you will have shorter retention time. Contact us at mail@allsep.com if you need Lc/MS compatible conditions.
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By Uwe Neue on Thursday, March 25, 2004 - 04:04 pm:
Both columns are working differently. ZIC HILIC has zwitterions on the surface, which should give you a neutral, but very hydrophilic surface. The silica HILIC column works via the same HILIC mechanism, but in addition it interacts via ion-exchange of the silanols with your amino sugars.
I personally would use the silica HILIC column first, since it has the ion-exchange function and thus the dual retention mechanism. More things to play with to get the right selectivity.
With respect to the comment by Allseptech, it needs to be said that sugars do not interact with C18, and thus only the ion-exchange would contribute to retention.
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By AllsepTech on Thursday, March 25, 2004 - 05:06 pm:
Dear Uwe,
I stated that Primesep columns has the reverse phase property to emphasize that you can do a lot of separations on it, not only ion-exchange. In many cases amino sugars are made through the route involving aldehydes, amines or other starting materials. So you might need a reverse phase property to monitor reaction, decomposition, etc. Plus if you are talking about formulations which involve amino sugars you might have other components which are not retained by ion-exchange mechanism by can be easily retained on our column by a reverse phase mechanism. The beauty of our approach is that you have combination of two columns with more capabilities for tough separations.
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By Raymond on Friday, March 26, 2004 - 12:55 am:
Hi,
What are the conditions i shld use on the waters atlantis HILIC column? Should I include ion pairing agents such as TFA in the mobile phase?
Thanks
-------------------------------------------------------------------------------------------------------
By Tobias Jonsson on Friday, March 26, 2004 - 05:56 pm:
Raymond and Uwe,
The zwitterionic ZIC®-HILIC column certainly has ion exchange properties, although it carries a zero net charge. It is true though, that the ion exchange properties are weaker compared to silica columns, but for positively charged analytes it is my experience that this is an advantage, since less salt in the mobile phase is required to disrupt the electrostatic interactions between the analyte and the stationary phase. Moreover is the charge on the ZIC®-HILIC column pH-independent, which the silanols are not. My first choice for the aminosugar separation problem would thus be ZIC®-HILIC, although I respect that Uwe at Waters prefers plain silica.
For isocratic method development on the ZIC®-HILIC column it is usually recommend to start with an eluent containing 80% acetonitrile, and using a few mM of a buffer such as ammonium acetate or formate (see pdf-presentation on www.sequant.com). For aminosugars though, I would recommend less acetonitrile, say 60%, and higher buffer strength, about 20 mM ammonium acetate, as a starting point, since aminosugars are very hydrophilic compounds and are positively charged under these conditions. These conditions should suit silica columns as well, but adding more buffer salt could be necessary.
Generally, phosphate buffer is not recommended in HILIC separations due to its low solubility in organic solvents. Another advice is to avoid TFA and other ion-pairing agents in HILIC separations, since these can “shieldâ€
I am currently analyzing an amino sugar. As i do not have much experience with HILIC, what conditions shld i try first to obtain seperation of these type of compounds. I have a zic HILIC column as well as a waters silica HILIC column.
Please Advice
Thank You
-------------------------------------------------------------------------------------------------------
By AllsepTech on Thursday, March 25, 2004 - 06:34 am:
Raymond,
After you try HILIC consider the following method specially developed for the retention of amino sugars.
http://www.allsep.com/makeChr.php?chr=Chr_012
This was performed on our Primesep 100 column. The column is a combination of reverse phase and ion-exchange columns. You can retain other compounds by any of two mechanisms (or by combination of both). If you change conditions (increase amount of acid in the mobile phase) you will have shorter retention time. Contact us at mail@allsep.com if you need Lc/MS compatible conditions.
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Thursday, March 25, 2004 - 04:04 pm:
Both columns are working differently. ZIC HILIC has zwitterions on the surface, which should give you a neutral, but very hydrophilic surface. The silica HILIC column works via the same HILIC mechanism, but in addition it interacts via ion-exchange of the silanols with your amino sugars.
I personally would use the silica HILIC column first, since it has the ion-exchange function and thus the dual retention mechanism. More things to play with to get the right selectivity.
With respect to the comment by Allseptech, it needs to be said that sugars do not interact with C18, and thus only the ion-exchange would contribute to retention.
-------------------------------------------------------------------------------------------------------
By AllsepTech on Thursday, March 25, 2004 - 05:06 pm:
Dear Uwe,
I stated that Primesep columns has the reverse phase property to emphasize that you can do a lot of separations on it, not only ion-exchange. In many cases amino sugars are made through the route involving aldehydes, amines or other starting materials. So you might need a reverse phase property to monitor reaction, decomposition, etc. Plus if you are talking about formulations which involve amino sugars you might have other components which are not retained by ion-exchange mechanism by can be easily retained on our column by a reverse phase mechanism. The beauty of our approach is that you have combination of two columns with more capabilities for tough separations.
-------------------------------------------------------------------------------------------------------
By Raymond on Friday, March 26, 2004 - 12:55 am:
Hi,
What are the conditions i shld use on the waters atlantis HILIC column? Should I include ion pairing agents such as TFA in the mobile phase?
Thanks
-------------------------------------------------------------------------------------------------------
By Tobias Jonsson on Friday, March 26, 2004 - 05:56 pm:
Raymond and Uwe,
The zwitterionic ZIC®-HILIC column certainly has ion exchange properties, although it carries a zero net charge. It is true though, that the ion exchange properties are weaker compared to silica columns, but for positively charged analytes it is my experience that this is an advantage, since less salt in the mobile phase is required to disrupt the electrostatic interactions between the analyte and the stationary phase. Moreover is the charge on the ZIC®-HILIC column pH-independent, which the silanols are not. My first choice for the aminosugar separation problem would thus be ZIC®-HILIC, although I respect that Uwe at Waters prefers plain silica.
For isocratic method development on the ZIC®-HILIC column it is usually recommend to start with an eluent containing 80% acetonitrile, and using a few mM of a buffer such as ammonium acetate or formate (see pdf-presentation on www.sequant.com). For aminosugars though, I would recommend less acetonitrile, say 60%, and higher buffer strength, about 20 mM ammonium acetate, as a starting point, since aminosugars are very hydrophilic compounds and are positively charged under these conditions. These conditions should suit silica columns as well, but adding more buffer salt could be necessary.
Generally, phosphate buffer is not recommended in HILIC separations due to its low solubility in organic solvents. Another advice is to avoid TFA and other ion-pairing agents in HILIC separations, since these can “shieldâ€
