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By HPLC newbie on Thursday, April 29, 2004 - 04:11 am:
Hi,
I am working on this unknown compound with very poor retentivity on C18 columns e.g. Atlantis dC18 that is supposed to work well even with polar compounds.
From expts, i have found that this compound has a free NH2 grp and that it has a sugar moiety.
I have tried strong ion exchangers (Q - strong anion exchanger) at pH of 4.0 and 9.0, and it did not bind to the column. I eluted with a buffer of the opposite pH ( load pH4.0, then elute with pH9.0).
However, i was not able to detect the compound when i used a strong cation exchanger (SP), in the load pH 9.0 or the elute under pH 4.0.
Can someone suggest what is going on and how I should proceed further to purify this compound?
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By MG on Thursday, April 29, 2004 - 06:55 am:
One option is hydrophilic interaction chromatography (HILIC). You can search this board and find several topics on HILIC. It is like normal phase but uses more polar solvents, like acetonitrile and water. I have used the Waters Atlantis HILIC silica column for this purpose.
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By Anonymous on Thursday, April 29, 2004 - 07:47 am:
Take the Atlantis and run a gradient from 95% of acidic water (slightly above pH 2) to 90% of ACN within 30 min.
If you can't use gradient mode, then begin with appr. 75% acidic water/25% ACN. If your compound eluates to fast, increase amout of acidic water for 5%. Repeat this until you get enough retention.
-------------------------------------------------------------------------------------------------------
By MG on Thursday, April 29, 2004 - 10:26 am:
To add to Anonymous' suggestion, I think the Atlantis dC18 can go to 100% aqueous, so you should try that as well if you haven't already, before switching to something else like HILIC.
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By Anonymous on Thursday, April 29, 2004 - 04:12 pm:
If it has an amine, why do you think it should bind to a strong anion exchanger? You need a strong cation exchanger and exactly the opposite load/eultion protocol.
If your compound is not retained on Atlantis in 100% water, you need to go to HILIC.
-------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, April 29, 2004 - 04:14 pm:
Also, with the strong cation exchanger, you used the opposite protocol that you should use: load at pH 4, elute at pH 9 or 10.
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By AllsepTech on Thursday, April 29, 2004 - 05:34 pm:
Another alternative to regular RP and HILIC ios mixed mode columns wgich have embedded ion-pairing reagent. You can control the retention of amines and other basic compounds by two mechanisms-reverse phase and cation-exchange. This gives you more flexebility to retain amines and to separate critical pairs of compounds which are otherwise coeluted or closely eluted. Check the following link:
http://www.allsep.com/Applications_By_Column.php
We developed a few methods for the retention of amines -check them out, may be you will find them helpful.
Disclaimer: we are manufacturing these mixed mode columns and there are few other companies out there maikin mixed mode columns.. You might want to do more reading and comparing.
-------------------------------------------------------------------------------------------------------
By HPLC newbie on Thursday, April 29, 2004 - 05:56 pm:
I have also tried the strong cation exchange with a load ph of 4.0 and a elute pH of 9.0 but still did not get any retention of the compound. The compund was detected in the load fraction.
When i used the reverse (load pH 9.0 and elute pH 4.0) i could detect my compound of i interest in both the load and the elute fractions.
-------------------------------------------------------------------------------------------------------
By HPLC newbie on Thursday, April 29, 2004 - 06:02 pm:
Another question i have is regarding reverse phase seperations, when you work at high pH for basic compound would you get better retention?
How bout for compounds that are amphoteric? Would changes in pH affect the retention of this compound and is this related to the pKa of the compound?
-------------------------------------------------------------------------------------------------------
By HPLC newbie on Thursday, April 29, 2004 - 06:02 pm:
I have read that for sugars there is peak spliting due to changes in configuration of the sugar moiety (alpha and beta anomeric forms of the sugar).
How do i avoid this situation without destroying the anomeric centre by reduction? Would changes in the temperature of the seperation or addition of buffers prevent the peak spliting?
-------------------------------------------------------------------------------------------------------
By Hugh on Thursday, April 29, 2004 - 11:48 pm:
Generally speaking, basic compounds chromatograph better at a higher pH in a reversed phase mode. If you can run at a pH above the pKa, then the unionized form will tend to exhibit better retention.
Again, this is a generalized statement, not necessarily true for all components.
-------------------------------------------------------------------------------------------------------
By HPLC Newbie on Friday, April 30, 2004 - 04:09 am:
What is the reason for the unionized form to have poor retention?
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By MG on Friday, April 30, 2004 - 06:34 am:
It's the ionized for that usually has less retention. But, if there's enough hydrophobic character on the molecule, there will still be enough retention to do separation by reverse phase. I have chromatographed plenty of bases in their ionized form on a C18, without ion pair reagents. Often the pH needed to get a basic compound into its neutral form would destroy the column, unless you have a special column designed for high pH (or a non-silica based column). Conversely, I have chromatographed sulfonic acids in their ionized form by RP as well, because the pH required to protonate them would be low enough to destroy the column.
Even if you can get the compound in a neutral form, if there's no hydrophobic character, it won't retain on a C18. Sugars are an example.
-------------------------------------------------------------------------------------------------------
By RH on Friday, April 30, 2004 - 10:46 am:
I agree with MG that it will be quite difficult to retain sugar derivatives well on RP18. Usually aminopropyl or polyamine columns based on silica are used. Much better separation can usually be achieved on special polymer-based columns working mainly with ion exclusion, ion exchange or ligand exchange modes. Have a look a webpages of BioRad, Macherey&Nagel, Dionex, etc.
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Friday, April 30, 2004 - 03:12 pm:
HPLC Newbie,
I want to pursue further the your statement that you observe no retention for your analytes using a strong cation exchange column at low pH. You should observe retention of an amino sugar on a strong cation exchange column at low pH. Perhaps there is some problem with your experimental conditions. What is your electrolyte in your mobile phase in this case? You should use an electrolyte which contains a weak eluent cation if you're analyte is really unretained under traditional experimental conditions. Lithium is generally the weakest eluent cation so if you're having trouble with retention try using lithium based eluents. What ionic strength did you use? In cation exchange separations, retention can be improved by reducing ionic strength. How long did you allow the column to reequilibrate prior to injection? If you haven't waited long enough after passing the pH 9 mobile phase over the column, the stationary phase may not yet be in equilibrium with your pH for buffer. Check to see that the column effluent pH matches the feed buffer pH. Also, how do you know for sure that you're analyte was unretained? A common problem is too misinterpret void volume artifacts as indication of no retention when in fact the analyte never eluted.
-------------------------------------------------------------------------------------------------------
By Einar Pontén - SeQuant AB on Sunday, May 2, 2004 - 03:00 pm:
Mr/Mrs HPLC Newbie, I also suspect that you need to repeat the experiment using a cation exchanger at low pH.
The trouble you are having with no retention on RP columns is a very good indicator that HILIC will solve your problem.
According to our experience amino-sugars have suitable retention on the ZIC®-HILIC column.
-------------------------------------------------------------------------------------------------------
By HPLC newbie on Monday, May 3, 2004 - 12:17 am:
Dear Chris,
I used a cell assay to detect for the presence of my compound, and showed that the compound eluted in the elute fraction with activity in the wash fraction as well.
The buffer system that i used was ammonia formate and ammonia carbonate the initial loading and sunsequent elute fraction.
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Tuesday, May 4, 2004 - 10:14 am:
HPLC newbie,
What did you use as your pH 4 eluent system in the case of the SCX work? When you say load at pH 4 and elute at pH 9 do you mean that the eluent is pH 9 and only your sample is pH 4 or do you mean that when you load, both the sample and the eluent are pH 4? If it is the former, this is probably not a very good strategy. Your eluent needs to be at a pH sufficient to allow for retention. Amino sugars should be separated at a pH < 8 if you want significant retention via SCX. Simply acidifying the sample will generally not be sufficient to achieve significant retention.
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, May 4, 2004 - 03:42 pm:
A different route...
Something you might try is a column like the Chirobiotic T. It is a chiral column, and rather expensive, but I have analysed several bases with this method in my reserach. Using the T column, and a mobile phase of 100% methanol, polar compounds, like bases and sugars, can be retained appreciably. Often 0.1% of an acid and/or base (acetic acid, formic acid, triethylamine, ammonium hydroxide are common) are added to modulate the degree of protonation/retention of the compounds.
If your compound is chiral, it may separate into enantiomer, which may or may not be deseriable.
The column is made buy a company called Astec. Web site is astecusa.com I don't work for them and I'm not trying to plug the products. I'm just offering advice that I would try if I were in your shoes. You can probably get method development advice from their website.
Good Luck.
-------------------------------------------------------------------------------------------------------
By Einar Pontén - SeQuant AB on Wednesday, May 5, 2004 - 03:10 pm:
Essentially this latter suggestion is a HILIC-mode separation on a bonded glycoprotein phase.
-------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, June 3, 2004 - 04:31 am:
u can use Refractive index detector since it contains a sugair moiety
Hi,
I am working on this unknown compound with very poor retentivity on C18 columns e.g. Atlantis dC18 that is supposed to work well even with polar compounds.
From expts, i have found that this compound has a free NH2 grp and that it has a sugar moiety.
I have tried strong ion exchangers (Q - strong anion exchanger) at pH of 4.0 and 9.0, and it did not bind to the column. I eluted with a buffer of the opposite pH ( load pH4.0, then elute with pH9.0).
However, i was not able to detect the compound when i used a strong cation exchanger (SP), in the load pH 9.0 or the elute under pH 4.0.
Can someone suggest what is going on and how I should proceed further to purify this compound?
-------------------------------------------------------------------------------------------------------
By MG on Thursday, April 29, 2004 - 06:55 am:
One option is hydrophilic interaction chromatography (HILIC). You can search this board and find several topics on HILIC. It is like normal phase but uses more polar solvents, like acetonitrile and water. I have used the Waters Atlantis HILIC silica column for this purpose.
-------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, April 29, 2004 - 07:47 am:
Take the Atlantis and run a gradient from 95% of acidic water (slightly above pH 2) to 90% of ACN within 30 min.
If you can't use gradient mode, then begin with appr. 75% acidic water/25% ACN. If your compound eluates to fast, increase amout of acidic water for 5%. Repeat this until you get enough retention.
-------------------------------------------------------------------------------------------------------
By MG on Thursday, April 29, 2004 - 10:26 am:
To add to Anonymous' suggestion, I think the Atlantis dC18 can go to 100% aqueous, so you should try that as well if you haven't already, before switching to something else like HILIC.
-------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, April 29, 2004 - 04:12 pm:
If it has an amine, why do you think it should bind to a strong anion exchanger? You need a strong cation exchanger and exactly the opposite load/eultion protocol.
If your compound is not retained on Atlantis in 100% water, you need to go to HILIC.
-------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, April 29, 2004 - 04:14 pm:
Also, with the strong cation exchanger, you used the opposite protocol that you should use: load at pH 4, elute at pH 9 or 10.
-------------------------------------------------------------------------------------------------------
By AllsepTech on Thursday, April 29, 2004 - 05:34 pm:
Another alternative to regular RP and HILIC ios mixed mode columns wgich have embedded ion-pairing reagent. You can control the retention of amines and other basic compounds by two mechanisms-reverse phase and cation-exchange. This gives you more flexebility to retain amines and to separate critical pairs of compounds which are otherwise coeluted or closely eluted. Check the following link:
http://www.allsep.com/Applications_By_Column.php
We developed a few methods for the retention of amines -check them out, may be you will find them helpful.
Disclaimer: we are manufacturing these mixed mode columns and there are few other companies out there maikin mixed mode columns.. You might want to do more reading and comparing.
-------------------------------------------------------------------------------------------------------
By HPLC newbie on Thursday, April 29, 2004 - 05:56 pm:
I have also tried the strong cation exchange with a load ph of 4.0 and a elute pH of 9.0 but still did not get any retention of the compound. The compund was detected in the load fraction.
When i used the reverse (load pH 9.0 and elute pH 4.0) i could detect my compound of i interest in both the load and the elute fractions.
-------------------------------------------------------------------------------------------------------
By HPLC newbie on Thursday, April 29, 2004 - 06:02 pm:
Another question i have is regarding reverse phase seperations, when you work at high pH for basic compound would you get better retention?
How bout for compounds that are amphoteric? Would changes in pH affect the retention of this compound and is this related to the pKa of the compound?
-------------------------------------------------------------------------------------------------------
By HPLC newbie on Thursday, April 29, 2004 - 06:02 pm:
I have read that for sugars there is peak spliting due to changes in configuration of the sugar moiety (alpha and beta anomeric forms of the sugar).
How do i avoid this situation without destroying the anomeric centre by reduction? Would changes in the temperature of the seperation or addition of buffers prevent the peak spliting?
-------------------------------------------------------------------------------------------------------
By Hugh on Thursday, April 29, 2004 - 11:48 pm:
Generally speaking, basic compounds chromatograph better at a higher pH in a reversed phase mode. If you can run at a pH above the pKa, then the unionized form will tend to exhibit better retention.
Again, this is a generalized statement, not necessarily true for all components.
-------------------------------------------------------------------------------------------------------
By HPLC Newbie on Friday, April 30, 2004 - 04:09 am:
What is the reason for the unionized form to have poor retention?
-------------------------------------------------------------------------------------------------------
By MG on Friday, April 30, 2004 - 06:34 am:
It's the ionized for that usually has less retention. But, if there's enough hydrophobic character on the molecule, there will still be enough retention to do separation by reverse phase. I have chromatographed plenty of bases in their ionized form on a C18, without ion pair reagents. Often the pH needed to get a basic compound into its neutral form would destroy the column, unless you have a special column designed for high pH (or a non-silica based column). Conversely, I have chromatographed sulfonic acids in their ionized form by RP as well, because the pH required to protonate them would be low enough to destroy the column.
Even if you can get the compound in a neutral form, if there's no hydrophobic character, it won't retain on a C18. Sugars are an example.
-------------------------------------------------------------------------------------------------------
By RH on Friday, April 30, 2004 - 10:46 am:
I agree with MG that it will be quite difficult to retain sugar derivatives well on RP18. Usually aminopropyl or polyamine columns based on silica are used. Much better separation can usually be achieved on special polymer-based columns working mainly with ion exclusion, ion exchange or ligand exchange modes. Have a look a webpages of BioRad, Macherey&Nagel, Dionex, etc.
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Friday, April 30, 2004 - 03:12 pm:
HPLC Newbie,
I want to pursue further the your statement that you observe no retention for your analytes using a strong cation exchange column at low pH. You should observe retention of an amino sugar on a strong cation exchange column at low pH. Perhaps there is some problem with your experimental conditions. What is your electrolyte in your mobile phase in this case? You should use an electrolyte which contains a weak eluent cation if you're analyte is really unretained under traditional experimental conditions. Lithium is generally the weakest eluent cation so if you're having trouble with retention try using lithium based eluents. What ionic strength did you use? In cation exchange separations, retention can be improved by reducing ionic strength. How long did you allow the column to reequilibrate prior to injection? If you haven't waited long enough after passing the pH 9 mobile phase over the column, the stationary phase may not yet be in equilibrium with your pH for buffer. Check to see that the column effluent pH matches the feed buffer pH. Also, how do you know for sure that you're analyte was unretained? A common problem is too misinterpret void volume artifacts as indication of no retention when in fact the analyte never eluted.
-------------------------------------------------------------------------------------------------------
By Einar Pontén - SeQuant AB on Sunday, May 2, 2004 - 03:00 pm:
Mr/Mrs HPLC Newbie, I also suspect that you need to repeat the experiment using a cation exchanger at low pH.
The trouble you are having with no retention on RP columns is a very good indicator that HILIC will solve your problem.
According to our experience amino-sugars have suitable retention on the ZIC®-HILIC column.
-------------------------------------------------------------------------------------------------------
By HPLC newbie on Monday, May 3, 2004 - 12:17 am:
Dear Chris,
I used a cell assay to detect for the presence of my compound, and showed that the compound eluted in the elute fraction with activity in the wash fraction as well.
The buffer system that i used was ammonia formate and ammonia carbonate the initial loading and sunsequent elute fraction.
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Tuesday, May 4, 2004 - 10:14 am:
HPLC newbie,
What did you use as your pH 4 eluent system in the case of the SCX work? When you say load at pH 4 and elute at pH 9 do you mean that the eluent is pH 9 and only your sample is pH 4 or do you mean that when you load, both the sample and the eluent are pH 4? If it is the former, this is probably not a very good strategy. Your eluent needs to be at a pH sufficient to allow for retention. Amino sugars should be separated at a pH < 8 if you want significant retention via SCX. Simply acidifying the sample will generally not be sufficient to achieve significant retention.
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, May 4, 2004 - 03:42 pm:
A different route...
Something you might try is a column like the Chirobiotic T. It is a chiral column, and rather expensive, but I have analysed several bases with this method in my reserach. Using the T column, and a mobile phase of 100% methanol, polar compounds, like bases and sugars, can be retained appreciably. Often 0.1% of an acid and/or base (acetic acid, formic acid, triethylamine, ammonium hydroxide are common) are added to modulate the degree of protonation/retention of the compounds.
If your compound is chiral, it may separate into enantiomer, which may or may not be deseriable.
The column is made buy a company called Astec. Web site is astecusa.com I don't work for them and I'm not trying to plug the products. I'm just offering advice that I would try if I were in your shoes. You can probably get method development advice from their website.
Good Luck.
-------------------------------------------------------------------------------------------------------
By Einar Pontén - SeQuant AB on Wednesday, May 5, 2004 - 03:10 pm:
Essentially this latter suggestion is a HILIC-mode separation on a bonded glycoprotein phase.
-------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, June 3, 2004 - 04:31 am:
u can use Refractive index detector since it contains a sugair moiety
