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By huang on Wednesday, December 24, 2003 - 05:30 am:
hi:everyone, happy new year.
I have looking for a HPLC method for risedronate sodium,( it is a diphosphonic acid salt).
Looked all over the net and couldn't get anything.
Can anyone help? Any information would be highly appreciated.
Thanks all.
-------------------------------------------------------------------------------------------------------
By huey on Thursday, December 25, 2003 - 06:09 pm:
My opinion:
In brief, if you're using an RP column (i.e. C8) then try Acidic mobile phases i.e. 0.1% Phosphoric in water, and Methanol. Use Tetrabutylammonium chloride as the ion-pairing agent. You may need to run the chromatography at 100% aqueous for a few minutes to retain the analyte, then a shallow organic gradient to elute it. I hope this help.
-------------------------------------------------------------------------------------------------------
By Zelechonok on Friday, December 26, 2003 - 06:58 am:
You will have no or very little retention on RP system without ion-pairing reagent. Good retention and full control of separation can be obtained on Primesep B or Primesep B2 columns with embedded ion-pairing reagent. See similar separation of Glyphosate which is also aminophosphoric acid, like risedronate, on our web site: http://allsep.com/makeChr.php?chr=Chr_001
-------------------------------------------------------------------------------------------------------
By huang on Tuesday, December 30, 2003 - 03:36 am:
huey:thanks.i use 2 mmol/L Tetrabutylammonium bromide as the ion-pairing agent.the mobile phase is (5mmol/L NH4H2PO4, 2 mmol/L Tetrabutylammonium bromide,1.5 mmol/LEDTA sodium salt, pH=7.2):methanol 75:25, column is waters symmetry C18, but the system peak is greater than 1%, how can i decrease it? if use diethylamine instead of EDTA , the peak of residronate sodium has heavy tail.can you help me? thanks all
-------------------------------------------------------------------------------------------------------
By A.Mouse on Tuesday, December 30, 2003 - 08:58 am:
Huang: what detection are you using?
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Tuesday, December 30, 2003 - 09:07 am:
Huang,
I haven't tried separating this compound using ion pair but I can tell you that your tailing problem is probably connected to metal contamination as this compound is a fairly good metal chelator. So, in the first place, it would be inadvisable to remove the EDTA as this is helping minimize the problem of metal contamination from various sources. However, since you still have tailing even in the presence of EDTA you may need to take additional steps to minimize the problem. First, I would try making several injections of 0.1% disodium EDTA to see whether or not this improves peak shape. If it helps, but doesn't get you all the way to where you want to go in terms of symmetry, make additional injections until peak symmetry is acceptable. If it helps, but only temporarily, then you should try increasing the amount of EDTA in your eluent or installing a chelating trap column before the injection valve. If it doesn't help at all, you're problem may be due to overloading and you should try reducing your injection quantity to see if this helps. FYI: it's generally not a great idea to use the bromide salt of tetrabutylammonium as this absorbs in the ultraviolet. It would be better to use a phosphate buffered tetrabutylammonium solution or to use tetrabutylammonium hydroxide and simply adjust the pH as necessary.
-------------------------------------------------------------------------------------------------------
By huang on Wednesday, December 31, 2003 - 03:56 am:
hi,everyone: I use UV-vis dectector.the peak of residronate sodium is not tail, but have a system peak which response is 1% at about 5 minitues when we use sodium EDTA. How can I decrease the system peak? thanks very much.
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Wednesday, December 31, 2003 - 08:45 am:
Huang,
Sorry, I didn't understand your question before. Your system peak is most likely caused by using the bromide salt of your tetrabutylammonium ion pair reagent. As I mentioned above, it's generally not advisable to use UV absorbing counterions as part of your eluent. I would recommend purchasing the puris grade tetrabutylammonium hydroxide from Fluka and using this to prepare your eluent. This will most likely solve the system peak problem. If the "system peak" is really caused by EDTA, I would suggest substituting another chelating agent with a different retention time such as NTA (which should elute earlier than EDTA) or CDTA or DETPA (which should elute later than EDTA) since I'm quite certain you need to have a chelating agent in the eluent in order to prevent formation of metal complexes with your analyte.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Wednesday, December 31, 2003 - 10:22 am:
Huang,
Chris is right. The problem is likely to be the bromide peak. Use a high-purity ion-pair reagent.
-------------------------------------------------------------------------------------------------------
By AllsepTech on Thursday, January 1, 2004 - 08:58 am:
Why would anybody want to use ion-pairing reagent (IPR) if we have a technology to retain polar compound without IPR. You will achieve same effects just using Primesep brand of columns. See the following link http://www.allsep.com/Technology_Retent ... pounds.php
Any Primesep column can be operated in reverse phase, normal phase, ion-exchange and ion-exclusion modes. You can do mixed mode separations or single mode depending on you compound and application.
-------------------------------------------------------------------------------------------------------
By huang on Thursday, January 1, 2004 - 10:25 pm:
Thank Chris Pohl and A. Mouse, I had done a test which replaced Tetrabutylammonium bromide by tetrabutylammonium hydroxide with the same other conditions, the elute is so quickly (The retention time is about 2 minutes),now I will try again with changing other conditions.
Sorry, AllsepTech, I have not your column now. and i couldn't visit your net yet.
Sorry, my English is not good, so may be there are some mistakes in my description. Please point out it.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Friday, January 2, 2004 - 08:39 am:
The hydroxide changes the pH. neutralize the hydroxide first with a suitable acid, for example phosphoric acid. Then use the same conditions as before.
-------------------------------------------------------------------------------------------------------
By HUANG on Saturday, January 17, 2004 - 06:59 am:
Hi:A.Mouse,I try to do it in according to you ,but the system peak is also apperence,and it incress bigger and bigger.How can I do?
-------------------------------------------------------------------------------------------------------
By A.Mouse on Saturday, January 17, 2004 - 08:15 am:
What did you neutralize it with? If you use phosphoric acid, then the system peak is an impurity in your reagent(s). It could be the purity of the tetrabutyl ammonium species, or it could be something else in you mobile phase or sample that is retained when the TBA is in its proper condition.
-------------------------------------------------------------------------------------------------------
By huang on Sunday, January 18, 2004 - 04:01 am:
A.Mouse:I neutralize it with phosphoric acid, and the tetrabutyl ammonium is HPLC grade(ACROSE brand), the system peak occur when the EDTA is added. And the phosphoric acid I used is analytical agent.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Sunday, January 18, 2004 - 09:40 am:
OK, then the system peak is from the EDTA. What happens when you leave the EDTA out?
-------------------------------------------------------------------------------------------------------
By AllsepTech on Sunday, January 18, 2004 - 10:36 am:
Ion-pairing, neutralization and other troubles and for what? Why not use Primesep column and forget about ion-pairing reagent in the mobile phase. I know that you have to buy new column and spend some money but aren't labor cost becoming an issue at some point? Before deciding on the column, please download our brochures on retention of polar compounds without ion-pairing reagent
http://www.allsep.com/Brochures_Home.php
-------------------------------------------------------------------------------------------------------
By A.Mouse on Sunday, January 18, 2004 - 07:31 pm:
Gee .... I don't think that there is a need to buy a new column. We have just figured out, that the system peak is from EDTA, which does not play a role at all in most applications. Why go through the trouble of redeveloping the method from scratch on a Primesep column? We try to give users some rational advise on how to solve application problems, while AllsepTech does nothing but selling columns.
-------------------------------------------------------------------------------------------------------
By AllsepTech on Sunday, January 18, 2004 - 09:39 pm:
A. Mouse: And what is wrong if the column we make is solving the problem? The forum here is to find time-saving solutions, and if somebody has an appropriate tool, why not use it? Huang posted his question almost a month ago and would have already resolved his issue on Primesep, while we're still "trying to give some rational advise". Method development takes a few hours - we provide it for free; Primeseps are LC/MS and preparative chromatography compatible and can be applied to future projects...again, what's wrong with it?
-------------------------------------------------------------------------------------------------------
By huang on Monday, January 19, 2004 - 05:31 am:
A.Mouse: The system peak disappeared when I leaved the EDTA out.
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Monday, January 19, 2004 - 08:27 am:
Huang,
Now that we know the system peak arises from the EDTA, I think you should consider one of the other chelators I suggested above. As I mentioned, it's important when chromatographically separating chelators that some chelating agent be added to your eluent in order to prevent formation of metal complexes with your analyte (which will show up as impurity peaks early in your chromatogram) which will bias your analytical results low. I'm sure that's the reason why EDTA is present in your eluent system. You might try NTA (which will elute earlier then EDTA) or CDTA (which will elute later than EDTA) to see if either of these are more workable in terms of location relative to your analyte. Another option would be to reduce the concentration of the EDTA as it will still probably be effective at a 10th or maybe even a 100th the concentration you mention. And, as I mentioned previously, a third option would be to use a metal chelating trap column ahead of the injection valve (Dionex sells such a column (the MFC-1) for this purpose) but in this case you need to be sure that there are no metal contaminants in your sample, otherwise you will still have the same problem unless you add a chelator to your sample.
-------------------------------------------------------------------------------------------------------
By Farid Samie on Thursday, June 17, 2004 - 07:10 am:
Hello,
Intrested about latest discussion I now ask you please explain what a system peak is and how/why is this caused???
Thank you,
Farid
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Thursday, June 17, 2004 - 11:38 am:
Farid,
The term: "system peak" is commonly employed in single column ion chromatography, particularly when a conductivity detector is used. The system peak is observed because of the fact that one is able to detect the eluent ion in this mode of detection. Essentially, the sample disturbs the equilibrium between the stationary phase and the mobile phase, causing a transient change in the amount of eluent ion in the solution at the head of the column. This disturbance then proceeds through the column as if it had been injected, giving a peak (either positive or negative, depending on a number of parameters including relative affinities of the ions in the sample and eluent) at the retention time of the eluent ion in the analytical system.
hi:everyone, happy new year.
I have looking for a HPLC method for risedronate sodium,( it is a diphosphonic acid salt).
Looked all over the net and couldn't get anything.
Can anyone help? Any information would be highly appreciated.
Thanks all.
-------------------------------------------------------------------------------------------------------
By huey on Thursday, December 25, 2003 - 06:09 pm:
My opinion:
In brief, if you're using an RP column (i.e. C8) then try Acidic mobile phases i.e. 0.1% Phosphoric in water, and Methanol. Use Tetrabutylammonium chloride as the ion-pairing agent. You may need to run the chromatography at 100% aqueous for a few minutes to retain the analyte, then a shallow organic gradient to elute it. I hope this help.
-------------------------------------------------------------------------------------------------------
By Zelechonok on Friday, December 26, 2003 - 06:58 am:
You will have no or very little retention on RP system without ion-pairing reagent. Good retention and full control of separation can be obtained on Primesep B or Primesep B2 columns with embedded ion-pairing reagent. See similar separation of Glyphosate which is also aminophosphoric acid, like risedronate, on our web site: http://allsep.com/makeChr.php?chr=Chr_001
-------------------------------------------------------------------------------------------------------
By huang on Tuesday, December 30, 2003 - 03:36 am:
huey:thanks.i use 2 mmol/L Tetrabutylammonium bromide as the ion-pairing agent.the mobile phase is (5mmol/L NH4H2PO4, 2 mmol/L Tetrabutylammonium bromide,1.5 mmol/LEDTA sodium salt, pH=7.2):methanol 75:25, column is waters symmetry C18, but the system peak is greater than 1%, how can i decrease it? if use diethylamine instead of EDTA , the peak of residronate sodium has heavy tail.can you help me? thanks all
-------------------------------------------------------------------------------------------------------
By A.Mouse on Tuesday, December 30, 2003 - 08:58 am:
Huang: what detection are you using?
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Tuesday, December 30, 2003 - 09:07 am:
Huang,
I haven't tried separating this compound using ion pair but I can tell you that your tailing problem is probably connected to metal contamination as this compound is a fairly good metal chelator. So, in the first place, it would be inadvisable to remove the EDTA as this is helping minimize the problem of metal contamination from various sources. However, since you still have tailing even in the presence of EDTA you may need to take additional steps to minimize the problem. First, I would try making several injections of 0.1% disodium EDTA to see whether or not this improves peak shape. If it helps, but doesn't get you all the way to where you want to go in terms of symmetry, make additional injections until peak symmetry is acceptable. If it helps, but only temporarily, then you should try increasing the amount of EDTA in your eluent or installing a chelating trap column before the injection valve. If it doesn't help at all, you're problem may be due to overloading and you should try reducing your injection quantity to see if this helps. FYI: it's generally not a great idea to use the bromide salt of tetrabutylammonium as this absorbs in the ultraviolet. It would be better to use a phosphate buffered tetrabutylammonium solution or to use tetrabutylammonium hydroxide and simply adjust the pH as necessary.
-------------------------------------------------------------------------------------------------------
By huang on Wednesday, December 31, 2003 - 03:56 am:
hi,everyone: I use UV-vis dectector.the peak of residronate sodium is not tail, but have a system peak which response is 1% at about 5 minitues when we use sodium EDTA. How can I decrease the system peak? thanks very much.
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Wednesday, December 31, 2003 - 08:45 am:
Huang,
Sorry, I didn't understand your question before. Your system peak is most likely caused by using the bromide salt of your tetrabutylammonium ion pair reagent. As I mentioned above, it's generally not advisable to use UV absorbing counterions as part of your eluent. I would recommend purchasing the puris grade tetrabutylammonium hydroxide from Fluka and using this to prepare your eluent. This will most likely solve the system peak problem. If the "system peak" is really caused by EDTA, I would suggest substituting another chelating agent with a different retention time such as NTA (which should elute earlier than EDTA) or CDTA or DETPA (which should elute later than EDTA) since I'm quite certain you need to have a chelating agent in the eluent in order to prevent formation of metal complexes with your analyte.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Wednesday, December 31, 2003 - 10:22 am:
Huang,
Chris is right. The problem is likely to be the bromide peak. Use a high-purity ion-pair reagent.
-------------------------------------------------------------------------------------------------------
By AllsepTech on Thursday, January 1, 2004 - 08:58 am:
Why would anybody want to use ion-pairing reagent (IPR) if we have a technology to retain polar compound without IPR. You will achieve same effects just using Primesep brand of columns. See the following link http://www.allsep.com/Technology_Retent ... pounds.php
Any Primesep column can be operated in reverse phase, normal phase, ion-exchange and ion-exclusion modes. You can do mixed mode separations or single mode depending on you compound and application.
-------------------------------------------------------------------------------------------------------
By huang on Thursday, January 1, 2004 - 10:25 pm:
Thank Chris Pohl and A. Mouse, I had done a test which replaced Tetrabutylammonium bromide by tetrabutylammonium hydroxide with the same other conditions, the elute is so quickly (The retention time is about 2 minutes),now I will try again with changing other conditions.
Sorry, AllsepTech, I have not your column now. and i couldn't visit your net yet.
Sorry, my English is not good, so may be there are some mistakes in my description. Please point out it.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Friday, January 2, 2004 - 08:39 am:
The hydroxide changes the pH. neutralize the hydroxide first with a suitable acid, for example phosphoric acid. Then use the same conditions as before.
-------------------------------------------------------------------------------------------------------
By HUANG on Saturday, January 17, 2004 - 06:59 am:
Hi:A.Mouse,I try to do it in according to you ,but the system peak is also apperence,and it incress bigger and bigger.How can I do?
-------------------------------------------------------------------------------------------------------
By A.Mouse on Saturday, January 17, 2004 - 08:15 am:
What did you neutralize it with? If you use phosphoric acid, then the system peak is an impurity in your reagent(s). It could be the purity of the tetrabutyl ammonium species, or it could be something else in you mobile phase or sample that is retained when the TBA is in its proper condition.
-------------------------------------------------------------------------------------------------------
By huang on Sunday, January 18, 2004 - 04:01 am:
A.Mouse:I neutralize it with phosphoric acid, and the tetrabutyl ammonium is HPLC grade(ACROSE brand), the system peak occur when the EDTA is added. And the phosphoric acid I used is analytical agent.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Sunday, January 18, 2004 - 09:40 am:
OK, then the system peak is from the EDTA. What happens when you leave the EDTA out?
-------------------------------------------------------------------------------------------------------
By AllsepTech on Sunday, January 18, 2004 - 10:36 am:
Ion-pairing, neutralization and other troubles and for what? Why not use Primesep column and forget about ion-pairing reagent in the mobile phase. I know that you have to buy new column and spend some money but aren't labor cost becoming an issue at some point? Before deciding on the column, please download our brochures on retention of polar compounds without ion-pairing reagent
http://www.allsep.com/Brochures_Home.php
-------------------------------------------------------------------------------------------------------
By A.Mouse on Sunday, January 18, 2004 - 07:31 pm:
Gee .... I don't think that there is a need to buy a new column. We have just figured out, that the system peak is from EDTA, which does not play a role at all in most applications. Why go through the trouble of redeveloping the method from scratch on a Primesep column? We try to give users some rational advise on how to solve application problems, while AllsepTech does nothing but selling columns.
-------------------------------------------------------------------------------------------------------
By AllsepTech on Sunday, January 18, 2004 - 09:39 pm:
A. Mouse: And what is wrong if the column we make is solving the problem? The forum here is to find time-saving solutions, and if somebody has an appropriate tool, why not use it? Huang posted his question almost a month ago and would have already resolved his issue on Primesep, while we're still "trying to give some rational advise". Method development takes a few hours - we provide it for free; Primeseps are LC/MS and preparative chromatography compatible and can be applied to future projects...again, what's wrong with it?
-------------------------------------------------------------------------------------------------------
By huang on Monday, January 19, 2004 - 05:31 am:
A.Mouse: The system peak disappeared when I leaved the EDTA out.
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Monday, January 19, 2004 - 08:27 am:
Huang,
Now that we know the system peak arises from the EDTA, I think you should consider one of the other chelators I suggested above. As I mentioned, it's important when chromatographically separating chelators that some chelating agent be added to your eluent in order to prevent formation of metal complexes with your analyte (which will show up as impurity peaks early in your chromatogram) which will bias your analytical results low. I'm sure that's the reason why EDTA is present in your eluent system. You might try NTA (which will elute earlier then EDTA) or CDTA (which will elute later than EDTA) to see if either of these are more workable in terms of location relative to your analyte. Another option would be to reduce the concentration of the EDTA as it will still probably be effective at a 10th or maybe even a 100th the concentration you mention. And, as I mentioned previously, a third option would be to use a metal chelating trap column ahead of the injection valve (Dionex sells such a column (the MFC-1) for this purpose) but in this case you need to be sure that there are no metal contaminants in your sample, otherwise you will still have the same problem unless you add a chelator to your sample.
-------------------------------------------------------------------------------------------------------
By Farid Samie on Thursday, June 17, 2004 - 07:10 am:
Hello,
Intrested about latest discussion I now ask you please explain what a system peak is and how/why is this caused???
Thank you,
Farid
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Thursday, June 17, 2004 - 11:38 am:
Farid,
The term: "system peak" is commonly employed in single column ion chromatography, particularly when a conductivity detector is used. The system peak is observed because of the fact that one is able to detect the eluent ion in this mode of detection. Essentially, the sample disturbs the equilibrium between the stationary phase and the mobile phase, causing a transient change in the amount of eluent ion in the solution at the head of the column. This disturbance then proceeds through the column as if it had been injected, giving a peak (either positive or negative, depending on a number of parameters including relative affinities of the ions in the sample and eluent) at the retention time of the eluent ion in the analytical system.
