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By Kaoula on Friday, - 07:30 am:
Hi all
Please I wont your opinions of this state it is correct or not :
The pH of mobile phase used in analytical method is about 3.5 and the pH of dissolution medium is 9.2
and what is the procedure when the pH of dissolution medium and mobile phase is very different
Thank you for your advise
Kaoula
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By Chris Pohl on Saturday, July 3, 2004 - 11:31 am:
Kaoula,
The answer to your question depends in part upon whether or not any of your analytes are weakly acidic. If all of your analytes are neutral or basic, then there there won't be any major problems with the wide disparity between your eluent pH and your sample pH (although it may be necessary to boost the buffer capacity of your mobile phase somewhat in order to prevent overloading your buffer system) since the higher pH of the sample is either irrelevant in the case of neutral compounds or suitable for "focusing" in the case of basic compounds. However, if one of your analytes is weakly acidic and you are using a low pH in order to achieve retention via ion suppression then injecting a high pH sample can significantly distort the peak shape of weakly acidic solutes because the ionized form of acidic analytes invariably are more weakly retained then the fully protonated form. In this case, it would be preferable to adjust the pH of the sample to more closely matched that of the mobile phase. If, however, the high pH for the sample is connected to difficulty of dissolving the sample, it might be worth trying a two-step protocol: preparing the sample at high pH and a somewhat higher concentration and then diluting the solution to the final injection concentration into a buffer system matching the eluent pH.
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By kaoula on Wednesday, July 7, 2004 - 06:12 am:
Hi Chris Pohl:
thank you for yor advice.
i wont to aske you if the Destroyed peak appear as tow peaks and the resolution between them is good 4.5 as example?
thank you
Kaoula
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By Chris Pohl on Saturday, July 10, 2004 - 12:56 pm:
Kaoula,
I'm not sure I understand your question regarding your question regarding a "destroyed peak which appears as two peaks and the resolution between them is good". Is this still in the context of sample pH being significantly different than mobile phase pH? Are you saying that you think the sample pH has caused peaks splitting? I guess I need to know that more about the analyte to say one way or the other.
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By Kaoula on Sunday, July 11, 2004 - 05:07 am:
Hi Chris Pohl:
My problem is as follow:
The analyte is weak acid material it exist tow method of analysis:
the first is based on ion suppression principle (pH about 3.5) and the second is based on ion pairing principle (pH about 7.5).
When we are used these tow methods to carried out the dissolution test which the pH of dissolution method is 9.2 the results is as follow:
Using ion pairing method: the chromatogram show one peak
Using ion suppression method: the chromatogram show tow peaks which the resolution between them is about 4.5.
Note: the tow peaks appear in the standard(prepared in dissolution medium) and the samples of dissolution but doesn't appear in the standard which prepared in methanol as solvent
Thank you very match for your advice
Kaoula
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By Chris Pohl on Monday, July 12, 2004 - 12:15 pm:
Kaoula,
Even if we consider all of the factors you have mentioned, it's still not possible to make a definitive assessment. Comparing two different retention modes like ion suppression and ion pairing, it's easy to imagine that an interference might be observed in one method and not in the other, so the discrepancy between the two methods could easily be due to either the sample pH or the fact that the two methods are fundamentally different in terms of retention mode. So this observation doesn't conclusively indicate a problem with your sample preparation on the chromatography. Furthermore, the fact you do not observe two peaks when injecting a sample which has been dissolved in methanol doesn't prove anything either since this discrepancy could be due to the fact that you are dissolving your sample in an "eluent" significantly more potent than your mobile phase. Generally, it's not advisable to dissolve your sample in pure solvent when using reversed phase (although you can sometimes get away with it by injecting a very small sample volume). My suggestion is that you try dissolving your sample in mobile phase (or better still, a solution containing a lower solvent content than your mobile phase) and see if you still see only one peak. If so, then this does suggest the problem is due to the high pH of your sample solution and I would suggest that you spike your sample with sufficient acid to bring the sample pH down to 3.
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By Kaoula on Tuesday, July 13, 2004 - 12:12 am:
Hi Chris Pohl:
Thank you for your advice.
I wont to ask you :
why it's not advisable to dissolve the sample in pure solvent when using reversed phase ?
Kaoula
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By DR on Tuesday, July 13, 2004 - 07:15 am:
Putting a sample on a (reverse phase) column with a solvent that is higher in organic content than your mobile phase can cause broader peaks, poor resolution and (sometimes) more tailing. These problems will be more pronounced with larger injection volumes. Using a weaker sample diluent results in narrower peaks, which allows for better resolution. This is because injecting sample in a slug of high organic solvent will allow some of the sample to move fairly far down the column before engaging in the normal mp/stationary phase partitioning that results in getting the separation in the first place.
Hi all
Please I wont your opinions of this state it is correct or not :
The pH of mobile phase used in analytical method is about 3.5 and the pH of dissolution medium is 9.2
and what is the procedure when the pH of dissolution medium and mobile phase is very different
Thank you for your advise
Kaoula
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Saturday, July 3, 2004 - 11:31 am:
Kaoula,
The answer to your question depends in part upon whether or not any of your analytes are weakly acidic. If all of your analytes are neutral or basic, then there there won't be any major problems with the wide disparity between your eluent pH and your sample pH (although it may be necessary to boost the buffer capacity of your mobile phase somewhat in order to prevent overloading your buffer system) since the higher pH of the sample is either irrelevant in the case of neutral compounds or suitable for "focusing" in the case of basic compounds. However, if one of your analytes is weakly acidic and you are using a low pH in order to achieve retention via ion suppression then injecting a high pH sample can significantly distort the peak shape of weakly acidic solutes because the ionized form of acidic analytes invariably are more weakly retained then the fully protonated form. In this case, it would be preferable to adjust the pH of the sample to more closely matched that of the mobile phase. If, however, the high pH for the sample is connected to difficulty of dissolving the sample, it might be worth trying a two-step protocol: preparing the sample at high pH and a somewhat higher concentration and then diluting the solution to the final injection concentration into a buffer system matching the eluent pH.
-------------------------------------------------------------------------------------------------------
By kaoula on Wednesday, July 7, 2004 - 06:12 am:
Hi Chris Pohl:
thank you for yor advice.
i wont to aske you if the Destroyed peak appear as tow peaks and the resolution between them is good 4.5 as example?
thank you
Kaoula
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Saturday, July 10, 2004 - 12:56 pm:
Kaoula,
I'm not sure I understand your question regarding your question regarding a "destroyed peak which appears as two peaks and the resolution between them is good". Is this still in the context of sample pH being significantly different than mobile phase pH? Are you saying that you think the sample pH has caused peaks splitting? I guess I need to know that more about the analyte to say one way or the other.
-------------------------------------------------------------------------------------------------------
By Kaoula on Sunday, July 11, 2004 - 05:07 am:
Hi Chris Pohl:
My problem is as follow:
The analyte is weak acid material it exist tow method of analysis:
the first is based on ion suppression principle (pH about 3.5) and the second is based on ion pairing principle (pH about 7.5).
When we are used these tow methods to carried out the dissolution test which the pH of dissolution method is 9.2 the results is as follow:
Using ion pairing method: the chromatogram show one peak
Using ion suppression method: the chromatogram show tow peaks which the resolution between them is about 4.5.
Note: the tow peaks appear in the standard(prepared in dissolution medium) and the samples of dissolution but doesn't appear in the standard which prepared in methanol as solvent
Thank you very match for your advice
Kaoula
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Monday, July 12, 2004 - 12:15 pm:
Kaoula,
Even if we consider all of the factors you have mentioned, it's still not possible to make a definitive assessment. Comparing two different retention modes like ion suppression and ion pairing, it's easy to imagine that an interference might be observed in one method and not in the other, so the discrepancy between the two methods could easily be due to either the sample pH or the fact that the two methods are fundamentally different in terms of retention mode. So this observation doesn't conclusively indicate a problem with your sample preparation on the chromatography. Furthermore, the fact you do not observe two peaks when injecting a sample which has been dissolved in methanol doesn't prove anything either since this discrepancy could be due to the fact that you are dissolving your sample in an "eluent" significantly more potent than your mobile phase. Generally, it's not advisable to dissolve your sample in pure solvent when using reversed phase (although you can sometimes get away with it by injecting a very small sample volume). My suggestion is that you try dissolving your sample in mobile phase (or better still, a solution containing a lower solvent content than your mobile phase) and see if you still see only one peak. If so, then this does suggest the problem is due to the high pH of your sample solution and I would suggest that you spike your sample with sufficient acid to bring the sample pH down to 3.
-------------------------------------------------------------------------------------------------------
By Kaoula on Tuesday, July 13, 2004 - 12:12 am:
Hi Chris Pohl:
Thank you for your advice.
I wont to ask you :
why it's not advisable to dissolve the sample in pure solvent when using reversed phase ?
Kaoula
-------------------------------------------------------------------------------------------------------
By DR on Tuesday, July 13, 2004 - 07:15 am:
Putting a sample on a (reverse phase) column with a solvent that is higher in organic content than your mobile phase can cause broader peaks, poor resolution and (sometimes) more tailing. These problems will be more pronounced with larger injection volumes. Using a weaker sample diluent results in narrower peaks, which allows for better resolution. This is because injecting sample in a slug of high organic solvent will allow some of the sample to move fairly far down the column before engaging in the normal mp/stationary phase partitioning that results in getting the separation in the first place.
