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By syx_gf on Tuesday, July 27, 2004 - 01:06 am:
Dear chromatographers,
I have a question:
What is mobile phase additive for chelating agent determination?
Our peak has a big tail, it is an acid chelating agent. We use ion-pair chromatography with 0.005M tetrabutylammonium HSO4.
Any suggestion will be appreciated.
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By Chris Pohl on Tuesday, July 27, 2004 - 12:20 pm:
There are several things to consider before answering this question including: which specific chelator you are using, whether or not metals may be present in your sample, whether or not metals may be present in your eluent or arising from your HPLC equipment and if you are interested in total chelating agent or only the fraction which is not complexed with metals present in the sample.
Without answers to these questions, if I assume that you are only interested in total chelating agent and that the chelating agent is an anion, I would advise the following steps: reduce the pH to minimize the stability of metal complexes, add a chelating trap column ahead of the injection valve (these are available from a number of sources including Dionex who sells a product called IonPac MFC-1) and make several repeated injections of a relatively concentrated solution of your chelating agent (for example, a 0.1% solution). These steps may be sufficient to eliminate the tailing problem you describe without the necessity of adding a chelating agent to your mobile phase.
However, the general rule of thumb is that a chelating additive should be chosen such that it has a higher stability constant for metals than your chelator of analytical interest. For example, 1,2-diaminocyclohexanetetraacetic acid or diethylenetriaminepentaacetic acid are good choices if you're analyte is EDTA. However, these are likely to raise the background, assuming you are using UV detection. Another option is to use amine chelating agents such as ethylenediamine or diethylenetriamine since these won't significantly increase your background if they are clean but this won't work for all metals. Generally, it's better to work at a pH where the chelator has limited chelating power and minimize exposure to metals than it is to add a mobile phase component to competitively bind the metals.
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By syx_gf on Tuesday, July 27, 2004 - 05:57 pm:
Our substance is a bisphosphonic acid. We cannot reduce the pH because the substance is unretained in low pH even though we use ion pair reagent.
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By Chris Pohl on Friday, July 30, 2004 - 12:39 am:
The idea is to reduce the pH until the chelating agent no longer has significant chelating power. It isn't necessary to lower the pH to the point that it is no longer an anion, so you should be able to eliminate the problem with metal contamination (which is the usual cause of peak tailing for chelating agents) and still retain the bisphosphonate via ion pair chromatography.
Dear chromatographers,
I have a question:
What is mobile phase additive for chelating agent determination?
Our peak has a big tail, it is an acid chelating agent. We use ion-pair chromatography with 0.005M tetrabutylammonium HSO4.
Any suggestion will be appreciated.
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Tuesday, July 27, 2004 - 12:20 pm:
There are several things to consider before answering this question including: which specific chelator you are using, whether or not metals may be present in your sample, whether or not metals may be present in your eluent or arising from your HPLC equipment and if you are interested in total chelating agent or only the fraction which is not complexed with metals present in the sample.
Without answers to these questions, if I assume that you are only interested in total chelating agent and that the chelating agent is an anion, I would advise the following steps: reduce the pH to minimize the stability of metal complexes, add a chelating trap column ahead of the injection valve (these are available from a number of sources including Dionex who sells a product called IonPac MFC-1) and make several repeated injections of a relatively concentrated solution of your chelating agent (for example, a 0.1% solution). These steps may be sufficient to eliminate the tailing problem you describe without the necessity of adding a chelating agent to your mobile phase.
However, the general rule of thumb is that a chelating additive should be chosen such that it has a higher stability constant for metals than your chelator of analytical interest. For example, 1,2-diaminocyclohexanetetraacetic acid or diethylenetriaminepentaacetic acid are good choices if you're analyte is EDTA. However, these are likely to raise the background, assuming you are using UV detection. Another option is to use amine chelating agents such as ethylenediamine or diethylenetriamine since these won't significantly increase your background if they are clean but this won't work for all metals. Generally, it's better to work at a pH where the chelator has limited chelating power and minimize exposure to metals than it is to add a mobile phase component to competitively bind the metals.
-------------------------------------------------------------------------------------------------------
By syx_gf on Tuesday, July 27, 2004 - 05:57 pm:
Our substance is a bisphosphonic acid. We cannot reduce the pH because the substance is unretained in low pH even though we use ion pair reagent.
-------------------------------------------------------------------------------------------------------
By Chris Pohl on Friday, July 30, 2004 - 12:39 am:
The idea is to reduce the pH until the chelating agent no longer has significant chelating power. It isn't necessary to lower the pH to the point that it is no longer an anion, so you should be able to eliminate the problem with metal contamination (which is the usual cause of peak tailing for chelating agents) and still retain the bisphosphonate via ion pair chromatography.
