Peptides with Perchlorate Mob.Phs. [August 5, 2004]
Posted: Sun Aug 29, 2004 6:25 pm
Peptide Seperation with Perchlorate Mobile Phase
Chromatography Forum: LC Message Board: Peptide Seperation with Perchlorate Mobile Phase
-------------------------------------------------------------------------------------------------------
By Eric Brown on Thursday, August 5, 2004 - 12:50 pm:
Dear Friends,
I have an old method that I was handed recently that uses sodium perchlorate 10 mM with 0.1% o-phosphoric acid for a peptide purity method. I was asked to run the method and optimize. I have no experience with perchlorate and understand it is not used very often because TFA is used in its place with the equivalent results.
Should I switch to TFA or is there an advantage to using perchlorate?
Should the perchlorate mobile phase be buffered to control available ions?
Any help is greatly appreciated!
Best regards,
Eric
-------------------------------------------------------------------------------------------------------
By Kostas Petritis on Friday, August 6, 2004 - 09:29 am:
Eric,
Personally I do not see any advantage of using perchlorate instead of TFA. I personally prefer to use perfluorinated carboxylic acids which are more volatile and depending on what are my separation objectives I can always choose between several of them (i.e. TFA, HFBA, NFPA, TDFHA, PDFOA...).
Kostas
-------------------------------------------------------------------------------------------------------
By Benjamin on Tuesday, August 10, 2004 - 06:07 am:
Dear Eric,
Your old method seems to be not well developed or very special. It is well known that different ion-pairing reagents yield slightly different separations of peptides. Therefore, it is possible that you will not obtain exactly the same separation with perchlorate or TFA.
If your mobile phase contains perchlorate and phosphate it is likely to have two competing ion-pair reagents in the separation. However, pep-tides have very difficult sobulities in some cases and that can be the reason for the combined presence of both ions (to keep the peptide in solution).
I recommend you try TFA alone or phosphate alone, it is likely you will obtain the same separation.
Good luck,
Benjamin
-------------------------------------------------------------------------------------------------------
By A.Mouse on Saturday, August 14, 2004 - 07:52 pm:
I think the bottom line is that there is really nothing that speaks against the mobile phase that you are starting off with. If a good separation has been obtained, and you are looking at some fine tuning or some method optimization or even qualification, then there is nothing wrong with the ingredients used. A change to TFA will change your elution pattern, because the ion-pairing properties of TFA and perchlorate are different. I would think that any buffering is achieved via the phosphoric acid, but at the pH that this method runs at, you do not need to worry about buffering (see the buffer discussions on this board for background information). Phosphate is not known to form ion pairs, but perchlorate does.
-------------------------------------------------------------------------------------------------------
By HW Mueller on Wednesday, August 18, 2004 - 01:08 am:
Tend to agree with A.Mouse (there is that extensive negative experience with TFA). But, does your column take this low pH? Maybe you need to add some H2PO4- to get the pH a bit higher, it doesn´t sound to me as if the original creators of this method bothered about optimizing pH.
A.Mouse, do you have a ref. on this ion pairing and phosphate? It´s somewhat surprising considering the relative low dissociation of H3PO4.
Chromatography Forum: LC Message Board: Peptide Seperation with Perchlorate Mobile Phase
-------------------------------------------------------------------------------------------------------
By Eric Brown on Thursday, August 5, 2004 - 12:50 pm:
Dear Friends,
I have an old method that I was handed recently that uses sodium perchlorate 10 mM with 0.1% o-phosphoric acid for a peptide purity method. I was asked to run the method and optimize. I have no experience with perchlorate and understand it is not used very often because TFA is used in its place with the equivalent results.
Should I switch to TFA or is there an advantage to using perchlorate?
Should the perchlorate mobile phase be buffered to control available ions?
Any help is greatly appreciated!
Best regards,
Eric
-------------------------------------------------------------------------------------------------------
By Kostas Petritis on Friday, August 6, 2004 - 09:29 am:
Eric,
Personally I do not see any advantage of using perchlorate instead of TFA. I personally prefer to use perfluorinated carboxylic acids which are more volatile and depending on what are my separation objectives I can always choose between several of them (i.e. TFA, HFBA, NFPA, TDFHA, PDFOA...).
Kostas
-------------------------------------------------------------------------------------------------------
By Benjamin on Tuesday, August 10, 2004 - 06:07 am:
Dear Eric,
Your old method seems to be not well developed or very special. It is well known that different ion-pairing reagents yield slightly different separations of peptides. Therefore, it is possible that you will not obtain exactly the same separation with perchlorate or TFA.
If your mobile phase contains perchlorate and phosphate it is likely to have two competing ion-pair reagents in the separation. However, pep-tides have very difficult sobulities in some cases and that can be the reason for the combined presence of both ions (to keep the peptide in solution).
I recommend you try TFA alone or phosphate alone, it is likely you will obtain the same separation.
Good luck,
Benjamin
-------------------------------------------------------------------------------------------------------
By A.Mouse on Saturday, August 14, 2004 - 07:52 pm:
I think the bottom line is that there is really nothing that speaks against the mobile phase that you are starting off with. If a good separation has been obtained, and you are looking at some fine tuning or some method optimization or even qualification, then there is nothing wrong with the ingredients used. A change to TFA will change your elution pattern, because the ion-pairing properties of TFA and perchlorate are different. I would think that any buffering is achieved via the phosphoric acid, but at the pH that this method runs at, you do not need to worry about buffering (see the buffer discussions on this board for background information). Phosphate is not known to form ion pairs, but perchlorate does.
-------------------------------------------------------------------------------------------------------
By HW Mueller on Wednesday, August 18, 2004 - 01:08 am:
Tend to agree with A.Mouse (there is that extensive negative experience with TFA). But, does your column take this low pH? Maybe you need to add some H2PO4- to get the pH a bit higher, it doesn´t sound to me as if the original creators of this method bothered about optimizing pH.
A.Mouse, do you have a ref. on this ion pairing and phosphate? It´s somewhat surprising considering the relative low dissociation of H3PO4.