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By Unsure on Wednesday, June 2, 2004 - 12:22 am:
Hi,
Can anyone sugguest what sort of method (column and mobile phase to use) i should use for the seperation of glycosylated peptides.
Problem:
1. Will there be peak spliting and how do i resolve this
2. The glycosylated peptides i want to seperate are very polar (contains tone or two amine groups as well as a carboxyl group)
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By Einar Pontén - SeQuant AB on Wednesday, June 2, 2004 - 12:25 pm:
Hydrophilic Liquid Interaction Chromatography is suitable for this problem.
The ZIC®-HILIC column is indeed suitable for glycosylated and glucoronated compounds. In addition, with the given information it is safe to recommend you this alternative.
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By Unsure on Wednesday, June 2, 2004 - 05:53 pm:
Will ion exchange work for the purification of this type of compound. What type of ion exchange shld be used?
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By Einar Pontén - SeQuant AB on Wednesday, June 2, 2004 - 11:25 pm:
Yes, ion exchange may work. It seems that you have a basic peptide and then a cation exchanger is an alternative.
Quite likely you will need relatively high ion strength (buffer) that -may- become a problem later on due to tedious work-up or interference in MS detection.
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By Uwe Neue on Thursday, June 3, 2004 - 04:09 pm:
We have run very polar peptides on HILIC column (in our case Atlantis HILIC silica) with a reverse gradient from high (90%) acetonitrile to 50% acetonitrile with 0.1% formate. this procedure gives good retention for peptides that are difficult to retain on a C18, and also good retention of mutiply posisitvely charged peptides. It is an interesting alternative to reversed-phase.
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By Einar Pontén - SeQuant AB on Friday, June 4, 2004 - 03:03 pm:
I can agree on what Uwe tell, but we have a more suitable alternative to offer (personal opinion).
Hi,
Can anyone sugguest what sort of method (column and mobile phase to use) i should use for the seperation of glycosylated peptides.
Problem:
1. Will there be peak spliting and how do i resolve this
2. The glycosylated peptides i want to seperate are very polar (contains tone or two amine groups as well as a carboxyl group)
-------------------------------------------------------------------------------------------------------
By Einar Pontén - SeQuant AB on Wednesday, June 2, 2004 - 12:25 pm:
Hydrophilic Liquid Interaction Chromatography is suitable for this problem.
The ZIC®-HILIC column is indeed suitable for glycosylated and glucoronated compounds. In addition, with the given information it is safe to recommend you this alternative.
-------------------------------------------------------------------------------------------------------
By Unsure on Wednesday, June 2, 2004 - 05:53 pm:
Will ion exchange work for the purification of this type of compound. What type of ion exchange shld be used?
-------------------------------------------------------------------------------------------------------
By Einar Pontén - SeQuant AB on Wednesday, June 2, 2004 - 11:25 pm:
Yes, ion exchange may work. It seems that you have a basic peptide and then a cation exchanger is an alternative.
Quite likely you will need relatively high ion strength (buffer) that -may- become a problem later on due to tedious work-up or interference in MS detection.
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Thursday, June 3, 2004 - 04:09 pm:
We have run very polar peptides on HILIC column (in our case Atlantis HILIC silica) with a reverse gradient from high (90%) acetonitrile to 50% acetonitrile with 0.1% formate. this procedure gives good retention for peptides that are difficult to retain on a C18, and also good retention of mutiply posisitvely charged peptides. It is an interesting alternative to reversed-phase.
-------------------------------------------------------------------------------------------------------
By Einar Pontén - SeQuant AB on Friday, June 4, 2004 - 03:03 pm:
I can agree on what Uwe tell, but we have a more suitable alternative to offer (personal opinion).
