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)
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).