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What causes peptides to have longer retention on a C4 vs C18
Posted: Wed Oct 15, 2008 1:01 am
by pipettemonkey
I have a peptide I'm trying to isolate. It was more highly retained on the Vydac C4 column than it was on a Zorbax C18 column (i.e., required higher % acetonitrile to elute).
Firstly, I'd like to ask if this is "real" (i.e., does this mean there is likely something wrong with the C18 column, and if it were new, would it be less likely to occur?)
How unusual is this?
What, physically, would cause this?
Posted: Wed Oct 15, 2008 2:00 am
by Uwe Neue
not unusual, could just be pore size...
Posted: Wed Oct 15, 2008 2:44 am
by pipettemonkey
What if the pore size were the same?
Posted: Wed Oct 15, 2008 3:19 am
by SIELC_Tech
Depending on pH of your mobile phase you can see more ion-exchange interaction on C4 in addition to reverse phase, as on C4 phase silanols are accessible than on C18.
Posted: Wed Oct 15, 2008 8:42 am
by danko
Pipettemonkey, are the dimensions of the Vydac and the Zorbax columns identical?
I can imagine that the Zorbax is a 2.1 mm (inner diameter) and maybe shorter as well.
Whereas the Vydac is the more traditional 4.6 mm. And if you applied the same flow rate on both columns, then you have the explanation.
Best Regards
Posted: Wed Oct 15, 2008 6:06 pm
by pipettemonkey
Pipettemonkey, are the dimensions of the Vydac and the Zorbax columns identical?
I can imagine that the Zorbax is a 2.1 mm (inner diameter) and maybe shorter as well.
Whereas the Vydac is the more traditional 4.6 mm. And if you applied the same flow rate on both columns, then you have the explanation.
Best Regards
Both columns are the same ID. I use the same flow rate (1 mL/min) and an acetonitrile gradient with 0.1 % TFA.
Posted: Wed Oct 15, 2008 10:29 pm
by danko
Same length as well?
Is the Zorbax column also a Poroshell variant?
Best Regards
Posted: Wed Oct 15, 2008 11:02 pm
by pipettemonkey
Same length as well?
Is the Zorbax column also a Poroshell variant?
Best Regards
They are different lengths. The C4 is 15 cm, the C18 is 10 cm
Posted: Thu Oct 16, 2008 9:10 am
by danko
There you go
Posted: Thu Oct 16, 2008 3:43 pm
by pipettemonkey
There you go
I don't think so. The difference in retention is too great to be accounted for by column volume.
Posted: Thu Oct 16, 2008 4:08 pm
by Kostas Petritis
Have you seen the same patten with any peptide or just the one that you are trying to isolate. Maybe there is something to do with the sequence/structure of the specific peptide that you are trying to isolate. In any case if you are not sure about the status of your Zorbax column (i.e. it is not a new one) maybe your results are not representative of what is really happening...
Having said that I know that the Zorbax 300A material has a pretty small specific surface area (i.e. 45 m2/g) while the Vydac I think it is higher (although I do not remember the exact number. The phenomenex Jupiter in comparison for 300A has 175 m2/g.