By Anonymous on Thursday, July 25, 2002 - 07:43 am:
Can something polar get stuck in normal C18 column? I just ran a reverse gradient from 100% acetonitrile to 60/40 Acetonitrile/water in 60 minutes to clean my column and something came out in 30 minutes. Is it something stuck in free -OH groups, or what? Is there more free -OH's in older columns than in new ones? Anybody washing C18 columns like this? Thank you.
By Anonymous on Thursday, July 25, 2002 - 08:31 am:
About your question:
"Can something polar get stuck in normal C18 column? "
I have not seen that.
But if the sample previously separated in your column had protein nature then probably you will see a peak(s) when you run a gradient in blank because proteins are not desorbed completely. Aditionally, proteins are desorbed from RP columns in a narrow range of % organic solvent. For example if your protein is desorbed at 68-70% acetonitrile, it does not no matter if your gradient is normal or reverse, the protein will elute at 68-70% not at 80% or 40%.
Anyway, to clean your column I recommend you to try gradients from H3PO4 25mM/water to 25mM H3PO4/1-propanol, decrease flow rate if pressure
increases too much.
I hope this can be useful and that you can understand my english writing.
By Anonymous on Thursday, July 25, 2002 - 08:52 am:
I use a similar gradient method to wash my columns. I usually see a gradient-related peak about halfway through the wash. If you see the same results when you run the wash program on different columns, then you may be seeing a gradient peak.
By Anonymous on Thursday, July 25, 2002 - 09:19 am:
Thanks for quick replies! I'm analysing polymer additives. I haven't gone this polar with my gradient before. I did this wash for two columns and something came out only with the older one? Any suggestions? Is there free -OH groups in C18 column with ability to bind something polar??
By Anonymous on Thursday, July 25, 2002 - 02:44 pm:
Are you using gradients in acetonitrile for your analysis, and not the standard reversed-phase water to acetonitrile gradients?
By Anonymous on Monday, July 29, 2002 - 06:46 am:
About your question:
"Is there free -OH groups in C18 column with ability to bind something polar?? "
Yes, it is possible the existence of free -SiOH,
I have read the endcapping is not complete, so free silanol groups can exist and they are the cause of electrostatic interaction with charged species since these are ionizable. But in very acidic solutions (TFA, etc) silanol groups are not ionized. Then other type of interaction may appear,between a polar group (SiOH) and polar or charged groups in compounds present in the sample. Hydrogen bonding can be one of the interactions.