C18 PepMap100 Column Cleanup

[bduffy]

I am looking for advice on the best way to regenerate a C18 PepMap100 Column. Does anyone have experience with this?

[Mark Tracy]

The PepMap stationary phase is a conventional monomerically bonded C18. (Carefully characterized and controlled for peptide mapping.) You would clean it in the usual ways. To remove hydrophobic contaminants, wash with 50:50 MeCN:H2O then acetone then MeCl2 then acetone then 50:50 MeCN:H2O.

If the problem is loss of bonded phase there is no cure. If there is a void, it is not practical to top off a capillary column. You have to replace the column.

[bduffy]

Thanks Mark for your input.

I am not sure why the column has lost capacity. It is a 300um capillary column that we are using for 2-D chromatography of peptides. Can the column retain or be effected by ionic species, such as the ammonium acetate we're using in the 1st dimension of our separation? If so, is there a cleanup that would clean the column of these ionic species?

Thanks

[Mark Tracy]

Exposure to ionic species will not cause contamination of the column. What is the pH of the ammonium acetate from the 1st dimension? How much organic in the 1st dimension mobile phase? How large an injection? Has the void peak shifted?

[bduffy]

Here's the info. you requested. Thanks for the help.

The pH is 7.2 of the 1000mM ammonium acetate solution. We are fractionating using salt slugs of 10ul each, with concentrations between 50 and 1000mM ammonium acetate.

The Switchos eluant is (2% acetonitrile/98% (0.05% TFA in water)).

The void peak has not shifted.

Thanks

[Mark Tracy]

Well, the pH is no problem. Since the void volume is unchanged, we can rule out de-wetting; not usually a problem with this system anyway. The 10µL injection volume is about equal to the void volume, so you may have retention loss and/or asymmetric peaks for the most hydrophilic peptides. Have you tested the column under standard 1-D isocratic conditions? If the column shows short retention times under standard conditions, then it is probably just worn out from loss of bonded phase.