retention problems using an amino column

[Andreas Karas]

I am using an amino-column under HILIC conditions for 2 months for the separation of weak acidic and basic compounds. The buffer salts I have employed were ammonium acetate/formate and ACN/H2O as solvents. 2 weeks ago I have also employed TFA and phosphoric acid. Returning the system to initial mobile phase conditions (that I have already worked with) the retention of the compounds is quite different. For example retention of weak acids is shortened and retention of basic compounds is increased! Is there a possibility that TFA is accumulated on the surface of the amino column and extensive wash is needed to resume chromatographic performance???

any suggestion would be helpful...

[Noser222]

Is there a possibility that TFA is accumulated on the surface of the amino column and extensive wash is needed to resume chromatographic performance???

They definitely are. Try washing with ammonium acetate or ammonium formate in 100% water; hopefully you have an amino phase that can handle that.

[Bryan Evans]

I suppose reasons for the reduced performance:
- sample gunk accumulated on column
- desorption of ligand phase
- anions are tightly bound to ligand

I think different column manufacturers will have different cleaning
procedures for their NH2 phases. So you may want to ask the
manufacturer.

For our NH2 phase, we would recommend 50mM NH4AcOH or (formate).
The less organic - the better (but not every NH2 phase can handle
100% aqueous eluent - so be mindful of that).

[Andreas Karas]

I suppose reasons for the reduced performance:
- sample gunk accumulated on column
- desorption of ligand phase
- anions are tightly bound to ligand

I think different column manufacturers will have different cleaning
procedures for their NH2 phases. So you may want to ask the
manufacturer.

For our NH2 phase, we would recommend 50mM NH4AcOH or (formate).
The less organic - the better (but not every NH2 phase can handle
100% aqueous eluent - so be mindful of that).

Thank you very much for your reply...I hope that disorption of ligand phase and sample gunk are not the cases here because I haven't ingected any "real samples" into the column. In addition I am working only with standards for approximately two months and at pH range 3-6. The truth is that I have tryed tetrahydrofuran to wash out any tightly bounded anions. A procedure employed for graphitized carbon columns..as far as 100% H2O, I am not sure if my column will handle such conditions, so I will communicate with the manufacturer.

[Noser222]

THF will not wash out anions. It is a weak solvent in this mode of HPLC.

[mohan_2008]

Yes. It seems to me that you have too many constituents such as TFA and phosphoric acid which can be avoided in your mobile phase.

Primarily, a HILIC operates in a totally different way as compared to your regular RP hplc.

Please try with this combination.

1. 90% ACN + 10% 10mM Amm.formate buffer @pH 4.0. Note: Here in Hilic, H2O is the stronger solvent.
2. Use a new Hilic column/ or flushed with atleast 80%ACN + 20% H2O prior to use.

Usually the selectivity in a HILIC is reverse to a traditional RP method. The more hydrophobic solutes should elute out fairly quickly and the more polar should stay longer.

Do not use more additives in a HILIC specially TFA as it can complicate the partition process equilibrium in the HILIC mode.