How to condition a new Glycan BEH Amide column - WKB111759

OBJECTIVE or GOAL

Condition a new Glycan BEH Amide column.

 

ENVIRONMENT

• UPLC
• HPLC
• HILIC
• Glycan analysis

PROCEDURE

In order to get the most out of a new (unused) Glycan BEH Amide column, it is best practice to condition the column and demonstrate performance before actual test sample analysis. Condition by sequential injection of a representative sample until a stable chromatographic profile is achieved.

Even if a Glycan BEH Amide column is to be used with ammonium formate mobile phase, it can still prove beneficial to first condition the column with a gradient of 0.1%TFA-containing mobile phase. TFA is effective in cleaning a column's stationary phase of both neutralizing and ion-pairing contaminants that in ionic forms might otherwise strongly adsorb to a HILIC stationary phase and ultimately give poor column performance.

ADDITIONAL INFORMATION

If you have issue with sialylated glycan, try the protocol below, conditioning with fetuin.

1. Preparation of 10 mg/mL fetuin solution:

- Weigh 10 mg of fetuin (Sigma F3004 or equivalent) in a 1.5-mL tube.

- Dissolve all solid chemicals with 1 mL of 18.2 MOhm water. Vortex to mix well.

- Transfer fetuin solution to LC vials.

2. Conditioning protocol:

- Equilibrate column with initial mobile phase at the column temperature used for LC gradient. Ensure that system pressure change is no more than 10 psi within 1 min.

- Make one blank injection of water on the Glycan column with the desired LC gradient.

- Make at least three injections of 10 mg/mL fetuin solution on the Glycan column with the desired LC gradient.

- Injection volume: 1 µL

- The column should now be conditioned and ready for use.