Column Tips Series # 2 – Normal Phase & HILIC Column Washing

To prolong column lifetimes, it is recommended to clean the HPLC column after every single analysis or whenever possible. Presence of buffer salts, acids or ion-pairing reagents in the column can cause hydrolysis of the stationary phase, leading to shorter column lifetimes.

Cleaning Normal-Phase Columns

Glantreo Silica, Diol, Amino and Cyano stationary phases can be used in normal-phase chromatography mode.

Frequent cleaning ensures that the process tends to be easier and quicker subsequently each time. Also, cleaning should always be performed after running a known “dirty” sample.

Normal-phase separations depend upon polar adsorptive interactions, where the stationary phase is polar and the mobile phase is non-polar. Polar analytes will be more strongly retained than non-polar analytes in normal-phase chromatography. It is recommended to clean the column with polar solvents to remove highly polar contaminants.

Solvent Polarity

A. Flush the column with mobile phase until all samples elute out completely.

B. If any buffer or modifiers are used, flush the column with 20 column volumes of intermediate mobile phase (original mobile phase without the buffer)

C. Flush with 20 column volumes of polar dry solvents like Ethanol, 2-Propanol, Hexane etc. The use of water for washing normal-phase columns is not recommended.

D. If high back pressure is observed, reduce and adjust the flow rate.

Cleaning HILIC Columns

Glantreo Silica, Aqua, Diol, Amino, Cyano, PFP and C1 stationary phases can be used in HILIC separation mode.

Under HILIC mode, polar analytes are retained with high organic mobile phases. The following describes the elution strength of solvents used in HILIC mode.

Solvent Polarity HILIC phase

A. Flush the column with the mobile phase until all samples elute out completely

B. Flush the column with 20 column volumes of intermediate mobile phase (original mobile phase without the buffer)

C. Flush with 20 column volumes of 50/50 Acetonitrile/Water solution, to remove highly polar components.

D. If the column still shows shift in retention time or distorted peak shapes, clean the column with 100 % water for at least 30 minutes

E. For Amino columns, if peak shape distortion or shift in retention times is still observed, clean the column with 20 volumes of 50 mM ammonium formate (or ammonium acetate) aqueous solution/acetonitrile = 50/50

F. If high backpressure is observed when increasing the organic solvent concentration, reduce and adjust the flow rate.

G. After cleaning the column, make sure to thoroughly equilibrate the column with the mobile phase to be used in the analysis prior to use.

H. For short-term storage, store the column in mobile phase (not containing any additives or buffers).

I. For long-term storage, store the column in 100% Acetonitrile.

Note: To maximise column lifetimes, always follow the operating guidelines described in the Glantreo Certificate of Analysis, supplied with the column.

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