Initial Use: When using either column to perform HILIC separations, flush the new column with 30 mL water, and then condition with at least 60 mL of a buffer solution with a salt concentration of 0.2 - 0.4 M and a pH in the range of 3 - 6 (exact figures are not important here). Flush again with another 20 ml water, then equilibrate with 30 ml of the mobile phase before injecting samples. (These volumes relate to 4.6 mm ID columns. For 9.4 mm ID columns, the volumes above should be multiplied by a factor of four).
Routine Use: Filter samples and mobile phases before use. Flush and store HILIC columns in water when not in use. Operation at room temperature is recommended, since elevated temperature shortens column lifetime.
General Mode of Operation: Salt is not required with solutes that are not electrolytes. In the case of electrolytes, use at least 25 mM buffer in the mobile phase. Gradient elution can be accomplished by a decreasing organic gradient (starting from 80-85% acetonitrile for peptides or 95% for phospholipids) or an increasing salt gradient (which typically gives flatter baselines). Solubility of salts can be a problem with mostly organic mobile phases, but sodium perchlorate works well and is transparent at low wavelengths (Fisher sells an HPLC grade). Buffer salts with reasonable solubility in 80% acetonitrile include triethylamine phosphate (TEAP) and sodium methylphosphonate (from methylphosphonic acid). Isocratic retention is typically several times greater with TEA salts than with the corresponding sodium or potassium salt. With 80% acetonitrile, concentrations of 75 mM (pH 5.0) or 100 mM (pH 2.8) TEAP are attainable. Gradient elution in HILIC generally requires one-fifth to one-tenth the concentration of salt required in ion-exchange chromatography.
A stock solution of TEAP can be prepared by making a concentrated aqueous solution of phosphoric acid, adding TEA until the desired pH is attained, then diluting to give a known final concentration (e.g., 2 M in phosphate). Similar methods are used with phosphonate buffers. Prepare stock solutions fresh monthly and store in the refrigerator. For preparation of the mobile phase, add the appropriate amount of stock solution and water to a volumetric flask. Next, add the acetonitrile to a level several mL short of the mark. Mix, then put the flask in a sonication bath for 5 minutes, this degasses and warms the solution. Finally, add acetonitrile to the mark and mix.
Organic solvents such as isopropanol can be used as alternatives to acetonitrile, but higher concentrations are usually required to attain the same degree of retention, and the resulting mobile phases are appreciably more viscous.