Description
General description
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Ascentis®Express 160Å ZIC®-cHILIC has a phosphorylcholinezwitterionic stationary phase covalently attached to Fused-Core® particle. The permanent and hydrophiliczwitterion functionality makes the column suitable forHydrophilic Interaction Liquid Chromatography (HILIC).Weak electrostatic interactions between charged analytesand the neutral zwitterionic stationary phase results ina unique selectivity, and especially suitable for analytesthat are poorly retained on reversed phase columns.
Application
The Ascentis® Express 160Å ZIC®-cHILIC column can be used as a tool to change the selectivity or to improve peak resolution for polar and hydrophilic compounds such as carbohydrates, metabolites, acids and bases, organic and inorganic ions, metal complexes, amino acids, peptides, and protein digests.
Preparation Note
Equilibrating the Column: Equilibrate the column with minimum 15 column volumesof initial mobile phase composition. Adjustments may beneeded depending on the analyte.
Storage: The column is delivered filled with 85% (v/v) acetonitrilein ammonium acetate buffer (25 mM, pH 6.8) and thatis also the recommended solvent for long term storage.Connect the end stop plugs when the column is removedfrom the system.
Sample Solvent and Solvent Strength: Sample solvents should consist of 60–100% organic solvent,or initial eluent composition. Water should be minimized.Weak HILIC solvents such as acetonitrile are favored.The relative solvent strength for HILIC is:Acetone Mobile Phase Considerations: To obtain reproducible results, maintain at least 3% waterin the mobile phase, to ensure sufficient hydration of thestationary phase particles.Suitable buffer systems for HILIC separations are formateand acetate, due to their excellent solubility even in veryhigh concentrations of organic solvent. Avoid phosphate,and other low solubility buffers, to prevent precipitationon the column bed. A buffer concentration in the range5–25 mM is recommended for most analytes, with anupper limit of 200–300 mM, depending on the solubilityin the eluent. TFA and other ion pair reagents should beavoided, as they can interfere with the HILIC separationmechanism, and suppress MS signals.
Typical Elution Protocols: Isocratic elution: 85 : 15 (v/v) acetonitrile/NH4Ac, (5–25 mM)or other suitable buffer salt.Gradient elution: 90% to 40% acetonitrile in 20 minutes(~2.5%/min).
Cleaning and Regeneration: Should the operating pressure of the column suddenlyincrease beyond normal levels, reversing the flow directionof the column may be attempted to remove debris on theinlet frit.In case of contamination from biological samples, use thefollowing recommended column wash procedure:An initial washing with 30 column volumes (CV) deionizedwater is used to remove organic solvent and polarimpurities, followed by a flush with 30 CV of 0.5 M sodiumchloride solution. Remove the salt solution with sufficientwater (30 CV) and equilibrate the column with 80%(v/v) acetonitrile, before finally equilibrating with therecommended mobile phase for storage or the intendedmobile phase. To conduct online cleaning of a HILIC column,increase both the aqueous component and ionic strengthof the eluent. While 100% aqueous eluent can be used, aconcentration of 50% is typically sufficient. This combinationenhances cleaning efficiency and facilitates faster column reequilibration.This procedure can be included at the end of agradient run and is especially useful for routine applicationswith samples containing appreciable amounts of salts, suchas urine and plasma.
Analysis Note
Column Hardware and Chemical Compatibility: The column and the frits are made from stainless steel with a bioinert coating to reduce non-specific adsorption. The Ascentis® Express ZIC®-cHILIC column can be operatedin the pH range 2 to 8, while strongly alkaline solutionsand washing with sodium hydroxide should be avoided. The column can be heated and operated up to 70 °C. Note that operating at high pH and high temperature maycompromise column lifetime.
Legal Information
Ascentis is a registered trademark of Merck KGaA, Darmstadt, Germany
Fused-Core is a registered trademark of Advanced Materials Technology, Inc.
ZIC is a registered trademark of Merck KGaA, Darmstadt, Germany
