Hydrophilic Interaction Liquid Chromatography (HILIC) is a specialized technique used within liquid chromatography for separating and analyzing complex mixtures. Chromatography separates components based on their differential interaction with a stationary phase and a mobile phase. HILIC was developed to address the analysis of highly polar compounds, such as certain metabolites and small pharmaceutical molecules, which are difficult to retain using conventional approaches. HILIC modifies the chemical environment inside the column to allow for stronger interactions with these water-soluble analytes.
Principles of Separation
HILIC separation relies on a highly polar stationary phase combined with a mobile phase rich in organic solvent. This combination creates a unique environment that facilitates the retention of hydrophilic analytes. The high concentration of organic solvent (typically 60% or more) causes the small amount of water in the mobile phase to be preferentially adsorbed onto the polar stationary phase surface. This adsorption forms a distinct, water-enriched stagnant layer.
Separation occurs as analytes introduced into the system partition between the organic-rich mobile phase and this water-enriched layer. Compounds that are highly hydrophilic, or water-loving, will spend more time partitioning into the stagnant aqueous layer. They are therefore retained longer within the column, exhibiting a later elution time. Conversely, less polar compounds remain predominantly in the organic-rich mobile phase and pass through the column more quickly.
The retention mechanism is a mixed-mode process, primarily driven by partitioning. Secondary interactions also play a role, including hydrogen bonding between the analyte and the stationary phase, or ionic interactions with charged groups on the column material. The balance of these forces dictates the final retention time. More hydrophilic analytes show increased retention until the aqueous content of the mobile phase is increased to elute them. The aqueous component acts as the strong solvent, meaning increased water concentration decreases retention time.
Key Components: Stationary and Mobile Phases
HILIC requires specific chemical compositions for both the stationary and mobile phases. The stationary phase must be highly polar to attract and hold the water required for forming the stagnant aqueous layer. Columns are often based on bare silica, which has exposed, highly polar silanol groups. Other common chemistries include bonded phases such as diol, amide, or zwitterionic groups, all presenting a hydrophilic surface to the mobile phase.
The mobile phase composition is inverted compared to many other liquid chromatography methods, utilizing a high concentration of organic solvent, typically acetonitrile. Acetonitrile is often selected because it is water-miscible and maintains a low viscosity, which helps reduce column back pressure. The organic component must constitute 60% up to approximately 95% of the total volume to ensure the water-enriched layer forms on the stationary phase surface.
The remaining component is water, often containing a buffer or salts. This aqueous portion establishes the stagnant water layer and controls the \(mathrm{pH}\) and ionic strength of the system. Volatile buffers, such as ammonium formate or ammonium acetate, are compatible with mass spectrometry detection. Controlling the \(mathrm{pH}\) manages the ionization state of both the analytes and the stationary phase functional groups, influencing secondary ionic interactions.
HILIC vs. Reverse Phase: A Comparative View
HILIC addresses limitations found in Reverse Phase Liquid Chromatography (RPLC), the most widely used separation technique. The fundamental difference lies in the polarity of their stationary and mobile phases, resulting in an inverse retention order. RPLC uses a non-polar stationary phase (e.g., C18) and a predominantly water-based mobile phase. This setup retains non-polar compounds longer, but highly polar compounds elute quickly, making their separation impossible.
HILIC employs a polar stationary phase and a mobile phase rich in organic solvent. This reversal of polarities means HILIC preferentially retains polar, hydrophilic compounds. The mobile phase strength is also inverted: in RPLC, increasing organic solvent concentration increases strength and speeds up elution. In HILIC, increasing the aqueous component increases mobile phase strength and speeds up the elution of highly polar analytes.
HILIC separates compounds based on hydrophilicity, while RPLC separates them based on hydrophobicity. HILIC is used for analyzing small, water-soluble molecules that exhibit poor or no retention under RPLC conditions. This distinction makes HILIC a complementary tool, rather than a replacement, for RPLC in analytical laboratories.
Primary Applications
HILIC is used for analyzing highly polar compounds that are poorly retained using standard reversed-phase methods. The technique is widely applied in metabolomics for analyzing small polar metabolites. Examples include the separation and quantification of amino acids, nucleotides, and various small carbohydrates or sugars.
The high organic content of the HILIC mobile phase provides an advantage when coupled with mass spectrometry (MS) detection. The highly organic solvent mixture evaporates more readily than the high-aqueous mixtures used in RPLC, enhancing the efficiency of the ionization process. This results in improved sensitivity for polar analytes, aiding trace analysis. HILIC is also applied in pharmaceutical analysis for separating polar drug compounds and impurities, and in biomarker discovery where target molecules are hydrophilic.