HILIC

The Technology:

HILIC (Hydrophilic Interaction Liquid Chromatography) is an advanced chromatographic method ideal for the separation and retention of polar species, including polar neutral and ionized analytes. It is a variation of normal-phase chromatography, but with key differences in the mobile phase composition and separation mechanism. While normal-phase chromatography typically employs 100% organic mobile phases, HILIC uses water-miscible organic solvents, creating a unique polar environment.

Historically, HILIC phases relied on unbound silica, but recent developments have introduced bonded polar phases classified into acidic, basic, and neutral chemistries. Zodiac’s HST HILIC columns encompass these modern bonded polar phases to address a wide range of analytical challenges.

HST B: A strong basic column with a pH range of 1 to 4.5, ideal for separating weak bases, amino acids, and metals. It works with mobile phases containing acids like trifluoroacetic, phosphoric, perchloric, or formic acid, ensuring strong retention of ionizable compounds under acidic conditions.
HST B2: A weak basic column featuring acidic carboxylic functional groups. With a broader pH range of 1 to 6.5, the carboxylic groups remain non-ionized below pH 5, making the column surface positively charged. It is well-suited for separations using ammonium acetate or ammonium formate buffered mobile phases.
HST SB: It has positive amine group with a alkyl chain, with working pH range from 1.0 to 4.5.

The Methodology:

Typical Mobile Phase Composition

The mobile phase commonly includes MeCN with a small water content.

Mechanism of Separation

A water-rich layer forms on the surface of the polar stationary phase, creating a liquid/liquid extraction system.
Analytes distribute between the stationary water layer and the mobile phase with low water content.
Polar compounds have stronger affinity to the water layer, penetrate the water rich layer on the stationary phase, where -as non-polar analytes remain in the organic mobile phase resulting in separation based on polarity.

Layer-Based Retention

Separation predominantly occurs between the water-rich layer on the stationary phase and the organic mobile phase.
The stationary phase is composed of ionized or polar functional groups but does not directly interact with analytes. Instead, it provides a polar environment, forming a water sub-layer that interacts with analytes.

Retention Control

- Retention time and selectivity can be fine-tuned by adjusting:
  - The concentration of MeCN (0–100%).
  - The buffer type, pH, and concentration.

HST S1, S2, and S3 Columns:

These are acidic phase Columns that form a negative charge on the stationary phase, depending on the pH of the mobile phase. Increasing the pH strengthens the negative charge, enhancing the column’s retention of cationic analytes.

Mechanism: Bases (positively charged) are retained through ion-exchange interactions, while acids (negatively charged) are repelled and separated via ion-exclusion chromatography.

HST N Column

This column is optimized for normal-phase separations and HILIC applications. Unlike other HILIC columns, the HST N’s unique surface chemistry involves functional groups such that positive and negative groups are optimally spaced apart, enabling electrostatic interactions with oppositely charged analytes. This distinctive configuration enhances retention of charged molecules and supports a wide range of organic modifiers, from 0% to 100% MeCN.

Notably, the HST N column performs exceptionally well in MeOH-based mobile phases, offering flexibility in methods requiring nitrogen-specific detection

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