A HILIC (Hydrophilic Interaction Liquid Chromatography) column is a type of analytical column used in liquid chromatography that separates analytes based on their hydrophilicity. It contains a polar stationary phase that attracts and retains hydrophilic (water-loving) compounds through mechanisms like hydrogen bonding, dipole-dipole interactions, and electrostatic forces. HILIC is complementary to reversed-phase chromatography and is particularly useful for separating highly polar and ionizable compounds that are poorly retained by non-polar stationary phases.

1. Excellent selectivity for polar and hydrophilic compounds:
HILIC provides superior separation of highly polar analytes like glycans, peptides, nucleic acids, metabolites, and polar drugs/metabolites that are poorly retained in reversed-phase LC.
2. Orthogonal selectivity to reversed-phase LC:
HILIC offers an orthogonal separation mechanism, making it ideal for 2D-LC setups and comprehensive analysis when combined with reversed-phase separations.
3. Compatible with mass spectrometry:
HILIC mobile phases containing high organic content are MS-friendly and facilitate efficient ionization and detection of analytes.
4. Improved peak shapes:
For ionizable compounds, HILIC can provide sharper peaks compared to ion-exchange chromatography by reducing secondary interactions.

There are several types of HILIC stationary phases available, each with its own advantages and disadvantages. The most common types include:

1.Bare silica:

This is the simplest type of HILIC stationary phase.

It is made from silica gel, which is a polar material that can interact with hydrogen bonding groups on analytes.

Bare silica is not very selective, but it can be a good choice for separating polar compounds that are difficult

to retain on reversed-phase columns.

 

Bare silica HILIC stationary phase

2.Bonded phases:

These stationary phases have a layer of bonded organic molecules on the surface of the silica gel.

The type of bonded molecule can have a significant impact on the selectivity of the column.

Some common bonded phases for HILIC include:

2.1 Diol phases:

These phases contain bonded diol groups, which can form hydrogen bonds with analytes.

Diol phases are a good choice for separating polar compounds such as carbohydrates,

peptides, and oligonucleotides.

 

Diol phases HILIC stationary phase

2.2 Amino phases:

These phases contain bonded amino groups, which can interact with analytes through hydrogen bonding and ion exchange.

Amino phases are a good choice for separating polar compounds such as amino acids and peptides.

Amino phases HILIC stationary phase

2.3 Zwitterionic phases:

These phases contain both positively and negatively charged groups.

Zwitterionic phases can be a good choice for separating polar compounds that are difficult

to retain on other types of HILIC columns.

Zwitterionic phases HILIC stationary phase

3.Hydride phases:

These phases are made from a special type of silica gel that is capped with a layer of hydride groups.

Hydride phases are very hydrophilic and can be a good choice for separating very polar compounds.

Hydride phases HILIC stationary phase

 

The choice of HILIC stationary phase will depend on the specific needs of the separation.

It is important to consider the polarity of the analytes, the desired selectivity, and the compatibility of

the stationary phase with the mobile phase.

Optimizing a HILIC separation can be an iterative process, but there are several key factors you can adjust to achieve the best results for your analytes. Here’s a breakdown of the key points:

1. Mobile Phase:

• Organic Modifier: This is the major factor influencing retention in HILIC. Unlike reversed-phase chromatography, increasing the organic content (like acetonitrile) strengthens interactions and increases retention time. Explore a range of organic modifier percentages (e.g., acetonitrile, methanol) to find the sweet spot for your analytes.
• Buffer and pH: Buffers like ammonium formate or acetate help control mobile phase pH and mitigate unwanted ionic interactions. Consider buffer type, concentration, and pH to fine-tune selectivity and peak shape.
• Additives: For specific analyte functionalities, additives like formic acid or modifiers with specific functional groups can enhance resolution or ionization for MS compatibility.

Stationary Phase:

• Column Selection: Different HILIC phases (bare silica, amide, diol, etc.) offer varying selectivity based on their surface chemistry. Experimenting with different column types might be necessary to achieve optimal separation for your analytes.

2. Chromatographic Conditions:

• Flow Rate: Adjusting flow rate can impact resolution and analysis time. Start with a moderate flow rate and explore a range to find the balance between peak efficiency and analysis speed.
• Gradient: HILIC often utilizes gradient elution with increasing organic content to elute retained analytes. Experiment with gradient slope and time to optimize peak separation while maintaining reasonable analysis time.
• Column Temperature: Temperature can influence analyte retention and peak shape. Consider a slightly elevated temperature (e.g., 30-40°C) compared to room temperature for some separations.

Additional Tips:

• Sample Preparation: Ensure proper sample cleanup to minimize matrix effects that can interfere with peak separation.
• System Equilibration: HILIC columns rely on a stable water layer for interaction. Allow sufficient equilibration time after injections and between runs for consistent retention times.
• Method Development Tools: Consider using software or statistical methods to design experiments and optimize multiple parameters simultaneously.
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But You can Remember, optimization is often an iterative process. Start by making small adjustments to one factor at a time, monitor the effects on your separation, and refine further based on your observations. 

Consulting application notes or technical resources from column manufacturers can also provide valuable insights for specific HILIC stationary phases.

HILIC mobile phases are quite unique compared to other chromatography techniques like reversed-phase HPLC. Here’s the breakdown:

Key Components:

• High Organic Content (60-70%+): The bulk of the mobile phase in HILIC is a strong organic solvent, typically acetonitrile (ACN). This creates a weak interaction with the polar stationary phase.
• Low Aqueous Content (3-10%+): A small percentage of water or another polar solvent like methanol is added. This forms a water-rich layer on the stationary phase for analyte partitioning.
• Buffers (Optional): Buffers can be included to control pH and influence analyte ionization and retention. Popular choices include ammonium formate and ammonium acetate due to their compatibility with MS detection and solubility in high-ACN environments.

Important Points:

• Water is the Strong Solvent: Unlike reversed-phase, where water is the weak solvent, water acts as the stronger solvent in HILIC, eluting more polar analytes later.
• Methanol Not Ideal: While methanol is a polar solvent, it’s generally not recommended in HILIC because it can disrupt the formation of the water layer.
• Mobile Phase Sensitivity: HILIC separations can be sensitive to small changes in mobile phase composition, especially the organic solvent percentage.