Hydrophilic Interaction Liquid Chromatography (HILIC) is a powerful and versatile chromatographic method in general used for the separation of polar compounds. It is especially powerful in applications where traditional reverse phase liquid chromatography (RPLC) fails to do separation and retention effectively, imparting particular advantages in terms of retention and selectivity for hydrophilic analytes. It is also known as Aqueous normal phase chromatography.
Hilic chromatography was first conceptualized and developed by Dr. Andrew Alpert. Dr. Alpert introduced the concept of HILIC in the mid-1990s, presenting it as a method particularly suited for the separation of polar compounds that are often difficult to retain using traditional Reversed-Phase Liquid Chromatography (RPLC).
Principle of Hilic chromatography
HILIC operates on a mechanism that combines aspects of both normal-phase chromatography and reversed-phase chromatography. It uses a polar stationary phase and a relatively non-polar mobile phase, typically consisting of a high concentration of organic solvent (such as acetonitrile) with a small proportion of water or aqueous buffer. The fundamental principle of HILIC involves the partitioning of analytes between the polar stationary phase and the aqueous layer adsorbed on the stationary phase surface.
Mechanism
Stationary Phase: The stationary phase in HILIC is usually a polar material, such as silica, or a bonded phase with functional groups like diol, amino, or zwitterionic groups.
Mobile Phase: The mobile phase often comprises a high percentage of organic solvent (e.g., 70-90% acetonitrile) mixed with water or an aqueous buffer.
Retention Mechanism: Analytes are retained based on their polarity. Polar compounds have stronger interactions with the stationary phase and are retained longer, while less polar compounds elute more quickly.
Present HILIC theory dictates that HILIC retention is caused by a partitioning of the injected analyte solute molecules between the mobile phase eluent and a water-enriched layer in the hydrophilic HILIC stationary phase. The more hydrophilic the analyte is, the more is the partitioning equilibrium shifted towards the immobilized water layer in the stationary phase, resulting in more retention of the analyte. Retaintion in Hilic chromatography also cause by hydrogen bonding and Diple-Dipole interaction.
Advantages of HILIC
Enhanced Retention of Polar Compounds: HILIC is particularly effective for compounds that are too polar to be retained on reversed-phase columns, such as small organic acids, bases, and peptides.
Compatibility with Mass Spectrometry (MS): The high organic solvent content in HILIC mobile phases improves ionization efficiency in electrospray ionization (ESI), making HILIC ideal for coupling with MS for sensitive detection.
Improved Peak Shape and Resolution: HILIC can provide better peak shapes and resolution for polar compounds compared to reversed-phase chromatography, reducing issues such as tailing and co-elution.
Flexibility in Method Development: The ability to adjust the water content and buffer strength in the mobile phase allows fine-tuning of retention times and selectivity.
Method Development in HILIC
Developing a robust HILIC method involves several key considerations:
Selection of Stationary Phase: Choosing the appropriate stationary phase is crucial. Silica-based columns are common, but bonded phases with specific functional groups can enhance selectivity for particular analytes.
Optimization of Mobile Phase: The ratio of organic solvent to water, as well as the type and concentration of buffer, must be optimized to achieve the desired retention and resolution.
pH and Ionic Strength: Adjusting the pH and ionic strength of the aqueous component can significantly impact analyte retention and peak shape.
Column Temperature: Temperature can affect the viscosity of the mobile phase and the interaction between analytes and the stationary phase, thus influencing retention and separation efficiency.
Example of Some Hilic HPLC coloumn available:
1) ACQUITY UPLC BEH Amide Column (Waters Corporation):
Stationary Phase: Ethylene-bridged hybrid (BEH) particles with amide bonding.
Features: High pH stability, excellent retention of polar compounds, and compatibility with a wide range of mobile phases.ZORBAX HILIC Plus (Agilent Technologies):
2) ZORBAX HILIC Plus (Agilent Technologies):
Stationary Phase: Silica-based particles with proprietary bonding.
Features: Enhanced retention and selectivity for polar compounds, robust performance in both isocratic and gradient elution modes.Atlantis HILIC Silica Column (Waters Corporation):
3) Atlantis HILIC Silica Column (Waters Corporation)
Stationary Phase: Silica particles.
Features: Strong retention of highly polar compounds, ideal for metabolomics and small molecule analysis, good peak shape and resolution.
4) SeQuant ZIC-HILIC (Merck Millipore):
Stationary Phase: Zwitterionic bonding on silica particles.
Features: Unique zwitterionic stationary phase providing balanced retention of both anionic and cationic compounds, high resolution, and low bleed characteristics for MS detection.
5) Kinetex HILIC (Phenomenex):
Stationary Phase: Core-shell silica particles with diol bonding.
Features: High efficiency and fast separations, suitable for high-throughput analysis, improved peak capacity and sensitivity.
6) Nucleodur HILIC (Macherey-Nagel):
Stationary Phase: Silica particles with polar modification.
Features: Suitable for separation of a wide range of polar compounds, high chemical stability, and excellent batch-to-batch reproducibility.
Some real time API analysis based on Hilic mode:
1.Vortioxetine
HILIC Method:
Column: ACQUITY UPLC BEH Amide
Mobile Phase: 85% Acetonitrile, 15% 20 mM Ammonium Formate (pH 3.0)
Flow Rate: 0.4 mL/min
Detection: MS detection
Reason: Vortioxetine is an antidepressant with polar characteristics. HILIC improves the retention and separation of vortioxetine and its metabolites, which might co-elute in RPLC, thus enhancing sensitivity and specificity, especially when coupled with mass spectrometry.
2. Adenosine
HILIC Method:
Column: Luna HILIC
Mobile Phase: 90% Acetonitrile, 10% 20 mM Ammonium Formate (pH 3.5)
Flow Rate: 0.6 mL/min
Detection: UV at 260 nm
Reason: Adenosine is a nucleoside with high polarity, making it difficult to retain and separate using RPLC. HILIC provides better retention and resolution, which is critical for accurate quantification and impurity profiling in pharmaceutical formulations.
3. Glycine
HILIC Method:
Column: SeQuant ZIC-HILIC
Mobile Phase: 75% Acetonitrile, 25% 10 mM Ammonium Formate (pH 4.0)
Flow Rate: 0.5 mL/min
Detection: UV at 210 nm or MS
Reason: Glycine is a simple amino acid with high polarity. HILIC is chosen for its ability to retain and separate glycine effectively, overcoming the limitations of RPLC where glycine elutes too early or not at all, thus ensuring better sensitivity and precision in analysis.
Commonly asked FAQs in Hilic chromatography:
1. What is HILIC chromatography?
Answer: Hydrophilic Interaction Liquid Chromatography (HILIC) is a chromatographic technique used to separate and analyze polar compounds. It employs a polar stationary phase and a predominantly organic mobile phase, typically consisting of a high percentage of acetonitrile with water or an aqueous buffer.
2. How does HILIC differ from Reversed-Phase Liquid Chromatography (RPLC)?
Answer: In RPLC, the stationary phase is non-polar, and the mobile phase is polar, primarily water with organic solvents like methanol or acetonitrile. In contrast, HILIC uses a polar stationary phase and a mobile phase with a high proportion of organic solvent (e.g., acetonitrile) mixed with water. This makes HILIC particularly suitable for retaining and separating highly polar compounds.
3. What types of stationary phases are used in HILIC?
Answer: Common stationary phases in HILIC include bare silica, and silica bonded with polar functional groups such as diol, amino, amide, or zwitterionic groups. Each type offers different selectivity and retention characteristics for various polar analytes.
4. What are the advantages of using HILIC?
Answer: The advantages of HILIC include enhanced retention and resolution of polar compounds, better peak shapes, compatibility with mass spectrometry, and flexibility in method development through adjustment of mobile phase composition and pH.
5. What kind of mobile phase is typically used in HILIC?
Answer: The mobile phase in HILIC usually consists of a high percentage (70-90%) of organic solvent like acetonitrile mixed with water or an aqueous buffer. The water content and buffer strength can be adjusted to optimize retention and separation.
6. Can HILIC be coupled with mass spectrometry (MS)?
Answer: Yes, HILIC is highly compatible with mass spectrometry. The high organic solvent content in HILIC mobile phases enhances ionization efficiency in electrospray ionization (ESI), leading to better sensitivity and lower detection limits.
7. Can I run 100% water as mobile phase in hillic chromatography.
Running 100% water as the mobile phase in Hydrophilic Interaction Liquid Chromatography (HILIC) is not recommended because it fundamentally contradicts the principles of HILIC. Here’s why?
1) Poor Retaintion:The high organic solvent content in HILIC mobile phases (usually 70-90% acetonitrile) is essential for retaining polar compounds. Using 100% water would lead to poor retention of polar analytes, causing them to elute very quickly or not be retained at all.
2) Stationary Phase Dehydration:
HILIC columns are designed to function with a substantial amount of organic solvent. Running 100% water can disrupt the equilibrium and interaction between the stationary phase and the mobile phase, potentially damaging the stationary phase over time or reducing its efficiency.
