Hydrophilic Interaction Liquid Chromatography (Hilic)

Ajay A, Prasanth S. S, Sanooja P. k, Jisha U, K. T. Akshara, Riya Rajan, Sibina M. K, Mohammed Farooq P,

doi:10.5281/zenodo.13292739

Review Paper | Open Access
Volume 02 | Issue 08 | Article Id IJPS/240208056

Hydrophilic Interaction Liquid Chromatography (Hilic)
Ajay A* Prasanth S. S Sanooja P. k Jisha U K. T. Akshara Riya Rajan Sibina M. K Mohammed Farooq P
Department Pharmaceutical Analysis Al-Shifa College Of Pharmacy, Perinthalmanna, Malappuram, Kerala Pin-676504.

Abstract

Hydrophilic interaction liquid chromatography or HILIC is a powerful analytical technique for the separation and analysis of polar and hydrophilic compounds, including small molecules, peptides, and proteins. HILIC is a variation of normal phase chromatography. It is also called as “Reverse reversed-phase” or “Aqueous normal-phase” chromatography. This chromatographic method capitalizes on the unique interactions between analytes and a polar stationary phase, which enhances the retention of hydrophilic compounds through mechanisms such as partitioning, hydrogen bonding and ion-exchange. The benefits of HILIC include significantly better solute diffusivity, enhanced sensitivity with ESI-MS, and very symmetrical peak shapes. However, a major downside is the heavy dependence on the aprotic solvent acetonitrile. This article mainly discusses the type of stationary phase, mobile phases, applicability and future prospective of HILIC in pharmaceuticals and biochemical research.

Keywords

Hydrophilic interaction liquid chromatography, hydrogen bonding, acetonitrile, mobile phase.

Introduction

Reversed-phase chromatography^1,2 is the widely adopted retention mechanism for the majority of separations. However, with respect to the analysis of small polar analytes this technique becomes difficult to apply. As an alternative to using normal-phase, Alpert investigated a technique he christened hydrophilic interaction chromatography³. HILIC^4,5,6or Hydrophilic Interaction Liquid Chromatography is a high-performance liquid chromatographic (HPLC) technique for separation of polar and hydrophilic compounds. HILIC^7,8is a variation of normal phase chromatography. It is also called as “Reverse reversed-phase” or “Aqueous normal-phase” chromatography. The stationary phase is a polar material like silica⁹, cyano, amino, amide and the mobile phase is highly organic with a smaller amount of aqueous mobile phase like water. The polar analytes¹⁰, interact with the polar particle surface and are retained in multiple ways, including hydrogen bonding, dipole-dipole, and ion exchange¹¹ Thus HILIC provides a column with a hydrophilic stationary phase and eluent with water, buffer and a high concentration of water-miscible organic solvent such as acetonitrile. Due to the more polar nature of the eluent, solubility issues of polar analytes¹²associated with normal phase could be solved.

Stationary Phase For Hilic

Typical HILIC stationary phases¹³ consist of classical bare silica¹⁴or silica gels modified with many polar functional groups. Polymer-based stationary phases can also be used. The first generation of HILIC¹⁵ mode separated carbohydrates by an amino-silica phase, in a mixture of acetonotrile and water (75:25 v/v). The next generation of stationary phases for HILIC used DIOL and amide-silica. They are usually prepared by chemically modifying the silica gel surface, like the C18 phases used for RP-LC. Chemically bonded DIOL phases demonstrate high polarity and hydrogen bonding properties. Basic analytes are in general strongly retained on silica gel by hydrogen bonding and ion-exchange interactions with silanol groups, acidic compounds show increased affinities to amino-silica columns¹⁶. Cyclodextrin-silica stationary phases that possess several linked glucopyranoside units and have chiral recognition properties are useful for HILIC chiral separations.

Mobile Phase For Hilic

Mobile phase for HILIC chromatography includes water-miscible polar organic solvents such as acetonitrile with a small amount of water. Aprotic solvents such trahydrofuran or dioxane. Ionic additives, such as ammonium acetate and ammonium formate, are typically used to control the mobile phase pH and ion strength. The use of other salts (such as 100–300 mM sodium perchlorate) that are soluble in high organic solvent mixtures (ca. 70?etonitrile) can be used to increase the polarity of the mobile phase in order to achieve elution. Less polar solvents are used for increasing the retention of polar analytes.

Mechanism Of Hilic

HILIC is used for the separation of polar analytes that cannot be separated by reverse phase chromatography. The mechanism includes the combination of partitioning, ion-exchange and hydrogen bonding. The polar analyte partitions between bulk mobile phase containing least polar solvent like acetonitrile and partially immobilized polar layer on material surface. The secondary interactions between surface silanols with the charged analyte leading to ion-exchange. The hydrogen bonding occurs between positively charged analyte and negatively charged surface silanols. Present theory proposes that HILIC retention is caused by partitioning. This phenomenon still lacks a thorough theoretical explanation. In this mode, the separation mechanism is based on the differential distribution of the injected analyte solute molecules between the acetonitrile-rich mobile phase and a water-enriched layer adsorbed onto the hydrophilic stationary phase. he more hydrophilic the analyte, the more the partitioning equilibrium is shifted towards the immobilized water layer on the stationary phase, and thus, the more the analyte is retained.

Figure No.1: mechanism of hilic

Advantages

HILIC helps in the retention of highly polar analytes that cannot be retained reversed-phase chromatographic method. Polar metabolites retain much more when compared with reversed-phase which increases the selectivity of the method. 1. HILIC separations can be easily paired with various detection methods like ultraviolet light absorbance (UV), fluorescence (FL), refractive index (RI), evaporative light scattering (ELSD), charged aerosol (CAD), and mass spectrometry¹⁷ (MS). 1. When HILIC is combined with MS detection, the sensitivity of ESI can be significantly higher (10-100 times) than in RPLC. This is because the mobile phase contains a high amount of organic solvent, which reduces surface tension and makes it easier for drops to form during the spraying process. As a result, the formation of ions in the gas phase is greatly enhanced, leading to improved sensitivity. HILIC^18,19 improves the sample throughput by direct injection of high organic extracts from liquid-liquid extraction or Solid-phase extraction without the need for dilution or evaporation and reconstitution.

Disadvantages

The main disadvantage in adopting HILIC is the reliance on acetonitrile, during times of shortage of this solvent. HILIC is best suited for highly polar compounds, on-polar or weakly polar compounds may not be effectively separated using this technique. Sample preparation for HILIC can be more complex than for other techniques, such as reversed-phase chromatography. The need for organic solvents and specific conditions can complicate sample handling. HILIC columns, especially those with silica-based stationary phases, can be less stable than those used in other chromatographic techniques. They may degrade over time, particularly in the presence of water or at extreme pH levels. Retention times in HILIC can be influenced by small changes in mobile phase composition or pH, leading to variability in results if conditions are not tightly controlled. The performance of HILIC columns can be sensitive to temperature changes, which may require precise temperature control during analysis. HILIC can generate high backpressure, which may require specialized equipment that can handle such conditions.

Applications Of Hilic

Hydrophilic Interaction Liquid Chromatography (HILIC) is a specialized chromatographic technique primarily used for the separation of polar and hydrophilic compounds. Here are some key applications of HILIC:

Analysis of Polar Compounds: HILIC is particularly effective for the separation and analysis of small polar molecules, such as sugars, amino acids, organic acids, and nucleotides, which are often challenging to analyze using traditional reversed-phase chromatography.
Biopharmaceuticals: HILIC is used in the analysis of biologics²⁰, including proteins and peptides, especially for assessing their glycosylation patterns and post-translational modifications. It helps in characterizing the heterogeneity of biopharmaceutical products.
Metabolomics: In metabolomics studies, HILIC can be employed to separate and analyze a wide range of metabolites in biological samples, providing insights into metabolic pathways and disease states.
Environmental Analysis: HILIC is applied in environmental chemistry for the analysis of polar contaminants in water and soil samples, including pesticides, pharmaceuticals, and other emerging contaminants.
Food Safety and Quality Control: HILIC can be used to analyze food components such as sugars, organic acids, and vitamins, aiding in quality control and ensuring food safety.
Drug Development: In pharmaceutical research, HILIC can be utilized to evaluate drug candidates' pharmacokinetics by analyzing their solubility and stability in aqueous environments.
Chiral Separations: While not its primary use, HILIC can also be adapted for chiral separations by using chiral stationary phases, which can help in the resolution of enantiomers.
Sample Preparation: HILIC can be incorporated into sample preparation techniques such as solid-phase extraction (SPE) for the enrichment of polar analytes from complex matrices.
Overall, HILIC is a versatile technique that complements other chromatographic methods, providing valuable insights across various fields including pharmaceuticals, environmental science, food safety, and biochemistry.

Future Prospective Of Hilic

Increased Automation and Integration: The integration of HILIC with automated systems and high-throughput techniques is likely to enhance its application in drug discovery, metabolomics, and food safety, allowing for faster and more efficient analyses.
Advancements in Stationary Phases: The development of new stationary phases with improved selectivity, stability, and reproducibility will expand the range of applications for HILIC. Innovations may include novel materials that enhance the retention of polar analytes or enable chiral separations.
Coupling with Mass Spectrometry: The combination of HILIC²¹ with mass spectrometry (MS) is expected to gain traction, particularly for the analysis of complex biological samples. This coupling can provide detailed structural information and improve sensitivity for low-abundance compounds.
Environmental Monitoring: With increasing regulatory scrutiny on environmental contaminants, HILIC may see expanded use in monitoring polar pollutants in water and soil, supporting efforts in environmental protection and sustainability.
Miniaturization and Microfluidics: Advances in microfluidics and miniaturized chromatography systems may enhance the applicability of HILIC for point-of-care testing and field analyses, making it more accessible for rapid assessments.
Education and Training: As HILIC becomes more widely recognized, there will be an increased focus on educating researchers and analysts about its principles, methodologies, and applications, leading to broader adoption across various industries.
Sustainability Initiatives: With a growing emphasis on green chemistry, researchers may explore more sustainable solvents and methods within HILIC to minimize environmental impact while maintaining analytical performance.
Interdisciplinary Research: HILIC's versatility will encourage interdisciplinary collaborations among chemists, biologists²², environmental scientists, and food technologists, fostering innovation in analytical techniques and applications.

CONCLUSION

HILIC is becoming more and more popular as a technique for separating polar and/or basic solutes. Compared to reversed-phase, HILIC offers several advantages, especially in terms of faster solute diffusivity, improved sensitivity with ESI-MS, and symmetrical peak shapes. It is now used for a wide range of polar compounds, both charged and uncharged. This method has gained attention due to the growing demand for analyzing polar compounds in complex mixtures. In recent years, HILIC has become popular in bioanalytical applications, particularly for polar drug and metabolite structures.

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