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Hydrophilic interaction chromatography

Hydrophilic interaction liquid chromatography (HILIC) is a high-performance liquid chromatography (HPLC) technique designed to separate polar and hydrophilic compounds using a polar stationary phase and a mobile phase consisting primarily of an organic solvent, such as acetonitrile, with a small percentage of water.^[1] The separation mechanism primarily involves partitioning of analytes between the organic-rich bulk mobile phase and a water-enriched layer adsorbed on the surface of the polar stationary phase, supplemented by secondary interactions like hydrogen bonding, dipole-dipole forces, electrostatic effects, and sometimes ion-exchange.^[1] Retention in HILIC increases with the polarity of the analytes, making it particularly effective for compounds that exhibit poor retention in reversed-phase liquid chromatography (RPLC).^[2] Introduced by Andrew J. Alpert in 1990 as an alternative to traditional normal-phase chromatography, HILIC builds on earlier applications from the 1970s, such as the separation of carbohydrates on amino-modified silica phases, but gained widespread adoption in the early 2000s due to its compatibility with mass spectrometry (MS) and the growing need for analyzing polar metabolites and biomolecules.^[3]^[1] Common stationary phases include unmodified silica, amide-bonded silica, zwitterionic materials (e.g., ZIC-HILIC), and diol phases, while mobile phases often incorporate buffers like ammonium acetate to stabilize pH and enhance selectivity.^[1] Despite advantages such as low mobile phase viscosity for high-efficiency columns and improved ESI-MS sensitivity (up to threefold higher than RPLC for polar analytes), HILIC can suffer from long column equilibration times and sensitivity to sample solvent composition.^[2] HILIC has become a cornerstone in fields like pharmaceutical analysis, proteomics, metabolomics, and glycomics, enabling the separation of challenging polar species such as peptides, nucleosides, sugars, and drug metabolites that are difficult to retain by other LC methods.^[1] Recent advances, particularly since the 2010s, have expanded its use to biopharmaceutical characterization, including intact monoclonal antibodies and oligonucleotides, often in hyphenated HILIC-MS workflows for impurity profiling and high-throughput screening.^[2] Its orthogonality to RPLC further enhances multidimensional separations in complex sample analyses.^[1]

Introduction

History

Hydrophilic interaction liquid chromatography (HILIC) builds on earlier applications from the 1970s, such as the separation of carbohydrates on amino-modified silica phases, but was formally introduced by Andrew J. Alpert in 1990 as a variant of normal-phase chromatography specifically designed for the separation of polar analytes, such as peptides, nucleic acids, and carbohydrates, which are poorly retained in reversed-phase systems.^[4]^[1] In his seminal paper published in the Journal of Chromatography A, Alpert described HILIC as utilizing polar stationary phases with predominantly organic mobile phases (typically >70% acetonitrile), where retention increases with the hydrophilicity of the solute, contrasting the behavior in reversed-phase liquid chromatography.^[4] This development addressed key limitations of traditional normal-phase chromatography, including poor reproducibility due to uncontrolled adsorption of atmospheric water onto the stationary phase and low solubility of polar compounds in non-polar mobile phases.^[5] HILIC improved these aspects by employing high-organic mobile phases that enhance the solubility of polar analytes while maintaining a water-enriched layer on the stationary phase surface, promoting more stable partitioning and reducing peak tailing.^[6] These innovations enabled reliable separations of hydrophilic biomolecules that were challenging with earlier techniques.^[5] Following its introduction, HILIC saw significant advancements, including the development of electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) by Alpert in 2007, which incorporated charged stationary phases to enhance selectivity for highly charged peptides and phosphopeptides through combined electrostatic and hydrophilic interactions.^[7] This extension built on HILIC's foundation to tackle separations intractable by standard methods, such as isolating phosphopeptides for proteomic analysis.^[8] A pivotal milestone came in 2012 with a comprehensive review by Bogusław Buszewski and Sylwia Noga, which synthesized experimental evidence to characterize HILIC's hybrid mechanism—blending elements of liquid-liquid partitioning, adsorption, and ionic interactions—solidifying its role as a versatile technique for polar compound analysis across fields like metabolomics and pharmaceuticals.^[9]

Overview and basic principles

Hydrophilic interaction chromatography (HILIC) is a liquid chromatography technique that employs polar stationary phases combined with predominantly organic mobile phases, typically containing more than 70% acetonitrile, to retain and separate hydrophilic analytes based on their polarity.^[4] This approach allows for the effective analysis of polar compounds such as peptides, nucleic acids, and carbohydrates that are often poorly retained in other chromatographic modes.^[4] The core principle of retention in HILIC involves the partitioning of analytes between the hydrophobic mobile phase and a water-enriched layer adsorbed onto the polar stationary phase surface.^[4] More hydrophilic analytes preferentially partition into this immobilized water layer, leading to stronger retention, in contrast to the hydrophobic interactions dominant in reversed-phase liquid chromatography.^[10] This mechanism enhances selectivity for polar solutes by leveraging hydrophilic interactions, including hydrogen bonding. HILIC offers several advantages, including improved solubility of polar compounds in the aqueous component of the mobile phase, orthogonality to reversed-phase LC for multidimensional separations, and high compatibility with mass spectrometry due to the high organic content, which minimizes ion suppression and improves sensitivity.^[10] Understanding HILIC requires familiarity with basic chromatography concepts, such as the retention factor k = \frac{t_R - t_0}{t_0}, where t_R is the retention time and t_0 is the void time; in HILIC, higher k values reflect greater partitioning into the polar layer for hydrophilic analytes.^[10]

Comparison to Other Techniques

Reversed-phase liquid chromatography

Reversed-phase liquid chromatography (RPLC) employs non-polar stationary phases, such as octadecylsilane (C18) or octylsilane (C8) bonded to silica particles, paired with mobile phases that are predominantly aqueous and often modified with organic solvents like acetonitrile or methanol.^[11] In this mode, retention is primarily driven by hydrophobic interactions, where non-polar analytes partition into the stationary phase and elute later than polar ones as the organic content in the mobile phase increases.^[11] This makes RPLC the most widely used liquid chromatography technique for separating non-polar to moderately polar compounds in fields like pharmaceutical analysis and small-molecule characterization.^[11] In contrast to hydrophilic interaction chromatography (HILIC), which uses polar stationary phases and high-organic mobile phases (typically >70% organic solvent with water as the strong eluent), RPLC inverts this composition to favor aqueous conditions.^[11] HILIC provides superior retention for highly polar and ionic analytes that exhibit poor or no retention in RPLC due to their limited hydrophobic interactions, often eluting in the void volume.^[11] Conversely, RPLC excels with hydrophobic molecules, offering robust separations where HILIC would yield minimal retention.^[11] Selection between the two depends on analyte polarity: RPLC serves as the default for non-polar species, while HILIC is preferred for polars to avoid co-elution with unretained matrix components.^[11] The orthogonality of RPLC and HILIC enables their integration in two-dimensional liquid chromatography (2D-LC) setups, enhancing separation coverage for complex samples.^[12] In such systems, RPLC in the first dimension separates based on hydrophobicity, while HILIC in the second targets hydrophilicity, as seen in proteomics where HILIC effectively fractionates polar peptides and glycopeptides from tryptic digests.^[12] For instance, this coupling has identified thousands of peptides from biological lysates, improving sensitivity and reducing ion suppression in mass spectrometry detection.^[12] A key limitation of RPLC for polar and ionic compounds arises from their weak interactions with the non-polar stationary phase, necessitating additives like ion-pairing reagents (e.g., trifluoroacetic acid or alkylsulfonates) to form neutral complexes and enhance retention.^[13] However, ion-pairing introduces challenges, including prolonged column equilibration times, gradient incompatibility, and interference with mass spectrometry due to signal suppression or contamination.^[13] HILIC addresses these issues by natively accommodating ionic interactions through its polar phases and water-rich adsorbed layer, avoiding the need for such additives.^[11]

Normal phase chromatography

Normal phase liquid chromatography (NP-LC) employs a polar stationary phase, such as bare silica, and a non-polar mobile phase, typically consisting of hydrocarbons like hexane or mixtures with chloroform, to retain polar analytes through adsorption onto the stationary phase surface.^[14] This mechanism relies on the competition between analytes and mobile phase components for polar sites on the silica, where retention increases with analyte polarity.^[14] However, NP-LC often suffers from irreversible binding of strongly polar compounds due to excessive adsorption, leading to poor recovery and column fouling.^[14] Additionally, the non-aqueous mobile phases result in low solubility for water-soluble polar analytes, limiting its applicability to hydrophilic samples and complicating coupling with techniques like mass spectrometry.^[15] Hydrophilic interaction liquid chromatography (HILIC) represents a modern evolution of normal phase chromatography, retaining the polar stationary phase (e.g., silica or amide-bonded silica) but incorporating water-miscible organic solvents in the mobile phase to establish a dynamic, immobilized aqueous layer on the stationary phase surface.^[16] This layer facilitates partitioning-based retention of polar analytes, supplemented by secondary polar and ionic interactions, while mitigating the dehydration effects prevalent in traditional NP-LC.^[16] By addressing inconsistencies in silanol ionization on bare silica—caused by trace moisture variations in non-polar solvents—HILIC enhances reproducibility, with relative standard deviations for selectivity often below 1%, and yields more symmetrical peak shapes due to reduced tailing from ionic interactions.^[14] Historically, HILIC emerged to overcome NP-LC's limitations in handling polar, ionizable compounds, building on normal phase principles while integrating aqueous components for better control over retention.^[16] NP-LC remains suitable for lipophilic separations involving non-polar solvents and moderately polar analytes, such as lipids or pharmaceuticals in organic matrices, where high organic content is advantageous.^[15] In contrast, HILIC is preferred for aqueous-compatible analyses of highly polar species, like metabolites, peptides, or glycans, offering improved solubility, efficiency, and compatibility with electrospray ionization mass spectrometry.^[14]

Separation Mechanism

Liquid-liquid partitioning

In hydrophilic interaction chromatography (HILIC), the primary retention mechanism involves liquid-liquid partitioning of analytes between the predominantly organic mobile phase and a water-rich stagnant layer formed on the surface of the hydrophilic stationary phase. This partitioning arises because the mobile phase, typically containing more than 70% organic solvent such as acetonitrile, promotes the adsorption of water molecules onto the polar stationary phase, creating a dynamic, enriched aqueous environment that functions as a pseudo-stationary phase.^[17] The water-rich layer typically occupies 4–13% of the stationary phase pore volume and has a thickness on the order of 1–3 nm, depending on the exact mobile phase composition and stationary phase properties; this layer grows thicker as the water proportion in the mobile phase increases but remains thin enough to be considered stagnant relative to the flow velocity. More hydrophilic analytes exhibit stronger retention because they preferentially distribute into this aqueous layer over the organic bulk phase, leading to longer migration times, while less polar compounds elute faster by remaining predominantly in the mobile phase.^[17]^[1] Mathematically, the partitioning process is characterized by the distribution coefficient K = \frac{C_{\mathrm{aq}}}{C_{\mathrm{org}}}, where C_{\mathrm{aq}} represents the analyte concentration in the water-rich layer and C_{\mathrm{org}} in the bulk mobile phase; higher K values indicate greater affinity for the aqueous layer. The observed retention factor k relates to this via k \approx \phi \cdot K, with \phi denoting the phase ratio (the volume of the water layer divided by the mobile phase volume in the column). This model highlights how retention scales with analyte polarity and layer volume, providing a framework for predicting separation behavior under varying conditions.^[18] Studies confirm the dominance of partitioning in typical HILIC operations through observations of small deuterium isotope effects on retention times, which align with bulk liquid-phase equilibria rather than localized surface adsorption, and through solvent composition gradients where increasing the water fraction progressively weakens retention in a manner consistent with shifting the partitioning equilibrium toward the mobile phase. For instance, gradients starting from high organic content (e.g., 95% acetonitrile) show linear decreases in log k with rising water levels, underscoring the role of the dynamic water layer in governing selectivity.^[1]

Adsorption and ionic interactions

In hydrophilic interaction chromatography (HILIC), adsorption serves as a secondary retention mechanism involving direct interactions between polar or charged functional groups on the analyte and the stationary phase surface, such as residual silanol groups on silica or polar ligands on bonded phases. These interactions primarily encompass hydrogen bonding, where analyte hydroxyl or amine groups form bonds with silanol oxygens or amide carbonyls; dipole-dipole forces between polar moieties like carbonyls and nitro groups on the analyte and electronegative sites on the phase; and ionic attractions between oppositely charged species.^[19]^[20] For charged analytes, ionic contributions arise from electrostatic attraction or repulsion, which can significantly alter selectivity beyond neutral partitioning. On bare or underivatized silica phases, deprotonation of silanol groups at higher pH (typically above 5) generates negatively charged sites that enhance retention of cationic analytes through ion-exchange-like attraction, while repelling anions and potentially causing reduced retention or peak distortion.^[21] These effects are particularly pronounced in low-water mobile phases, where the thin water layer on the stationary phase may modulate the accessibility of these ionic sites, but direct surface interactions dominate for ionized species.^[20] HILIC retention exhibits a hybrid nature, with liquid-liquid partitioning into the enriched water layer as the primary mechanism, while adsorption contributes notably, especially on underivatized silica where surface silanols are abundant. Thermodynamic analysis via van't Hoff plots often reveals non-linear relationships between retention and temperature (ln k vs. 1/T), indicative of mixed enthalpic (adsorption-driven) and entropic (partitioning-influenced) processes, contrasting with the linearity expected for pure partitioning.^[22]^[23] Unlike pure partitioning, which yields symmetric peaks for polar neutrals, adsorption introduces differences such as peak tailing for basic compounds due to heterogeneous ionic interactions with ionized silanols, leading to prolonged secondary retention. This tailing can be mitigated through endcapping of the stationary phase with non-polar groups to reduce silanol activity and minimize unwanted electrostatic effects.^[21]^[19]

Stationary Phase

Surface types and chemistry

Hydrophilic interaction chromatography (HILIC) employs a variety of stationary phase surface types, each designed to enhance retention of polar analytes through specific chemical interactions such as hydrogen bonding, dipole-dipole forces, and ionic exchanges. These surfaces are broadly categorized into five main types: neutral polar, ionic, zwitterionic, mixed-mode, and underivatized silica. Neutral polar phases, including amide and cyano functionalities, are widely used for their ability to form hydrogen bonds and polar interactions without introducing charge-based effects.^[14] Amide-bonded phases feature carbonyl and amide groups that act as hydrogen bond acceptors and donors, promoting retention of polar neutral compounds like carbohydrates and metabolites. Cyano phases, with nitrile groups, provide moderate polarity through dipole interactions, offering complementary selectivity to amides for small polar molecules. Ionic stationary phases incorporate cationic or anionic exchangers to facilitate separation of charged species alongside hydrophilic interactions. Cationic exchangers typically include sulfoalkyl chains, such as sulfopropyl or sulfonate groups, which enable cation exchange by attracting positively charged analytes while maintaining polar retention.^[14] Anionic exchangers, conversely, feature quaternary ammonium or amino groups for anion exchange, suitable for negatively charged polar compounds like nucleotides. These phases balance electrostatic and hydrophilic mechanisms to improve selectivity for ionizable analytes. Zwitterionic phases, exemplified by sulfobetaine structures, contain both positive and negative charges in close proximity, such as a quaternary ammonium cation paired with a sulfonate anion, which minimizes secondary ionic interactions and enhances pure hydrophilic retention. This balanced charge distribution reduces analyte adsorption to silanol sites on the underlying silica support, leading to more reproducible separations. Mixed-mode phases combine hydrophilic elements with additional functionalities, such as reversed-phase or ion-exchange groups, allowing tunable interactions for complex samples.^[14] Underivatized silica serves as a foundational phase, relying on native silanol groups for polar adsorption, though it is prone to secondary interactions and often end-capped to mitigate this. Selection of surface types depends on analyte properties: neutral polar phases are preferred for non-ionic polar compounds to avoid charge interference, while ionic phases are chosen for charged species to leverage electrostatic retention. Commercial examples include the Waters BEH Amide column, which uses ethylene-bridged hybrid particles bonded with amide groups for stable hydrogen bonding in polar separations, and the Thermo Scientific Accucore HILIC column, featuring a polar-modified silica surface optimized for basic and neutral polar analytes.^[14] Zwitterionic phases, in particular, provide higher selectivity for carbohydrates due to multiple interaction sites that accommodate hydroxyl groups through combined hydrogen bonding and ionic pairing.

Preparation and properties

Hydrophilic interaction liquid chromatography (HILIC) stationary phases are primarily prepared by chemically modifying silica particles through silane-based bonding to introduce polar functional groups that enhance hydrophilic interactions. This process typically involves the reaction of silanol groups on the silica surface with organosilane reagents, such as chlorosilanes or alkoxysilanes, under anhydrous conditions to form stable Si-O-Si linkages. For instance, amide-functionalized phases, which are widely used for their neutral polarity and stability, are synthesized by first attaching 3-aminopropyltriethoxysilane (APTES) to the silica surface, followed by acylation with agents like acryloyl chloride or succinic anhydride to form the amide moiety.^[24] This stepwise silanization ensures uniform coverage and minimizes unreacted silanols, which could otherwise lead to nonspecific interactions. Alternative supports, such as hybrid organic-inorganic particles, are prepared via ethylene-bridged silsesquioxane sol-gel chemistry, incorporating organic spacers into the silica matrix to improve mechanical strength and pH tolerance. These hybrid particles, exemplified by bridged ethyl hybrid (BEH) materials, exhibit stability across a broad pH range of 1 to 12, enabling robust performance in varied mobile phase conditions.^[25]^[26] Key physical properties of HILIC stationary phases include particle sizes ranging from 1.7 to 5 μm, which balance efficiency and pressure requirements in both HPLC and UHPLC formats. Smaller particles, such as 1.7 μm, enable higher resolution but demand specialized instrumentation, while larger 5 μm particles suit conventional systems with lower backpressures. Pore diameters typically span 90 to 300 Å, with narrower pores (e.g., 90-120 Å) providing higher surface areas for small-molecule separations and wider pores (up to 300 Å) facilitating access for larger biomolecules like peptides and oligonucleotides. To mitigate residual silanol activity, which can cause peak tailing for basic analytes, many phases undergo endcapping with trimethylsilyl groups after primary bonding; this reduces ion-exchange effects while preserving the hydrophilic character essential for HILIC retention.^[27]^[28]^[29] Despite these optimizations, traditional silica-based HILIC phases are susceptible to hydrolytic degradation under aqueous or high-pH conditions, where siloxane bonds hydrolyze, leading to particle dissolution, void formation, and efficiency loss of 30-70% after prolonged exposure. This instability is exacerbated at pH >8 due to increased surface area exposure to water, with half-lives as short as 0.3 hours for some amino-modified silica columns under accelerated testing at pH 11.3 and 70°C. Recent advances from 2023 to 2025 have addressed these limitations through the development of metal-organic frameworks (MOFs) and polymer-based phases; for example, MOF@COF composites offer enhanced durability via covalent linkages and tunable porosity, while amine-modified polymeric monoliths provide hydrolytic resistance without silica degradation. As of 2025, further progress includes molecular dynamics simulations elucidating retention mechanisms and enhanced stationary phases for oligonucleotide analysis.^[26]^[30]^[31]^[32] These innovations extend column lifetimes under demanding conditions, with polymer phases demonstrating minimal efficiency loss over thousands of injections. In terms of performance, well-designed HILIC stationary phases achieve theoretical plate counts exceeding 100,000 plates per meter, with UHPLC-compatible formats reaching up to 30,000–40,000 plates for 15 cm columns due to reduced eddy diffusion and longitudinal broadening. These efficiencies stem from the uniform particle morphology and optimized pore structures that promote rapid mass transfer. Additionally, hybrid and core-shell particles tolerate operational pressures up to 15,000 psi, enabling faster separations with flow rates of 0.5-2 mL/min while maintaining peak integrity.^[33]

Mobile Phase and Additives

Composition and role of water layer

The mobile phase in hydrophilic interaction chromatography (HILIC) is predominantly organic, typically comprising 70–95% acetonitrile as the primary solvent, with 5–30% water to facilitate the formation of a hydrophilic environment on the stationary phase surface. This high organic content contrasts with reversed-phase liquid chromatography and promotes the adsorption of water onto the polar stationary phase. Alternative organic solvents, such as methanol or isopropanol, can replace acetonitrile to modulate selectivity, while aprotic options like tetrahydrofuran (THF) or dioxane are employed for specialized separations requiring distinct solvophobic interactions. A critical feature of HILIC retention is the dynamic adsorption of water molecules from the mobile phase onto the hydrophilic stationary phase, which forms a thin, water-enriched layer—approximately 1–2 nm thick, depending on the organic solvent proportion.^[34] This immobilized water layer acts as a pseudo-stationary phase, enabling analyte partitioning primarily based on hydrophilicity; retention correlates inversely with the octanol-water partition coefficient (log P), favoring more hydrophilic (lower log P) compounds that preferentially enter the layer over the organic bulk phase. The layer's composition and stability are influenced by the mobile phase's water content, with sufficient hydration (at least 3–5% water) essential to maintain the partitioning mechanism without collapsing into normal-phase behavior. The proportion of organic solvent in the mobile phase directly impacts the water layer's thickness and overall retention. Higher acetonitrile concentrations (e.g., 80–95%) shrink the water layer, increasing retention times for polar analytes by enhancing their partitioning drive into the diminishing aqueous domain. Conversely, gradient elution strategies typically start with high organic content (e.g., 90% acetonitrile) and progressively reduce it to lower levels (e.g., 60–70%), promoting the elution of hydrophilic compounds as the water layer expands and the bulk phase becomes more aqueous.^[35] Incorporating alcohols like methanol or isopropanol into the mobile phase (e.g., 10–20% as co-solvents) disrupts hydrogen bonding networks within the water layer, which can fine-tune selectivity for compounds involved in such interactions. This effect is particularly pronounced for glycosylated biomolecules, where alcohol addition reduces retention of glycopeptides by weakening silanol-analyte hydrogen bonds, enabling better resolution of structural isomers.

Buffers and ionic additives

In hydrophilic interaction liquid chromatography (HILIC), buffers and ionic additives are essential for maintaining pH stability, modulating ionic strength, and enhancing separation selectivity of polar analytes. Volatile buffers such as ammonium acetate or ammonium formate, typically at concentrations of 10-100 mM, are commonly employed due to their compatibility with mass spectrometry (MS) detection, where they minimize ion suppression and facilitate electrospray ionization (ESI).^[36] These buffers help form a stable water layer on the stationary phase while shielding electrostatic interactions between analytes and residual silanol groups on silica-based columns. For instance, in the analysis of polar antibiotics like gentamicin, higher buffer concentrations around 80 mM ammonium formate improve peak sharpness and resolution by competing for ionic binding sites, thereby reducing retention times and baseline noise.^[37] Non-volatile buffers, such as phosphate at 20-50 mM, are preferred in applications coupled with ultraviolet (UV) detection, where MS compatibility is not required, as they provide robust pH control and enhance peak shapes for metabolites and peptides.^[36] By suppressing silanol ionization, particularly at mildly acidic pH, these additives mitigate secondary ionic interactions that cause peak tailing, especially for basic compounds, leading to more symmetric elution profiles and improved selectivity.^[27]^[38] Ionic additives like trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA), often at 0.1-1% concentrations, serve as ion-pairing agents to enhance retention and selectivity for basic and charged analytes in HILIC. These fluorinated acids form dynamic ion pairs with positively charged species, reducing peak tailing through electrostatic shielding and promoting better separation of glycopeptides or nucleotides.^[39] However, their use requires careful consideration of volatility for ESI-MS interfacing, as TFA can cause significant ion suppression, though alternatives like HFBA offer improved MS performance in some cases. Recent advancements, including ion-pair HILIC methods from 2023, have applied these additives for profiling impurities in therapeutic phosphorothioate oligonucleotides, achieving baseline resolution of n-1 deletions and phosphodiester variants without excessive organic solvent demands.^[40]^[41]

Operational Parameters

pH selection

In hydrophilic interaction chromatography (HILIC), pH selection is crucial for modulating the ionization states of analytes and the charge on the stationary phase surface, thereby influencing retention and selectivity.^[35] For silica-based stationary phases, the typical operational pH range is 3–7 to prevent silica dissolution at higher pH values, where silanol groups deprotonate and increase solubility; low pH below 3 can protonate silanols, reducing negative surface charge and minimizing unwanted ionic interactions with cationic analytes.^[42] Hybrid or organo-silica phases extend this range to pH 2–12, offering greater stability for broader analyte compatibility, while polymeric phases like those with zwitterionic functionalities maintain performance across pH 3–8 with reduced secondary electrostatic effects.^[43] Optimal pH for ionic surfaces often falls between 3.5 and 8.5, where balanced surface charge enhances selectivity without excessive repulsion or attraction.^[35] The pH profoundly affects analyte behavior through protonation or deprotonation, altering their net charge and hydrophilicity in the aqueous-enriched layer of the stationary phase. For ionizable compounds such as peptides or nucleotides, retention increases when pH shifts the analyte away from its isoelectric point, promoting charged forms that interact more strongly via partitioning and adsorption; for instance, operating 1–2 pH units above the pKa of acidic analytes enhances deprotonation, promoting charged forms that increase retention and improve peak shapes.^[42] In contrast, low pH protonates basic analytes, increasing retention through enhanced ionic interactions with negatively charged silanols.^[43] This pH-dependent charge modulation is particularly vital for polar biomolecules, where acidic conditions (pH 2.8–4.8) favor separation of bases by suppressing silanol ionization, while higher pH (up to 9) suits acids by promoting surface deprotonation.^[35] Practical pH scouting involves testing at least three values spanning the analyte's pKa range to optimize selectivity, often using gradients from low to high pH to identify conditions that minimize tailing and maximize resolution.^[42] Buffers such as ammonium formate or acetate are selected to maintain stable pH within their effective ranges (e.g., 2.8–5.8 for formate) without introducing ions that suppress mass spectrometry signals in coupled analyses.^[44] For silica phases, avoiding pH >6 during extended runs prevents degradation, while hybrid materials allow scouting up to pH 11 for robust method development.^[42]

Elution modes and optimization

In hydrophilic interaction chromatography (HILIC), elution modes are selected based on sample complexity to achieve efficient separation of polar analytes. Isocratic elution, where the mobile phase composition remains constant, is suitable for simple mixtures with analytes exhibiting similar retention factors, typically starting at high organic solvent percentages such as 70-95% acetonitrile (ACN) to maintain the water-enriched layer on the stationary phase.^[35] For more complex samples, gradient elution is preferred, involving a progressive decrease in organic solvent content (e.g., from 95% to 70% ACN over 10-20 minutes) to elute less polar compounds first and highly polar analytes last, enhancing resolution while minimizing analysis time.^[35] This reversed gradient compared to reversed-phase liquid chromatography (RP-LC) exploits the partitioning mechanism dominant in HILIC, where retention decreases as water content increases.^[2] Optimization of operational parameters is crucial for robust HILIC methods, focusing on flow rate and column temperature to balance efficiency, pressure, and selectivity. Flow rates typically range from 0.2 to 2 mL/min, adjusted according to column dimensions (e.g., 0.4 mL/min for a 2.1 mm × 100 mm column), to optimize linear velocity and minimize band broadening while managing the higher viscosity of organic-rich mobile phases.^[35]^[27] Column temperatures of 20-50°C are commonly employed, as moderate heating reduces mobile phase viscosity, enhances mass transfer, and slightly decreases retention, thereby improving peak efficiency without compromising stationary phase stability.^[45]^[46] Troubleshooting common issues like peak tailing and secondary interactions ensures method reliability in HILIC. Peak tailing often arises from insufficient buffering, leading to analyte interactions with residual silanol groups or metal impurities; this can be mitigated by increasing buffer concentration (e.g., 10-50 mM ammonium acetate) to promote hydrogen bonding and shield active sites, or by incorporating additives like 0.1% formic acid.^[47]^[35] Secondary interactions, such as ionic or electrostatic effects, are minimized by selecting appropriate stationary phases (e.g., zwitterionic or amide-bonded silica) that match analyte polarity and reduce non-specific adsorption.^[47] Retention modeling aids in systematic optimization of gradient elution profiles for HILIC methods. The linear solvent strength (LSS) theory, adapted from RP-LC, models retention as a linear function of mobile phase composition, using parameters like slope (S) and intercept (log k_w) derived from scouting gradients to predict elution times and optimize conditions for complex mixtures.^[48] While LSS provides a straightforward approach, more advanced models like adsorption-based ones may offer higher accuracy (e.g., <2% prediction error) for polar compounds on diol phases, enabling algorithmic method development with minimal experiments.^[48]

Applications

Separation of polar biomolecules

Hydrophilic interaction chromatography (HILIC) plays a crucial role in the separation of polar biomolecules, particularly in glycosylation analysis of glycoproteins, where it enables the detailed profiling of N-glycans released from therapeutic proteins. This technique leverages polar stationary phases, such as amide-bonded silica, to retain hydrophilic glycans through partitioning into a water-enriched layer and hydrogen bonding interactions, facilitating the resolution of complex glycan structures that are poorly retained in reversed-phase liquid chromatography (RPLC).^[49] In proteomics, HILIC supports peptide mapping by providing orthogonal selectivity to RPLC, allowing the separation of polar and hydrophilic peptides that co-elute or exhibit low retention in hydrophobic systems.^[50] Additionally, HILIC is widely applied to the separation of nucleotides and carbohydrates, where it effectively distinguishes isomers and charged species based on their polarity and ionic interactions.^[51] A prominent example is the retention of sialylated glycans on amide phases, which enhances the separation of acidic N-glycans differing in sialic acid linkages (α2,3- vs. α2,6-linked), critical for assessing glycan heterogeneity in biologics like monoclonal antibodies.^[52] This capability is essential for quality control in biopharmaceutical production, where HILIC-based methods quantify glycan variants to ensure product consistency and monitor post-translational modifications that impact efficacy and immunogenicity.^[53] For instance, in glycoprotein analysis, HILIC resolves sialylated structures from neutral glycans, providing insights into site-specific glycosylation patterns that influence protein folding and biological activity.^[54] Compared to RPLC, HILIC offers superior orthogonality for polar peptides, enabling better resolution of hydrophilic sequences in tryptic digests that are often unretained or poorly separated in reversed-phase conditions.^[55] Furthermore, HILIC demonstrates higher loading capacity at preparative scales for polar biomolecules, attributed to the organic-rich mobile phase that maintains solubility and minimizes band broadening during large-scale purifications of carbohydrates and nucleotides.^[1] This advantage supports efficient isolation of polar compounds for downstream applications in drug development and biochemical research. HILIC also improves mass spectrometry sensitivity for these analytes through enhanced ionization in organic solvents, though detailed workflows are beyond this scope.^[36] Early applications in the 1990s established HILIC's utility in the separation of sugars, with pioneering work demonstrating baseline resolution of complex carbohydrates like oligosaccharides on polyhydroxy stationary phases, differing by linkage position and residue composition.^[51] By 2012, expansions to nucleosides highlighted HILIC's expanded role, as shown in methods simultaneously separating 16 nucleobases and nucleosides with high throughput and specificity using zwitterionic phases.^[56] These case studies underscore HILIC's evolution as a robust tool for polar biomolecule analysis, prioritizing selectivity for isomeric and charged species in biological matrices.

Integration with mass spectrometry and recent uses

Hydrophilic interaction liquid chromatography (HILIC) exhibits strong compatibility with mass spectrometry (MS), particularly electrospray ionization (ESI)-MS, due to its mobile phases containing high proportions of organic solvents like acetonitrile, which facilitate efficient desolvation and reduce ion suppression compared to reversed-phase liquid chromatography (RPLC).^[57]^[58] This results in an average sensitivity improvement of approximately 10-fold for polar analytes, with gains up to 100-fold observed for certain weak bases, enabling lower limits of detection in complex matrices.^[57]^[58] To maintain MS compatibility, volatile additives such as ammonium acetate or formate are essential in HILIC mobile phases, minimizing adduct formation and source contamination while supporting stable ionization.^[2] Common workflows leverage HILIC-MS/MS for targeted and untargeted metabolomics, particularly for profiling polar metabolites in biological fluids like human plasma, where it achieves comprehensive coverage of compounds such as amino acids, nucleotides, and organic acids with high selectivity.^[59] For instance, HILIC coupled with Fourier transform MS has been applied to simultaneous extraction and analysis of polar metabolites and lipids from plasma, enhancing throughput and reproducibility in clinical studies.^[59] In proteomics, ultra-high-performance liquid chromatography (UHPLC)-HILIC enables high-throughput separations, often integrated with MS for lipidomics or glycomics, supporting the analysis of thousands of features per run in vacuum-jacketed systems to minimize band broadening.^[60] Recent applications from 2023 to 2025 highlight HILIC's expanding role in biopharmaceutical analysis. A 2024 review emphasizes HILIC-MS for impurity profiling of therapeutic oligonucleotides, such as phosphorothioate-modified antisense strands, offering orthogonal selectivity to ion-pair RPLC and improved resolution of diastereomers and metabolites in serum, with miniaturization boosting MS sensitivity by over 3-fold.^[20] In 2023, HILIC-MS/MS methods advanced quantitative plasma analysis of pharmaceuticals, including caffeine and its metabolites in premature infants, achieving rapid, sensitive detection with limits of quantification below 1 ng/mL for pharmacokinetic monitoring.^[61] For natural product isolation, a 2025 study utilized HILIC in traditional Chinese medicine analysis to separate polar bioactive components like flavonoids and alkaloids from complex extracts, promoting efficient purification with MS-guided fractionation.^[62] Emerging strategies include two-dimensional (2D) HILIC-RPLC setups for shotgun proteomics, where HILIC prefractionates hydrophilic peptides orthogonally to RPLC, increasing the number of identified proteins by 17% to 34% in low-flow configurations coupled to high-resolution MS, ideal for deep sequencing of complex samples like cell lysates.^[63]

Variants

Electrostatic repulsion hydrophilic interaction chromatography (ERLIC)

Electrostatic repulsion hydrophilic interaction chromatography (ERLIC) is a specialized variant of hydrophilic interaction chromatography (HILIC) that integrates electrostatic repulsion mechanisms to enhance selectivity for charged analytes in complex mixtures. Introduced by Andrew J. Alpert in 2008, ERLIC employs strong cation or anion exchange stationary phases, such as Polysulfoethyl A for cations or PolyWAX LP for anions, combined with high-organic mobile phases containing buffers to exploit both hydrophilic partitioning and charge-based repulsion.^[8] This approach allows for isocratic separations of polar, charged solutes like peptides and nucleotides, which are often challenging in conventional HILIC due to poor resolution from co-eluting interferents.^[8] The core mechanism of ERLIC relies on electrostatic repulsion between the charged stationary phase and similarly charged analytes or interferents, which reduces retention of highly charged species while promoting hydrophilic interactions for less charged or neutral polars. For instance, in phosphopeptide analysis, a positively charged anion exchanger repels non-phosphorylated peptides (which carry positive charges at low pH) but retains negatively charged phosphopeptides through both electrostatic attraction and hydrophilic partitioning in high acetonitrile (ACN) content (>60%).^[8] Mobile phases typically consist of 60-80% ACN with ammonium or phosphate buffers at 10-80 mM concentrations and pH 2-6, enabling the shielding of repulsive forces at appropriate ionic strengths to fine-tune selectivity; for example, 20 mM ammonium formate at pH 3 has been used to exclude common phosphate ions from tryptic peptides, thereby improving resolution of post-translationally modified species.^[8] This dual-mode retention contrasts with standard HILIC by actively excluding like-charged interferents, such as basic peptides in cation-exchange ERLIC setups.^[8] ERLIC finds key applications in proteomics, particularly for peptide mapping, where it facilitates the characterization of therapeutic antibodies like denosumab by separating glycoforms and modified peptides with high orthogonality to reversed-phase LC.^[64] It is also widely used for glycopeptide enrichment, leveraging anion-exchange phases to attract sialylated or negatively charged glycans while repelling unmodified peptides, as demonstrated in analyses of complex tryptic digests from cell lines.^[65] In biologics quality control (QC), ERLIC supports the isolation of phosphopeptides for site-specific modification analysis, enabling robust detection in monoclonal antibody digests that standard methods overlook due to charge heterogeneity.^[8] Additionally, it separates neutral polar compounds from ionic interferents in nucleotide mixtures, aiding downstream mass spectrometry workflows. Recent applications include quantitative analysis of polyamines (as of 2024) and improved separation of phosphorylated peptides in nanoscale LC-MS (as of 2025).^[66]^[67] A primary advantage of ERLIC is its orthogonality to conventional HILIC and reversed-phase chromatography, providing complementary selectivity that enhances multidimensional separations for post-translational modifications like phosphorylation and glycosylation.^[8] This results in superior resolution for highly charged biomolecules, such as multi-phosphorylated peptides, which elute isocratically without the peak broadening seen in gradient-based HILIC, and supports selective enrichment that improves detection sensitivity in low-abundance analytes in mass spectrometry-coupled assays.^[8]

Cationic and anionic enhanced HILIC (eHILIC)

Enhanced hydrophilic interaction liquid chromatography (eHILIC), also known as ERLIC, utilizes charge-specific stationary phases to modulate retention of charged analytes through electrostatic repulsion while preserving hydrophilic interactions.^[68] This approach employs oppositely charged surfaces relative to target interferents to minimize excessive retention of strongly interacting charged groups, thereby enhancing selectivity for neutral or oppositely charged species in complex mixtures. In cationic eHILIC, negatively charged stationary phases, such as those featuring sulfonate groups (e.g., PolySulfoethyl A), are utilized to repel anionic species like phosphate groups, thereby reducing their over-retention and allowing improved separation of basic or neutral polar compounds.^[8] This repulsion counteracts the strong hydrophilic affinity of anions, enabling isocratic or shallow gradient elution where retention is primarily governed by partitioning and hydrogen bonding for less charged analytes.^[68] Conversely, anionic eHILIC employs positively charged stationary phases, such as quaternary ammonium-functionalized materials (e.g., PolyWAX LP), to repel cationic analytes, which minimizes their interference and facilitates enhanced resolution of acidic or neutral polar species.^[8] By antagonizing cationic retention, this variant promotes elution based on hydrophilic mechanisms for anions, improving orthogonality in multidimensional separations. Operationally, eHILIC modes typically use low ionic strength buffers, such as 12.5 mM acetate or formate at pH 2–4, combined with high organic solvent content (70–80% acetonitrile) to maintain HILIC conditions while enabling MS compatibility through volatile additives.^[68] These conditions support gradient elution by increasing aqueous content, with flow rates of 0.5–1 mL/min on columns of 100–200 mm length and 5 μm particle size, yielding high peak capacities in optimized setups.^[8] Key applications of cationic eHILIC include phosphopeptide enrichment and analysis, where repulsion of phosphate anions from negatively charged surfaces allows selective fractionation of singly to multiply phosphorylated peptides from tryptic digests, enabling effective identification of phosphorylation sites in cell lysates with good recovery. For anionic eHILIC, separations of sialic acid-containing glycans and glycopeptides benefit from cationic repulsion, enabling baseline resolution based on sialylation degree and integration with electrospray ionization mass spectrometry (ESI-MS) for enhanced signal intensity due to low-water mobile phases. Overall, these variants improve peak capacity and reproducibility in MS-coupled workflows, particularly for post-translational modification profiling in proteomics.^[8]

References

[1]
Hydrophilic interaction liquid chromatography (HILIC) - NIH
Aug 31, 2011 · This paper covers fundamental developments in hydrophilic interaction liquid chromatography. The objective of the present work is to review ...
[2]
Hydrophilic-Interaction Chromatography: An Update
This review addresses recent ideas on the separation mechanism of HILIC, the advantages and disadvantages of the technique, and new applications.
[3]
https://doi.org/10.1016/S0021-9673(00)96972-3
[4]
Hydrophilic-interaction chromatography for the separation ... - PubMed
J Chromatogr. 1990 Jan 19:499:177-96. doi: 10.1016/s0021-9673(00)96972-3. Author. A J Alpert. Affiliation. 1 PolyLC, Columbia, MD 21045. PMID: 2324207; DOI ...Missing: Andrew original paper
[5]
Hydrophilic interaction chromatography (HILIC) in the analysis of ...
... HILIC, which overcomes the drawbacks of the poor solubility often encountered in normal phase LC. HILIC is suitable for analyzing compounds in complex ...
[6]
[PDF] HYDROPHILIC-INTERACTION CHROMATOGRAPHY FOR ... - PolyLC
The data in this paper suggest that the mechanism of HILIC involves partition- ing between the (hydrophobic) mobile phase and a layer of mobile phase enriched ...Missing: original | Show results with:original
[7]
Electrostatic repulsion hydrophilic interaction chromatography for ...
ERLIC affords convenient separations of highly charged peptides that cannot readily be resolved by other means. In addition, phosphopeptides can be isolated ...
[8]
Electrostatic Repulsion Hydrophilic Interaction Chromatography for ...
Since 1990, HILIC has been applied to a wide variety of peptides, 5-10 complex carbohydrates, 11 and some proteins 12-15 and is increasingly being applied to ...Missing: original | Show results with:original
[9]
Hydrophilic interaction liquid chromatography (HILIC) - PubMed
The purpose of this work was to review the options for the characterization of HILIC stationary phases and their applications for separations of polar compounds ...
[10]
https://doi.org/10.1007/s00216-011-5308-5
[11]
Reverse-Phase Liquid Chromatography vs. Hydrophilic Interaction ...
Oct 15, 2025 · Reverse-phase chromatography is the most commonly used mode of liquid chromatography due to its broad applicability and reproducibility. In ...
[12]
https://doi.org/10.1007/s00216-010-3991-2
[13]
Ion Pair Chromatography – How IPC Works, Strengths, Limitations ...
Mar 12, 2024 · Chromatography of charged analytes is often challenging as they are not retained effectively on the commonly used reversed phase columns and ...
[14]
https://doi.org/10.1016/j.aca.2011.02.047
[15]
Hydrophilic Interaction Chromatography - ScienceDirect.com
Adsorption chromatography or normal-phase chromatography (NPC) uses a polar adsorbent (silica) and a nonpolar mobile phase such as petroleum ether and ...
[16]
https://doi.org/10.1016/j.aca.2019.09.010
[17]
https://doi.org/10.1016/j.chroma.2008.03.049
[18]
https://doi.org/10.3390/molecules28186459
[19]
https://doi.org/10.1016/j.aca.2017.01.060
[20]
https://doi.org/10.1016/j.trac.2024.117758
[21]
https://doi.org/10.1016/j.chroma.2010.03.011
[22]
https://doi.org/10.1016/j.aca.2021.339025
[23]
https://doi.org/10.1021/acsomega.9b02689
[24]
A novel amide stationary phase for hydrophilic interaction liquid ...
A novel amide-based HILIC SP was prepared via clicking asparagine on azide-silica gel. The SP has very simple structure and is easy to be prepared. The SP-based ...Missing: silane | Show results with:silane
[25]
https://www.waters.com/nextgen/us/en/library/application-notes/2021/introducing-atlantis-beh-z-hilic-a-zwitterionic-stationary-phase-based-on-hybrid-organic-inorganic-particles.html
[26]
Further Evaluation of the Base Stability of Hydrophilic Interaction ...
Mar 6, 2023 · The results show a clear trend of stability increasing as the surface area decreases. Several commercially available HILIC columns that are ...
[27]
[PDF] HILIC separations - Thermo Fisher Scientific
For most columns the test compound has little retention. (mean k of 0.2) when the acetonitrile content is less than. 60%. Acclaim HILIC-10, Acclaim Mixed Mode ...
[28]
Characterization of a highly stable zwitterionic hydrophilic ...
Dec 22, 2021 · The ethylene-bridged hybrid particles used for the BEH Z-HILIC stationary phase have an average pore diameter of 95 Å, smaller than the 130 Å ...
[29]
Advances in the understanding of stationary phases for HILIC
Apparently, the surface silanols on silica are sufficient to create a surface water layer and the material can provide "HILIC retention." It is well known that ...
[30]
Construction of MOFs@COFs composite material as stationary ...
A new stationary phase (SP) with multiple retention mechanisms was developed. The SP showed excellent separation performance for various compounds.Missing: advances | Show results with:advances
[31]
Ultrahigh-Pressure LC in Pharmaceutical Analysis: Performance ...
In this article, UHPLC refers to with the use of system pressures of ~15,000 psi rather than the original term used to describe research work using capillary ...
[32]
Preparation of High-Efficiency HILIC Capillary Columns Utilizing ...
Perhaps the highest performance by HILIC used a 4 m long monolithic column to achieve a plate height minimum (Hmin) of 12 μm, efficiency of 300,000 theoretical ...Missing: metrics | Show results with:metrics
[33]
http://www.chromforum.org/viewtopic.php?t=12873
[34]
Hydrophilic interaction chromatography – mass spectrometry for ...
Other major benefits revolve around the lower mobile phase viscosity (2–3 times lower than RPLC), which results in lower back pressure and allows for the use of ...
[35]
An Ultra Performance Liquid Chromatography Mass Spectrometry Method for the Analysis of Non-derivatized Gentamicin Components
### Summary of Buffers and Ionic Additives in HILIC for Gentamicin Analysis
[36]
Study of retention and peak shape in hydrophilic interaction ...
Hydrophilic interaction chromatography (HILIC) has over the last 10 years become accepted as a complimentary LC separation mechanism to reversed-phase (RP) ...
[37]
Influence of ion-pairing reagents on the separation of intact ...
Jan 11, 2023 · In this paper, we report on a study aimed at minimizing the presence and the effects of TFA in HILIC-MS of intact (glyco-) proteins. ... and one ...
[38]
Ion-Pairing Hydrophilic Interaction Chromatography for Impurity ...
Jul 17, 2025 · Ion-Pairing Hydrophilic Interaction Chromatography for Impurity Profiling of Therapeutic Phosphorothioated Oligonucleotides. Click to copy ...Experimental Section · Hilic And Ip-Hilic · Ip-Hilic: Effect Of...
[39]
Ion-pair reversed-phase and hydrophilic interaction chromatography ...
Dec 6, 2023 · We recommend a 20–30 mM ammonium acetate buffer concentration for HILIC oligonucleotide separations. Further minor improvements were ...Ion-Pair Reversed-Phase And... · 3. Results And Discussion · 3.2. Hilic Application To Ps...<|control11|><|separator|>
[40]
None
### Summary of pH Selection in HILIC (Hydrophilic Interaction Chromatography)
[41]
[PDF] Technical Note - YMC America
Applications at extreme pH values put chemical stress on the stationary phase which can lead to faster degradation and loss of column performance. At pH values ...
[42]
Hydrophilic Interaction Liquid Chromatography
### Summary of pH Dependence in HILIC, Stationary Phases Stability, and Analyte Charge Effects
[43]
Buffer Choices for HILIC
### Summary of Buffer and pH Choices for HILIC
[44]
Column temperature in HILIC - Separation Science
In HILIC, column temperature is usually set between 10 and 60°C. Higher temperature values can be used depending on both stationary phase stability and analyte ...
[45]
[PDF] Column Temperature in HILIC - AFIN-TS
During the development of a HILIC method the chromatographic parameters should be optimized in the following order: (1) organic content in the mobile phase, (2).
[46]
HILIC Troubleshooting | Thermo Fisher Scientific - US
Insufficient buffer concentration can result in increased secondary interactions that cause peak tailing. Increasing the buffer concentration can enhance ...
[47]
Applicability of retention modelling in hydrophilic-interaction liquid ...
Dec 29, 2017 · The model most commonly used in reversed-phase LC (RPLC) involves a linear relationship between the logarithm of the retention factor (k) and ...
[48]
N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid ...
Sep 25, 2021 · We present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with ...
[49]
Peptide separation by Hydrophilic-Interaction Chromatography
HILIC has proven quite useful for the analysis and purification of peptides, as well as the separation of polar compounds such as sugars, oligosaccharides and ...
[50]
Hydrophilic-interaction chromatography of complex carbohydrates
HILIC readily resolved carbohydrates differing in residue composition and position of linkage. Complex carbohydrate mixtures could be resolved using ...
[51]
HILIC–MRM–MS for Linkage-Specific Separation of Sialylated ...
Mar 6, 2020 · The method shows the feasibility of the use of a HILIC stationary phase for the separation of isomeric glycopeptides to detect specific glyco-proteoforms.
[52]
Characterization of Monoclonal Antibody Glycan Heterogeneity ...
Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) is an invaluable technology for the characterization of protein glycosylation.
[53]
Protein glycosylation analysis by HILIC-LC-MS of Proteinase K ...
This approach appears to be a valuable technique for in-depth analysis of the site-specific N-glycosylation and O-glycosylation of individual glycoproteins.
[54]
Evaluating the potential of hydrophilic interaction liquid ...
Dec 6, 2024 · This study presents a comprehensive and systematic approach aimed at developing a specific HILIC method for collagen peptide mapping, while also ...
[55]
Hydrophilic interaction liquid chromatography (HILIC)—a powerful ...
Aug 31, 2011 · This paper covers fundamental developments in hydrophilic interaction liquid chromatography. The objective of the present work is to review ...
[56]
What You Need to Know About HILIC | LCGC International
Below a proportion of 60% acetonitrile, the water layer at the surface of ... You mentioned that at least 3% water is required for HILIC. Why isn't it ...
[57]
https://www.chromatographyonline.com/view/what-you-need-know-about-hilic
[58]
Concurrent profiling of polar metabolites and lipids in human plasma ...
Nov 7, 2016 · The HILIC-FTMS method was developed using mixed standards of polar metabolites and lipids, the separation efficiency of which is better in HILIC ...
[59]
High Throughput UHPLC-MS-Based Lipidomics Using Vacuum ...
Dec 30, 2021 · In this study vacuum jacketed columns (VJC), positioned at the source of the mass spectrometer, were applied to the lipidomic analysis of plasma extracts.
[60]
Determination of caffeine, paraxanthine, theophylline and ... - PubMed
A HILIC-MS/MS method was developed and validated to determine caffeine and its major metabolites in plasma of premature infants.Missing: quantitative | Show results with:quantitative<|separator|>
[61]
Recent progress in the application of hydrophilic interaction liquid ...
Oct 1, 2025 · This review summarizes the major types of HILIC stationary phases and their recent applications in the separation of polar components in TCM.
[62]
Antibody characterization using novel ERLIC-MS/MS-based peptide ...
The ERLIC-MS/MS method can enhance biologic therapeutics analysis with more reliability and confidence for bottom-up peptide mapping-based characterization.Missing: QC | Show results with:QC
[63]
Comparison of Enrichment Methods for Intact N- and O-Linked ...
Oct 10, 2017 · We studied three different enrichment methods for enrichment of N- and O-linked glycopeptides. It was found that removal of N-glycans prior to enrichment of O- ...Missing: biologics | Show results with:biologics
[64]
Electrostatic Repulsion-Hydrophilic Interaction Chromatography ...
ERLIC is a subset of HILIC using charged interactions to reduce retention of highly charged groups, allowing isocratic separation of less charged compounds.