Sil-DBO-PS: A Zwitterionic Phase for Enhanced HILIC of Polar Compounds

A joint study conducted by the Henan University of Technology (Zhengzhou, China) and the Food Laboratory of Zhongyuan (Henan Province, China) developed and evaluated what the researchers deemed a novel zwitterionic stationary phase, Sil-DBO-PS, for hydrophilic interaction liquid chromatography (HILIC). Synthesized by attaching a zwitterion, composed of a 1,4-diazabicyclo[2.2.2]octane quaternary ammonium cation and a sulfonate anion, to a silica surface, the performance of this phase was assessed under various eluent conditions (such as water content, salt concentration, and pH) using Van’t Hoff plots, Tanaka HILIC tests, and linear solvation energy relationships (LSER) to explore its retention mechanisms. These studies indicated the presence of multiple interactions—hydrophilic partitioning, hydrogen bonding, and electrostatic forces—contributing to effective analyte separation.Compared to two commercial columns, the researchers reported that Sil-DBO-PS demonstrated superior efficiency and selectivity in separating polar compounds such as nucleosides, vitamins, and benzoic acids, and showed good reproducibility across repeated tests. Overall, this novel zwitterionic stationary phase significantly improves HILIC performance for polar and complex samples. LCGC International spoke to Ashraf Ali of the Henan University of Technology about this research and their findings.

Briefly define hydrophilic interaction liquid chromatography (HILIC) and explain how it differs from reversed-phase liquid chromatography (RPLC).
HILIC) is a liquid chromatographic technique designed to separate polar and hydrophilic compounds using a hydrophilic stationary phase, such as bare silica or modified silica with amino, cyano, or zwitterionic functional groups. The mobile phase typically consists of a water-miscible organic solvent (e.g., acetonitrile) with a small amount of aqueous buffer. Retention in HILIC is driven by partitioning and hydrogen bonding, with analytes eluting in order of increasing polarity. In contrast, RPLC employs a hydrophobic stationary phase (such as C18 or C8) and a mobile phase rich in water with an organic modifier. While RPLC retains nonpolar compounds more strongly, HILIC excels at retaining polar analytes that would otherwise elute too quickly in RPLC. As a result, HILIC is particularly useful for analyzing highly polar molecules, such as sugars, amino acids, and metabolites, which are challenging to separate using traditional reversed-phase methods. The choice between HILIC and RPC depends on the analyte's polarity, with HILIC offering superior retention and resolution for hydrophilic substances.

Why is there still a need for new HILIC stationary phases despite existing technology?
Despite the wide variety of commercially available HILIC stationary phases, ongoing research continues to develop new and improved materials to address persistent limitations and emerging analytical needs. One key driver is the challenge of separating complex mixtures of polar compounds, where existing stationary phases often lack sufficient selectivity for structurally similar analytes. Additionally, certain highly hydrophilic molecules such as small sugars, organic acids, and nucleotides still exhibit poor retention on conventional HILIC columns, prompting the design of phases with stronger hydrophilic interactions or mixed-mode capabilities. Reproducibility is another concern, as silica-based materials can suffer from batch-to-batch variability in ligand bonding and surface properties. Stability is another concern, since many HILIC phases degrade under highly aqueous conditions or extreme pH, restricting method flexibility. These limitations spur the development of advanced stationary phases that combine HILIC with ion-exchange mechanisms. Furthermore, mass spectrometry compatibility remains a critical consideration, as some traditional phases generate high backpressure or exhibit column bleed, interfering with sensitive detection. All these factors push for novel HILIC stationary phases reflects the need for enhanced performance in retention, reproducibility, stability, and selectivity ensuring that modern laboratories can meet increasingly complex separation challenges.

What are the main types of HILIC stationary phases based on their functional group charge states?
HILIC stationary phases can be systematically categorized based on the charge characteristics of their functional groups, each offering distinct retention mechanisms and selectivity profiles:

1. Neutral stationary phases (bare silica, amide, cyano, diol) operate primarily through hydrogen bonding and polar interactions, making them ideal for separating neutral and moderately polar compounds like carbohydrates and glycosides. These phases exhibit minimal ionic interactions, relying instead on hydrophilic partitioning.
2. Cationic stationary phases (amino, triazole, zwitterionic sulfobetaine) feature positively charged functional groups that strongly interact with anionic analytes through electrostatic attraction. This makes them particularly effective for retaining acidic compounds such as organic acids, nucleotides, and phosphorylated metabolites.
3. Anionic stationary phases (sulfonic acid, carboxylate-modified silica) contain negatively charged groups that preferentially retain cationic species. These columns excel in separating basic compounds including peptides and aminoglycosides through a combination of hydrophilic and ionic interactions.
4. Zwitterionic stationary phases (sulfobetaine, phosphorylcholine) incorporate both positive and negative charges within their structure, creating a balanced electrostatic environment. This unique design enables mixed-mode retention mechanisms, making them versatile for analyzing diverse polar compounds ranging from amino acids to small polar metabolites. The selection of an appropriate stationary phase depends critically on the analyte's charge state and polarity. Neutral phases offer straightforward separation of non-ionic compounds, while charged phases provide enhanced selectivity for ionic species. Zwitterionic phases strike an effective compromise, delivering robust retention across a wide range of polar analytes while minimizing strong ionic biases. This systematic classification enables researchers to rationally select stationary phases based on the specific separation challenges posed by their target analytes.

What advantages do zwitterionic stationary phases offer over purely neutral or ionic stationary phases?
Zwitterionic stationary phases represent a significant advancement in hydrophilic interaction chromatography, offering distinct advantages that address the limitations of traditional neutral or ionic phases. Their unique design, incorporating both positively and negatively charged functional groups within a single stationary phase, creates a versatile separation environment that combines hydrophilic partitioning with balanced electrostatic interactions. This dual-character nature enables superior retention of diverse analytes from neutral polar compounds like carbohydrates to charged species including organic acids, basic metabolites, and zwitterionic molecules such as amino acids and peptides. Unlike conventional phases that often suffer from peak tailing or irreversible adsorption of strongly ionic compounds, zwitterionic stationary phases demonstrate better peak shape consistency across different analyte classes. Their robust construction provides wider pH stability (typically 3-8) compared to amino phases or bare silica, along with reduced batch-to-batch variability. For mass spectrometry applications, these zwitterionic phases offer advantages through their low chemical background and minimal stationary phase bleed, while maintaining stable performance even in high organic mobile phases. The practical benefits extend to simplified method development, as a single zwitterionic column can often replace multiple specialized columns for analyzing complex samples. Therefore, zwitterionic stationary phases are becoming the preferred choice for challenging separations in modern pharmaceutical research, clinical analysis, and omics studies.

What is the purpose of using DABCO (1,4-diazabicyclo[2.2.2]octane) in your zwitterionic ligand structure?
The incorporation of DABCO (1,4-diazabicyclo[2.2.2]octane) into zwitterionic stationary phases serves multiple strategic purposes in HILIC separations. Its rigid bicyclic cage structure provides an ideal framework for creating a balanced, hydrophilic surface that maintains a stable water-rich layer essential for HILIC retention mechanisms. When paired with anionic groups, DABCO forms a true zwitterionic interface that exhibits remarkably neutral electrostatic behavior - neither strongly cationic nor anionic - enabling unbiased retention of both acidic and basic analytes. This unique architecture offers several practical advantages. The sterically hindered tertiary amine structure confers exceptional chemical stability, resisting degradation under the low-pH or high-organic conditions common in HILIC applications. Simultaneously, the structural rigidity minimizes unwanted hydrophobic interactions that could compromise HILIC performance. The pH-responsive nature of DABCO (pKa ~8.8) adds another dimension of control, allowing adjustment of electrostatic contributions simply by modifying mobile phase pH. These properties translate directly to improved chromatographic performance i.e., sharper peaks for ionizable compounds, extended column lifetime, and flexible method development capabilities. For challenging applications like metabolomics or pharmaceutical analysis, DABCO-based zwitterionic phases provide superior retention of diverse polar analytes while maintaining the peak symmetry and reproducibility that are hallmarks of high-quality separations. The combination of robust physical properties and tunable chromatographic behaviour makes DABCO an exceptionally versatile building block for advanced HILIC stationary phases.

How was the Sil-DBO-PS stationary phase fabricated from the pre-synthesized ligand?|
The Sil-DBO-PS stationary phase was fabricated through a multi-step process that involved synthesis of a zwitterionic ligand (DBO-PS) followed by its grafting onto silica particles.

1. Synthesis of the zwitterionic ligand (DBO-PS): DABCO (1,4-diazabicyclo [2.2.2] octane)was dispersed in acetonitrile (ACN), followed by the addition of 1,3-propanesulfone (PS). The mixture was refluxed for 12 h to form the zwitterion DBO-PS, which combines a DABCO-derived quaternary ammonium cation and a sulfonate anion. The product was filtered, washed with ACN/ethyl acetate, and dried.
2. Functionalization of silica particles with zwitterionic ligand (DBO-PS): Silica particles (5 μm, 10 nm pores) were dispersed in anhydrous toluene. 3-Chloropropyltrimethoxysilane (CPTMS) was added as a linker, and the mixture was stirred at 110°C for 24 h under nitrogen to yield chloropropyl-modified silica. The product was washed with toluene, methanol, and water, then dried. Then, the chloropropyl-modified silica was reacted with DBO-PS and potassium iodide (catalyst) in a methanol/water mixture (70:30 v/v). The reaction was refluxed for 24 hours, enabling nucleophilic substitution of the chloro group by DBO-PS. The final Sil-DBO-PS product was washed and dried.

What experimental variables did you investigate to study the retention behavior of the Sil-DBO-PS column?
The effect of various variables on the retention behavior of Sil-DBO-PS column were studied such as mobile phase, composition, buffer salt concentration, mobile phase pH, and temperature. The impact of mobile phase composition by testing water content ranging from 10% to 35% in acetonitrile, observing characteristic HILIC behavior where analyte retention decreased with increasing aqueous content. Buffer conditions were explored by varying ammonium acetate concentration (2-20 mM) and pH (3.4-6.5), revealing how ionic strength and pH influence both hydrophilic partitioning and electrostatic interactions. Temperature effects (20-50°C) were analyzed using Van't Hoff plots, confirming the exothermic nature of HILIC retention. The retention mechanism was further elucidated through advanced characterization techniques including modified linear solvation energy relationships (LSER) and Tanaka HILIC tests, which quantified contributions from hydrophilic, hydrogen bonding, and electrostatic interactions. Comparative studies against commercial ZIC-HILIC and Atlantis HILIC columns demonstrated Sil-DBO-PS's superior performance, achieving higher plate numbers (31,000-49,000 N/m) and improved resolution (Rs = 2.05-2.88) for challenging polar analytes like nucleosides and benzoic acid derivatives. The column exhibited excellent reproducibility, with retention time RSDs <0.18% and peak area RSDs <1.06% across multiple runs. These comprehensive investigations, detailed in Sections 3.2-3.4 of the study, established Sil-DBO-PS as a high-performance stationary phase with tunable selectivity for polar compound separations.

What are the dominant interactions governing analyte retention on the Sil-DBO-PS phase?
The retention of analytes on the Sil-DBO-PS stationary phase is governed by a sophisticated interplay of three key interactions that collectively enable exceptional separation of polar compounds. Hydrophilic partitioning forms the foundation of the retention mechanism, with analytes distributing between the mobile phase and a structured water layer immobilized on the polar stationary phase surface this was clearly demonstrated by the characteristic decrease in retention with increasing water content (10-35% in ACN) and confirmed through thermodynamic analysis. Complementing this partitioning effect, electrostatic interactions between charged analytes and the zwitterionic DABCO-sulfonate groups provide additional selectivity, as evidenced by the pH-dependent retention of ionizable compounds and the distinctive behavior observed when varying buffer ionic strength. Hydrogen bonding further refines the separation, with the stationary phase's polar groups engaging in specific interactions that enhance resolution of hydrogen-bonding analytes like nucleosides and vitamins. What sets Sil-DBO-PS apart is how these three mechanisms - hydrophilic partitioning, electrostatic interactions, and hydrogen bonding - work in concert. The zwitterionic architecture creates a uniquely balanced environment where neutral polar compounds are retained primarily through partitioning, while charged species benefit from tailored electrostatic effects that can be fine-tuned by adjusting pH or salt concentration. This multimodal retention capability explains why Sil-DBO-PS consistently outperforms commercial columns, delivering superior peak shapes, enhanced resolution of structural isomers, and greater flexibility in method development.

What types of analytes were used to assess the separation performance of the Sil-DBO-PS column?
The Sil-DBO-PS column's separation performance was comprehensively evaluated using selected suite of polar analytes designed to challenge its retention capabilities across multiple interaction modes. Nucleosides and nucleobases including cytosine, cytidine, adenosine, and uridine were used as primary probes to assess hydrophilic partitioning and hydrogen bonding interactions, leveraging their inherent polarity and sugar-backbone structures. To test electrostatic interactions and isomer selectivity, a series of benzoic acid derivatives (benzoic acid, 4-aminobenzoic acid, salicylic acid) and their positional isomers were employed, with the column demonstrating exceptional resolution of structurally similar compounds. Water-soluble vitamins provided insight into the stationary phase's handling of biologically relevant, multifunctional molecules. The evaluation also incorporated standardized Tanaka test reagents like uridine and deoxyguanosine isomers to enable direct comparison with commercial columns, while a diverse set of 30 LSER test compounds with systematically varied physicochemical properties allowed quantitative analysis of retention mechanisms. This rigorous testing protocol not only confirmed the column's superior performance in separating challenging polar compounds but also highlighted its advantages over existing commercial options, particularly in resolution of nucleosides (Rs = 2.05-2.88) and benzoic acid isomers where conventional columns failed.

How did the Sil-DBO-PS stationary phase compare to commercial columns in terms of separation efficiency and selectivity?
The Sil-DBO-PS stationary phase demonstrated clear advantages over commercial HILIC columns (ZIC-HILIC and Atlantis HILIC) in both separation efficiency and selectivity, as revealed by comprehensive comparative testing. In terms of efficiency, Sil-DBO-PS generated significantly sharper peaks, achieving 31,000-49,000 theoretical plates per meter for nucleosides, a marked improvement over the 25,000-35,000 N/m range of ZIC-HILIC and 22,000-32,000 N/m for Atlantis HILIC. This enhanced efficiency translated to superior resolution capabilities, with Sil-DBO-PS delivering baseline separation (Rs = 2.05-2.88) for eight nucleosides that commercial columns struggled to resolve completely. The selectivity advantages were particularly evident in challenging separations, where Sil-DBO-PS successfully distinguished benzoic acid isomers like 3,5-dinitrobenzoic acid and salicylic acid that co-eluted on ZIC-HILIC, while also providing better retention and peak shapes for water-soluble vitamins compared to Atlantis HILIC. These performance improvements stem from Sil-DBO-PS's unique zwitterionic architecture, which enables a balanced combination of hydrophilic partitioning, electrostatic interactions, and hydrogen bonding - a multimodal retention mechanism that outperforms the more limited interaction capabilities of single-mode commercial columns. The Sil-DBO-PS's robust performance across varying pH (3.4-6.5) and ionic strength (2-20 mM ammonium acetate) conditions, coupled with exceptional reproducibility (RSD < 0.18% for retention times), further establishes its superiority for demanding applications in polar compound analysis.

What was your approach to testing the reproducibility and stability of the Sil-DBO-PS stationary phase?
For reproducibility testing, five consecutive injections of standardized mixtures containing nucleosides and benzoic acids under consistent chromatographic conditions (85% ACN/15% 10 mM ammonium acetate, pH 6.3), achieving exceptional retention time repeatability with RSDs below 0.18% and peak area RSDs under 1.06%. Column-to-column consistency was verified by evaluating multiple production batches, all demonstrating uniform performance characteristics thanks to tightly controlled synthetic protocols and consistent ligand bonding densities (1.49 µmol/m²). Long-term stability assessments subjected the stationary phase to extended operation across challenging conditions, including 100+ injections at varying pH (3.4-6.5) and prolonged exposure to high organic mobile phases (90% ACN), with no observable degradation in chromatographic performance even after 500 injections.

What potential applications do you envision for the Sil-DBO-PS column beyond the current study?
Owing to the exceptional separation performance for a diverse class of analytes, Sil-DBO-PS column may be used for the separation of pharmaceutical industries to separate polar drugs and their metabolites including antibiotics, antivirals, and zwitterionic therapeutics. Moreover, the Sil-DBO-PS column could also be used in metabolomics for separation of polar metabolites like nucleotides and organic acids, particularly for biomarker discovery in complex biological samples. The developed column could be used for the separation of polar contaminants like perfluorinated compounds and algal toxins in environmental samples. The food industry could utilize its robust performance for simultaneous analysis of water-soluble vitamins and mycotoxins in nutritional products. Its compatibility with mass spectrometry and stability across pH extremes make it particularly valuable for emerging omics technologies, potentially enabling novel 2D-LC workflows that combine its HILIC capabilities with reversed-phase separations.

How could this new stationary phase impact the analysis of biological samples like plasma or urine?
The unique zwitterionic structure of Sil-DBO-PS stationary phase enabled efficient separation of challenging polar compounds from amino acids and nucleosides to organic acids and neurotransmitters that often evade detection in conventional reverse-phase analysis. I hope that Sil-DBO-PS stationary phase can be able to separate different kinds of target analytes in biological samples such as urine and plasma after sample pre-treatment. Moreover, we are working on this stationary phase to further evaluate its performance for the separation of peptides and proteins.

What future research directions would you recommend based on your findings?
Future work will focus on the evaluation of Sil-DBO-PS column for multiple research avenues spanning clinical diagnostics (polar biomarker detection), multi-omics integration, and single-cell analysis to environmental monitoring and biopharmaceutical applications.

References

1. Hu, Y.; Zhang, P.; Ali, A. et al. Preparation of a 1,4-Diazabicyclo[2.2.2]octane Sulfonate Betaine Zwitterionic Stationary Phase and Comparative Evaluation of its Separation Performance in Hydrophilic Interaction Chromatography. Anal. Chim. Acta 2025, 1356, 344028. DOI: 10.1016/j.aca.2025.344028

Ashraf Ali received his PhD degree in Chemistry with specialization in Analytical Chemistry from Advanced Separation Science Lab, Inha University, South Korea in February 2018 and worked as a postdoctoral fellow at the same university until May 2020. Then, he joined the University of Haripur, Pakistan as Assistant Professor in Chemistry and worked until January 2024. In February 2024, he joined Department of Chemistry & Chemical Engineering Henan University of Technology (Zhengzhou China) as a Senior Research Associate. Ali has published more than 40 research articles in international journals of well repute and one patent as primary inventor. He attended several international conferences in USA, Italy, Switzerland, Belgium, Japan, Singapore, and China. He is working on the synthesis of mesoporous silica, silica monoliths, surface functionalization of mesoporous materials, preparation of HPLC stationary phases, and the development of capillary electrochromatography columns for the separation of peptides, proteins, etc. Photo courtesy of Ali.