Zwitterionic Stationary Phase Research Articles

Chiral zwitterionic stationary phases based on Cinchona alkaloids and dipeptides: Application in chiral separation of dipeptides under reversed phase conditions

Chromatographic behavior of novel chiral stationary phases with bonded selectors based on Cinchona alkaloids modified with dipeptides was studied using dipeptides as probe molecules. Buffer-free and salt containing hydro-organic solutions were used as the mobile phases. The selectors exhibit pseudoenantiomeric behavior with respect to the L/D or LL/DD enantiomers and do not behave so with respect to the LD/DL enantiomers. The alkaloid part of the selectors is the driver of enantioselectivity, while the dipeptide substituent plays a modulating role. The quinidine-based selectors demonstrate stronger adsorption affinity and higher enantioselectivity as compared to the quinine-based selectors. The dipeptide analytes containing a glycyl fragment are weaker retained and their enantiomers are worse separated comparing to dipeptides with both units being larger amino acids. Moreover, a phenyl group in the structure of a dipeptide analyte facilitates enantioseparation. The effect of the mobile phase composition on retention depends on the hydrophobicity of an analyte. Hydrophobic dipeptides are better eluted by methanol-rich solvents, hydrophilic dipeptides are better eluted with water-rich solvents, and dipeptides with an intermediate hydrophobicity demonstrate a U-shaped or more complicated dependence of the retention factor on the percentage of methanol. Even a small buffer addition to the mobile phase decreases retention, but the ion-exchange mechanism was not confirmed. The effect of an electrolyte is rather due to the shielding of the charged groups of the selector reducing thereby electrostatic interaction between the selector and analyte. Efficiency of the novel columns is comparable to that of other brush-type chiral columns, the highest achieved number of the theoretical plates per 1 m varying between 30,000 and 40,000.

Chiral zwitterionic stationary phases based on Cinchona alkaloids and dipeptides – design, synthesis and application in chiral separation

Chiral resolution of polar organic compounds such as amino acids and peptides represents an important chromatographic task due to increasing significance of natural species, which play important signaling and regulatory roles in the living organisms. Despite the number of available chiral stationary phases, this task remains challenging, since not many of the commercially available systems are capable to resolve non-derivatized zwitterionic species. In this study, we present a target-oriented design of a new class of chiral selectors. Pursuing the goal to separate amino acids, and especially short peptides, we have combined Cinchona alkaloids – quinine and quinidine – with three different biogenic dipeptides. We have synthesized six different chiral stationary phases, with selector loading of ∼200 μmol g−1, and tested their chiral recognition capabilities for acidic, basic and zwitterionic analytes using various mobile phases. We have observed that all chiral stationary phases retain the chiral anion exchange capability known for commercially available Cinchona-based columns leading to baseline or partial resolution of six out of ten analytes. The performance in chiral resolution of basic analytes is not optimum due to the weak cation exchange character of the peptidic residue. However, we report on encouraging results in the chiral resolution of short peptides, for which, depending on their structure, we see the chiral resolution of up to three stereoisomers (from four possible) in a preliminary screening.

Simultaneous chromatographic separation of the anomers of saccharides on a polymer sulfobetaine zwitterionic stationary phase.

Simultaneous chromatographic separation of the anomers of saccharides was achieved by using a polymer zwitterionic stationary phase functionalized by acrylamide-type sulfobetaine. By optimization of separation parameters including column temperature, pH, and flow rate, the column operated in hydrophilic interaction chromatography mode exhibited excellent separation selectivity toward five monosaccharides and their anomers (including ribose, xylose, galactose, glucose, and arabinose) and two disaccharides (lactose and maltose). Baseline separation could be achieved at mild operation conditions such as 20-30°C of column temperature or typical mobile phase composition (85% acetrontrile-15% 20mM ammonium formate [NH4 FA]) with wide pH tolerance range of 2-8. This offers a rapid way to determine the configuration of α or β anomer of the saccharides.

Preparation of two zwitterionic polymer functionalized stationary phases and comparative evaluation under mixed-mode of reversed phase/ hydrophilic interaction/ion exchange chromatography

Zwitterions are a promising choice to prepare separation materials because of their hydrophilicity and biocompatibility. We described the preparation of two zwitterionic polymer functionalized stationary phases and evaluation under mixed-mode chromatography. A zwitterionic monomer, S-(4-vinylbenzyl) cysteine (SVC), was synthesized and bonded to silica via reversible addition fragmentation chain transfer (RAFT) polymerization to afford a zwitterionic stationary phase, Sil-SVC. A hydrophobic monomer, N-(4-phenylbutan-2-yl) acrylamide (NPA), was copolymerized with SVC onto the stationary phase (Sil-SVCNPA) for comparison. The stationary phases were characterized with FT-IR, TGA, EA, and zeta-potential measurements. Mobile phase composition (ACN content, pH and salt concentration) was varied to study the retention property. Linear solvation energy relationship and Van't Hoff plot were used to investigate the retention mechanism and how chromatographic conditions influenced it. Both stationary phases showed a mixed-mode of RPLC/HILIC/IEC and satisfactory performance in separating hydrophobic analytes (alkylbenzenes and polycyclic aromatic hydrocarbons), hydrophilic nucleotide and bases, and anions, high column efficiency of 60,000 plates·m−1 was achieved. In summary, zwitterionic polymers are attractive options to prepare stationary phases and the retention property can be easily regulated by copolymer.

Liquid Chromatography–Tandem Mass Spectrometry Analysis of Aminoglycosides in Foods Using an Ethylene-Bridged Hybrid Zwitterionic Stationary Phase and Hydrophilic–Lipophilic-Balanced Solid-Phase Extraction Cartridges

This work aimed to develop an analytical method for the screening of multiple aminoglycoside residues in foods of animal origin using an ethylene-bridged hybrid (BEH) particle-based sulfoalkylbetaine stationary phase. The effects of chromatographic conditions on the separation of 17 aminoglycosides have been systematically investigated. Sample preparation and mass spectrometry detection have also been investigated and optimized. In contrast to high buffer concentrations in the mobile phase required for silica-based sulfoalkylbetaine stationary phases, a moderate buffer concentration (20 mM) provided the optimal separation of 17 aminoglycosides with the BEH sulfoalkylbetaine stationary phase. The developed method has been evaluated in milk, beef, pork, liver, and honey samples with good performance for retention, selectivity, sensitivity, linearity, precision, and accuracy. The majority of the limit of quantitation estimated with the matrix was less than 25 μg/kg. The overall accuracy across five matrices was in the range from 96 to 111%, with standard deviations of less than 19%.

Hydrophilic Interaction Chromatography with Sulfobetaine Zwitterionic Polymer-Bonded Stationary Phases

A rapid method for simultaneous determination of two statin drugs based on zwitterionic chromatography (ZIC) had been developed and validated. The development method included examining the effects of chromatographic conditions, including the percentage of organic modifier, pH values, ionic strength of the acetate buffer, and the predominant retention mechanismand#39;s experimental determination. Separation developed by two zwitterionic stationary phases (100 mm and#215; 4.6 mm I.D., 3.5and#181;). The influence of various spacer lengths was being used as an examination tool on atorvastatin and rosuvastatin retention behaviours. Two zwitterionic stationary phases and a mobile phase consisting of acetonitrile and acetate (pH = 4.75, 40 mM) in a ratio of 80:20 V/V were used to achieve optimum chromatographic conditions. The methods had been validated for linearity, accuracy, and precision. This validation shows that the ZIC-HILIC methods proposed were sufficient for quantification analysis of atorvastatin and rosuvastatin. Quantifications were achieved with U.V. detection at 240 nm over the concentration range of 0.1–7.0 μg mL-1 for atorvastatin and rosuvastatin, respectively.

Efficient enantioresolution of aromatic α-hydroxy acids with Cinchona alkaloid-based zwitterionic stationary phases and volatile polar-ionic eluents

Single enantiomers of mandelic acid (1), 3-phenyllactic acid (2), and 3-(4-hydroxyphenyl)lactic acid (3) are the subject of many fields of investigation, spanning from the pharmaceutical synthesis to that of biocompatible and biodegradable polymers, while passing from the interest towards their antimicrobial activity to their role as biomarkers of particular pathological conditions or occupational exposures to specific xenobiotics. All above mentioned issues justify the need for accurate analytical methods enabling the correct determination of the individual enantiomers. So far, all the developed liquid chromatography (LC) methods were not or hardly compatible with mass spectrometry (MS) detection. In this paper, a commercially available Cinchona-alkaloid derivative zwitterionic chiral stationary phase [that is, the CHIRALPAK® ZWIX(−)] was successfully used to optimize the enantioresolution of compounds 1–3 under polar-ionic (PI) conditions with a mobile phase consisting of an acetonitrile/methanol 95/5 (v/v) mixture with 80 mM formic acid. With the optimized conditions, enantioseparation and enantioresolution values up to 1.46 and 4.41, respectively, were obtained. In order to assess the applicability of the optimized enantioselective chromatography conditions in real-life scenarios and on MS-based systems, a proof-of-concept application was efficiently carried out by analysing dry urine spot samples spiked with 1 by means of a LC-MS system. The (S)<(R) enantiomer elution order (EEO) was established for compounds 1 and 2 by analysing a pure enantiomeric standard of known configuration. This was not possible for 3 because not commercially available. For this compound, the same EEO was identified applying a procedure based on ab initio time-dependent density-functional theory simulations coupled to electronic circular dichroism analyses. Moreover, a molecular dynamics simulation unveiled the role of the phenolic OH in compound 3 in the retention mechanism.

Analysis of adsorption energy distribution in selected hydrophilic-interaction chromatography systems with amide, amine, and zwitterionic stationary phases.

Adsorption mechanisms of caffeine, quercetin, and phenol as test substances in various chromatographic systems have been analyzed. The investigations were conducted using three different chromatographic columns packed with polar bonded stationary phases, that is, amide, amine, and zwitterionic. Methanol-water and acetonitrile-water systems with different organic solvent contents have been used as mobile phases. On the basis of adsorption isotherms obtained for the tested systems, Scatchard plots and adsorption energy distributions have been determined. The most likely retention mechanisms have been discussed. The results of investigations indicate that (i) the surfaces of tested adsorbents are energetically heterogeneous, and (ii) the main role in sorption mechanism is played by low-energy sites.

Preparation and evaluation of a polymer-based sulfobetaine zwitterionic stationary phase

We describe a polymer-based sulfobetaine zwitterionic stationary phase for hydrophilic interaction liquid chromatography (HILIC). It was prepared by grafting acrylamide-type sulfobetaine monomer instead of common methacrylate-type sulfobetaine onto hydrolysed poly (glycidyl methacrylate-divinylbenzene) (GMA-DVB) microspheres via pendant double bonds of DVB. The phase has been characterized by elemental analysis, scanning electron micrograph and N2 adsorption-desorption experiment. It shows wider pH tolerance range (from 2 to 12) and excellent separation ability towards common strong polar analytes such as nucleosides and nucleic bases, water-soluble vitamins, amino acids, inorganic anions and cations. Notably, it exhibits negligible baseline noise level (~0.15 pA) under typical HILIC mobile phase. Excellent selectivity in separation of α- and β-anomers of reducing sugars and lactose/lactulose has also been observed.

A new high-throughput method based on hydrophilic interaction liquid chromatography-tandem mass spectrometry to determine 18 short-chain carboxylic acids in foods

In this work, an interesting analytical method to determine 18 short-chain carboxylic acids in food samples was developed using Hydrophilic Interaction Liquid Chromatography coupled to mass spectrometry (HILIC-MS/MS). The method was optimized through univariate and multivariate designs. The chromatographic SeQuant® ZIC®-HILIC column and mobile phase comprised of 75:25 (v/v) ACN and ammonium formate buffer (8 mmol L−1, pH 3) were selected. The analytical performance was assessed obtaining limits of quantification that varied from 0.04 to 2 mg L−1, and precision values less than 14%. Five different food samples were analyzed using calibration by standard addition with internal standard and sample preparation by dilute-and-shoot, which allowed for an efficient determination of the analytes in matrices of high complexity. In summary, the proposed method provided high-throughput, a great selectivity given by the HILIC-MS/MS system, an adequate sensitivity for the tested samples and interesting information about HILIC mechanisms for short-chain carboxylic acids as analytes and zwitterionic stationary phase.

PH-dependent selective separation of acidic and basic proteins using quaternary ammoniation functionalized cysteine-zwitterionic stationary phase with RPLC/IEC mixed-mode chromatography

In this paper, a cysteine-functionalized zwitterionic stationary phase (Cys-silica) was prepared based on the “thiol-ene” click chemistry between cysteine and vinyl-functionalized silica, and was further modified with bromoethane, 1-bromooctane and 1-bromooctadecane, respectively, to obtain a series of quaternary ammoniation-functionalized stationary phases (Cys-silica-Cn, n = 2, 8 and 18). These zwitterionic stationary phases were regarded as reversed-phase/ion-exchange (RP/IEC) mixed-mode chromatography (MMC) stationary phases for protein separation. The retention behaviors of proteins on these zwitterionic stationary phases were carefully investigated. The results indicated that the retentions of acidic and basic proteins on these zwitterinonic stationary phases were significantly influenced by the acetonitrile and salt concentrations, pH of mobile phase as well as the hydrophobicity of the ligand. The separation selectivity of proteins on these zwitterionic stationary phases strongly depended on the pH value of mobile phase. The baseline separation of 6 kinds of basic proteins can be achieved at pH 8.0 using Cys-silica-C2 or Cys-silica-C8 column, and 5 kinds of acidic proteins can also be separated completely at pH 4.0 with Cys-silica-C2 column. Moreover, owing to the quaternary ammoniation-functionalization on Cys-silica by using appropriately hydrophobic bromoalkanes, the selectivity and separation efficiency of proteins can be enhanced greatly. As a result, the acidic and basic proteins can be separated completely step by step from the complex sample by adjusting pH of mobile phase using a single Cys-silica-C2 column, which illustrates that the cysteine-functionalized zwitterionic stationary phase has a great potential for protein separation.

An exploratory approach for an oriented development of an untargeted hydrophilic interaction liquid chromatography-mass spectrometry platform for polar metabolites in biological matrices

The analysis of polar metabolites based on liquid chromatography-mass spectrometry (LC-MS) methods should take into consideration the complexity of interactions in LC columns to be able to cover a broad range of metabolites of key biological pathways. Therefore, in this study, different chromatographic columns were tested for polar metabolites including reversed-phase and hydrophilic interaction liquid chromatography (HILIC) columns. Based on a column screening, two new generations of zwitterionic HILIC columns were selected for further evaluation. A tree-based method optimization was applied to investigate the chromatographic factors affecting the retention mechanisms of polar metabolites with zwitterionic stationary phases. The results were evaluated based on a scoring system which was applied for more than 80 polar metabolites with a high coverage of key human metabolic pathways. The final optimized methods showed high complementarity to analyze a wide range of metabolic classes including amino acids, small peptides, sugars, amino sugars, phosphorylated sugars, organic acids, nucleobases, nucleosides, nucleotides and acylcarnitines. Optimized methods were applied to analyze different biological matrices, including human urine, plasma and liver cell extracts using an untargeted approach. The number of high-quality features (< 30% median relative standard deviation) ranged from 3,755 for urine to 5,402 for the intracellular metabolome of liver cells, showing the potential of the methods for untargeted purposes.

Evaluation of Different Tandem MS Acquisition Modes to Support Metabolite Annotation in Human Plasma Using Ultra High-Performance Liquid Chromatography High-Resolution Mass Spectrometry for Untargeted Metabolomics.

Ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) is a powerful and essential technique for metabolite annotation in untargeted metabolomic applications. The aim of this study was to evaluate the performance of diverse tandem MS (MS/MS) acquisition modes, i.e., all ion fragmentation (AIF) and data-dependent analysis (DDA), with and without ion mobility spectrometry (IM), to annotate metabolites in human plasma. The influence of the LC separation was also evaluated by comparing the performance of MS/MS acquisition in combination with three complementary chromatographic separation modes: reversed-phase chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) with either an amide (aHILIC) or a zwitterionic (zHILIC) stationary phase. RPLC conditions were first chosen to investigate all the tandem MS modes, and we found out that DDA did not provide a significant additional amount of chemical coverage and that cleaner MS/MS spectra can be obtained by performing AIF acquisitions in combination with IM. Finally, we were able to annotate 338 unique metabolites and demonstrated that zHILIC was a powerful complementary approach to both the RPLC and aHILIC chromatographic modes. Moreover, a better analytical throughput was reached for an almost negligible loss of metabolite coverage when IM-AIF and AIF using ramped instead of fixed collision energies were used.

A Study of Retention Behavior and Method Development of Salbutamol Sulfate in Hydrophilic Interaction Chromatography

For chromatographical separation and estimation of salbutamol sulfate, zwitterionic stationary phases with large capacity were obtained by zwitter-molecules attached to a polystyrene-divinylbenzene (PS/DVB) particle. Salbutamol sulfate retention activity was studied with eluent at various pH mobile phase, mobile phase concentrations, and acetonitrile (ACN) percentage. The methods of separation are based on separating the salbutamol sulfate into hydrophobic and cations interactions. Linearity of 0.01 to 0.9 μg.mL-1 for two columns was developed with direct calibration curves, %RSD percent (0.48 ± 0.12 and 0.49 ± 0.22), LoD (0.058 and 0.04 μg.mL-1), and LoQ (0.203 and 0.14 μg.mL-1) were created, respectively.

Simultaneous determination of vitamin B6 and catechins in dietary supplements by ZIC-HILIC chromatography and their antioxidant interactions

Hydrophilic interaction liquid chromatography coupled to mass spectrometry was employed for simultaneous determination of vitamin B6 and catechins in dietary supplements. The obtained results clearly shows the potential of the application of alcohol eluent (instead mainly used acetonitrile) as a component of an with zwitterionic stationary phase. The limits of detection on ZIC-HILIC column were 0.01 mg/L for catechins and vitamins B (only for pyridoxal phosphate was 0.10 mg/L). The investigations between green tea extract (GTE) and vitamin B were also evaluated using isobolographic analysis as well as the interaction indexes. Antioxidant activities of single components and their mixtures were determined by DPPH assay. It was found that the mixtures of GTE and vitamin B acted synergistically. In comparison to GTE alone, faster DPPH radical bleaching of the mixtures was observed in the presence of different forms of vitamin B6 (pyridoxine, pyridoxal, or pyridoxal phosphate), particularly for pyridoxal.

Application of hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for the retention and sensitivity studies of antisense oligonucleotides

The aim of the present investigation was application of hydrophilic interaction liquid chromatography as an alternative chromatographic approach for the study of antisense oligonucleotides. The influence of several mobile phases, differing with the salt type, their concentration and pH value on the retention and the separation of antisense oligonucleotides has been examined for this purpose. Four different stationary phases were also applied including unmodified silica, silica modified with the use of sulfobetaine groups, polyhydroxy and aminopropyl groups. Such wide range of tested conditions has been useful in better understanding of the retention mechanism of tested compounds. The results obtained during this investigation indicated that greater retention, greater peaks symmetry, as well as more effective separation of oligonucleotides, were obtained for the zwitterionic stationary phase. Moreover, the optimization of tandem mass spectrometry parameters with the use of Central Composite Design was performed and different mobile phases were tested to choose that one, which provided the greatest antisense oligonucleotides peak areas in Multiple Reaction Monitoring mode and consequently, the greatest possible sensitivity. Hydrophilic interaction liquid chromatography was compared with the ion pair chromatography, commonly used in the analysis of oligonucleotides. Both techniques were compared in terms of selectivity of separation as well as the sensitivity of their determination. Obtained results proved that ion pair chromatography provided better results in terms of separation efficiency and peak areas in Multiple Reaction Monitoring for tested conditions. However, these results do not preclude application of hydrophilic interaction liquid chromatography as an alternative chromatographic approach for the oligonucleotides analysis especially when a mobile phase without ion pair reagents is required.

Fragment-based Design of Zwitterionic, Strong Cation- and Weak Anion-Exchange Type Mixed-mode Liquid Chromatography Ligands and their Chromatographic Exploration

The role of individual functional groups has been assessed with regard to surface charge and chromatographic retention. Coatings were prepared from various fragments of the chiral zwitterionic materials Chiralpak ZWIX(+) and ZWIX(-). The different chromatographic ligands allowed fine tuning of the surface charge. Chiralpak ZWIX phases showed strongly negative ζ-potentials over the entire pH-range. Zwitterionic congeners with quinuclidine and sulfonic acid moieties but lacking the quinolone ring in the ligand structure exhibited shifted ζ-potentials of around + 5 to 20 mV depending on the surrounding residues. Capillary electrophoretic mobilitiy measurements with the chromatographic ligands and molecular dynamics simulations were carried out to offer some explanation of these surface charge differences of the distinct zwitterionic stationary phases. The new mixed-mode phases were also chromatographically characterized by simple RP and HILIC tests. The results allowed their positioning within a large variety of different commercially available RP, HILIC and mixed-mode phases, which were evaluated as well, by multivariate data processing using principal component analysis. The new mixed-mode phases overall exhibit reasonable hydrophilicity-lipophilicity balance and enable retention of ionic compounds by additional ionic interactions through weak anion-exchange (WAX-type), strong cation-exchange (SCX-type) or both (RP/ZWIX-type). Hence, the new RP/ZWIX phases can be flexible tools for selectivity tuning in RP and HILIC separations.

A click tyrosine zwitterionic stationary phases for hydrophilic interaction liquid chromatography

New zwitterionic (ZIC) stationary phases (SPs) are synthesized with the click and conventional bonding of tyrosine to silica gel. Infrared spectra and elemental analysis demonstrate the successful click and conventional bonding of this ZIC group on silica particles by the surface coverage including 2.36 and 0.75 µm m−2, respectively. Given the above-mentioned explanation, the present study evaluated the retention mechanism and chromatographic manners of polar compounds on these new materials under hydrophilic interaction liquid chromatography (HILIC) conditions. Based on the results, the Click-Tyrosine Stationary Phase provided good HILIC characteristics when it was applied to separate phenolic compounds, amino acids, alkaloids, and nucleobases compared to bare silica gel SP and even conventional tyrosine SPs. Further, this new Click-Tyrosine-SP represented appropriate HILIC features and column efficiency (the theoretical plate number was up to 50,000 plates m−1 for thebaine). Furthermore, the study investigated the effect of solute polarity (the number of the hydroxyl group of phenolic compounds) and hydrophobicity (the number of the side chain of aliphatic amino acids) on retention behaviors. Finally, some important factors were studied as the potential variables for guiding the retention behavior of the polar compound in HILIC condition including solvent composition, salt concentration, and the buffer pH of the mobile phase.

A polymer-based zwitterionic stationary phase for hydrophilic interaction chromatography

We describe a novel polymer-based zwitterionic stationary phase for hydrophilic interaction chromatography (HILIC). It is prepared by chemical modification of poly (glycidyl methacrylate-divinylbenzene) (GMA-DVB) by converting epoxide groups of microsphere surface to diol groups via hydrolysis, then clicking cysteine onto the microsphere with pendant double bonds of microsphere surface via “thiol-ene” click chemistry. The phase has been characterized by scanning electron micrograph, elemental analysis and zeta potential measurement. Diol and zwitterionic group (carboxylate and amine group associated with cysteine) onto the surface of GMA-DVB microspheres make them possess good hydrophilicity, as supported by effective separation towards common polar analytes. It shows good stability at alkaline solution (e.g. pH 10) and negligible bleed (e.g. only 1.7-fold blank and ~55-fold lower than a commercial silica-based polar phase tested with the minimal bleed level). Such phase exhibits specific separation selectivity to ionic analytes and simultaneous separation of anions and cations is achieved in the retention order of I− < NO3− < Choline < Br− < Cl− < K+< Na+.

Preparation and comparison of three zwitterionic stationary phases for hydrophilic interaction liquid chromatography.

Surface-bonded zwitterionic stationary phases have shown highlighted performances in separation of polar and hydrophilic compounds under hydrophilic interaction chromatography mode. So, it would be helpful to evaluate the characteristics of zwitterionic stationary phases with different arranged charged groups. The present work involved the preparation and comparison of three zwitterionic stationary phases. An imidazolium ionic liquid was designed and synthesized, and the cationic and anionic moieties respectively possessed positively charged imidazolium ring and negatively charged sulfonic groups. Then, the prepared ionic liquid, phosphorylcholine and an imidazolium-based zwitterionic selector were bonded on the surface of silica to obtain three zwitterionic stationary phases. The selectivity properties were characterized and compared through the relative retention of selected solute pairs, and different kinds of hydrophilic solutes mixtures were used to evaluate the chromatographic performances. Moreover, the zwitterionic stationary phases were further characterized by the modified linear solvation energy relationship model to probe the multiple interactions. All the results indicated that the types and arrangement of charged groups in zwitterionic stationary phases mainly affect the retention and separation of ionic or ionizable compounds, and for interaction characteristics the contribution from n and π electrons and electrostatic interactions displayed certain differences.