Preparation and evaluation of n-octadecylphosphonic acid-modified magnesia-zirconia stationary phases for reversed-phase liquid chromatography

Three n-octadecylphosphonic acid-modified magnesia-zirconia reversed stationary phases (C₁₈PZM) are prepared via the strong Lewis base interactions between organophosphonate and magnesia-zirconia composite. And two of them are end-capped by using trimethylchlorosilane as end-capping agent in different procedures. Stability studies at extreme high pH conditions (pH 9-12) show that both the non-endcapped and endcapped columns are quite stable at pH 12 mobile phase. The reversed-phase liquid chromatographic behavior of three C₁₈PZM stationary phases are comparatively investigated in detail using a variety of basic compounds as probes. The retention of basic compounds on the three phases is studied over a wide range of pHs. And the possible retention mechanisms of basic compounds on the three stationary phases are discussed. The results show that the basic solutes retain by a hydrophobic and cation-exchange interaction mixed mechanism on three stationary phases when they are operated in eluents at pH values near to the pK_a of the Brönsted conjugate acid form of the analyte, suggesting that inherent zirconol groups on ZM are not expected to interact with bases via cation-exchange interaction at lower pH. Nonetheless, the non-endcapped phase differs markedly from the edncapped ones in retention and selectivity of basic solutes using eluents at pH 4.1, implying a complex retention mechanism at this pH. The cation-exchange sites under such conditions are more likely due to the adsorbed Lewis base anionic buffer constituents (acetate) on accessible ZM surface sites than the chemisorbed phosphonate. Although the three phases exhibit very similar chromatographic behavior with eluents at pH 10.1, and show in general satisfactory separation of basic compounds and alkaloids studied, the performance for a specific analyte, however, differs largely from column to column.     

Retention behavior of basic compounds on alkylphosphonate-modified magnesia-zirconia composite stationary phase in RPHPLC

Summary The chromatographic properties of an alkylphosphonate-modified magnesia-zirconia composite stationary phase have been investigated by reversed-phase high-performance liquid chromatography with basic compounds as probes. The influence of organic modifier composition and mobile phase pH was studied. The new stationary phase, similar to a silica-based reversed-phase stationary phase, has hydrophobic properties, but greater pH stability. Use of the phase results in more symmetric peaks for basic compounds. A possible mechanism of retention of basic solutes on the new stationary phase is discussed. The chromatographic behavior of the basic solutes depends mainly on hydrophobic interactions between the solutes and the hydrophobic moiety of the stationary phase. Br?nsted acidic and basic sites on the surface of the new stationary phase play an important role in the retention of ionized solutes by ion-exchange interaction. Promising separations of some basic compounds have been achieved by use of methanolic TRIS buffer, pH 10.0, as the mobile phase.     

1-萘甲膦酸改性氧化锆固定相分离芳胺类化合物的色谱性能研究

研究了一些芳胺类化合物在1-萘甲膦酸改性氧化锆固定相上的色谱行为。分别考察了流动相中甲醇含量、缓冲液pH值和离子强度等对芳胺类化合物色谱保留的影响,并对这类化合物在该固定相上的保留机理进行了探讨。研究结果表明,芳胺类化合物在该固定相上表现出反相和阳离子交换的混合保留模式。以pH 10.1的Tris-甲醇(60/40,V/V)溶液为流动相,在1-萘甲膦酸改性氧化锆固定相上成功分离了间苯二胺、邻甲苯胺、N-甲苯胺、对硝基苯胺、邻硝基苯胺和α-甲萘胺6种芳胺类化合物。     

十二烷基键合氧化锆固定相的制备与性能评价

以自制5μm球形氧化锆为基质，制备了十二烷基键合氧化锆HPLC固定相，考察了正烷基取代苯、稠环芳烃、苯胺及吡啶衍生物、苯酚和硝基苯酚异构体等不同性质化合物在固定相上的保留行为，并与十二烷基键合硅胶固定相进行了比较。结果表明：中性和碱性化合物在固定相上主要为反相色谱保留机理；酸性化合物在固定相上以反相色谱保留机理为主，但是氧化锆表面的Lewis酸性中心对溶质也存在一定程度吸附作用，导致色谱峰拖尾。     

Characterization of a mixed-mode reversed-phase/cation-exchange stationary phase prepared by thermal immobilization of poly(dimethylsiloxane) onto the surface of silica

A novel stationary phase prepared by the thermal immobilization of poly(dimethylsiloxane) onto the surface of silica (PDMS–SiO₂) has been described, evaluated and compared with 229 commercially available RP-LC stationary phases using the Tanaka column classification protocol. The phase exhibited many unique chromatographic properties and, based on the phases in the database, was most similar to the fluoroalkylated phases (aside from the obvious lack of fluoro selectivity imposed by the C–F dipole). The phase exhibited classic reversed-phase behaviour in acid mobile phase conditions and mixed-mode reversed-phase/cation-exchange retention behaviour in neutral mobile phase conditions. The phase exhibited acceptable stability at both low and intermediate pH, conditions which should impart optimum chromatographic selectivity to the phase. Retention of basic analytes was shown to occur by a “three site model” as proposed by Neue. This new PDMS–SiO₂ stationary phase is extremely interesting in that the dominancy of its hydrophobic and ion-exchange interactions can be controlled by the influence of mobile phase pH, buffer type and concentration. The PDMS–SiO₂ stationary phase may provide a complementary tool to reversed-phase and HILIC stationary phases. The present results highlight the fact that the type of buffer, its concentration and pH can not only affect peak shape but also retention, selectivity and hence chromatographic resolution. Therefore, in method development and optimization strategies it is suggested that more emphasis should be given to the evaluation of these mobile phase operating parameters especially when basic solutes are involved.     

The role of the dual nature of ionic liquids in the reversed-phase liquid chromatographic separation of basic drugs

The cationic nature of basic drugs gives rise to broad asymmetrical chromatographic peaks with conventional C18 columns and hydro-organic mixtures, due to the ionic interaction of the positively charged solutes with the free silanol groups on the alkyl-bonded reversed-phase packing. Ionic liquids (ILs) have recently attracted some attention to reduce this undesirable silanol activity. ILs are dual modifiers (with a cationic and anionic character), which means that both cation and anion can be adsorbed on the stationary phase, giving rise to interesting interactions with the anionic free silanols and the cationic basic drugs. A comparative study of the performance of four imidazolium-based ILs as modifiers of the chromatographic behaviour of a group of β-blockers is shown. The ILs differed in the adsorption capability of the cation and anion on C18 columns. Mobile phases without additive and containing a cationic (triethylamine, TEA) or anionic (sodium dodecyl sulphate, SDS) additive were used as references for the interpretation of the behaviours. The changes in the nature of the chromatographic system, at increasing concentration of the additives, were followed based on the changes in retention and peak shape of the β-blockers. The silanol suppressing potency of the additives, and the association constants between the solutes and modified stationary phase or additive in the mobile phase, were estimated. The study revealed that SDS and the ionic liquid 1-hexyl-3-methylimidazolium tetrafluoroborate are the best enhancers of chromatographic peak shape among those studied.     

稠环芳烃在烷基膦酸改性锆镁复合氧化物固定相上的反相液相色谱保留行为

 以稠环芳烃为探针,考察了烷基膦酸改性锆镁复合氧化物材料的反相色谱性能。研究了稠环芳烃类化合物的结构与其保留值的关系,比较了烷基膦酸改性锆镁复合氧化物固定相和十八烷基键合硅胶ZorbaxODS对稠环芳烃异构体的选择性,并对可能的保留机理进行了讨论。以甲醇-水(体积比为75∶25)为流动相,在烷基膦酸改性锆镁复合氧化物固定相上分离了8种稠环芳烃类化合物。     

Selectivity of some basic solutes on a poly(methyltetradecylsiloxane)-silica stationary phase

Complex analyses of polar compounds, especially basic ones, require more selective stationary phases. The present paper describes a stationary phase prepared by thermal immobilization of poly(methyltetradecylsiloxane) onto chromatographic silica (PMTDS-SiO(2)). This stationary phase presents hydrophobic and ion-exchange interactions that confer both high retention and unique selectivities for basic solutes. The influence of ion-exchange interactions is confirmed by the increase in retention factors of basic solutes when the mobile-phase pH changes from acidic to neutral and by the decrease in retention factors when the mobile-phase pH changes from neutral to alkaline. The ion-exchange properties of the stationary phase are enriched in neutral mobile phase (pH 7-7.5) using soft Lewis bases such as tricine and tris as buffers but are suppressed in both acidic (pH 2.5-6) and highly alkaline mobile phases (pH≤10). Increasing both temperature and flow rate permits more rapid separations while maintaining the selectivity. The stability of the stationary phase is evaluated with acid, neutral and alkaline mobile phases.     

阴离子型胶束液相色谱的溶质保留行为

以SDS阴离子表面活性剂作流动相,酸性、中性及两性药物为受试药物,运用三相平衡理论考察影响阴离子型胶束液相色谱（AMLC）溶质保留行为的几个因素。保留由溶质与胶束相及修饰后固定相的综合作用决定。有机调节剂正丙醇的加入改变了溶质从水相到固定相或到胶束相的平衡,保留取决于溶质疏水性和静电性间的平衡。此外对羟基苯甲酸酯类同系物的亲脂性与3种细菌最小抑菌浓度具有显著相关性,提示其抑菌机理主要取决于药物与生物膜的亲和性。     

Chromatographic evaluation of reversed-phase/anion-exchange/cation-exchange trimodal stationary phases prepared by electrostatically driven self-assembly process

This work describes chromatographic properties of reversed-phase/cation-exchange/anion-exchange trimodal stationary phases. These stationary phases were based on high-purity porous spherical silica particles coated with nano-polymer beads using an electrostatically driven self-assembly process. The inner-pore area of the material was modified covalently with an organic layer that provided both reversed-phase and anion-exchange properties while the outer surface was coated with nano-sized polymer beads with strong cation-exchange characteristics. This design ensured spatial separation of the anion-exchange and the cation-exchange regions, and allowed reversed-phase, anion-exchange and cation-exchange retention mechanisms to function simultaneously. Chromatographic evaluation of ions and small molecules suggested that retention of ionic analytes was influenced by the ionic strength, pH, and mobile phase organic solvent content, and governed by both ion-exchange and hydrophobic interactions. Meanwhile, neutral analytes were retained by hydrophobic interaction and was mainly affected by mobile phase organic solvent content. Depending on the specific application, selectivity could be optimized by adjusting the anion-exchange/cation-exchange capacity ratio (selectivity), which was achieved experimentally by using porous silica particles with different surface areas.     

Analysis of selected ionic liquid cations by ion exchange chromatography and reversed-phase high performance liquid chromatography

The chromatographic behavior of 8 ionic liquids - 7 homologues of 1-alkyl-3-methylimidazolium and 4-methyl-N-butylpyridinium - has been investigated with a strong cation exchange adsorbent. In particular, the dependence of the retention properties of these solutes on mobile phase composition, pH, and buffer concentration was evaluated with the aim of optimizing and improving the selectivity and retention of solute separation. While using the SCX stationary phase, several interactions occurred with varying strengths, depending on the mobile phase composition. Cation exchange, nonspecific hydrophobic interactions, and adsorption chromatography behavior were observed. Reversed phase chromatography occurred at low concentrations of acetonitrile, electrostatic and adsorption interactions at higher organic modifier concentrations. Elevated buffer concentrations lowered the retention factors without affecting the selectivity of ionic liquids. Obtained results were further compared to the chromatographic behaviour of ionic liquids in the reversed phase system. All analyzed ionic liquids follow reversed-phase behavior while being separated. Much lower selectivity in the range of highly hydrophilic compounds is obtained. This suggests preferred use of ion chromatography for separation and analysis of compounds below 4 carbon atoms in the alkyl side chain.     

Single-step preparation and characterization of polymeric monolith for pressurized capillary electrochromatography of typical homologs

A monolithic stationary phase was prepared in a single step by in situ copolymerization of iso-butyl methacrylate (IBMA), ethylene dimethacrylate (EDMA), and N,N-dimethylallylamine (DMAA) in a binary porogenic solvent consisting of N,N-dimethylformamide (DMF) and 1,4-butanediol. As the frame structures of monoliths, the amino groups are linked to support the EOF necessary for driving the mobile phase through the monolithic capillary, while the hydrophobic groups are introduced to provide the nonpolar sites for the chromatographic retention. To evaluate the column performance, separations of typical kinds of neutral or charged homologs, such as alkylbenzenes, phenols (including isomeric compounds of hydroquinone, resorcin, and catechol), and anilines (including isomeric compounds of o-phenylenediamine and 1,4-phenylenediamine), were performed, respectively on the prepared column under the mode of pressurized pCEC. Effects of the buffer pH and the mobile phase composition on the linear velocity of mobile phase and the retention factors of these compounds were investigated. It was found that the retention mechanism of charged solutes could be attributed to a mixed mode of hydrophobic interaction and electrophoresis, while an RP chromatographic behavior on the monolithic stationary phases was exhibited for neutral solutes. Especially, basic compounds such as anilines were well separated on the monolithic columns in the "counterdirectional mode," which effectively eliminated the electrostatic adsorption of basic analytes on the charged surface of the stationary phases.     

Mixed-mode reversed-phase and ion-exchange separations of cationic analytes on polybutadiene-coated zirconia

The retention and selectivity of the chromatographic separation of basic (cationic) analytes on a polybutadiene-coated zirconia (PBD-ZrO2) stationary phase have been studied in greater detail than in previous studies. These separations are strongly influenced by the chemistry of the accessible surface of zirconia. In the presence of buffers which contain hard Lewis bases (e.g., phosphate, fluoride, carboxylic acids) zirconia's surface becomes negatively charged due to adsorption of the buffer anion at the hard Lewis acid sites. Consequently, under most conditions (e.g., neutral pH), cationic analytes undergo both hydrophobic and cation-exchange interactions. This mixed-mode retention process generally leads to greater retention factors for cations relative to those on silica-based reversed phases despite the lower surface areas of the zirconia phase, but, more importantly, adsorption of hard Lewis bases can be used to control the chromatographic selectivity for cationic analytes on these zirconia-based stationary phases. In contrast to our prior work, here we show that when mixed-mode retention takes place, both retention and selectivity are easily adjusted by changing the type of hard Lewis base buffer anion, the type of buffer counter-ion (e.g., sodium, potassium, ammonium), the pH, and the ionic strength of the eluent as well as the type and amount of organic modifier.     

On the chromatography mechanism of reversed-phase liquid chromatography

Summary An equation is derived which can describe how the retention of solutes is influenced by the composition of the mobile phase in reversed-phase liquid chromatography, the retention of solutes in alkyl bonded stationary phase regarded as the complexation between solute molecule and the active sites on the surface of the stationary phase. When the stationary phase is not fully saturated by the organic modifier, the activity of the active sites, the activity coefficient of the adsorbed solute as well as the activity coefficient of the solute in the mobile phase depend on the composition of the mobile phase. However, when the stationary phase is fully saturated, the composition of the mobile phase mainly influences the activity coefficient of the solute in the mobile phase. In addition, the selectivity of retention is discussed in terms of the derived equation.     

Hexylacrylate-based mixed-mode monolith,a stationary phase for the nano-HPLC separation of structurally related enkephalins

The potential of an in situ photopolymerized hexylacrylate-based monolithic stationary phase-bearing sulfonic acid groups was investigated by studying the chromatographic retention of small structurally related peptides (enkephalins) by nano-LC. Several retention mechanisms were highlighted. First, a reverse-phase chromatographic behavior toward neutral solutes due to hexylacrylate-moieties was demonstrated. Second, an evaluation of the influences of buffer pH suggested the involvement of a cation-exchange mechanism due to the presence of 2-acrylamido-2-methyl-1-propanesulfonic acid. This cation-exchange phenomenon was confirmed by the clear influence of Na⁺ concentration in the mobile phase on peptide retention.     

Ion-exchange and hydrophobic interactions affecting selectivity for neutral and charged solutes on three structurally similar agglomerated ion-exchange and mixed-mode stationary phases

The nature and extent of mixed-mode retention mechanisms evident for three structurally related, agglomerated, particle-based stationary phases were evaluated. These three agglomerated phases were Thermo Fisher ScientificIon PacAS11-HC – strong anion exchange, Thermo Fisher Scientific IonPac CS10 – strong cation-exchange PS-DVB, and the Thermo Fisher Scientific Acclaim Trinity P1silica-based substrate, which is commercially marketed as a mixed-mode stationary phase. All studied phases can exhibit zwitterionic and hydrophobic properties, which contribute to the retention of charged organic analytes. A systematic approach was devised to investigate the relative ion-exchange capacities and hydrophobicities for each of the three phases, together with the effect of eluent pH upon selectivity, using a specifically selected range of anionic, cationic and neutral aromatic compounds. Investigation of the strong anion-exchange column and the Trinity P1 mixed-mode substrate, in relation to ion-exchange capacity and pH effects, demonstrated similar retention behaviour for both the anionic and ampholytic solutes, as expected from the structurally related phases. Further evaluation revealed that the ion-exchange selectivity of the mixed-mode phase exhibited properties similar to that of the strong anion-exchange column, with secondary cation-exchange selectivity, albeit with medium to high anion-exchange and cation-exchange capacities, allowing selective retention for each of the anionic, cationic and ampholytic solutes. Observed mixed-mode retention upon the examined phases was found to be a sum of anion- and cation-exchange interactions, secondary ion-exchange and hydrophobic interactions, with possible additional hydrogen bonding. Hydrophobic evaluation of the three phases revealed log P values of 0.38–0.48, suggesting low to medium hydrophobicity. These stationary phases were also benchmarked against traditional reversed-phase substrates namely, octadecylsilica YMC-Pac Pro C18 and neutral μPS-DVB resin IonPac NS1-5u, yielding log P values of 0.57 and 0.52, respectively.     

溶质在动态改性氧化锆液相色谱柱上的保留行为

分别采用硬脂酸、环糊精和十二烷基磺酸钠动态改性自制的ZrO2微球,研究了流动相中甲醇和改性剂浓度对苯酚及苯甲酸的衍生物、苯胺衍生物及芳香烃类化合物的色谱保留行为的影响。中性及碱性化合物的保留时间较短,色谱峰对称;酸性化合物保留时间较长,色谱峰拖尾较严重。改性氧化锆表现出反相色谱性能。     

Role of ion pairing in anionic additive effects on the separation of cationic drugs in reversed-phase liquid chromatography

Mobile phase additives can significantly affect the separation of cationic drugs in reversed-phase liquid chromatography (RPLC). Although there are many applications for anionic additives in RPLC separations, the retention mechanism of basic drugs in the presence of inorganic and highly hydrophilic anionic species in the mobile phase is not at all well understood. Two major retention mechanisms by which anionic additives can influence the retention of cations are: (1) ion pair formation in the mobile phase with subsequent retention of the neutral ion pair; (2) pre-sorption of anionic additives on the stationary phase followed by "dynamic ion-exchange" or "electrostatic interaction" with the analytes. Because the use of ion pair chromatography in the separation of proteins, peptides, and basic drugs is rapidly increasing, understanding the retention mechanism involved is becoming more important, especially for the smaller commonly used hydrophilic anionic additives (e.g., formate HCOO, chloride Cl-, trifluoroacetate CF3COO-, perchlorate ClO4-, and hexafluorophosphate PF6-). In this work, we compared various anionic additives in light of their effects on the retention of basic drugs. As did many others we found that the addition of anionic additives (Cl-, CF3COO-, ClO4-, PF6-) profoundly influences the retention of basic drugs. In order to explain the data and differentiate the mechanisms by which the anionic additives perturb the chromatography, we used ion pair formation constants independently measured by capillary electrophoresis (CE) under the mobile phase conditions (pH, solvent composition) identical to those used in chromatography. Agreement between the predicted and experimental chromatographic data under various conditions was evaluated. Under specific circumstances (e.g., pH, stationary phase, and nature of anionic additive), we conclude that the ion pair mechanism is more important than the dynamic ion-exchange and at other conditions it remains a significant contribution.     

Comparison of the performance of non-ionic and anionic surfactants as mobile phase additives in the RPLC analysis of basic drugs

Surfactants added to the mobile phases in reversed-phase liquid chromatography (RPLC) give rise to a modified stationary phase, due to the adsorption of surfactant monomers. Depending on the surfactant nature (ionic or non-ionic), the coated stationary phase can exhibit a positive net charge, or just change its polarity remaining neutral. Also, micelles in the mobile phase introduce new sites for solute interaction. This affects the chromatographic behavior, especially in the case of basic compounds. Two surfactants of different nature, the non-ionic Brij-35 and the anionic sodium dodecyl sulfate (SDS) added to water or aqueous-organic mixtures, are here compared in the separation of basic compounds (β-blockers and tricyclic antidepressants). The reversible/irreversible adsorption of the monomers of both surfactants on the stationary phase was examined. The changes in the nature of the chromatographic system using different columns and chromatographic conditions were followed based on the changes in retention and peak shape. The study revealed that Brij-35 is suitable for analyzing basic compounds of intermediate polarity, using "green chemistry", since the addition of an organic solvent is not needed and Brij-35 is a biodegradable surfactant. In contrast, RPLC with hydro-organic mixtures or mobile phases containing SDS required high concentrations of organic solvents.