Retention pattern profiling of fungal metabolites on mixed-mode reversed-phase/weak anion exchange stationary phases in comparison to reversed-phase and weak anion exchange separation materials by liquid chromatography-electrospray ionisation-tandem mass spectrometry

Retention properties of 79 fungal metabolites (including neutral, acidic, basic, and amphoteric compounds) were evaluated on distinct mixed-mode reversed-phase/weak anion exchange (RP/WAX)-type stationary phases by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) in gradient as well as in isocratic elution mode. The RP/WAX separation materials were prepared by functionalising thiol-modified silica with N-(10-undecenoyl)-3-aminoquinuclidine and N-(10-undecenoyl)-3-alpha-aminotropane, respectively. To evaluate complementarity in chromatographic selectivity the physico-chemically heterogeneous solute set was analysed also on a RP phase (C(18)), an amino-type WAX phase, and a commercially available RP/WAX-like mixed-mode phase. Analytes may interact with the RP/WAX ligands via (attractive/repulsive) ionic, RP-like hydrophobic, as well as hydrophilic (HILIC) retention mechanisms. Individual interactive increments were found to be basically controlled by the nature and amount of organic modifier, pH value of eluent, and ionic strength of buffer additives. It could be demonstrated that RP/WAX columns offer the potential to separate compounds by exploiting a combination of various chromatographic interaction modes, which is not accessible with conventional RP and WAX columns. Such multi-modal properties increase both versatility and degrees of freedom for adjustment of chromatographic selectivity. For example, highly polar mycotoxins such as moniliformin were well retained on RP/WAX-type phases without compromising RP-selectivity for neutral (e.g. aflatoxins) and most basic solutes (e.g. epimer separation of ergot alkaloids) under fully MS-compatible conditions like a hydro-organic eluent with acetonitrile as organic modifier and an acetic acid/ammonium acetate buffer. Flexibility of the employed mixed-mode separation materials may be of value particularly for LC-ESI-MS/MS-based bioanalytics involving analytes with widely varying physico-chemical properties or applications prone to matrix effects.     

Hydrophilic interaction chromatography separation mechanisms of tetracyclines on amino-bonded silica column

Effects of mobile-phase variations on the chromatographic separation on amino-bonded silica column in hydrophilic interaction chromatography (HILIC) were investigated for four zwitterionic tetracyclines (TCs): oxytetracycline, doxycycline, chlortetracycline, and tetracycline. A mixed-mode retention mechanism composed of partitioning, adsorption, and ion exchange interactions was proposed for the amino HILIC retention process. Buffer type and pH significantly influenced the retention of TCs, but showed similar separation selectivity for the tested analytes. Experiments varying buffer salt concentration and pH demonstrated the presence of ion exchange interactions in TCs retention. The type and concentration of organic modifier also affected the retention and selectivity of the analytes, providing direct evidence supporting the Alpert retention model for HILIC. The retention time of the analytes increased in the following order of organic modifiers: tetrahydrofuran < methanol < isopropanol < acetonitrile. The linear relationships of logk' versus %water (v/v) curve and logk' versus logarithm of %water (v/v) in the mobile phase indicated that TCs separation on the amino phase was controlled by partitioning and adsorption. The developed method was successfully utilized in the detection of TCs in both river water and wastewater samples using solid-phase extraction (SPE) for sample cleanup.     

The effect of buffer concentration and cation type in the mobile phase on retention of amino acids and dipeptides in hydrophilic interaction liquid chromatography

The current work is focused on exploring the effect of buffer cation type and its concentration on retention of amino acids, dipeptides and their blocked analogues on two stationary phases, i.e., bare silica and amide-based in hydrophilic interaction liquid chromatography. Five different buffers of pH 4.0 composed of Tris/acetic acid, triethylamine/acetic acid, ammonium/acetic acid, Li⁺/acetic acid and Ba²⁺/acetic acid were used in various concentrations. Interestingly, an increase of the buffer concentration caused increasing, decreasing or stable retention of analytes, according to the cation type in the buffer. The buffers containing barium cations provided the highest retention of all the analytes in comparable mobile phases, i.e., buffers with the same ionic strength and pH on both columns. Moreover, using buffers with barium cation different selectivity for dipeptides was observed. The chromatographic systems with buffers consisting of triethylamine behaved differently compared to others.     

磷霉素改性氧化锆固定相色谱性能研究

本文利用磷霉素与氧化锆表面的强Ｌｅｗｉｓ酸碱作用,分别采用静态和动态两种途径以磷霉素对自制ＺｒＯ２固定相进行改性,考察了改性前后固定相色谱性能的变化。通过磷霉素改性,能够较好地覆盖氧化锆表面存在的Ｌｗｅｉｓ酸活性中心点,从而减少对酸性化合物的不可逆吸附及拖尾现象。磷霉素动态改性氧化锆表现出一定的反相色谱性能,静态改性氧化锆则表现出较强的极性。     

High-performance liquid chromatography of some basic drugs on a n-octadecylphosphonic acid modified magnesia-zirconia stationary phase

The high-performance liquid chromatographic behavior of some basic drugs was studied on a n-octadecylphosphonic acid modified magnesia-zirconia (C18PZM) stationary phase. The effect of mobile phase variables such as methanol content, ionic strength, and pH on their chromatographic behavior was investigated. The retention mechanism of basic drugs on the stationary phase was elucidated. The results indicate that both hydrophobic and cation-exchange interactions contribute to solute retention under most chromatographic conditions. The inherent Br?nsted-acid sites and also the adsorbed Lewis base anionic buffer constituents on accessible ZM surface Lewis acid sites play a role in the retention of ionized solutes by cation-exchange interaction. However, especially at high mobile phase pH, the retention of basic drugs depends mainly on hydrophobic interactions between solutes and support. Separations of the basic drugs on the C18PZM phase by a predominantly reversed-phase retention mode were very promising. The mixed-mode retention feature on this phase, as a result of the adsorbed Lewis base anionic buffer constituents acting as sites for cation-exchange, could also be very useful, e.g. for enhancing the chromatographic selectivity of such analytes. The C18PZM seems to be an excellent alternative to silica-based reversed-phase stationary phase for the separation of strongly basic solutes.     

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.     

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.     

Chromatographic properties of zirconia-based stationary phases for ion exchange chromatography having surface bound cationic functions

A series of non-porous, microspherical zirconia-based stationary phases with surface bound cationic functions have been introduced and evaluated in ion exchange chromatography of proteins and small acidic solutes. Different surface modification procedures were evaluated in the covalent attachment of weak, strong or hybrid anion exchange moieties on the surface of non-porous zirconia micropar-ticles. N,N-Diethylaminoethanol (DEAE) was used as the weak anion exchange ligand while glycidyltrimethylammonium chloride, which was covalently attached to poly(vinyl alcohol) layer (PVAN) on the zirconia surface, constituted the strong anion exchange moiety. Partially quaternarized poly(ethyleneimine) hydroxyethylated (PEI) was used as the hybrid type of anion exchange coating. DEAE-zir-conia microparticles acted as purely cation exchange stationary phases toward basic proteins indicating the predominance of electron donor-electron acceptor interaction (EDA) with surface exposed zirconium sites as well as cation exchange mechanism via electrostatic interaction with unreacted and unshielded hydroxyl groups. PVAN-zirconia stationary phase exhibited anion exchange chromatographic properties toward acidic proteins, but EDA interaction has stayed as an important contributor to solute retention despite the presence of a relatively thick layer of poly(vinyl alcohol) on the surface of the zirconia particles. The modification of zirconia surface with partially quaternarized PEI proved to be the most effective approach to minimize Lewis acidic metallic properties of the support. In fact, PEI-zirconia stationary phase operated as an anion exchanger toward acidic proteins and other small acidic solutes.     

氧化锆及铈-锆复合氧化物的阴离子交换和配体交换色谱性能

高效液相色谱法;氧化锆及铈-锆复合氧化物的阴离子交换和配体交换色谱性能     

Chromatographic characterization of a phosphate-modified zirconia support for bio-chromatographic applications.

A phosphate-modified zirconia was investigated for its potential use as a high-performance inorganic cation-exchange support for the separation of proteins. This phosphate modification effectively blocks the sites responsible for the strong interactions of certain Lewis bases with the zirconia surface. It provides a more "bio-compatible" stationary phase, resulting in high recoveries for proteins and enzymes and retention of their enzymatic activity. The stability, loading capacity, selectivity, efficiency and separation mechanism on the phosphate-modified zirconia are reported. These studies have shown that phosphate-modified zirconia is a useful high-performance ion-exchange support for the separation of cationic proteins and for blocking the sites responsible for the high affinity of zirconia towards certain anions. This makes the phosphate modification interesting in its own right and as an intermediate stage for the development of other zirconia-based chromatographic supports.     

High-performance anion-exchange chromatography of proteins using aza-ether bonded silica-based phases

The use of wide-pore silica-based hydrophilic aza-ether bonded phases for the chromatographic separation of proteins under anion-exchange conditions was studied. Polyether silanes containing terminal morpholine or piperazine derivatives are synthesized for attachment to the silica surface and provide a flexible approach to bonded phase design. In one instance, a quaternized amine support may be prepared by further derivatization of the methylpiperazine bonded phase. The supports provide high-performance anion-exchange chromatographic separations of proteins using gradients of increasing salt content, e.g., to 1.0 M sodium acetate at pH 7.0. The salt type and concentration can be varied to control protein retention while the buffer system used at pH 7.0 exerts a minimal influence on the separation. The anion exchangers may be reproducibly prepared and exhibit chromatographic retention stability at pH 7.5 for at least 2 months of operation. Acceptable capacity for protein on the bonded phase is demonstrated with high recovery of solute mass. The flexibility in anion exchanger design provides a probe of bonded ligand hydrophobic effects which can contribute in an undefined and deleterious manner to the desired ion-exchange separation. Taken together, these results provide a greater insight into the operating characteristics of anion exchangers, especially with regard to competing retention mechanisms.     

Comparison of the chromatography of octadecyl silane bonded silica and polybutadiene-coated zirconia phases based on a diverse set of cationic drugs

In this study, we compare the separation of basic drugs on several octadecyl silane bonded silica (ODS) phases and a polybutadiene-coated zirconia (PBD-ZrO2) phase. The retention characteristics were investigated in detail using a variety of cationic drugs as probe solutes. The ODS phases were selected to cover a relatively wide range in silanol activity and were studied with ammonium phosphate eluents at pH 3.0 and 6.0. Compared to any of the ODS phases, the PBD-ZrO2 phase showed very significant differences in selectivities towards these drugs. Due to the presence of both reversed-phase and ion-exchange interactions between the stationary phase and the basic analyte on ODS and PBD-ZrO2, mixed-mode retention takes place to some extent on both types of phases. However, very large differences in the relative contributions from ion-exchange and reversed-phase interactions on the two types of phases led to quite different selectivities. When phosphate is present in the eluent and adsorbs on the surface, the PBD-ZrO2 phase takes on a high negative charge over a wide pH range due to phosphate adsorption on its surface. On ODS phases, ion-exchange interactions result from the interactions between protonated basic compounds and ionized residual silanol groups. Since the pH of the eluent influences the charge state of the silanol groups, the ion-exchange interactions vary in strength depending on pH. At pH 6.0, the ion-exchange interactions are strong. However, at pH 3.0 the ion-exchange interactions on ODS are significantly smaller because the silanol groups are less dissociated at the lower pH. Thus, not only are the selectivities of the ODS and PBD-ZrO2 phases different but quite different trends in retention are observed on these two types of phases as the pH of the eluent is varied. More importantly, by using the large set of "real" basic analytes we show the extreme complexity of the chromatographic processes on the reversed stationary phases. Both the test condition and solute property influence the column performance. Therefore, use of only one or two probe solutes is not sufficient for column ranking.     

一种可控离子比例的两性亲水色谱固定相的制备及性能研究

将不同比例的氨基和巯基的硅烷偶联剂键合到硅胶表面，再利用巯基与乙烯基膦酸之间的点击化学反应将膦酸基团引入到硅胶表面，制备了一种可调节正负离子比例的两性亲水色谱固定相。通过测定固定相中C、H、N、P元素的含量，证明了氨基与膦酸基团已成功键合到固定相的表面，同时通过N元素与P元素的质量分数确定固定相表面氨基与膦酸基团的比例。制备了3种不同电荷比例的氨基膦酸固定相，将其作为亲水模式下的固定相填料填装在150 mm×4.6 mm不锈钢色谱柱中。以一系列经典的极性小分子作为探针，研究了流动相中乙腈含量、缓冲盐pH值及缓冲盐浓度等因素对探针分子在3种色谱柱上的保留的影响，结果表明，分析物在固定相上是多重保留机理。最后通过比较核苷、水溶性维生素、碱性化合物、苯甲酸这几类标准物质在3种色谱柱上的保留行为来对比3种不同电荷比例的固定相的分离选择性与色谱性能。结果表明，对于不同的分析物，3种固定相表现出完全不同的分离选择性和色谱行为。可以根据分析物的特征选取不同电荷比例的固定相，表明此种固定相在极性化合物的分离上具有良好的应用前景。     

Selectivity and efficiency of different reversed-phase and mixed-mode sorbents to preconcentrate and isolate aroma molecules

A comparison of the ability of different sorbent systems, including mixed-mode resins and reversed-phase sorbents, to extract and isolate volatile molecules from hydroalcoholic medium has been carried out by means of the determination of liquid–solid distribution coefficients. Eighteen volatile compounds covering a wide range of physicochemical properties (acids, bases and neutrals) and chemical functionalities, and thirteen different sorbents have been tested. LiChrolut EN and Isolute ENV (both polymeric with high surface area) showed the highest retention capability for nearly all analytes at all pHs tested. Exceptions were 2,3,5-trimethylpyrazine, most efficiently extracted with Strata XC at acidic pH, and indole best retained with Oasis MCX and Strata XC at any pH. Although nearly all basic compounds were most selectively extracted with cationic mixed-mode resins at acid pHs and 3-mercaptohexyl acetate and m-cresol show maxima α at pH 10 with Oasis MAX, the α values obtained have been relatively low, which suggests that retention is not particularly driven by ionic forces. The study has also shown that selectivity depends on the pH, the exact kind of mixed-mode sorbent and on the polarity of the analyte. High selectivity towards ionogenic compounds can be obtained by combining retention in mixed mode, a rinsing with a non-polar solvent and further elution with a solvent containing a neutralizing agent. However, not all the ionogenic molecules seem to be retained in ionic mode in the conditions tested and the complete elution of some analytes can be difficult, which suggests that analyte-specific isolation procedures should be analyzed case by case.     

A rational approach in the design of selective mesoporous adsorbents

Two MCM-41 derived adsorbents have been tailor-made for the separation of silver and copper ions using the hard-soft, acid-base (HSAB) principle as the design guideline. NH2-MCM-41 containing "hard" Lewis base adsorption sites (i.e., RNH2) was prepared for the adsorption of the "hard" Lewis acid, Cu2+, and SH-MCM-41 with a grafted "soft" thiolpropyl base was prepared for the selective removal of Ag+, a "soft" Lewis acid. Single- and binary-component adsorption studies were conducted at different metal concentrations, solution compositions, and pH values. The experimental results showed that SH-MCM-41 has excellent affinity and capacity for silver adsorption and adsorbed only the silver ions with copper remaining in the solution. The selectivity was not affected by the metal concentration and composition, anion, and pH. Under similar experimental conditions, NH2-MCM-41 selectively adsorbed copper from the binary solution. The selectivity of NH2-MCM-41 remained for the copper at different pH values, although the adsorption capacity diminished at lower pH values. The type of anions used affected copper adsorption on NH2-MCM-41 with an increased copper uptake in the presence of the sulfate ions. A simple Freundlich adsorption model was sufficient to describe metal adsorption on SH-MCM-41 and NH2-MCM-41, and the LeVan and Vermeulen model was successfully used to predict the adsorption capacity and selectivity for binary-component adsorptions.     

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.     

木糖醇和麦芽糖醇型亲水作用色谱固定相的制备及其分离性能的评价

利用-NCO和-OH的加成反应,通过简单的两步反应将木糖醇和麦芽糖醇成功地键合于硅胶表面,制备了两种新型糖醇类亲水作用色谱固定相。流动相中乙腈含量对保留的影响曲线表明,这两种糖醇固定相具有典型的亲水作用色谱固定相性质,对极性和亲水性化合物有很强的保留作用。利用这两种固定相成功分离了水溶性维生素、水杨酸及其类似物、碱基及其相应的核苷和淫羊藿苷类似物等模型混合物,同时糖醇固定相展现了新颖的选择性,特别是相对于线形的木糖醇键合固定相,非线形的麦芽糖醇键合固定相表现出了对糖基的独特保留能力。此外,缓冲盐的pH和浓度对保留的影响表明静电作用在这两种糖醇固定相的保留机理中也发挥着一定的作用。本文所发展的糖醇类固定相具有良好的分离性能,有望在亲水作用色谱分离领域发挥潜在的应用价值。     

Chromatographic behavior of epirubicin and its analogues on high-purity silica in hydrophilic interaction chromatography

Hydrophilic interaction chromatography has been applied for the separation of epirubicin and its analogues using high-purity silica column with aqueous-organic mobile phase. Parameters affecting the chromatographic behavior of the solutes such as organic modifier, buffer pH, ionic strength and sample size, have been investigated. Of utmost importance for successful separation of these analogues is the choice of organic modifier, since it impacts both the solvent selectivity and the ionization of silica silanols as well as buffer solution, and consequently the retention behavior of solutes. Acetonitrile was shown to offer superior separation of these analogues to methanol, isopropanol or tetrahydrofuran. Results of the effects of organic modifier, buffer pH and ion strength indicate that the retention mechanism is a mixed-mode of adsorption and ion exchange. In addition, an irreversible adsorption of these compounds was found on silica in the weakly acidic or neutral mobile phases, and the effect of various factors on irreversible adsorption was also preliminarily discussed. More significantly, these basic compounds have exhibited peaks with a slanted front and a sharp tail, a typical overloading peak profile belonging to the behavior of competitive anti-Langmuir isotherm by increasing the sample size at the experimental conditions.     

Investigation of the ion-exchange properties of methacrylate-based mixed-mode monolithic stationary phases employed as stationary phases in capillary electrochromatography

The potential of methacrylate-based mixed-mode monolithic stationary phases bearing sulfonic acid groups for the separation of positively charged analytes (alkylanilines, amino acids, and peptides) by capillary electrochromatography (CEC) is investigated. The retention mechanism of protonated alkylanilines as positively charged model solutes on these negatively charged mixed-mode stationary phases is investigated by studying the influence of mobile phase and stationary phase parameters on the corrected retention factor which was calculated by taking the electrophoretic mobility of the solutes into consideration. It is shown that both solvophobic and ion-exchange interactions contribute to the retention of these analytes. The dependence of the corrected retention factor on (1) the concentration of the counter ion ammonium and (2) the number of methylene groups in the alkyl chain of the model analytes investigated shows clearly that a one-site model (solvophobic and ion-exchange interactions take place simultaneously at a single type of site) has to be taken to describe the retention behaviour observed. Comparison of the CEC separation of these charged analytes with electrophoretic mobilities determined by open-tubular capillary electrophoresis shows that mainly chromatographic interactions (solvophobic and ion-exchange interactions) are responsible for the selectivity observed in CEC, while the electrophoretic migration of these analytes plays only a minor role.