Evaluation of different column types for the hydrophilic interaction chromatographic separation of iron-citrate and copper-histidine species from plants

Hydrophilic interaction chromatography (HILIC) has emerged as a very useful separation method for polar analytes, including non-covalent metal species. Several types of stationary phases are available for HILIC applications, differing mainly in their chemical functionalities that supply additional interaction modes and alternative selectivities for the separation of special analytes. With regard to the separation of metal species only few of these stationary phases have been applied to date, and it is not completely clear what are their differences with respect to the chromatographic separation of metal species, but also with respect to species stability during chromatography. Here, a comparison of different column types for the HILIC separation of iron citrate and copper histidine species is presented and the results are discussed with respect to retention mechanisms and chromatographic stability of these metal species. It is shown that different stationary phases display very different separation patterns. In particular, three types of HILIC columns enable successful separation of iron citrates and copper histidine at pH 5.5, namely a crosslinked diol phase, a zwitterionic phase, and an amide phase. Two groups of iron-citrates are separated on all three columns, consisting of a species of 3:3 stoichiometry and another one of mainly 3:4 stoichiometry (plus 1:2 and 2:2 species). For copper-histidine only one stable species is found based on the 1:2 stoichiometry. Detection and unambiguous identification of the different species is possible by employing electrospray mass spectrometry in the negative ionization mode. Species found in standard solutions are consistent with species found in spiked plant samples. Also in unspiked solutions iron citrate of 3:4 stoichiometry (plus 1:2 and 2:2) is detectable, but no species of 3:3 stoichiometry. Significant differences of related species patterns are found in real plant samples.     

Simultaneous separation of inorganic anions and metal-citrate complexes on a zwitterionic stationary phase with on-column complexation

The retention and separation selectivity of inorganic anions and on-column derivatised negatively charged citrate or oxalate metal complexes on reversed-phase stationary phases dynamically coated with N-(dodecyl-N,N-dimethylammonio)undecanoate (DDMAU) has been investigated. The retention mechanism for the metal-citrate complexes was predominantly anion exchange, although the amphoteric/zwitterionic nature of the stationary phase coating undoubtedly also contributed to the unusual separation selectivity shown. A mixture of 10 inorganic anions and metal cations was achieved using a 20 cm monolithic DDMAU modified column and a 1 mM citrate eluent, pH 4.0, flow rate equal to 0.8 mL/min. Selectivity was found to be strongly pH dependent, allowing additional scope for manipulation of solute retention, and thus application to complex samples. This is illustrated with the analysis of an acidic mine drainage sample with a range of inorganic anions and transition metal cations, varying significantly in their concentrations levels.     

The effects of temperature and mobile phase on the retention of aliphatic carboxylic acids in hydrophilic interaction chromatography on zwitterionic stationary phases

The influence of the mobile phase and temperature, on the retention behavior of seven aliphatic acids (pyruvic, gluconic, 2‐oxoglutaric, tartaric, malic, oxalic, and citric acid) in hydrophilic interaction liquid chromatography on zwitterionic stationary phases with sulfobetaine and phosphorylcholine ligands is investigated. In agreement with the van't Hoff model, most acids show linear ln k versus 1/T plots. However, the retention of structurally symmetrical oxalic and tartaric dicarboxylic acids is almost independent of temperature, or slightly increases at rising temperature. The experimental parameters of the van't Hoff plots suggest positive entropic contributions to the retention of these symmetrical acids, possibly connected with changes in molecular symmetry on their adsorption. The type of the zwitterionic stationary phase and the mobile phase composition (the molar concentration of acetate buffer and the volume fraction of acetonitrile) affect the retention and the selectivity of the separation of the acids.     

The synthesis and characterization of chemically bonded silica-based zwitterion-exchangers for HPLC

The synthesis and characterization of a zwitterionic stationary phase bonded onto microparticulate silica is described. The bonded zwitterionic phase was characterized by elemental analysis, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), and quantitative analysis of the ligands by high performance liquid chromatography (HPLC) following chemical cleavage from the silica backbone. Chromatographic evaluation of this novel bonded phase indicates that it functions as a weak cation exchanger at pH values above 4.5, an anion exchanger at pH values below 7, and as a zwitterionic phase between these two values. The simultaneous separation of a mixture of cationic, anionic and zwitterionic solutes with this novel bonded phase is shown. Using nucleotides as model compounds, a correlation was developed between maximum solute retention and the pH values corresponding to maximum solute/stationary phase zwitterion overlap. The possibility for a quadrupolar retention mechanism of the bonded zwitterionic phase for zwitterionic solutes is explored.     

Analysis of iron(II)/iron(III) phytosiderophore complexes by nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (nano-ESI-FTICRMS) was employed for the analysis of the phytosiderophore 2'-deoxymugineic acid (DMA) and the candidate ligand for the intracellular iron transport in plants nicotianamine (NA). Due to the zwitterionic nature of NA and DMA, complementary mass spectra were obtained in positive and negative ionization modes. The technique was also used for speciation of their complexes with Fe(II) and Fe(III), respectively. The species observed at pH 7.3 are the 1:1 Fe-ligand complexes and no evidence for the existence of dimeric complexes was observed. NA and DMA differ only by one mass unit. Consequently, in the system NA + DMA + Fe(II)/Fe(III), there are pairs of iron species (i.e. NA-Fe(II) and DMA-Fe(III)) with the same nominal mass, which differ only by approximately 0.02 mass units. It is shown that high-resolution MS accompanied by accurate mass data analysis allows the unequivocal identification of all four iron species (NA-Fe(II), NA-Fe(III), DMA-Fe(II), DMA-Fe(III)) in one solution without separation. We also addressed the possible alteration of the oxidation state of chelated iron under nano-ESI conditions, but no redox reactions were observed under optimized conditions.     

Isoelectric focusing of small non-covalent metal species from plants

IEF is known as a powerful electrophoretic separation technique for amphoteric molecules, in particular for proteins. The objective of the present work is to prove the suitability of IEF also for the separation of small, non-covalent metal species. Investigations are performed with copper-glutathione complexes, with the synthetic ligand ethylenediamine-N,N'-bis(o-hydroxyphenyl)acetic acid (EDDHA) and respective metal complexes (Fe, Ga, Al, Ni, Zn), and with the phytosiderophore 2'-deoxymugineic acid (DMA) and its ferric complex. It is shown that ethylenediamine-N,N'-bis(o-hydroxyphenyl)acetic acid and DMA species are stable during preparative scale IEF, whereas copper-glutathione dissociates considerably. It is also shown that preparative scale IEF can be applied successfully to isolate ferric DMA from real plant samples, and that multidimensional separations are possible by combining preparative scale IEF with subsequent HPLC-MS analysis. Focusing of free ligands and respective metal complexes with di- and trivalent metals results in different pIs, but CIEF is usually needed for a reliable estimation of pI values. Limitations of the proposed methods (preparative IEF and CIEF) and consequences of the results with respect to metal speciation in plants are discussed.     

CE of phytosiderophores and related metal species in plants

Phytosiderophores (PS) and the closely related substance nicotianamine (NA) are key substances in metal uptake into graminaceous plants. Here, the CE separation of these substances and related metal species is demonstrated. In particular, the three PS 2'-deoxymugineic acid (DMA), mugineic acid (MA), and 3-epi-hydroxymugineic acid (epi-HMA), and NA, are separated using MES/Tris buffer at pH 7.3. Moreover, three Fe(III) species of the different PS are separated without any stability problems, which are often present in chromatographic analyses. Also divalent metal species of Cu, Ni, and Zn with the ligands DMA and NA are separated with the same method. By using a special, zwitterionic CE capillary, even the separation of two isomeric Fe(III) chelates with the ligand ethylenediamine-N,N'-bis(o-hydroxyphenyl)acetic acid (EDDHA) is possible (i.e., meso-Fe(III)-EDDHA and rac-Fe(III)-EDDHA), and for fast separations of NA and respective divalent and trivalent metal species, a polymer CE microchip with suppressed EOF is described. The proposed CE method is applicable to real plant samples, and enables to detect changes of metal species (Cu-DMA, Ni-NA), which are directly correlated to biological processes.     

The retention behaviour of amino acids in hydrophilic interaction liquid chromatography on zwitterionic stationary phases†

The retention behaviour of amino acids was studied in hydrophilic LC on zwitterionic stationary phases. Evaluation of the influences of acetonitrile/water content, ammonium acetate (NH₄Ac) concentration and mobile phase pH values was performed. Fourteen amino acids were tested and they were all retained to varying extents, with poorer retention in high water content eluents. The linear relationship between the logarithm of retention factor and log(water content) indicated that adsorption dominated or at least was partly involved in the separation mechanism. Electrostatic and hydrophilic interactions also contributed to the retention of these amino acids under different separation conditions with various mobile phase pH values and NH₄Ac concentrations. Thus, the overall retention mechanism could be explained as a combination of adsorption, electrostatic and hydrophilic interactions. The magnitude and contribution of each mechanism is dependent on the nature of the analyte and the separation conditions applied.     

Stationary phases for hydrophilic interaction chromatography, their characterization and implementation into multidimensional chromatography concepts

Hydrophilic interaction chromatography (HILIC) is becoming increasingly popular for separation of polar samples on polar columns in aqueous-organic mobile phases rich in organic solvents (usually ACN). Silica gel with decreased surface concentration of silanol groups, or with chemically bonded amino-, amido-, cyano-, carbamate-, diol-, polyol-, or zwitterionic sulfobetaine ligands are used as the stationary phases for HILIC separations, in addition to the original poly(2-sulphoethyl aspartamide) strong cation-exchange HILIC material. The type of the stationary and the composition of the mobile phase play important roles in the mixed-mode HILIC retention mechanism and can be flexibly tuned to suit specific separation problems. Because of excellent mobile phase compatibility and complementary selectivity to RP chromatography, HILIC is ideally suited for highly orthogonal 2-D LC-LC separations of complex samples containing polar compounds, such as peptides, proteins, oligosaccharides, drugs, metabolites and natural compounds. This review attempts to present an overview of the HILIC separation systems, possibilities for their characterization and emerging HILIC applications in 2-D off-line and on-line LC-LC separations of various samples, in combination with RP and other separation modes.     

Chiral separation of amino acids by copper(II) complexes of tetradentate diaminodiamido-type ligands added to the eluent in reversed-phase high-performance liquid chromatography: a ligand exchange mechanism

In this paper we report a study on the mechanism of the enantiomeric separation of unmodified D,L-amino acids in RP-HPLC by copper(II) complexes of two tetradentate diaminodiamido ligands, (S,S)-N,N'-bis(phenylalanyl)ethanediamine (PheNN-2) and (S,S)-N,N'-bis(methylphenylalanyl)ethanediamine (Me2PheNN-2), added to the eluent. The aim is to investigate whether and how a copper(II) complex with no free equatorial positions can perform chiral discrimination of bidentate analytes such as unmodified amino acids. The problem is approached in a systematic way by: (a) varying the different chromatographic parameters (pH, selector concentration, eluent polarity); (b) performing chiral separation with the selector adsorbed on the stationary phase; (c) studying the ternary complex formation of these ligands with D- and L-amino acids in solution by glass electrode potentiometry and electrospray ionization MS. All the experimental data are consistent with a mechanism of chiral recognition, based on ligand exchange, which involves as selectors the species [Cu2L2H(-2)]2+ and [CuLH(-2)] and proceeds by displacement of two binding sites from the equatorial positions, giving rise to the ternary species [CuLA]+ and [CuLH(-1) A]. The most important factor responsible for chiral discrimination seems to be the affinity of the diastereomeric ternary complexes for the stationary phase since no enantioselectivity is observed in solution.     

Separation of negatively charged nonsteroidal anti-inflammatory drugs by reversed-phase capillary electrochromatography

Reversed-phase capillary electrochromatography in a 5-microm C18 fully packed capillary was employed to optimize the separation of negatively charged nonsteroidal anti-inflammatory drugs. The effect of the physico-chemical parameters and different analysis modes on the separation of 2-arylpropionic acids was studied and evaluated. The mobile phase composition, buffer type, concentration and pH differently influenced the peak efficiency and resolution, selectively modulating the analytes interaction with the stationary phase. The use of zwitterionic MES or acetate mobile phases strongly modulated the analytes migration order and peak efficiency. The optimum experimental conditions were found in MES buffer, pH 5.0, containing the 75% acetonitrile-methanol (1:1). All the analytes were baseline separated in a mixture in less than 13 min with peak efficiencies in the range of 78,500-84,200 N/m. Under these conditions the analytes were negatively charged and their effective electrophoretic mobilities played a role in the separation. The analysis of different pharmaceutical preparations containing anti-inflammatory drugs, e.g. drops and tablets, is also presented after a very simple sample pretreatment.     

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。     

Retention of ionizable compounds in high-performance liquid chromatography. IX. Modelling retention in reversed-phase liquid chromatography as a function of pH and solvent composition with acetonitrile-water mobile phases

The influence of pH and solvent composition of acetonitrile-water mobile phases on the retention of acids and bases on a polymeric stationary phase is studied. Very good relationships between retention and mobile phase pH are obtained if the pH is measured in the proper pH scale. The fit of retention to pH for a particular solvent composition provides the pKa values of the equilibria between the different acid-base species and the retention parameters of these species at this solvent composition. Several models are tested that relate these parameters to solvent composition and properties in order to propose a general model to predict retention for any mobile phase pH and composition.     

原子转移自由基聚合技术制备新型手性配体交换色谱固定相及对手性化合物的拆分

采用原子转移自由基聚合(ATRP)技术, 以溴代硅胶为引发剂, CuCl/2,2'-联吡啶(Bpy)为催化体系, 水为溶剂, N-丙烯酰基-L-脯氨酸为单体, 室温下在硅胶表面进行聚合反应, 制得硅胶接枝聚N-丙烯酰基-L-脯氨酸分子刷. 通过改变ATRP反应体系中单体的量, 制备了3种不同键合量且键合量可控的手性配体交换色谱固定相, 利用元素分析和热重分析对其进行表征. 考察了配体接枝率、 流动相Cu²⁺浓度、 pH值和柱温等对DL-氨基酸和α-羟基酸拆分的影响, 优化了色谱分离条件, 探讨了拆分过程的热力学. 结果表明, 所合成的手性配体交换色谱固定相能够分离9种DL-氨基酸和α-羟基酸, 其中DL-酪氨酸、 DL-色氨酸和DL-苏氨酸3种氨基酸可同时进行拆分, 且拆分过程由熵控制.     

Preparation and application of novel zwitterionic monolithic column for hydrophilic interaction chromatography

A novel zwitterionic hydrophilic porous monolithic stationary phase was prepared based on the thermal‐initiated copolymerization of N,N‐dimethyl‐N‐(3‐methacryl‐amidopropyl)‐N‐(3‐(sulfopropyl)ammonium betaine and ethylene glycol dimethacrylate. A typical hydrophilic separation mechanism was observed at a highly organic mobile phase (ACN >60%) on this optimized zwitterionic hydrophilic interaction chromatography (HILIC) monolithic stationary phase. Good permeability, stability, and column efficiency were observed on the final monolithic column. Additionally, a weak electrostatic interaction for charged analytes was confirmed in analysis of six benzoic acids by studying the influence of mobile phase pH and salt concentration on their retention behaviors on the obtained zwitterionic HILIC monolithic column. The optimized zwitterionic HILIC monolith exhibited good selectivity for a range of polar test analytes.     

Separation efficiencies in hydrophilic interaction chromatography

Hydrophilic interaction chromatography (HILIC) is important for the separation of highly polar substances including biologically active compounds, such as pharmaceutical drugs, neurotransmitters, nucleosides, nucleotides, amino acids, peptides, proteins, oligosaccharides, carbohydrates, etc. In the HILIC mode separation, aqueous organic solvents are used as mobile phases on more polar stationary phases that consist of bare silica, and silica phases modified with amino, amide, zwitterionic functional group, polyols including saccharides and other polar groups. This review discusses the column efficiency of HILIC materials in relation to solute and stationary phase structures, as well as comparisons between particle-packed and monolithic columns. In addition, a literature review consisting of 2006-2007 data is included, as a follow up to the excellent review by Hemstr?m and Irgum.     

Buffer Choice for Tuning the Selectivity in Reverse Phase Chromatography

Abstract

Retention behaviour of ionogenic species in high-performance liquid chromatography on reversed phase materials was studied, specifically dependence of buffer quality applied to mobile phases. The buffers' effect on retention of organic acids, amino acids and dipeptides is quantified by modelling capacity factors as a function of pH-values. At constant ionic strength, increasing capacity factors were observed going from phosphate to less polar citrate buffer, modification of accessible silanol groups of the stationary phase being responsible for this effect. Application of citrate buffer for separation of a seven-component mixture is demonstrated on the basis of a computerized search for optimum chromatographic performance. The evaluated factor levels (pH, methanol content and ionic strength) differ from those found using phosphate buffer-containing mobile phases.     

Development and evaluation of new zwitterionic hydrophilic interaction liquid chromatography stationary phases based on 3-P,P-diphenylphosphonium-propylsulfonate

New zwitterionic stationary phases were synthesized by covalently bonding 3-P,P-diphenylphosphonium-propylsulfonate to silica gel. The resulting materials possess both a negatively charged sulfonate group and a positively charged quaternary phosphonium group, which means that there is no net charge over a wide pH range. The retention mechanism and chromatographic behavior of polar solutes under HILIC conditions were studied on these zwitterionic phases. Compared to the commercial ZIC-HILIC column and a bare silica gel stationary phase, the newly synthesized zwitterionic stationary phases provided greater retention, higher peak efficiency and better peak symmetry in the HILIC mode. The analytes examined included: β-blockers, nucleic acid bases and nucleosides, salicylic acid and its analogues, and water soluble vitamins. Factors, such as the type of organic modifiers, solvent composition, pH and the buffer concentration of the mobile phase, have been considered as potential variables for controlling the chromatographic retention of polar analytes.     

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.     

Use of surfactant-mediated phase separation (cloud point extraction) with affinity ligands for the extraction of hydrophilic proteins

The feasibility of utilizing a zwitterionic surfactant, 3-(nonyldimethylammonio)propylsulfate, or nonionic surfactant, Triton X-114, mediated phase separation in conjunction with affinity ligands was studied for hydrophilic protein extractions. Below (or above) its critical temperature (so-called cloud point), aqueous solutions of zwitterionic (or nonionic) surfactants separate into two immiscible phases, a surfactant-rich phase and an aqueous phase. Avidin was successfully extracted into the zwitterionic surfactant-rich phase when a small amount of the affinity ligand, N- biotinoyl)dipalmitoyl- l -alpha- phosphatidyl ethanolamine, was added to the system. It was not possible to extract hexokinase into the surfactant-rich phase of the nonionic surfactant, Triton X-114, even if a considerable amount of octyl-beta-d-glucoside was added to the solution as an affinity ligand. In contrast, the use of the zwitterionic surfactant and octyl-beta-d-glucoside as an affinity ligand proved to be effective for the extraction of hexokinase. The hexokinase extraction efficiency was found to depend upon the solution pH and the concentration of the affinity ligand in the system. The results clearly indicate that hydrophilic proteins can be successfully extracted with surfactant mediated phase separations (cloud point extractions) via use of the zwitterionic surfactant, 3-(nonyldimethylammonio)propylsulfate, and appropriate affinity ligands. Some advantages of zwitterionic surfactants in such extractive processes relative to that of nonionic surfactants are delineated.