Contributions of commercial sorbents to the selectivity in immobilized metal affinity chromatography with Cu(II)

Immobilized copper(II) affinity chromatography [Cu(II)-immobilized metal affinity chromatography (IMAC)] has been used in proteomics to simplify sample mixtures by selecting histidine-containing peptides from proteolytic digests. This paper examines the specificity of four different support materials with an iminodiacetic acid (IDA) stationary phase in the selection of only histidine-containing peptides in the single step capture-release mode. Three of the sorbents examined were commercially available: HiTrap Chelating HP (agarose), TSK Chelate-5PW, and Poros 20MC. IDA was also immobilized on CIM discs (monolithic glycidylmethacrylate-ethylene dimethacrylate). Tryptic digests of transferrin and beta-galactosidase were used as model samples to evaluate these sorbents. It was found that among the examined matrices, the TSK Chelate-5PW sorbent bound histidine-containing peptides the strongest, while Poros matrix was found to have a high degree of non-specific bindings. Agarose-based columns showed relatively high selectivity and specificity.     

丁基胺丙基硅胶毛细管整体柱的制备及其电色谱性能研究

采用溶胶-凝胶技术制备了丁基胺丙基硅胶毛细管整体柱,此整体固定相表面同时含有能产生阳极的电渗流的仲胺官能团和产生疏水作用的正丁基和丙基官能团。对所制备的整体柱电色谱性能进行了详细的表征和分析。考察了流动相pH值对电渗流的影响;对烷基苯同系物、有机酸酸性化合物和苯胺类碱性化合物保留行为进行了研究,并对其可能的保留机理进行了探讨。实验结果表明,对于中性化合物的保留机理主要基于反相作用;而对于酸性化合物的保留行为则是基于混合模式作用机理,即除了电泳作用外,还包括阴离子交换和疏水作用。碱性化合物在丁基胺丙基硅胶毛细管整体柱上的峰形较好,没有明显的峰拖尾现象。     

Preparation of monolithic silica columns for high-performance liquid chromatography

Preparation methods of monolithic silica columns for HPLC including the surface modification were reviewed. Chemical modification methods recently reported to obtain stationary phases for reversed-phase (RP), chiral, ion-exchange, and hydrophilic interaction chromatography (HILIC) separations were discussed. Recent results related to preparation methods of monolithic silica were also covered. The characteristics and properties of silica monoliths and some applications of monolithic silica columns for different analytical and bioanalytical fields will be commented.     

Photografted methacrylate‐based monolithic columns coated with cellulose tris(3,5‐dimethylphenylcarbamate) for chiral separation in CEC

A chiral capillary monolithic column for enantiomer separation in capillary electrochromatography was prepared by coating cellulose tris(3,5‐dimethylphenylcarbamate) on porous glycidyl methacrylate‐co‐ethylene dimethacrylate monolith in capillary format grafted with chains of [2(methacryloyloxy)ethyl] trimethylammonium chloride. The surface modification of the monolith by the photografting of [2(methacryloyloxy)ethyl] trimethylammonium chloride monomer as well as the coating conditions of cellulose tris(3,5‐dimethylphenylcarbamate) onto the grafted monolithic scaffold were optimized to obtain a stable and reproducible chiral stationary phase for capillary electrochromatography. The effect of organic modifier (acetonitrile) in aqueous mobile phase for the enantiomer separation by capillary electrochromatography was also investigated. Several pairs of enantiomers including acidic, neutral, and basic analytes were tested and most of them were partially or completely resolved under aqueous mobile phases. The prepared monolithic chiral stationary phases exhibited a good stability, repeatability, and column‐to‐column reproducibility, with relative standard deviations below 11% in the studied electrochromatographic parameters.     

Pressurized CEC of neutral and charged solutes using silica monolithic stationary phases functionalized with 3-(2-aminoethylamino)propyl ligands

A novel silica monolithic stationary phase functionalized with 3-(2-aminoethylamino)propyl ligands for pressurized CEC has been presented. The monolithic capillary columns were prepared by a sol-gel process in 75 microm id fused-silica capillaries and followed by a chemical modification. The diamino groups on the surface of the stationary phase are meant to generate the chromatographic surface and a substantial anodic EOF as well as to provide electrostatic interaction sites for charged solutes. The electrochromatographic characterization and column performance were evaluated by a variety of neutral and charged solutes. It was observed that the anodic EOF for the diamine-bonded monolith was greatly affected by the reaction time with 3-(2-aminoethylamino)propyltrimethoxysilane and the PEG amount in the sol-gel reaction mixture in addition to the mobile phase conditions. The monolithic stationary phase exhibited hydrophilic interaction chromatographic behavior toward neutral solutes. Good separations of various solutes including phenols, nucleic acid bases, nucleosides and nucleotides were achieved under different experimental conditions. Fast and efficient separations were obtained with high plate counts reaching more than 130,000 plates/m.     

有机-无机杂化硅胶基质整体柱的制备及其电色谱性能

采用溶胶-凝胶法,以四乙氧基硅烷和苯基三乙氧基硅烷作为反应单体,通过酸碱两步催化在毛细管中进行原位缩聚反应,制备了新型有机-无机杂化硅胶基质毛细管整体柱,制备过程简单。整体柱基质中均匀分布的苯基基团可直接用于反相毛细管电色谱的分离,因而不需要对基质再进行衍生化。优化了整体柱的制备条件,采用扫描电镜和压汞法对整体柱的微观结构和孔径分布进行了表征。分别考察了溶胶-凝胶初始反应液中水的用量对柱床结构的影响和两种单体的配比对材料孔径分布的影响。研究了稠环芳烃类化合物在整体柱上的保留行为,用所制备的整体柱分离了7种苯酚类化合物,平均柱效达100000塔板/m。     

Chemically L-prolinamide-modified monolithic silica column for enantiomeric separation of dansyl amino acids and hydroxy acids by capillary electrochromatography and mu-high performance liquid chromatography

A silica-based chiral monolithic column prepared by sol-gel process and chemical modification of chiral selector was used for enantioseparation of dansyl amino acids and hydroxy acids by capillary electrochromatography (CEC) and mu-high-performance liquid chromatography (mu-HPLC). L-Prolinamide was modified as a chiral selector. The chiral stationary phase (CSP), the chiral complex of Cu(II) with L-prolinamide, provides an anodic electroosmotic flow (EOF) in CEC. The EOF was found to be dependent on applied electric field strength, the pH, and the composition of mobile phases. Scanning electron micrograph showed that monolithic columns have the morphology of continuous skeleton and large through-pore. D-Enantiomers migrated before L-enantiomers except for dansyl-(Dns)-DL-Ser. The separation efficiencies of up to 17600 (D) and 13,200 plates m(-1) (L) were achieved for the separation of DL-indole-3-lactic acid.     

Thermoresponsive polymer brush on monolithic-silica-rod for the high-speed separation of bioactive compounds

Poly(N-isopropylacrylamide), one of the most utilized thermoresponsive polymers, brush-grafted monolithic-silica columns were prepared through surface-initiated atom transfer radical polymerization (ATRP) for effective thermoresponsive-chromatography matrices. ATRP initiator was grafted on monolithic silica-rod surfaces by flowing a toluene solution containing ATRP initiator into monolithic silica-rod columns. N-Isopropylacrylamide (IPAAm) monomer and CuCl/CuCl(2)/Me(6)TREN, an ATRP catalytic system, were dissolved in 2-propanol, and the reaction solution was pumped into the preprepared initiator-modified columns at 25 °C for 16 h. The constructed PIPAAm-brush structure on the monolithic silica-rod surface was confirmed by XPS, elemental analysis, SEM observation, and GPC measurement of grafted PIPAAm. The prepared monolithic silica-rod columns were also characterized by chromatographic analysis. PIPAAm-brush-modified monolithic silica-rod columns were able to separate hydrophobic steroids with a short analysis time (10 min), compared to PIPAAm-brush-modified silica-beads-packed columns, because of the horizontally limited diffusion path length of monolithic supporting materials. Additionally, diluted PIPAAm-brush monolithic silica-rod column gave a further shorting analysis time (5 min). These results indicated (1) surface-initiated ATRP constructed PIPAAm-brush structures on monolithic silica-rod surfaces and (2) PIPAAm-brush grafted monolithic silica-rod column prepared by ATRP was a promising tool for analyzing hydrophobic-bioactive compounds with a short analysis time.     

Steroid profiles determined by capillary electrochromatography, laser-induced fluorescence detection and electrospray-mass spectrometry

Macroporous, monolithic capillary electrochromatography (CEC) columns, featuring a hydrophobic stationary phase, have been applied to the separations of steroids with good column efficiency. Using isocratic and gradient elution runs, mixtures of neutral or conjugated steroids could be resolved. While dansylated ketosteroids were detectable through laser-induced fluorescence at attomole levels, the CEC columns coupled to electrospray-ion-trap mass spectrometry featured femtomole detection limits.     

Characterization of some physical and chromatographic properties of monolithic poly(styrene-co-divinylbenzene) columns

Monolithic capillary columns were prepared by copolymerization of styrene and divinylbenzene inside a 200 microm i.d. fused silica capillary using a mixture of tetrahydrofuran and decanol as porogen. Important chromatographic features of the synthesized columns were characterized and critically compared to the properties of columns packed with micropellicular, octadecylated poly(styrene-co-divinylbenzene) (PS-DVB-C18) particles. The permeability of a 60 mm long monolithic column was slightly higher than that of an equally dimensioned column packed with PS-DVB-C18 beads and was invariant up to at least 250 bar column inlet pressure, indicating the high-pressure stability of the monolithic columns. Interestingly, monolithic columns showed a 3.6 times better separation efficiency for oligonucleotides than granular columns. To study differences of the molecular diffusion processes between granular and monolithic columns, Van Deemter plots were measured. Due to the favorable pore structure of monolithic columns all kind of diffusional band broadening was reduced two to five times. Using inverse size-exclusion chromatography a total porosity of 70% was determined, which consisted of internodule porosity (20%) and internal porosity (50%). The observed fast mass transfer and the resulting high separation efficiency suggested that the surface of the monolithic stationary phase is rather rough and does not feature real pores accessible to macromolecular analytes such as polypeptides or oligonucleotides. The maximum analytical loading capacity of monolithic columns for oligonucleotides was found to be in the region of 500 fmol, which compared well to the loading capacity of the granular columns. Batch-to-batch reproducibility proved to be better with granular stationary phases compared to monolithic stationary phase, in which each column bed is the result of a unique column preparation process.     

High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry

High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.     

A study of the effects of column porosity on gradient separations of proteins

The type of the stationary phase for reversed-phase liquid chromatography significantly affects the sample elution. Hydrodynamic properties, efficiency and gradient elution of proteins were investigated on five commercial C18 columns with wide-pore totally porous particles, with superficially porous layer particles, non-porous particles and a silica-based monolithic bed. The efficiency in the terms of reduced plate height is higher for low-molecular ethylbenzene than for proteins, but depends on the character of the pores in the individual columns tested. The superficially porous Poroshell and the non-porous Micra columns provide the best efficiency for proteins at high mobile phase flow rates, probably because of similar pore architecture in the stationary phase. The Zorbax column with similar pore architecture as the Poroshell active layer, i.e. narrow pore distribution of wider pores shows better efficiency than the packed column with narrow pores and broad pore distribution. The monolithic column shows lower efficiency for proteins at high flow rates, but it performs better than the broad-pore distribution totally porous particulate columns. Different pore architecture affects also the retention and selectivity for proteins on the individual columns. The retention times on all columns can be predicted using the model for reversed-phase gradient elution developed originally for low-molecular compounds. Consideration of the limited pore volume accessible to the biopolymers has negligible effect on the prediction of retention on the columns packed with non-porous or superficially porous particles, but improves the accuracy of the predicted data for the totally porous columns with broad pore distribution.     

Preparation and characterization of alkylated polymethacrylate monolithic columns for micro-HPLC of proteins

Protein separations were carried out by micro-high performance liquid chromatography (micro-HPLC) with surface alkylated monolithic columns, which were prepared by in situ copolymerization of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) in the presence of dodecanol and toluene as porogens. First, glycidyl groups at the surface of the porous monolith were hydrolyzed with sulfuric acid. The hydroxyl groups thus formed were then reacted with n-alkyl chloride to form alkylated stationary phase. Separation performance for proteins on columns with C18 and C8 stationary phases was compared. The results showed that a poly(GMA-EGDMA) support derivatized with octadecyl moieties could achieve much better resolution than one with octyl groups. A protein mixture was separated with the octadecylated poly-(GMA-EGDMA) monolithic column, and the effluent peaks were collected and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The physical properties of the monolithic columns such as column morphology, surface area, mesopore size distribution, and column permeability were further characterized by scanning electron microscopy (SEM), multipoint BET nitrogen adsorption/desorption, and Darcy's law, respectively.     

Iminodiacetic acid functionalised organopolymer monoliths: application to the separation of metal cations by capillary high-performance chelation ion chromatography

Lauryl methacrylate-co-ethylene dimethacrylate monoliths were polymerised within fused silica capillaries and subsequently photo-grafted with varying amounts of glycidyl methacrylate (GMA). The grafted monoliths were then further modified with iminodiacetic acid (IDA), resulting in a range of chelating ion-exchange monoliths of increasing capacity. The IDA functional groups were attached via ring opening of the epoxy group on the poly(GMA) structure. Increasing the amount of attached poly(GMA), via photo-grafting with increasing concentrations of GMA, from 15 to 35 %, resulted in a proportional and controlled increase in the complexation capacity of the chelating monoliths. Scanning capacitively coupled contactless conductivity detection (sC⁴D) was used to characterise and verify homogenous distribution of the chelating ligand along the length of the capillaries non-invasively. Chelation ion chromatographic separations of selected transition and heavy metals were carried out, with retention factor data proportional to the concentration of grafted poly(GMA). Average peak efficiencies of close to 5,000 N/m were achieved, with the isocratic separation of Na, Mg(II), Mn(II), Co(II), Cd(II) and Zn(II) possible on a 250-mm-long monolith. Multiple monolithic columns produced to the same recipes gave RSD data for retention factors of <15 % (averaged for several metal ions). The monolithic chelating ion-exchanger was applied to the separation of alkaline earth and transition metal ions spiked in natural and potable waters.     

整体柱离子色谱的研究进展

该文介绍了离子色谱的分类,整体柱的分类、制备及特点,并以此为依据归纳总结了常规整体柱在离子色谱中的应用和毛细管整体柱在毛细管离子色谱中的应用,其中包括硅胶基质整体柱和聚合物基质整体柱,评述并展望了整体柱离子色谱的发展前景.     

Development of a new C14 monolithic silica column containing embedded polar groups for pressurized capillary electrochromatography

Monolithic columns have been prepared with a novel bonded silica stationary phase, tetradecylamine bonded silica (TDAS), and used in pressurized capillary electrochromatography (pCEC). The monolithic silica column matrix was prepared by a sol-gel process and then chemically modified with the spacer (3-glycidoxypropyl)trimethoxysilane and tetradecylamine. The introduced embedded polar amine groups dominated the charge on the surface of the monolithic stationary phase and generated an EOF from cathode to anode under acidic conditions. The tetradecyl hydrophobic chains in TDAS provide chromatographic interactions. The chromatographic characteristics of the prepared monolithic column were studied. Some aromatic compounds including alkylbenzenes, aromatic hydrocarbons, phenols, and anilines were successfully separated on the TDAS monolithic column in pCEC mode. As expected, the TDAS monolithic stationary phases exhibit typical reversed-phase electrochromatographic behavior toward neutral solutes due to the introduced tetradecyl groups. Hydrophobic as well as electrophoretic migration processes within the monoliths were observed in the separation of basic anilines. Symmetrical peaks can be obtained for anilines because the embedded polar amine groups on the surface can effectively shield the adsorption of positively charged analytes onto the stationary phase.     

New Stationary Phase Prepared by Immobilization of a Copper-Amine Complex on Silica and Its Use for High Performance Liquid Chromatography

Chromatographic silica (10 μm) was chemically modified with the silylating agent: [3-(2-aminoethyl)aminopropyl]trimethoxysilane (AEAPTS). The reaction product was characterized by elemental analysis and infrared and ¹³C and ²⁹Si NMR spectra. The chemically modified silica was treated with Cu(II) in methanol medium. This cation was strongly adsorbed through complexation by the pendant ethylenediamine groups attached to the silica surface. The complex formed on the silica surface was shown to be stable in both aqueous and non-aqueous media. The aim of Cu(II) immobilization is to use this new material as a stationary phase in High Performance Liquid Chromatography (HPLC). Separations of synthetic mixtures of aromatic amines and of polyaromatic hydrocarbons were undertaken using 150×3.9 mm HPLC columns packed with the modified silica, with and without copper ions, to follow the influence of the cation on the chromatographic separation and to verify the efficiency of the new stationary phase for HPLC.     

Preparation of High‐capacity,Monodisperse Polymeric Weak Cation Exchange Packings Using Surface‐initiated Atom Transfer Radical Polymerization and Its Chromatographic Properties

A novel stationary phase for weak cation exchange (WCX) chromatography was prepared by "grafting from" strategy. Surface initiated atom transfer radical polymerization (ATRP) of acrylic acid (AA) was conducted in toluene medium, starting from the macromolecule initiators of poly(4‐vinylbenzyl chloride‐co‐divinylbenzene) (P_CMS/DVB) beads. The amounts of poly(acrylic acid) grafted chains with different ATRP formulations were calculated based on the elemental analyses. The poly(acrylic acid) grafted beads obtained with different ATRP formulations were tried as chromatographic packings in the separation of proteins by ion‐exchange chromatography. The effect of the poly(acrylic acid) grafted chain lengths on P_CMS/DVB beads for the separation of proteins was investigated in details. Simultaneously, characterization of the column was investigated as ion chromatographic stationary phase for the separation of inorganic cations. The results show that poly(acrylic acid) grafted columns had excellent performance for separation of proteins and inorganic cations. The highest of the dynamic capacity of the column was 35.55 mg/mL. The columns were provided with high column efficiency.     

Polymeric monolithic stationary phases for capillary electrochromatography

This review summarizes the contributions of a number of groups working in the rapidly growing area of monolithic columns for capillary electrochromatography (CEC), with a focus on those prepared from synthetic polymers. Monoliths have quickly become a well-established stationary phase format in the field of CEC. The simplicity of their in situ preparation method as well as the good control over their porous properties and surface chemistries make the monolithic separation media an attractive alternative to capillary columns packed with particulate materials. A wide variety of approaches as well as materials used for the preparation of the monolithic stationary phases are detailed. Their excellent chromatographic performance is demonstrated by numerous separations of different analytes.     

Adamantyl-functionalized polymer monolith for capillary electrochromatography

An adamantyl (ADM)-functionalized monolithic stationary phase was newly synthesized by a single-step copolymerization of 1-adamantyl-(α-trifluoromethyl) acrylate, ethylene dimethacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid in order to prevent the peak tailing of basic solutes in capillary electrochromatography and was compared with butyl methacrylate (BMA)-based one. The ADM structure shields the negatively charged groups on the surface of monolith from basic solutes, resulting in better peak shapes than BMA-based monolithic stationary phase. As the monomers ratio decreased, the monolithic column had lower retention and higher column efficiency which was likely due to lower phase ratio and smaller globule size of monolith, respectively. The ADM-functionalized monolithic columns exhibited a good repeatability and reproducibility of column preparation with relative standard deviation values below 9% in the studied chromatographic parameters.