多齿配体-硅胶色谱介质的制备与评价

以新型环保多齿螯合剂——亚氨基二琥珀酸(IDS)为配体,在优化条件下,合成了IDS-Silica固定相。用电位滴定法测定了固定相上IDS的键合量。考察了IDS-Silica柱的色谱特性以及金属螯合特性。使用制备柱成功地分离了标准蛋白质混合物,该制备柱展现出了典型的阳离子交换特性。用电感耦合等离子体原子发射光谱法考察了金属离子在IDS-Silica固定相上的键合特性。结果表明,金属离子在IDS-Silica固定相上键合量的变化规律与它们同该固定相螯合的强弱顺序一致。通过比较金属Cu~(2+)在4种不同氨羧类配体硅胶柱上的键合量,发现IDS对金属离子具有强的螯合能力。IDS对金属离子的强螯合特性为其今后作为固定金属亲和色谱填料奠定了基础,为缓解亲和柱在使用过程中固定金属离子的流失提供了一种有效的解决方法。     

Complexation ion chromatography —an overview of developments and trends in trace metal analysis

Complexation ion chromatography (IC), including all ion chromatographic modes in which complexation is exploited for the separation and detection of metal ions in different ways, is now a widely accepted method of trace metal analysis. Some of the significant advances in the theoretical aspects and practical applications of complexation IC modifications (non-suppressed cation chromatography with complex- forming mobile phases, coordination chromatography with chelate-forming bonded phases, ion-exchange and ion-pair chromatography of anionic metal chelates) recently developed in the authors' laboratories are reviewed. The retention behaviour and separation mechanism of non-complexed and completed metal analytes are discussed from the point of view of basic coordination chemistry (stability of metal complexes, effective charge of metal atom, ligand completing ability, etc.). Comparisons and contrasts between various metal complexation IC techniques and their common features and advantages relative to other methods used in analyses for transition and heavy metal ions are evaluated.     

Selective Pre-concentration and Solid Phase Extraction of Mercury（Ⅱ） from Natural Water by Silica Gel-loaded （E）-N-（1-Thien-2＇-ylethylidene）-1,2-phenylenediamine Phase

Silica gel-loaded （E）-N-（1-thien-2＇-ylethylidene）-1,2-phenylenediamine （TEPDA） phase was synthesized based on physical adsorption approaches. The stability of a chemically modified TEPDA especially in concentrated hydrochloric acid that was then used as a recycling and preconcentration reagent allowed the further uses of silica gel-loaded immobilized TEPDA phase. The application of this silica gel-loaded phase to sorption of a series of metal ions was performed by using different controlling factors such as the pH of the metal ion solution and the equilibration shaking time by the static technique. This difference was interpreted on the basis of selectivity incorporated in these sulfur containing silica gel-loaded TEPDA phases. Hg（Ⅱ） was found to exhibit the highest affinity towards extraction by these silica gel-loaded TEPDA phases. The pronounced selectivity was also confirmed by the determined distribution coefficients （Kd） of all the metal ions, showing the highest value reported for mercury（Ⅱ） extraction by the silica gel immobilized TEPDA phase. The potential applications of the silica gel immobilized TEPDA phase to selective extraction of mercury（Ⅱ） from aqueous solution were successfully accomplished and preconcentration of low concentration of Hg（Ⅱ） （30 pg·mL^-1） from natural tap water with a preconcentration factor of 200 for Hg（Ⅱ） off-line analysis was conducted by cold vapor atomic absorption analysis.     

Effects of Immobilized Metal Ion on Retention Behaviors of Proteins in Metal Chelate Affinity Chromatography

The chromatographic behaviors of proteins on iminodiacetic acid (IDA) column with and without immobilized metal ion were examined in detail. Comparing the effects of pI, solution pH, and salt concentration on retention of proteins in cation‐exchange chromatography (CEC) and metal chelate affinity chromatography (MCAC), the retention mechanism of proteins was investigated in MCAC. By aid of observing the retention characteristics of proteins on naked IDA and metal chelate columns in high concentration salt‐out salt solution, the hydrophobic interaction in MCAC and the influence of metal ion on it were proved. In terms of the comparison of the thermodynamics of proteins in CEC and MCAC, the thermostability, the conformational change entropy Δ(ΔS⁰) and enthalpy Δ(ΔH⁰), compensation temperature β, the driving force and caloritic effect of proteins in MCAC were discussed. The identity of retention mechanism at protein thermal denaturation in CEC and MCAC was demonstrated by using the compensation relationship between ΔH⁰ and ΔS⁰.     

Ion‐exchange vs reversed‐phase chromatography for separation and determination of basic psychotropic drugs

Ion exchange chromatography, an alternative to reversed‐phase (RP) chromatography, is described in this paper. We aimed to obtain optimal conditions for the separation of basic drugs because silica‐based RP stationary phases show silanol effect and make the analysis of basic analytes hardly possible. The retention, separation selectivity, symmetry of peaks and system efficiency were examined in different eluent systems containing different types of buffers at acidic pH and with the addition of organic modifiers: methanol and acetonitrile. The obtained results reveal a large influence of the salt cation used for buffer preparation and the type of organic modifier on the retention behavior of the analytes. These results were also compared with those obtained on an XBridge C₁₈ column. The obtained results demonstrated that SCX stationary phases can be successfully used as alternatives to C₁₈ stationary phases in the separation of basic compounds. The most selective and efficient chromatographic systems were applied for the quantification of some psychotropic drugs in fortified human serum samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Preparation of Immobilized Metal Affinity Chromatographic Packings by Immobilization of Carboxymethylated Asparate (CM-Asp) Based on Monodisperse Hydrophilic Non-porous Beads and Their Application

The hydrophilic immobilized metal affinity chromatographic packing was prepared by immobilization of carboxymethylated asparate (CM‐Asp) as chelating ligand and Ni²⁺ as center ion on the base of monodispersed, 3.0 µm non‐porous monodisperse poly(glycidylmethacrylate‐co‐ethylenedimethacrylate) (P_GMA/EDMA) particles. The retention behavior of proteins and the effect of pH on the retention in the range from 4.0 to 9.0 were investigated on both the naked and metal ion chelated columns. Four proteins were quickly separated in 2.0 min with linear gradient elution at a flow rate of 3.0 mL·min^?1 by using the synthesized Ni²⁺‐CM‐Asp‐P_GMA/EDMA packings. The separation time was shorter than other immobilized metal affinity chromatography reported in the literature. The Ni²⁺‐CM‐Asp‐P_GMA/EDMA column was further investigated for the rapid separation and purification of copper‐zinc superoxide dismutase (Cu,Zn‐SOD) from the blood of pig in 3.0 min with only one step. The results obtained were satisfactory.     

Synthesis, characterization and structure effects on selectivity properties of silica gel covalently bonded diethylenetriamine mono- and bis-salicyaldehyde and naphthaldehyde Schiff(,)s bases towards some heavy metal ions

Four silica gel-immobilized new metal chelate Schiff(,)s bases were synthesized (I-IV). Silica gel chemically bonded diethylenetriamine mono-naphthaldehyde and mono-salicyaldehyde Schiff's bases (phases I and III) were produced via the interaction of silica gel-modified diethylenetriamine with naphthaldehyde and salicylaldehyde, respectively. However, phases II and IV arose through the interaction of bis-naphthaldehyde and bis-salicylaldehyde Schiff(,)s bases of diethylenetriamine with 3-chloropropyltrimethoxysilane modified silica gel. The characterization of such new phases, their capabilities towards selective extraction or separation of Fe(III), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II) ions were studied and evaluated by both batch and column techniques as a function of pH and time of contact. Phases III and I showed high performance towards Cu(II) extraction, where their Cu(II) sorption determined to be 0.957 and 0.940 mmol g(-1), respectively. However, for phases IV and II, the great affinity was devoted to Fe(III) extraction followed by Cu(II) ions. The reactivity of metal ion sorption was discussed in the light of effects of bulkiness as well as orientation of immobilized chelate on sorbent reactivity. Donor sites of phases III and I (diethylenetriamine and azomethene nitrogens along with phenolic hydroxyl group oxygen) are fully active, whereas phases IV and II are partially active with only participation of oxygen and azomethene nitrogen. The order of increasing thermal stability (IV相似文献   

Phosphoprotein electrophoresis in the presence of Fe(III) ions

Preparation of affinity polyacrylamide gels containing immobilized Fe(III) ions for the separation of proteins exhibiting metal ion binding properties is described. The presented method enables uniform distribution of immobilized metal ions in the affinity part of the polyacrylamide separating gel. Affinity gels prepared by this way are suitable to follow the effect of different concentrations of metal ions immobilized in polyacrylamide gel on a protein electrophoretic behavior. Polyacrylamide gels containing immobilized Fe(III) ions were used to study the electrophoretic behavior of two model proteins differing in their phosphate group content: chicken ovalbumin and bovine α‐casein. For the electrophoretic separation, both the native and the denaturating conditions were used.     

Preparation of Immobilized Metal Affinity Chromatographic Packings Based on Monodisperse Hydrophilic Non‐porous Beads and Their Application

Three hydrophilic immobilized metal affinity chromatographic packings for HPLC have been synthesized by chemical modification of 3.0 µm monodisperse non‐porous poly(glycidyl methacrylate‐co‐ethylenedimethacrylate) (P_GMA/EDMA) beads. The retention behavior of proteins on the metal ion chelated columns loaded with copper(II), nickel(II) and zin(II) ion was studied. The effect of pH on the protein retention was investigated on both the naked and metal ion chelated columns in the range from 4.0 to 9.0. Four proteins were quickly separated in 3.0 min with linear gradient elution at a flow rate of 3.0 mL/min by using the synthesized Ni²⁺‐IDA (iminodiacetic acid) packings. The separation time was shorter than other immobilized metal affinity chromatography reported in the literature. Purification of lysozyme from egg white and trypsin on the commercially available trypsin was performed on the naked‐IDA and Cu²⁺‐IDA columns, respectively. The purities of the purified trypsin and lysozyme were more than 92% and 95%, respectively.     

Silica-immobilized formylsalicylic acid as a selective phase for the extraction of iron(III)

The immobilization of formylsalicylic acid compounds on the surface of amino group-containing silica gel phases is described. The resulting phases were tested for the extraction of iron(III) and showed an exchange capacity of 0.95-0.96 mmol g(-1). The other metal ions tested showed lower metal capacity values than iron(III). The selectivity of the phases tested for the extraction of iron(III) from a mixture containing several other metal ions was evaluated using atomic absorption spectrometry. A method for the recycling of immobilized silica gel after metal extraction is described for practical applications.     

一种混合模式反相强阳离子交换色谱固定相的制备及其保留机理的考察

采用一种简单的“混合配体”方法将辛基和磺酸基键合到硅胶表面,制备了辛基-磺酸基共同键合硅胶(OSS)材料。通过元素分析和吸附容量检测对OSS材料进行表征,证明辛基和磺酸基已成功键合到硅胶表面上。将该OSS硅胶材料作为混合模式反相强阳离子交换(RP/SCX)色谱固定相,在反相液相色谱(RPLC)流动相条件下,采用几种碱性探针分子定量地考察了该固定相的混合模式RP/SCX保留机理。通过改变流动相中缓冲盐的浓度,考察了溶质保留因子和盐浓度的对数和倒数的关系,得到了几种碱性探针分子在该混合模式OSS固定相上的单点和两点保留机理的数学模型。对两种数学模型的方程进行了线性拟合,结果表明两点保留机理更加符合实验的结果。此外,根据混合模式两点保留机理的数学方程,可以得到单一的疏水或离子交换作用力对总保留的影响,对混合模式色谱的实际分离应用可提供有价值的参考。     

Dipyridyl‐immobilized ionic liquid type hybrid silica monolith for hydrophilic interaction electrochromatography

A pyridinium‐based immobilized ionic liquid type multifunctional hybrid silica monolith was prepared by the in situ polymerization of 3‐chloropropyl‐silica matrix and 4,4′‐dipyridyl for hydrophilic interaction CEC. The obtained hybrid monolith possessed of high stable skeletal microstructures with obviously hydrophilic retention mechanism under ACN content >50% in the mobile phase. Strong and stable anodic EOF could be observed under a broad pH range from pH 3.0 to 9.0. Due to the immobilized dipyridyl groups bonded to the silica matrix surface, the resulting hydrophilic hybrid monolith possessed multiple separation interactions including hydrogen bond, π–π, and anion exchange. Excellent separations of various polar analytes including electroneutral phenols, charged acid nucleotides, and basic analytes were successfully achieved. The highest column efficiencies up to 120 000, 164 000, and 106 000 plates/m were obtained for nucleotides, nucleic acid bases, and nucleosides and nicotines, respectively. These results demonstrated that the dipyridyl‐immobilized ionic liquid functionalized hybrid monolith possessed highly mechanical stability and good chromatographic performance for hydrophilic interaction electrochromatography.     

Comparison of Reversed Stationary Phases for the Chromatographic Separation of Inorganic Analytes Using Hydrophobic Ion Mobile Phase Additives

Abstract

Alkyl-modified silica (RSi) and polystyrenedivinylbenzene (PRP-1) stationary phases are compared for the chromatographic separation of inorganic analyte anions and cations using hydro-phobic ions of opposite charge as mobile phase additives. Tetra-alkylammonium salts were used for anion separations and alkyl sulfonate salts for cation separations. Two major equilibria influence the retention of analyte ions on PRP-1. These are: retention of the hydrophobic ion on PRP-1 and an ion exchange selectivity between the hydrophobic counterion and the analyte ion. When using RSi retention is also influenced by ion exchange at residual silanol groups, which act as weak cation exchange sites. Mobile and stationary phase variables that influence analyte retention are identified. Optimization of these provides favorable eluting conditions for the separation of inorganic ionic analytes. Of particular interest is the potential use of PRP-1 and RSi columns for the separation of inorganic cations; conditions for the separation of alkali metals and alkaline earths are discussed.     

Use of a novel cation-exchange restricted-access material for automated sample clean-up prior to the determination of basic drugs in plasma by liquid chromatography

A new kind of silica-based restricted-access material (RAM) has been tested in pre-columns for the on-line solid-phase extraction (SPE) of basic drugs from directly injected plasma samples before their quantitative analysis by reversed-phase liquid chromatography (LC), using the column switching technique. The outer surface of the porous RAM particlescontains hydrophilic diol groups while sulphonic acid groups are bound to the internal surface, which gives the sorbent the properties of a strong cation exchanger towards low molecular mass compounds. Macromolecules such as proteins have no access to the internal surface of the pre-column due to their exclusion from the pores and are then flushed directly out. The retention capability of this novel packing material has been tested for some hydrophilic basic drugs, such as atropine, fenoterol, ipratropium, procaine, sotalol and terbutaline, used as model compounds. The influence of the composition of the washing liquid on the retention of the analytes in the pre-column has been investigated. The elution profiles of the different compounds and the plasma matrix as well as the time needed for the transfer of the analytes from the pre-column to the analytical column were determined in order to deduce the most suitable conditions for the clean-up step and develop on-line methods for the LC determination of these compounds in plasma. The cationic exchange sorbent was also compared to another RAM, namely RP-18 ADS (alkyl diol silica) sorbent with respect to retention capability towards basic analytes.     

Preparation and characterization of a novel dual‐retention mechanism mixed‐mode stationary phase with PEG 400 and succinic anhydride as ligand for protein separation in WCX and HIC modes

A novel dual‐retention mechanism mixed‐mode stationary phase based on silica gel functionalized with PEG 400 and succinic anhydride as the ligand was prepared and characterized by infrared spectra and elemental analysis. Because of the ligand containing PEG 400 and carboxyl function groups, it displayed hydrophobic interaction chromatography (HIC) characteristic in a high‐salt‐concentration mobile phase, and weak cation exchange chromatography (WCX) characteristic in a low‐salt‐concentration mobile phase. As a result, it can be employed to separate proteins with both WCX and HIC modes. The resolution and selectivity of the stationary phase was evaluated under both HIC and WCX modes with protein standards, and its performance was comparable to that of conventional ion‐exchange chromatography and HIC columns. The results indicated that the novel dual‐retention mechanism column, in many cases, could replace two individual WCX and HIC columns as a ‘2D column’. In addition, the mixed retention mechanism of proteins on this ‘2D column’ was investigated with stoichiometric displacement theory for retention of solute in liquid chromatography in detail in order to understand why the dual‐retention mechanism column has high resolution and selectivity for protein separation under WCX and HIC modes, respectively. Based on this ‘2D column’, a new 2DLC technology with a single column was developed. It is very important in proteome research and recombinant protein drug production to save column expense and simplify the processes in biotechnology. Copyright © 2013 John Wiley & Sons, Ltd.     

Chromatographic separation simulation of metal‐chelating peptides from surface plasmon resonance binding parameters

Some metal‐chelating peptides have antioxidant properties, with potential nutrition, health, and cosmetics applications. This study aimed to simulate their separation on immobilized metal ion affinity chromatography from their affinity constant for immobilized metal ion determined in surface plasmon resonance, both technics are based on peptide‐metal ion interactions. In our approach, first, the affinity constant of synthetic peptides was determined by surface plasmon resonance and used as input data to numerically simulate the chromatographic separation with a transport‐dispersive model based on Langmuir adsorption isotherm. Then, chromatographic separation was applied on the same peptides to determine their retention time and compare this experimental t_R with the simulated t_R obtained from simulation from surface plasmon resonance data. For the investigated peptides, the relative values of t_R were comparable. Hence, our study demonstrated the pertinence of such numerical simulation correlating immobilized metal ion affinity chromatography and surface plasmon resonance.     

Retention Behavior of Proteins on Glutamic Acid-Boned Silica Stationary Phase

A glutamic acid-bonded silica (Glu-silica) stationary phase with cation-exchange properties was synthesized using l-glutamic acid as ligand and silica gel as matrix. The effects of solution pH value, salt concentration and metal ion on the retention of proteins were examined. The standard protein mixture was separated with a prepared chromatographic column and an iminodiacetic acid column, and compared. The influence of the binding capacity of an immobilized metal ion on the complexation of metal chelate column was studied. The results indicate that the obtained column displays cation-exchange characteristic and better separation ability for proteins. As fixing metal ion on the Glu-silica column, retention of proteins on the column is a cooperative interaction of metal chelate and cation-exchange. The stationary phase shows the typical metal chelate properties with the increase of the sorption capacity of immobilized metal ion.     

Evaluation and optimization of the metal‐binding properties of a complex ligand for immobilized metal affinity chromatography

The simultaneous determination of two binding parameters for metal ions on an immobilized metal affinity chromatography column was performed by frontal chromatography. In this study, the binding parameters of Cu²⁺ to l ‐glutamic acid were measured, the metal ion‐binding characteristics of the complex ligand were evaluated. The linear correlation coefficients were all greater than 99%, and the relative standard deviations of two binding parameters were 0.58 and 0.059%, respectively. The experiments proved that the frontal chromatography method was accurate, reproducible, and could be used to determine the metal‐binding parameters of the affinity column. The effects of buffer pH, type, and concentration on binding parameters were explored by uniform design experiment. Regression, matching and residual analyses of the models were performed. Meanwhile, the optimum‐binding conditions of Cu²⁺ on the l ‐glutamic acid‐silica column were obtained. Under these binding conditions, observations and regression values of two parameters were similar, and the observation values were the best. The results demonstrated that high intensity metal affinity column could be effectively prepared by measuring and evaluating binding parameters using frontal chromatography combined with a uniform design experiment. The present work provided a new mode for evaluating and preparing immobilized metal affinity column with good metal‐binding behaviors.     

Purification and determination of the binding site of lactate dehydrogenase from chicken breast muscle on immobilized ferric ions.

Lactate dehydrogenase from chicken breast muscle was purified to homogeneity in one step by immobilized metal ion affinity chromatography. The purified enzyme was used to localize the binding site to immobilized Fe(III) ions. After cyanogen bromide degradation and digestion with trypsin, small enzyme fragments capable of binding to immobilized Fe(III) ions were obtained. It is proposed that several histidyl groups are involved in the binding.     

One-pot preparation of silica-supported hybrid immobilized metal affinity adsorbent with macroporous surface based on surface imprinting coating technique combined with polysaccharide incorporated sol--gel process

A simple and reliable one-pot approach using surface imprinting coating technique combined with polysaccharide incorporated sol-gel process was established to synthesize a new organic-inorganic hybrid matrix possessing macroporous surface and functional ligand. Using mesoporous silica gel being a support, immobilized metal affinity adsorbent with a macroporous shell/mesoporous core structure was obtained after metal ion loading. In the prepared matrix, covalently bonded coating and morphology manipulation on silica gel was achieved by using one-pot sol-gel process starting from an inorganic precursor, -glycidoxypropyltrimethoxysiloxane (GPTMS), and a functional biopolymer, chitosan (CS) at the atmosphere of imprinting polyethylene glycol (PEG). Self-hydrolysis of GPTMS, self-condensation, and co-condensation of silanol groups (Si-OH) from siloxane and silica gel surface, and in situ covalent cross-linking of CS created an orderly coating on silica gel surface. PEG extraction using hot ammonium hydroxide solution gave a chemically and mechanically stabilized pore structure and deactivated residual epoxy groups. The prepared matrix was characterized by using X-ray energy dispersion spectroscopy (EDX), scanning electron microscopy (SEM) and mercury intrusion porosimetry. The matrix possessed a high capacity for copper ion loading. Protein adsorption performance of the new immobilized metal affinity adsorbent was evaluated by batch adsorption and column chromatographic experiment using bovine serum albumin (BSA) as a simple model protein. Under the optimized coating conditions, the obtained macroporous surface resulted in a fast kinetics and high capability for protein adsorption, while the matrix non-charged with metal ions offered a low non-specific adsorption.