螯合离子色谱的研究进展

 讨论了近 10年来发展较为活跃的螯合离子色谱 (CIC)的原理及其系统组成和分析流程 ,高效螯合离子色谱(HPCIC)的原理 ,高效螯合固定相的类型、应用范围及优缺点等 ,其中包括化学键合、永久涂敷或动态涂敷的高效螯合固定相 , 5 3篇。     

金属螯合亲和色谱中固定金属与蛋白质的作用

在不同PHNaCl的磷酸缓冲体系，比较了牛血清蛋白（BSA）、核糖核酸酶（RNase)、细色素C（Cyt-C)和溶菌酶（Lys)在IDA裸柱和一些金属螯合柱上的保留特性，考察了固定金属对蛋白质保留行为的影响，指出蛋白质在强结合IDA－Cu柱上的保留主要受固定金属和蛋白质间配位作用支配，在弱亲和的IDA－Ni,IDA-Co和IDA－Zn柱上的保留主要受静电作用控制，配位作用为辅，讨论了金属螯合亲和色谱中影响蛋白质和金属配位的主要因素，金属离子的电荷和半径，配位原子对中心离子外层d轨道的影响，以及蛋白质表面配位的组氨酸数目，离解常数和取向，影响金属螯合配体和蛋白质静电作用的主要因素为溶液的PH和蛋白质的等电点pI.     

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.     

Optimization of working conditions for polyelectrolyte sorbents

Matters affecting the working stability of polyelectrolyte sorbents are considered. The decrease in retention time during exploitation is found not to relate to desorption of the modifying polymer. It is found that dissociation of surface silanol groups influences significantly the extra retention of ionene on the surface of silica-based sorbents. Sorption of heavy metals and microbial contamination of the sorbent are identified as causes of decreased efficiency of polyectrolyte sorbents. The influence of organic additives on the chromatography behavior of a number of inorganic anions is studied. Finally, exploitational conditions for chromatography polyelectrolyte-containing columns are elaborated, allowing the initial parameters of separation to be maintained for a period of 1.5 months or longer.     

Recent advances in gas chromatography for solid and liquid stationary phases containing metal ions

This review is devoted to the application of metal complexes as column packings and liquid stationary phases in gas chromatography. Particular attention is paid to the stationary phases with nitrogen-containing functional groups (e.g., amine and ketoimine) and β-diketonates on the modified silica surface. The review also concerns the results of the research on metallomesogenes and chiral stationary phases. The factors influencing the retention mechanism in complexation gas chromatography are discussed. Practical application of the metal chelate-containing chromatographic packings for analytical separation of organic substances is considered.     

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.     

Mixed-mode chromatography for fractionation of peptides, phosphopeptides, and sialylated glycopeptides

A mixed-mode chromatographic (MMC) sorbent was prepared by functionalizing the silica sorbent with a pentafluorophenyl (PFP) ligand. The resulting stationary phase provided a reversed-phase (RP) retention mode along with a relatively mild strong cation-exchange (SCX) retention interaction. While the mechanism of interaction is not entirely clear, it is believed that the silanols in the vicinity of the perfluorinated ligand act as strongly acidic sites. The 2.1 mm x 150 mm column packed with such sorbent was applied to the separation of peptides. Linear RP gradients in combination with salt steps were used for pseudo two-dimensional (2D) separation and fractionation of tryptic peptides. An alternative approach of using linear cation-exchange gradients combined with RP step gradients was also investigated. Besides the attractive forces, the ionic repulsion contributed to the retention mechanism. The analytes with strong negatively charged sites (phosphorylated peptides, sialylated glycopeptides) eluted in significantly different patterns than generic tryptic peptides. This retention mechanism was employed for the isolation of phosphopeptides or sialylated glycopeptides from non-functionalized peptide mixtures. The mixed-mode column was utilized in conjunction with a phosphopeptide enrichment solid phase extraction (SPE) device packed with metal oxide affinity chromatography (MOAC) sorbent. The combination of MOAC and mixed-mode chromatography (MMC) provided for an enhanced extraction selectivity of phosphopeptides and sialylated glycopeptides peptides from complex samples, such as yeast and human serum tryptic digests.     

金属螯合亲和色谱中的疏水作用

通过考察盐溶盐和盐析盐浓度对蛋白质在IDA裸柱和金属螯合柱上保留行为的影响,详细研究了金属螯合色谱中的疏水作用,疏水作用的发生、形成的条件以及不同条件下对蛋白质保留值的贡献。实验结果表明,在高浓度和低浓度的盐溶盐以及低浓度盐析盐中,蛋白质在金属螯合柱上的保留主要受静电和配位作用控制,而疏水作用对蛋白质的保留影响很小。对弱亲和性的金属螯合柱以静电作用为主,其大小可用参数Q表征;对强亲和性的IDA-Cu（Ⅱ）柱以配位作用为主。仅在高浓度的盐析盐中,金属螯合柱才呈现较强的疏水作用,支配蛋白质保留。实验证明,金属螯合色谱中疏水作用主要来自固定相间隔臂中的疏水碳链和盐析盐对蛋白质的增疏作用,利用这种疏水作用有可能改善金属螯合色谱分离的选择性。     

Use of complexing reagents as additives to the eluent for optimization of separation selectivity in high-performance chelation ion chromatography

This paper details a study of the selectivity characteristics of high-performance chelation ion chromatography when separating a range of metal ions with a number of complexing eluents. It shows that exploitation of competitive metal complexation between ligands in the eluent and surface bonded chelating groups allows a wide range of control over the retention order and selectivity coefficients of groups of metal ions for specific applications. An indication of the metal separation characteristics found for simple non-complexing eluents on iminodiacetic acid (IDA) silica bonded substrates is given first, followed by an illustration of the selectivity changes that can be achieved by using complexing eluents. Using a novel approach, plots of logbeta(1) of the metal complexes of a chosen eluent ligand against the surface bonded IDA metal complexes were found to be useful indicators of which metals may show unusual selectivity changes during separation. Example chromatograms of metal separations are given for three selected complexing eluent reagents, namely, oxalic acid, picolinic acid, and chloride, either singly or in admixture. For special mention it was found that very specific retention control could be achieved for Cu(II) with picolinic acid, Fe(III) and Fe(II) speciation with oxalic acid, Pb with dipicolinic acid and Cd with chloride.     

Reversed-phase liquid chromatography-mass spectrometry of some organic tellurium compounds

The chromatographic behavior of some organic tellurium compounds (OTCs) was studied under the conditions of high-performance liquid chromatography with mass spectrometric detection. The retention characteristics (retention factor, relative retention, and differential molar Gibbs energy difference) of the compounds were obtained under the conditions of gradient elution reversed-phase liquid chromatography. The chemical ionization mass spectra of OTCs at atmospheric pressure were considered. It was shown that there was a relation between the structure of the compounds studied and their chromatographic retention.     

New alpha-amino phenylalanine tetrazole ligand for immobilized metal affinity chromatography of proteins

A new chelating compound has been developed for use in the immobilized metal affinity chromatographic (IMAC) separation of proteins. The bidentate ligand, alpha-amino phenylalanine tetrazole, 4, was synthesized via a five-step synthesis from N-fluorenylmethoxycarbonyl phenylalanine and then immobilized onto silica through the epoxide coupling procedure. The binding behavior of the resulting IMAC sorbent, following chelation with Zn2+ to a density of 183 micromol Zn2+ ions/g silica, was characterized by the retention of proteins in the pH range of 5.0-8.0, and by the adsorption behavior of lysozyme with frontal chromatography at pH 6.0 and 8.0. The prepared column showed the separation ability to four test proteins and the retention time of these proteins increased with an increase in pH. From the derived isotherms, the adsorption capacity, qm, for the binding of lysozyme to immobilized Zn2+-alpha-amino phenylalanine tetrazole-silica was found to be 1.21 micromol/g at pH 6.0 and 1.20 micromol/g sorbent at pH 8.0, respectively, whilst the dissociation constants KD at these pH values were 5.22x10(-6) and 3.49x10(-6) M, respectively, indicating that the lysozyme was retained more stable under alkaline conditions, although the binding capacity in terms of micromole protein per gram sorbent remained essentially unchanged.     

Separation of chitosan oligomers by immobilized metal affinity chromatography

A novel approach for chitosan oligosaccharide (COS) separation by immobilized metal affinity chromatography (IMAC) based on the differences in the interactions of chelated copper (II) ions with various COS (dimers, trimers, tetramers) is described. Polyhydroxylic chromatographic supports (agarose CL-6B and silica) were functionalized with various chelating functions such as iminodiacetate (IDA), carboxymethyl-aspartate (CM-Asp) and tris(carboxymethyl)ethylenediamine (TED). The COS retention capacities of the columns were between 2 and 6 mg/cm(3), depending on the chelating group. The COS were separated and/or enriched up to 95% for dimer and trimer and 90% for the tetramer, with yields of 60-95%.     

Retention properties of a cyanopropylsiloxane-bonded silica-based sorbent for solid-phase extraction

The characteristic kinetic and retention properties of a silica-based cyanopropylsiloxane-bonded sorbent for solid-phase extraction are described. Abraham′s solvation parameter model is used to characterize the contribution of individual intermolecular interactions to retention under liquid chromatographic and sample processing conditions with aqueous methanol mixtures as the mobile phase. The main features governing retention by the sorbent are the solute's size and hydrogen-bond basicity; interactions of a dipole type are not significant when aqueous methanol solutions are employed as the mobile phase. Compared to typical silica-based octadecylsioxane-bonded sorbents the greater difficulty of forming a cavity in the solvated cyanopropylsiloxane-bonded sorbent more than offsets the more favorable dipole-type and solute hydrogenbond base interactions of the cyanopropylsiloxane-bonded sorbent. It is shown that there are no practical circumstances for which a cyanopropylsiloxane-bonded sorbent would be more useful than a typical ODS sorbent for the isolation of organic non-electrolytes from water by solid-phase extraction.     

流动相组成、浓度和pH对蛋白质在金属螯合柱上的保留特性的影响

 系统研究了流动相中盐的性质和浓度、溶液 pH以及竞争配体对蛋白质在金属螯合色谱中保留值的影响。导出了描述蛋白质在金属螯合色谱中保留特征的数学表达式 ,提出用洗脱强度指数ε表征盐溶液的洗脱能力。根据不同色谱条件下蛋白质的保留特性 ,发现蛋白质在金属螯合色谱中的保留是配位、静电和疏水的协同作用。对与蛋白质强结合的金属螯合柱 ,以配位作用为主 ,静电作用为辅 ;对弱结合的金属柱 ,以静电作用为主 ,配位作用为辅。在流动相中加入高浓度非成络盐 ,可增强蛋白质和固定相间的疏水作用。     

Chromatographic behaviors of proteins on cation-exchange column

A weak cation-exchanger (XIDACE-WCX) has been synthesized by the indirect method. The chromatographic characteristics of the synthesized packing was studied in detail. The standard protein mixture and lysozyme from egg white were separated with the prepared chromatographic column. The chromatographic thermodynamics of proteins was studied in a wide temperature range. Thermodynamic parameters standard enthalpy change (deltaH0) and standard entropy change (deltaS0) and compensation temperature (beta) at protein denaturation were determined in the chromatographic system. By using obtained deltaS0, the conformational change of proteins was judged in the chromatographic process.The linear relationship between deltaH0 and deltaS0 can be used to identify the identity of the protein retention mechanism in the weak cation-exchange chromatography. The interaction between weak cation-exchanger and metal ions was investigated. Several metal chelate columns were prepared. The effects of introducing metal ion into the naked column on protein retention and the retention mechanism of proteins in the metal chalet affinity chromatography were discussed.     

Retention of Cr(III) by high-performance chelation ion chromatography interfaced to inductively-coupled plasma mass spectrometric detection with collision cell

High-performance chelation ion chromatography (HPCIC) was employed to retain cationic Cr(III) on an anion-exchange column and hence allow the separation of the two most prevalent forms of chromium, Cr(II) and Cr(VI). A mobile phase of nitric acid was utilized at pH = 1.5; additionally, 2,6-pyridinedicarboxylic acid was used at a concentration of 6 mM. Additives with different structural characteristics were used in an effort to elucidate retention mechanisms. Inductively-coupled plasma mass spectrometry was used for chromium detection. A collision cell was utilized to reduce chloride-based polyatomic ions that may interfere with the detection of Cr(III), and a detection limit study yielded levels in the low part-per-billion range. The newly developed method was applied to the chromatographic analysis of samples of an incubation medium containing Cr(VI) incubated with cell nuclei.     

Nonlinear gas chromatography as a way of studying inhomogeneous sorbents

A way of organizing and processing the results from gas–chromatographic experiments to obtain chromatographic retention characteristics for a fixed concentration of sorbate in the gas phase or on the surface of the sorbent is proposed and substantiated. The suitability and expediency of such retention characteristics for describing the sorption properties of inhomogenous sorbents is demonstrated using a wide variety of adsorbents of different natures (activated carbons, swelling and nonswelling polymers, silicas and their silver derivatives) as examples.

     

Liquid chromatography of dextrans on porous silica beds

Kinetics, equilibrium isotherms and chromatography retention times for sorption of dextrans T-10, T-20, T-40, T-70, T-110, T-161, T-250 and T-500 on porous silica were measured at 25 degrees C. The Henry constant and retention factors for the dextrans were obtained. The values of the partition coefficient for the distribution of the dextrans between the bulk solution and the pore space were calculated within the framework of a pore volume filling model with consideration given to the ratio between the sizes of the macromolecular coils and the pore inlet. The measurements showed that this parameter depends on the structure of the sorbent and the molecular mass distribution of the dextran. The interaction of aqueous dextran solution with porous silica is characterized by the sieve effect. Large macromolecular coils of dextran T-161 cannot penetrate into the pore space of the silica sorbent with pore diameter 14 nm. The difference in Henry law constants calculated from adsorption and chromatographic data for dextrans T-70 and T-110 can be explained by the slow diffusion of dextran macromolecules into silica pores under chromatographic conditions.     

Catching the elusive persistent and mobile organic compounds: Novel sample preparation and advanced analytical techniques

Persistent and Mobile Organic Compounds (PMOCs) are emerging pollutants in the environment that have only been rarely detected in previous years due to the lack of analytical techniques available for their analysis. These compounds, being very polar, are able to spread through the surface waters, and reach groundwaters and drinking water sources. The challenges in the analysis of these compounds in water include their poor extraction efficiencies from environmental matrices and weak retention in conventional chromatographic columns. As a result, the toxicity and environmental fate of PMOCs are largely unknown. This review will examine recent developments in sorbent and chromatographic column technologies, and other sample preparation strategies that will enable analysis of PMOCs and advance our knowledge on their fate and transport in the environment. In addition, analysis of these compounds in water via liquid chromatography with tandem mass spectrometry (LC–MS/MS) are discussed in this review.     

Characterisation by immobilised metal ion affinity chromatographic procedures of the binding behaviour of several synthetic peptides designed to have high affinity for Cu(II) ions.

In this investigation, several peptides containing an increasing number of histidine residues have been designed and synthesised. The peptides involved repeat units of either the pentameric EAEHA or the tetrameric HLLH sequence motifs. Adsorption isotherms for these synthetic peptides and hexahistidine (hexa-His) as a control substance were measured under batch equilibrium binding conditions with an immobilised Cu(II)-iminodiacetic acid (IDA) sorbent. The experimental data were analysed in terms of Langmuirean binding behaviour. In common with previous studies with synthetic peptides, these investigations have demonstrate that the sequential organisation of the histidine side chains in these peptides can affect the selectivity of the coordination interactions with borderline metal ions in immobilised metal ion affinity chromatographic systems. The results also confirm that peptides selected on the basis of their potential to form amphipathic secondary structures with their histidine residues presented on one face of the molecule can exhibit equivalent or higher affinity constants towards copper ions than hexa-His, although they contain fewer histidine residues. These findings are thus relevant to the selection of peptides produced inter alia by combinatorial synthetic procedures to have enhanced binding properties for Cu(II) or Ni(II) ions, or intended for use as peptide tags in the fusion handle approach for the affinity chromatographic purification of recombinant proteins.