Protein Loading Capacity and Textural Properties of Column Packings in Reversed-Phase HPLC

Abstract

The relationship between the textural properties (pore size, pore volume and surface area) of reversed-phase silica gel packings for HPLC and the dynamic loading capacity of large biomolecules was studied by using silica gels manufactured by similar processes. Several silica gels whose unbonded pore diameters range from 100 to 250 A and whose pore volumes range from 1.0 to 1.4 ml/g have been prepared and characterized. The bonded phase is monomeric C18. The textural properties of the bonded silica gels are also presented and related to the properties of the unbonded silica gels.

Chromatographic evaluation with typical proteins in an underload-to-overload condition was performed in order to relate the influence of textural properties of silica gel to loading capacity and resolution. The packings with larger pore size and pore volume produced better column performance and higher loading of proteins.     

Effect of the structure and density of chemically bonded C18 phase on the recovery of tryptophan and its metabolites from human urine

A series of materials with chemically bonded C18 phase for use as the packings in clean-up columns for solid-phase extraction were prepared. The effects of the monomeric and/or polymeric structure of the chemically bonded phase and of the porous structure of the silica gel support on the recovery of tryptophan and two of its metabolites used as test substances were considered. It appeared that the best recoveries of at least 60% of the three test substances were obtained on material of the "monomer" type containing chemically bonded C18 phase characterized by a high coverage density of alpha RP approximately 3.8 mumol/m2. The use of a silica gel support with a larger pore size and volume permits not only the effective isolation of individual substances, e.g., from urine, but also their 5-fold concentration.     

聚烯烃催化剂载体硅胶研发进展

以Ziegler-Natta催化剂和茂金属催化剂为例,介绍了聚烯烃均相催化剂的负载化技术以及载体选择的影响因素,明确了载体的强度、密度、总孔容、孔分布、孔径、粒度和颗粒形状等物理性质是选择载体要考虑的主要因素。综述了聚烯烃催化剂载体硅胶的结构特征和性能特征,并对聚烯烃催化剂载体硅胶的合成机理、影响因素进行了详细介绍。此类载体硅胶比表面积280~320m2/g、堆密度0.28~0.32g/cm3、孔容1.40~1.70cm3/g、平均孔径18~22nm。建议通过开展对不同性能硅胶(如更小粒径、不同元素改性等)的研究开发,以满足不同催化剂对载体硅胶的特殊要求。     

高分子覆盖型硅胶填料的制备与色谱保留行为

 采用甲基丙烯酸甲酯 (MMA)或MMA 亚乙基二甲基丙烯酸酯 (EDMA)在硅胶表面聚合的方法 ,制备生成了新型高分子覆盖型硅胶填料C或D。借助红外光谱、元素分析、尺寸排阻色谱和反相液相色谱分析对反应过程、覆盖程度、交联剂的影响和填料的色谱保留行为进行了评价和讨论。结果显示 ,在合成时可以通过控制聚合单体的量控制生成高分子层的厚度 ,而合成中加入交联剂可以改变填料表面的微孔构造。通过考察这种色谱填料的疏水性和对芳香族化合物的分离性能 ,认为其柱效和分离效果接近C18填料的性能。     

高效液相色谱快速分离生物大分子的大孔硅质固定相的研制及应用

介绍了不同孔径的大孔硅胶基质的制备、二醇固定相的合成及其对蛋白质的色谱分离；考察了焙烧温度对硅胶孔隙度的影响；探讨了流动相中盐浓度和ｐＨ对蛋白质保留时间、柱效、分离度的影响以及硅胶孔径对键合密度和分离性能的影响。该种大孔固定相相对于常规多孔固定相具有小的比表面积、柱容量低、从而有利于生物大分子的微量分离分析。     

Changes in the pore character of silicas caused by surface bonding and polymer coating

The pore character of packings for liquid chromatography, especially reversed-phase (RPLC) packings, has been studied by means of the nitrogen adsorption method (BET method). Micro-spherical silica gels with 9, 12, 30, 40 and 50 nm average pore diameter have been used as carriers. These silica gels have been modified with monochlorodimethyloctadecylsilane and several polymers (polyoctadecylmethacrylate-methylmethacrylate co-polymer, polyacrylamide gel, polyvinyl alcohol, poly-2-hydroxyethyl-methacrylate). A larger decrease in the specific surface area values was observed in the case of the polymer coating with the polyoctadecylmethacrylate-methylmethacrylate co-polymer compared with the derivatization by silanes or the modification with polymers without C18-groups. A new approach has been suggested to explain some questions concerning the interpretation of the data obtained during the measurements of the pore characteristics of the derivatized packings. An attempt has been made to reveal peculiarities connecting values of the measured surface of RP-packings with the specific surface area values of the initial silica, as well as with the chromatographically accessible surface.     

锆基色谱填料的制备方法和物理化学性质

氧化锆基质色谱填料适合于碱性物质,特别是生物大分子的分离,因而具有良好的应用前景。它的制备方法对其物理化学性质有很大的影响,从而影响填料的色谱性能。该文对氧化锆微球的制备方法及其物理化学性质进行了综述。分析表明,目前氧化锆基质色谱填料的制备方法存在着难以克服的缺点,要制备理想的锆基色谱填料需要新的思路。     

几种手性醇在纤维素键合的SBA-15基手性固定相上的直接拆分

在强酸性条件下, 以三嵌段聚醚P123为模板, 合成了孔径大且粒径均匀的SBA-15介孔二氧化硅微球. 将含有少量三乙氧硅丙基氨基甲酸酯残基的纤维素-三(3,5-二甲基苯基氨基甲酸酯)通过分子间缩聚作用固载到氨丙基化的SBA-15微球上, 制得手性固定相; 采用常规和非常规的流动相模式, 对一些芳香醇的消旋体进行了手性拆分. 实验结果表明, 所制备的SBA-15微球不仅分散性良好, 具有规则的二维六方孔道结构, 而且消除了微孔; 所制备的键合手性固定相不仅固载手性选择剂的量大, 而且经六甲基二硅胺烷封端后可有效改善拖尾现象, 对实验选用的手性醇具有较高的拆分能力; 与大孔硅胶为基质的同类纤维素键合手性固定相相比, 该固定相对同种手性消旋体的分离因子明显提高.     

Application of liquid phase deposited titania nanoparticles on silica spheres to phosphopeptide enrichment and high performance liquid chromatography packings

A novel core-shell composite (SiO(2)-nLPD), consisting of micrometer-sized silica spheres as a core and nanometer titania particles as a surface coating, was prepared by liquid phase deposition (LPD). Here, we show the resulting core-shell composite to have better efficient and selective enrichment for mono- and multi-phosphopeptides than commercially available TiO(2) spheres without any enhancer. The material exhibited favorable characteristics for HPLC, which include narrow pore size distribution, high surface area and pore volume. We also show that the core-shell composite can efficiently separate adenosine phosphate compounds due to the Lewis acid-base interaction between titania and phosphate group when used as HPLC packings. After coating the silica sphere with titania by LPD, the silanol of silica spheres will be shielded and that the stationary phase, C(18) bonded SiO(2)-3LPD, could be used under extreme pH condition.     

Influence of the pressure on the properties of chromatographic columns .III. retention volume of thiourea, hold-up volume, and compressibility of the C18-bonded layer

The influence of the average column pressure (ACP) on the elution volume of thiourea was measured on two RPLC columns, packed with Resolve-C18 (surface coverage 2.45 micromol/m2) and Symmetry-C18 (surface coverage 3.18 micromol/m2), and it was compared to that measured under the same conditions on an underivatized silica (Resolve). Five different methanol-water mixtures (20, 40, 60, 80 and 100% methanol, v/v) were used. Once corrected for the compressibility of the mobile phase, the data show that the elution volume of thiourea increases between 3 and 7% on the C18-bonded columns when the ACP increases from 50 to 350 bar, depending on the methanol content of the eluent. No such increase is observed on the underivatized Resolve silica column. This increase is too large to be ascribed to the compressibility of the stationary phase (silica + C18 bonded chains) which accounts for less than 5% of the variation of the retention factor. It is shown that the reason for this effect is of thermodynamic origin, the difference between the partial molar volume of the solute in the stationary and the mobile phase, Delta V, controlling the retention volume of thiourea. While Delta V is nearly constant for all mobile phase compositions on Resolve silica (with Delta V approximately equal to -4 mL/mol), on RPLC phases, it significantly increases with increasing methanol content, particularly above 60% methanol. It varies between -5 mL/mol and -17 mL/mol on Resolve-C18 and between -9 mL/mol and -25 mL/mol on Symmetry-C18. The difference in surface coverage between these two RP-HPLC stationary phases increases the values of Delta V by about 5 mL/mol.     

极稀溶液体系中自组装合成纳米介孔氧化硅及其铝修饰材料

在极稀溶液中, 通过改变反应溶剂去离子水的量或原料中铝源的量, 可控合成了不同粒径(20~70 nm)、形貌和孔道结构的纳米介孔氧化硅颗粒和纳米介孔铝掺杂氧化硅材料. 这种材料具有高比表面积(BET比表面积1000 m²/g)和较大的孔容(1.1~1.8 cm³/g). 反应物浓度降低或反应物中添加铝源后, 介孔材料的有序性下降, 粒径减小, 孔容增大, 并产生大量的间隙孔. 通过小角X射线衍射(SAXRD)、透射电镜和氮气吸附-脱附实验表征了样品.     

Alkyl-Diol Silica (ADS): restricted access precolumn packings for direct injection and coupled-column chromatography of biofluids

The direct and repetitive injection of untreated biological fluids (e.g., hemolyzed blood, plasma, serum, cell culture and tissue homogenates) onto an HPLC-system and the subsequent analysis of low-molecular weight compounds (e.g. drugs, xenobiotics, metabolites) is rendered possible by a coupled-column configuration and special precolumn packings. For this purpose a new family of chemically and enzymatically tailored reversed-phase packing materials have been prepared. The LC-integrated sample clean-up with these restricted access (bimodal) phases is based on the complete nonadsorptive size exclusion of macromolecules (e.g. proteins) and on the simultaneous dynamic partitioning of the target molecules. The bonded phase which exclusively covers the internal pore surface of a glyceryl-modified silica is a butyryl-(C-4), capryloyl-(C-8) or stearoyl-(C-18) moiety. These ligands allow a classical reversed-phase or ion-pair chromatography during the sample work-up step. The capacity of the n-alkyl phase is comparable with conventional silica based RP-materials. The broad hydrophobic retentive capability of these packings allows the extraction of a wide variety of compounds of biomedical interest. The electroneutral and hydrophilic particle exterior (glyceryl-residues) was generated using either soluble or immobilized enzymes (lipase, esterase) which cleave the fatty acid esters exclusively at the outer surface. Unwanted macromolecular components of a sample (e.g. proteins) are quantitatively eluted in the void volume due to the restricted access given by the pore size (6 nm) and the nonadsorptive external diol coverage. The lifetime of a precolumn (25 × 4 mm I.D.) packed with these novel bimodal, i.e. RP-SEC phases exceeds more than 200 injections of 500 l plasma. In addition to the synthesis, this paper describes an application of each of these Alkyl-Diol Silica (ADS) precolumn packings in fully automated coupled-column HPLC systems for the analysis of drugs and endogenous compounds in different biological matrices.Dedicated to Professor Dr. Dr. h.c. mult. J.F.K. Huber on the occasion of his 70th birthday     

Characterization of the properties of stationary phases for liquid chromatography in aqueous mobile phases using aromatic sulphonic acids as the test compounds

We investigated the effects of the concentration of naphthalene sulphonic acids (NSAs) as anionic test compounds in the injected sample and of the salt additives to the mobile phase on ion-exclusion. The retention behaviour of NSAs sensitively reflects even minor changes in the ionic and hydrophobic interactions and can be useful for predicting the effects of the stationary phases in reversed-phase chromatography of polar and ionic compounds, both small ones and biopolymers, e.g., oligonucleotides. We studied chromatographic properties of several stationary phases intended for separations in aqueous mobile phases: a C18 column end-capped with polar hydrophilic groups, a densely bonded C8 column doubly end-capped with short alkyl groups, a short alkyl stationary phase designed to keep full pore accessibility in highly-aqueous mobile phases and a Bidentate column with “bridged” C18 groups attached to the silica hydride support. The chemistry and pore structure of various types of column packing materials and of the salt additives to the mobile phase affect the proportion of the pore volume non-accessible to anions due to ion-exclusion and consequently the peak asymmetry and hydrophobic selectivity in reversed-phase chromatography of organic acids. We also addressed the problems connected with the determination of column hold-up volume in aqueous mobile phases. The accessibility of the stationary phase for anionic compounds in contact with the sample zone is affected by ion-exclusion due to repulsive interactions with the negatively charged surface in the pores of the stationary phase. The accessible part of the stationary phase increases and consequently the migration velocity along the column decreases with increasing concentration of the sample in the zone moving along the column. Because of a limited access to the stationary phase, its capacity can be easily overloaded. The combination of the column overload and ion-exclusion effects may result in fronting or tailing peak asymmetry. To explain this behaviour, we proposed a modified Langmuir model, respecting the variation of the column capacity due to the effects of sample concentration on ion-exclusion.     

Large-pore mesoporous SBA-15 silica particles with submicrometer size as stationary phases for high-speed CEC separation

A new mesoporous sphere-like SBA-15 silica was synthesized and evaluated in terms of its suitability as stationary phases for CEC. The unique and attractive properties of the silica particle are its submicrometer particle size of 400 nm and highly ordered cylindrical mesopores with uniform pore size of 12 nm running along the same direction. The bare silica particles with submicrometer size have been successfully employed for the normal-phase electrochromatographic separation of polar compounds with high efficiency (e.g., 210,000 for thiourea), which is matched well with its submicrometer particle size. The Van Deemeter plot showed the hindrance to mass transfer because of the existence of pore structure. The lowest plate height of 2.0 microm was obtained at the linear velocity of 1.1 mm/s. On the other hand, because of the relatively high linear velocity (e.g., 4.0 mm/s) can be generated, high-speed separation of neutral compounds, anilines, and basic pharmaceuticals in CEC with C18-modified SBA-15 silica as stationary phases was achieved within 36, 60, and 34 s, respectively.     

Comparative studies of gas chromatographic properties of new packings with chemically bonded complexes

Specific interactions of aliphatic linear and branched hydrocarbons as well as cyclic and aromatic hydrocarbons with new packings containing chemically bonded complexes of transition metals are studied. The possibility of using these packings to separate these compounds is also discussed. The packings under study contain complexes of Cu(II) and Cr(III) chemically bonded to the silica surface. Chlorides of these metals are bonded to the silica surface by the use of the ketoimine group originally from 2-(3-triethoxysililpropylimino)-3-(n-buthyl)-pentanon-4. In order to determine an influence of the performed modification to gas chromatographic properties of the packings, such retention parameters as retention factor, retention index, molecular retention index, and specific retention volume are measured for these compounds. Based on the obtained values, a trial is taken to determine an influence of the nature of the bonded metal from the complex on the retention of the adsorbates under study and a dependence between a structure of an adsorbate molecule and values of charge-transfer interactions with the bonded metal complexes.     

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.     

Evaluation of high-performance liquid chromatography column retentivity using macromolecular probes. II. Silanophilic interactivity traced by highly polar polymers

A new method of HPLC column retentivity testing utilizes polymeric probes instead of conventional sets of low molar mass substances. The procedure allows at least semiquantitative, separate and independent evaluation of adsorption and partition properties of column packings. In this present work, the method is applied for comparison of the polar interactivities of selected silica gel C18 HPLC columns. It is shown that free silanols which remained on the surface of the end-capped silica C18 column packings are accessible for interaction with highly polar macromolecules. High molar mass polymeric test probes are adsorbed on the surface silanols and their retention volumes increase. As result, deviations from regular size-exclusion chromatographic (SEC) behavior are observed. The extent of retention volume changes depends on both the nature of polymer probes and on column packing type. Adsorption of macromolecules can be suppressed by addition of a highly polar substance to the mobile phase. The amount of polar additive which is needed to attain regular SEC elution of the polymer probe depends on the column packing type and can be used as a characteristic of silanophilic column interactivity. Courses of dependences of retention volumes on sizes of macromolecules indicate the presence of "U-turn" adsorption which allows two and more silanols situated among C18 groups to be occupied simultaneously with the same macromolecule.     

Characterization of internal surface reversed-phase silica supports for liquid chromatography

Internal surface reversed-phase (ISRP) supports synthesized from commercially available porous silica particles with a variety of nominal pore diameters and specific surface areas are characterized with regard to physical and chromatographic properties. Bonded phase coverage, pore size, capacity and efficiency measurements are made upon the various ISRP supports in order to evaluate the effect that the physical properties of silica have upon the chromatographic performance of ISRP packings. In addition, various models that describe the pore structure of silica supports are discussed.     

Evaluation of protein, peptide, and amino acid retention on C5 hydride-based stationary phases

A pentyl (C5) stationary phase is synthesized on a hydride surface using both 100 and 300 A silica particles. The aqueous normal phase properties of these new phases are evaluated using amino acids at high concentrations of ACN in the mobile phase containing 0.1% formic acid. The RP properties of the materials are tested by using peptides and proteins in order to evaluate the effect of pore size on retention. Comparisons of retention as well as efficiencies and peak symmetry with a C18 phase are used to determine the influence of the hydrophobic properties of the bonded material. An evaporative light scattering detector (ELSD) is used in all phases of the study and compared with UV detector for gradient elution of proteins.     

CP/MAS NMR investigations of silica gel surfaces modified with aminopropylsilane

Summary Aminopropyl chemically bonded phases for high performance liquid chromatography (HPLC) have been prepared using mono- and trifunctional methoxyor ethoxysilanes. Three types of silica gel with different surface characteristics were used as support for the chemically bonded phases (CBPs). Surface characteristics of the packings before and after chemical modification were determined by porosity parameters, elemental analysis and CP/MAS NMR spectroscopy.²⁹Si and¹³C CP/MAS NMR investigations gave informations about different interactions between aminosilyl ligands and/or these ligands and/or water molecules condensed in the pores of the silica gel surface. With decreasing pore diameter of the silica gel the proportion of protonated aminopropyl ligand increases.