Preparation of a weak anion exchange/hydrophobic interaction dual‐function mixed‐mode chromatography stationary phase for protein separation using click chemistry

In this study, 3‐diethylamino‐1‐propyne was covalently bonded to the azide‐silica by a click reaction to obtain a novel dual‐function mixed‐mode chromatography stationary phase for protein separation with a ligand containing tertiary amine and two ethyl groups capable of electrostatic and hydrophobic interaction functionalities, which can display hydrophobic interaction chromatography character in a high‐salt‐concentration mobile phase and weak anion exchange character in a low‐salt‐concentration mobile phase employed for protein separation. As a result, it can be employed to separate proteins with weak anion exchange and hydrophobic interaction modes, respectively. The resolution and selectivity of the stationary phase were evaluated in both hydrophobic interaction and ion exchange modes with standard proteins, respectively, which can be comparable to that of conventional weak anion exchange and hydrophobic interaction chromatography columns. Therefore, the synthesized weak anion exchange/hydrophobic interaction dual‐function mixed‐mode chromatography column can be used to replace two corresponding conventional weak anion exchange and hydrophobic interaction chromatography columns to separate proteins. Based on this mixed‐mode chromatography stationary phase, a new off‐line two‐dimensional liquid chromatography technology using only a single dual‐function mixed‐mode chromatography column was developed. Nine kinds of tested proteins can be separated completely using the developed method within 2.0 h.     

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.     

Gold nanoparticle decorated graphene oxide/silica composite stationary phase for high‐performance liquid chromatography

In the initial phase of this study, graphene oxide (GO)/silica was fabricated by assembling GO onto the silica particles, and then gold nanoparticles (GNPs) were used to modify the GO/silica to prepare a novel stationary phase for high‐performance liquid chromatography. The new stationary phase could be used in both reversed‐phase chromatography and hydrophilic interaction liquid chromatography modes. Good separations of alkylbenzenes, isomerides, amino acids, nucleosides, and nucleobases were achieved in both modes. Compared with the GO/silica phase and GNPs/silica phase, it is found that except for hydrophilicity, large π‐electron systems, hydrophobicity, and coordination functions, this new stationary phase also exhibited special separation performance due to the combination of 2D GO with zero‐dimensional GNPs.     

硅胶基质高效液相色谱填料研究进展

高效液相色谱(HPLC)不仅是一种有效的分析分离手段,也是一种重要的高效制备分离技术。色谱柱是HPLC系统的核心,不同性能的填料是HPLC广泛应用的基础。硅胶是开发最早、研究最为深入、应用最为广泛的HPLC固定相基质,其制备方法主要有喷雾干燥法、溶胶-凝胶法、聚合诱导胶体凝聚法及模板法等。近年来,亚2μm小粒径硅胶、核-壳型硅胶、双孔径硅胶、介孔性硅胶、有机杂化硅胶等新型硅胶应用于HPLC并取得了色谱分离技术的飞速发展,例如基于亚2μm填料的超高压液相色谱技术、基于核-壳型填料的快速分离技术、基于杂化硅胶填料的高温液相色谱技术等。硅胶经表面化学键合、聚合物包覆等有机改性可制得先进的大分子限进填料、温敏性填料、手性填料等,大大扩展了HPLC的应用范围。本文对液相色谱用硅胶的制备方法、改性与修饰方法以及硅胶基质固定相的评价方法加以系统综述,概述了新型硅胶在HPLC中的应用进展,并对硅胶基质填料的发展方向与应用前景进行了展望。     

新型吡咯烷键合反相高效液相色谱固定相的制备与研究（英文）

正A new ionic liquid-based high-performance liquid chromatography stationary phase is reported.A derivative of N-methyl pyrrolidinium tetrafluoroborate was covalently immobilized on the surface of silica particles to prepare silica-based N-methyl pyrrolidinium tetrafluoroborate(SilprMP BF4)stationary phase.The obtained ionic liquid-modified silica was evaluated and confirmed by elemental analysis,infrared spectroscopy,and thermogravimetric analysis.A column was packed with the modified particles.The retention behavior of aromatic compounds,alkyl benzenes,and acidic and basic compounds on the SilprMP BF4 stationary phase was studied under reversed-phase liquid chromatography conditions.The effect of the eluent pH on the separation of the acidic and basic compounds was also studied.The new stationary phase involves multiple retention mechanisms,such as electrostatic,hydrophobic,ion-dipole,and anion-exchange interactions,which might lead to multipurpose separation media.     

Poly(vinylpyrrolidone)-coated silica: A versatile,polar stationary phase for HPLC

Summary Poly(vinylpyrrolidone) (PVP) is immobilized on both, small- and large-pore silicas by thermal treatment, γ-radiation, or peroxide initiated polymerization. The hydrolytic stability of such a highly polar stationary phase significantly exceeds that of a comparable phase prepared by the chemical reaction of silica with a pyrrolidone ethyl dimethylchlorosilane silanization reagent. The properties of the different PVP-silicas are evaluated by elemental analysis, spectroscopy, and chromatography. Columns of PVP-silica packings can be used in several modes: a) under normal-phase conditions as a polar bonded stationary phase, b) under reversed-phase conditions, for the separation of organic proton-donor and hydrogen-bonding compounds, c) for the aqueous size exclusion chromatography of proteins, and d) with salt gradients for the hydrophobic interaction chromatography of proteins. The minimum observable reduced plate-height of PVP-silica columns is about 3. Double-layer polymer coating experiments using PVP-silica covered with poly(methyloctadecylsiloxane) have been performed to study diffusion and shielding effects of different polymer layers in the stationary phase. Depending on separation conditions, one or the other polymer governs the retention process. A mixed selectivity was observed in a reversed phase mode with acidic eluents.     

葫芦[6]脲单轮烷键合固定相的制备、表征及色谱性能

将超分子自组装技术与色谱键合硅胶固定相制备技术相结合,采用γ-[(2,3)-环氧丙氧]丙基三甲氧基硅烷(KH-560)为偶联剂,首次将一种葫芦[6]脲单轮烷(CB6MR)键合到硅胶上,制备了一种新型的葫芦[6]脲单轮烷键合固定相(CB6MRBS)。通过元素分析、红外光谱和热分析对该固定相进行了结构表征。以中性、酸性、碱性化合物和二取代苯位置异构体等溶质为探测因子,分别在反相和正相色谱模式下对固定相的色谱性能和保留机理进行了研究。结果表明：CB6MRBS是一种多模式键合固定相,具有良好的正相和反相色谱性能,对位置异构体具有较高的识别能力,尤其是可有效地用于碱性化合物的分离分析。其保留机理存在氢键、静电、π-π和疏水作用等多种作用力机制,协同作用提高了CB6MRBS对溶质的分离选择性。由于CB6MR配体中含有酰胺基和众多极性羰基,CB6MRBS可能在络合色谱面有应用前景。     

Imidazolium‐embedded iodoacetamide‐functionalized silica‐based stationary phase for hydrophilic interaction/reversed‐phase mixed‐mode chromatography

A novel imidazolium‐embedded iodoacetamide‐functionalized silica‐based stationary phase has been prepared by surface radical chain‐transfer polymerization. The stationary phase was characterized by Fourier transform infrared spectrometry, thermogravimetric analysis, and element analysis. Fast and efficient separations of polar analytes, such as nucleosides and nucleic acid bases, water‐soluble vitamins and saponins, were well achieved in hydrophilic interaction chromatography mode. Additionally, a mixed mode of hydrophilic interaction and reversed‐phase could be also obtained in the analysis of polar and nonpolar compounds, including weak acidic phenols, basic anilines and positional isomers, with high resolution and molecular‐planarity selectivity, outperforming the commercially available amino column. Moreover, simultaneous separation of polar and nonpolar compounds was also achieved. In conclusion, the multimodal retention capabilities of the imidazolium‐embedded iodoacetamide‐functionalized silica‐based column could offer a wide range of retention behavior and flexible selectivity toward hydrophilic and hydrophobic compounds.     

Fast separation of hen egg white protein with a phosphorylcholine type zwitterionic ion chromatography stationary phase

In this work,a kind of preparation method of zwitterionic ion chromatography(ZIC) stationary phase modified with phosphorylcholine(PC) was obtained by hydrolyzing after bonding phosphorylcholine dichloride to diol-silica to better explore the characteristics of the PC groups as ZIC stationary phase ligand in simultaneous separation of acidic proteins and basic proteins. The results showed that tw’o kinds of acidic proteins and three kinds of basic proteins can be separated completely,meanwhile,hen egg white was separated and purified and three kinds of egg white components ovalbumin,G2 ovoglobulin and ovotransfemin proteins were separated completely by one single step on PC-ZIC column,the purity of all proteins reached above 95%.PC-ZIC stationary phase was successfully improved with better separation capacity and selectivity than previously reported in this paper.     

高效液相色谱槲皮素键合硅胶固定相分离极性化合物

槲皮素是一种植物体中含量丰富、价格较便宜的黄酮类化合物,本研究以γ-[(2,3)-环氧丙氧]丙基三甲氧基硅烷(KH-560)为偶联剂,将其化学键合到硅胶上,得到一种含天然配体的槲皮素键合硅胶固定相(QUSP)。 采用红外光谱、热重分析、元素分析及固体核磁对其结构进行表征,测得硅胶表面槲皮素的键合量为0.139 mmol/g。 采用不同结构的溶质作探针,在评价固定相反相液相色谱疏水作用性能的基础上,侧重研究新固定相对极性芳香族化合物的分离能力,探讨了新固定相的色谱分离机理。 研究表明,仅采用甲醇或乙腈-水简单流动相,无需用缓冲液精确控制pH值,QUSP就能分别实现吡啶类、芳胺类、苯酚类、苯甲酸类和黄酮类等极性化合物的快速基线分离。 QUSP键合的槲皮素除含疏水性的C6-C3-C6骨架外,黄酮环还能为溶质提供氢键、偶极、π-π、电荷转移等多种作用位点,各种协同作用有利于提高色谱分离选择性,尤其对极性的可离子化的酸性和碱性化合物。     

基于硅胶整体材料研磨颗粒的多功能聚乙二醇键合固定相的制备及其在高效液相色谱多模式分离中的应用

研究通过对溶胶-凝胶法制备的硅胶整体材料进行研磨、浮选、假晶相转换和水热处理，最终获得了粒径为2~5 μm、孔径为20~60 nm的硅胶颗粒。利用部分含氟的阴离子表面活性剂Capstone FS-66和常用的阳离子表面活性剂十六烷基三甲基溴化铵（CTAB）组成的双胶束模板体系对硅胶基质进行假晶相转换处理；再采用碳酸钠溶液水热处理的方式，进一步扩大孔径。用扫描电镜（SEM）和N₂吸附-解吸等温线测量对扩孔处理前后的硅胶整体材料研磨颗粒进行表征，结果清楚地显示了处理前后的形貌变化和差异。随后将含有长链聚乙二醇（PEG）的硅烷键合到扩孔后的硅胶颗粒表面，分别利用元素分析、红外光谱以及热重分析对固定相进行表征，并对固定相进行色谱性能评价。对键合固定相的元素分析和热重分析数据进行分析表明，硅胶表面键合PEG的含量约为8%。研究揭示了利用假晶相转换法与碳酸钠溶液水热处理和长链PEG硅烷修饰的硅胶整体材料颗粒在尺寸排阻色谱分离蛋白质方面的良好分离效果。同时进一步的高效液相色谱评价结果表明，该键合固定相还可用于疏水作用色谱模式分离核糖核酸酶A和溶菌酶，以及可用于亲水作用色谱模式分离吡啶甲酸、左旋多巴、三聚氰胺和邻苯二酚等极性比较强的化合物。研究显示了PEG键合固定相具有多功能性，及其在多模式高效液相色谱分离中的应用潜力。     

Investigation of polar stationary phases for the separation of sympathomimetic drugs with nano-liquid chromatography in hydrophilic interaction liquid chromatography mode

In this study, the retention and selectivity of a mixture of basic polar drugs were investigated in hydrophilic interaction chromatographic conditions (HILIC) using nano-liquid chromatography (nano-LC). Six sympathomimetic drugs including ephedrine, norephedrine, synephrine, epinephrine, norepinephrine and norphenylephrine were separated by changing experimental parameters such as stationary phase, acetonitrile (ACN) content, buffer pH and concentration, column temperature. Four polar stationary phases (i.e. cyano-, diol-, aminopropyl-silica and Luna HILIC, a cross-linked diol phase) were selected and packed into fused silica capillary columns of 100 μm internal diameter (i.d.). Among the four stationary phases investigated a complete separation of the all studied compounds was achieved with aminopropyl silica and Luna HILIC stationary phases only. Best chromatographic results were obtained employing a mobile phase composed by ACN/water (92/8, v/v) containing 10 mM ammonium formate buffer pH 3. The influence of the capillary temperature on the resolution of the polar basic drugs was investigated in the range between 10 and 50 °C. Linear correlation of ln k vs. 1/T was observed for all the columns; ΔH° values were negative with Luna HILIC and positive with aminopropyl- and diol-silica stationary phases, demonstrating that different mechanisms were involved in the separation.To compare the chromatographic performance of the different columns, Van Deemter curves were also investigated.     

Polar-copolymerized approach based on horizontal polymerization on silica surface for preparation of polar-modified stationary phases

A new approach for preparation of polar-modified reversed-phase liquid chromatography stationary phases was developed by using horizontal polymerization technique on silica surface, which was defined as “polar-copolymerized” approach. Based on this new approach, a representative polar-copolymerized stationary phase composed of mixed n-octadecyl and chloropropyl (C18–C3Cl) was synthesized. The resulting stationary phase named C18HCE was characterized with elemental analysis and solid phase ¹³C and ²⁹Si NMR, which proved the chemistry of polar-copolymerized stationary phases. Chromatographic evaluation and application of the C18HCE were also investigated. The results of preliminary chromatographic evaluation demonstrated that the C18HCE stationary phase exhibited 100% aqueous mobile phase compatibility, low silanol activity. In addition, the application results demonstrated that the C18HCE had superior separation performance in alkaloids separation at acidic conditions compared to some commercial stationary phases.     

Synthesis and characterization of phenylpropanolamine bonded silica for multimode liquid chromatography of small molecules

Summary A multimodal silica support carrying both anionexchange and hydrophobic functions was prepared by bonding phenylpropanolamine to epoxysilane-modified silica. This support was characterized with regard to its physical and chromatographic properties and has a surface coverage of 150 mol g^–1. The reversed-phase and the anion-exchange behaviour of the new stationary phase were investigated by injection of acidic, neutral and basic drugs and inorganic anions. The retention of the tested compounds was manipulated by modifying the pH and proportion of organic modifier in the mobile phase in isocratic mode. The mixed-mechanism column provided a flexible and versatile method for the simultaneous separation of neutral, acidic and basic compounds.     

Coating and fusing cell membranes onto a silica surface and their chromatographic characteristics

Summary A new cell membrane stationary phase (CMSP) consisting of porous silica coated with active cell membranes is presented for affinity chromatography. By immersing, silica into a suspension of cell membranes, the whole surface of silica was covered by the cell membranes due to the irreversible adsorption of silanol groups (Si−OH) on the silica surface and the self-fusion of the cell membranes. CMSP can be used directly as a chromatographic packing material without any additional chemical modification. The surface characteristics, enzymatic activity, and chromatographic behavior of CMSP were investigated. The results obtained from scanning electron microscope, surface energy spectrometer, enzyme assay, and liquid chromatography showed that the surface characteristics of CMSP were very different from that of normal and reversed stationary phases. CMSP was found to have the characteristics of both cell membrane activity and chromatographic separation. Moreover, CMSP, as a chiral stationary phase, could be used for the enantiomeric separation of (±) Bay-K8644. The capacity factor of some calcium antagonists on CMSP was found to have a good correlation with their pharmacological actions. It is concluded that CMSP may be used not only as a kind of packing material in bio-affinity chromatography, but also as a tool for studying the interactions between a drug and its receptor.     

Evaluation of a silicon oxynitride hydrophilic interaction liquid chromatography column in saccharide and glycoside separations

The retention characteristics of a silicon oxynitride stationary phase for carbohydrate separation were studied in hydrophilic interaction chromatography mode. Four saccharides including mono‐, di‐, and trisaccharides were employed to investigate the effects of water content and buffer concentration in the mobile phase on hydrophilic interaction liquid chromatography retention. For the tested saccharides, the silicon oxynitride column demonstrated excellent performance in terms of separation efficiency, hydrophilicity, and interesting separation selectivity for carbohydrates compared to the bare silica stationary phase. Finally, the silicon oxynitride hydrophilic interaction liquid chromatography column was employed in the separation of complex samples of fructooligosaccharides, saponins, and steviol glycoside from natural products. The resulting chromatograms demonstrated good separation efficiency and longer retention compared with silica, which further confirmed the advantages and potential application of silicon oxynitride stationary phase for hydrophilic interaction liquid chromatography separation.     

双阳离子型离子液体和十八烷基修饰的混合模式硅胶固定相的制备及色谱性能

通过分步键合反应制备了一种咪唑基双阳离子型离子液体和十八烷基共同修饰的混合模式硅胶色谱固定相(Sil-C18-IL-C4); 采用元素分析和红外光谱对其进行了表征. 分别评价了该固定相在反相色谱模式(RPLC)、 亲水色谱模式(HILIC)和RPLC/HILIC混合色谱模式下的色谱分离性能, 并在HILIC模式下对6种碱基核苷类化合物进行分离, 考察了流动相中有机相体积分数和水相中甲酸铵浓度对分离效果的影响. 此外, 还考察了该固定相的分离重复性. 制备的Sil-C18-IL-C4固定相的元素分析结果表明, 氮元素含量为1.65%, 碳元素含量为11.16%, 氢元素含量为2.44%. 该固定相的红外光谱中, 2928和2856 cm^?1处出现了—CH的不对称和对称伸缩振动峰, 1440 和660 cm^?1处出现了咪唑环上C=C的伸缩振动峰和C=N的弯曲振动峰, 说明十八烷基和1,5-双(咪唑-1-基)戊烷均已接枝到硅胶表面. 色谱性能评价结果表明, Sil-C18-IL-C4固定相可表现出反相色谱模式和亲水色谱模式分离性能, 对6种碱基核苷类物质能够实现完全分离, 而且在一定的色谱条件下可以在单根色谱柱单次运行中实现RPLC/HILIC混合模式色谱分离, 对于处理复杂样品中的碱基核苷类化合物等亲水物质具有良好的应用潜能.     

一种聚合型弱阳离子交换/亲水相互作用色谱固定相的制备及色谱性能

通过亲核取代反应将聚乙烯马来酸酐键合到氨基硅胶表面,然后将残余的马来酸酐水解,制备了一种弱阳离子交换/亲水相互作用高效液相色谱固定相(Sil-PolyCOOH),通过固体核磁、ζ-电势及元素分析对固定相进行了表征。选取核苷和核酸碱为模型化合物,通过考察流动相组成,离子强度和pH等因素对溶质保留的影响,探讨了固定相的分离性能和保留机理,结果表明,该固定相的保留机理同时涉及亲水分配相互作用和多重主客体作用力。该固定相还对糖类、敌草快与百草枯等化合物具有良好的分离性能。上述研究结果表明该固定相在极性化合物的分离上具有良好的应用前景。     

A Sulfonic‐Azobenzene‐Grafted Silica Amphiphilic Material: A Versatile Stationary Phase for Mixed‐Mode Chromatography

A novel sulfonic‐azobenzene‐functionalized amphiphilic silica material was synthesized through the preparation of a new sulfonic azobenzene monomer and its grafting on mercaptopropyl‐modified silica by a surface‐initiated radical chain‐transfer reaction. The synthesis was confirmed by infrared spectra, elemental analysis, and thermogravimetric analysis. This new material was successfully applied as a new kind of mixed‐mode stationary phase in liquid chromatography. This allows an exceptionally flexible adjustment of retention and selectivity by tuning the experimental conditions. The distinct separation mechanisms were outlined by selected examples of chromatographic separations in the different modes. In reversed‐phase liquid chromatography, this new stationary phase presented specific chromatographic performance when evaluated using a Tanaka test mixture. Seven dinitro aromatic isomers, four steroids, and seven flavonoids were separated successfully in simple reversed‐phase mode. This stationary phase can also be used in hydrophilic interaction chromatography because of the existing polar functional groups; for this, nucleosides and their bases were used as a test mixture. Interestingly, the same nucleosides and bases can also be separated in per aqueous liquid chromatography using the same stationary phase. Three ginsenosides including Rg1, Re, and Rb1 were successfully separated in hydrophilic mode. There is the potential for more applications to benefit from this useful column.     

Using subcritical/supercritical fluid chromatography to separate acidic,basic, and neutral compounds over an ionic liquid-functionalized stationary phase

We packed an ionic liquid (IL)-functionalized stationary phase – based on 1-octyl-3-propylimidazolium chloride covalently bounded to silica gel – into a 3.2 mm × 250 mm column for the simultaneous separation of acidic, basic, and neutral compounds using carbon dioxide subcritical/supercritical fluid chromatography (SFC), and examined the effects of the pressure, temperature, co-solvents, and additives on the retention behavior of the analytes. The model compounds tested for SFC separation are acetaminophen, metoprolol, fenoprofen, ibuprofen, naphthalene, and testosterone. The data indicate that hydrogen-bonding and hydrophobic interactions between the analytes and the IL-modified stationary phase seem to involve in the separation process. Simultaneous separation of acidic, basic, and neutral compounds via SFC was successful at a co-solvent content of 20% MeOH, a pressure of 110 bar, and a column temperature of 35 °C. The relative standard deviations of the retention times and peak areas at 50 ppm were all less than 4 and 8% (n = 6), respectively.