反相毛细管整体柱的制备及其在多肽混合物分离中的应用

采用甲基丙烯酸月桂酯为基础功能单体,乙二醇二甲基丙烯酸酯为交联剂,正十二醇、1,4-丁二醇及二甲基亚砜为致孔剂,在内径为75 μm的石英毛细管内制备了具有良好机械性能及化学稳定性的反相毛细管整体柱。考察了致孔剂的种类、比例以及交联剂在单体混合物中的比例对柱压和分离效果的影响;以单体15%、交联剂15%、致孔剂70%(均为质量分数)作为优化配方,在70 ℃条件下反应24 h;并对所合成的毛细管整体柱进行了电镜表征,测试了流速、柱长与柱压的关系。结果表明,毛细管整体柱的通透性良好,可通过延长柱长的方法提高分离效果。将所制备的毛细管整体柱装于纳升级高效液相色谱仪上进行牛血清白蛋白及血浆样本的胰蛋白酶酶切液的分离,获得了比较理想的分离效果。     

聚(4-乙烯基苯硼酸-季戊四醇三丙烯酸酯)亲和整体柱的制备与应用

以4-乙烯基苯硼酸为单体,季戊四醇三丙烯酸酯为交联剂,偶氮二异丁腈为引发剂,乙二醇和二甘醇为致孔剂,经原位聚合制备了聚(4-乙烯基苯硼酸-季戊四醇三丙烯酸酯)毛细管整体柱.以单体、交联剂、致孔剂和引发剂的用量为4种因素,最大吸附量、柱效和保留时间为3个考察标准,利用实验设计软件(DOE)优化了其合成条件,验证了正交实验最优结果,得到了整体柱合成的最佳配比为单体7.32 mg,交联剂6 mg,致孔L剂100 μL,引发剂1 mg.在最佳条件下合成整体柱,并进行表征,在微径液相色谱(μHPLC)和加压毛细管电色谱(pCEC)实验中考察了分离特性.结果表明,整体柱固定相表面的硼酸基团在碱性条件下能特异性吸附邻苯二酚、腺苷等含有邻二羟基结构的化合物,具有亲和色谱的特性;同时由于交联剂的性质使其具有反相分离机理,在含有20％有机相的条件下能够将极性不同的苯系物分开.     

以聚乙二醇/甲醇为二元致孔剂的新型磺酸甜菜碱型两性离子亲水毛细管整体柱的合成与色谱评价

使用新型二元致孔剂聚乙二醇(PEG)/甲醇,以N,N-二甲基-N-甲基丙烯酰胺基丙基-N,N-二甲基-N-丙烷磺酸内盐(SPP)为单体,季戊四醇三丙烯酸酯(PETA)为交联剂,偶氮二异丁腈(AIBN)为引发剂,通过原位聚合法制备磺酸甜菜碱型两性离子亲水毛细管整体柱。对各反应物的配比进行了优化。结果表明,当单体与致孔剂的质量比为1∶2.5,并且致孔剂中PEG与甲醇的质量比为1∶2,单体内部SPP与PETA的质量比为1∶1,AIBN为总质量的0.1%时为最优配比;PEG/甲醇二元致孔剂的加入实现了对整体柱内部孔径大小的调节,得到了结构更为均一,渗透性、机械稳定性良好的毛细管整体柱,并且理论塔板数与传统制备方法相比有显著提高,在毛细管液相色谱模式下最高可达2.4×105塔板/m。将制备的整体柱应用于毛细管液相色谱和加压毛细管电色谱分离酚类、核苷类等极性小分子混合物,得到了很好的分离效果。     

联二萘酚印迹的电色谱整体柱的制备

讨论了分子印迹毛细管电色谱整体柱的制备.联二蔡酚作为印迹分子在热引发条件下聚合,其中甲基丙烯酸为功能单体,二甲基丙烯酸乙二醇酯为交联剂,甲苯和异辛烷为致孔剂.考察了聚合参数对整体柱通透性及手性分离的影响.联二萘酚对映体在印迹整体柱上得到了基线分离,Rs值可达1.8.     

聚十二烷基甲基丙烯酸酯整体柱的制备及其在毛细管电色谱法分离肌红蛋白酶解产物中的应用

以十二烷基甲基丙烯酸酯(LMA)为功能单体,乙叉二甲基丙烯酸酯(EDMA)为交联剂,正丙醇、1,4-丁二醇和水为三元致孔剂,以及2-丙烯酰胺-2-甲基丙磺酸(AMPS)为电渗流产生剂,制备了聚十二烷基甲基丙烯酸酯整体柱。系统考察了AMPS含量和单体-致孔剂比例对柱性能的影响。结果表明,单体溶液和致孔剂的最佳聚合溶液质量比为35:65,其中单体溶液组成为59.5%（质量分数,下同）LMA、40%EDMA和0.5%AMPS,致孔剂溶液组成为60%正丙醇、30%1,4-丁二醇和10%水。在优化的流动相条件下应用制备的整体柱采用毛细管电色谱法成功地分离了肌红蛋白酶解产物。     

混合模式毛细管聚合物整体柱的制备及应用

混合模式毛细管整体色谱柱由于保留机理多样,具有很好的应用前景。本文以[2-（甲基丙烯酰基氧基）乙基]二甲基-（3-磺酸丙基）氢氧化铵（SPE）为单体,乙二醇二甲基丙烯酸酯（EDMA）为交联剂,偶氮二异丁腈（AIBN）为引发剂,正丙醇/1,4-丁二醇/水三元体系为致孔剂,制备了聚合物基质SPE-co-EDMA毛细管液相色谱整体柱。通过系统优化致孔剂和反应物种类和配比、引发剂的用量、反应时间和反应温度等因素,提高了整体柱的柱效、机械强度、渗透性和重复性。结果表明该毛细管整体柱在10 MPa内具有良好的机械强度,渗透性为2.17×10^-14 m²,而且批次内和批次间峰面积的重现性（RSD）分别为1.0%和4.6%。以极性和非极性的多种化合物评价了该毛细管整体柱的色谱性能,结果表明该柱在高有机相中具有亲水相互作用机理,在低有机相中具有反相作用机理,显示出混合模式分离特性。     

新型有机-无机杂化整体柱的制备及其在毛细管液相色谱及加压毛细管电色谱中的应用

以丙烯酰胺(AM)为单体,八乙烯基倍半硅氧烷(POSS)为交联剂,偶氮二异丁腈(AIBN)为引发剂,四氢呋喃(THF)为致孔剂,通过原位聚合法制备了poly(POSS-co-AM)有机-无机杂化整体柱,并对各反应物的配比进行了优化。结果表明,当功能单体与致孔剂、POSS与AM的质量比均为1.0: 5.0, AIBN的质量分数为0.1%时,杂化整体柱的柱效最高。无机材料的引入使整体柱结构均匀并具有良好的渗透性,该整体柱既能用于亲水色谱模式,也能用于反相色谱模式。将制备的整体柱用于毛细管液相色谱和加压毛细管电色谱分离核苷类、胺类、硝基苯胺类等化合物,获得了良好的效果。     

新型亲水整体柱的制备及其在毛细管液相色谱和加压电色谱中的应用

以N,N-二甲基-N-甲基丙烯酰胺基丙基-N,N-二甲基-N-丙烷磺酸内盐(SPP)为单体,季戊四醇三丙烯酸酯(PETA)为交联剂,偶氮二异丁腈(AIBN)为引发剂及两类不同的致孔剂(乙醇/乙二醇和甲醇/1,4-丁二醇)制备了两种新型亲水性整体柱。为了获得理想的柱效、电渗流速度和渗透性,对制备整体柱的各反应物配比进行了研究和优化。比较了两种整体柱在渗透性和分离样品方面的性能,结果表明,以乙醇/乙二醇为致孔剂制备的整体柱在柱效、分离度方面优于以甲醇/1,4-丁二醇为致孔剂制备的整体柱,但在渗透性方面不及后者。探讨了流动相中盐浓度对核苷类样品保留的影响,发现当甲酸铵浓度从10 mmol/L增加到70 mmol/L时,核苷样品的保留因子呈现先增加后减小的状态。将制备的整体柱用于毛细管液相色谱和加压电色谱分别分离胺类、酚类和核苷类样品,获得了理想的分离效果。在分离酚类和核苷类混合样品时,发现加压毛细管电色谱在分离度和分离速度上均优于毛细管液相色谱。     

弱阴离子交换整体柱对白介素-18的分离

制备了弱阴离子交换整体柱,并建立了白介素-18在该柱上分离纯化的方法。以色谱柱管为模具,通过原位聚合法制备了一种以甲基丙烯酸缩水甘油酯(GMA)为单体,乙二醇二甲基丙烯酸酯(EDMA)为交联剂的整体色谱柱,并用二乙胺将其修饰为弱阴离子交换柱。考察了交联剂及致孔剂用量对色谱柱压力的影响,发现:随着交联剂用量的减小,致孔剂用量的增大,柱压下降;当交联剂和致孔剂用量达到一定水平后,柱压变化不再明显,但柱体机械强度降低。用该柱对白介素-18进行了分离,同时考察了不同固定相、缓冲液体系、pH值、盐的类型以及有机添加剂对白介素-18分离效果的影响。方法简单、灵敏、快速,重现性好。     

辛基型毛细管液相色谱整体柱的制备、表征及评价

本研究采用以甲基丙烯酸辛酯为单体,乙二醇二甲基丙烯酸酯为交联剂,偶氮二异丁腈为引发剂,正丙醇、1,4丁二醇和水三元混合物为共溶剂,制备了内径为0．53mm的毛细管整体柱材料。详细考察了单体／交联剂比例、单体混合物与致孔剂之间的比例,对所制得材料的通透性、孔径分布、粒度大小等性能的影响;应用包括扫描电镜和压汞法对其进行表征;在毛细管液相色谱(c—HPLC)操作模式进行了初步色谱评价,结果表明：所制得的整体柱具有优良的通透性能,可在高达100μL／min的流速下进行快速分离,同时在离子对模式下对3种金属离子进行了分离,取得了较理想的效果。     

Preparation of glycerol dimethacrylate-based polymer monolith with unusual porous properties achieved via viscoelastic phase separation induced by monodisperse ultra high molecular weight poly(styrene) as a porogen

The preparation of polymer-based monolith capillary was examined by the use of glycerol dimethacrylate (GDMA) as monomer and monodisperse standard polystyrene (PS) solution in chlorobenzene as porogen. Poly-GDMA monoliths were prepared in situ in test tubes with standard PS having the variety of molecular weight (defined as Mw hereafter) from 50,000 to 3,840,000, and their morphology was compared to that of poly-GDMA monolith prepared in situ with a poor porogenic solvent of GDMA. According to scanning electron micrograph (SEM) observation, the structure of poly-GDMA monolith prepared in situ with toluene as a poor porogenic solvent showed a typical agglomerated globular structure, whereas the morphology of poly-GDMA monolith prepared in situ with the polymer (PS) porogenic solution was transformed from the aggregated globule form to three dimensionally (3D) continuous skeletal structure with the increase of Mw of standard PS utilized. Along with this morphological transformation or change, in the case of poly-GDMA monolith prepared in situ with ultra high Mw standard PS porogenic solution, the pore size distribution showed a sharp bimodal distribution, with one peak being located around 4 nm in the mesopore range (2-50 nm) and the other peak located around 1-2 microm in the macropore range (>50 nm), respectively. The poly-GDMA capillaries were prepared in situ with toluene, low Mw (50,000, 600,000) PS solution in chlorobenzene and the above mentioned ultra high Mw PS solution in chlorobenzene as a porogen, respectively, and measured by mu-HPLC with benzene and n-alkyl phenyl ketone as solutes for the evaluation in aqueous methanol (MeOH/H(2)O = 50/50-80/20, v/v). The permeability of capillaries prepared in situ with ultra high Mw standard PS polymer porogenic solution was much larger, compared to those of the capillaries prepared in situ with low Mw standard PS polymer porogenic solution or with toluene as porogen. On the other hand, the column efficiency was better in the case of the capillary prepared in situ with the ultra high Mw PS solution than in the latter capillaries. Those observations indicated that the ultra high Mw standard PS polymer porogenic solution should delay dynamically the phase separation of polymerizing mixture because of its visco-elasticity and should contribute to the creation of three dimensionally continuous skeletal monolith structure better to afford high separation efficiency.     

Preparation and evaluation of hydroxylated poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic capillary for in-tube solid-phase microextraction coupled to high-performance liquid chromatography

A hydroxylated poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMA-co-EDMA) monolithic capillary was prepared and investigated for in-tube solid-phase microextraction (SPME). The polymer monolith was synthesized by in-situ polymerization of GMA and EDMA in the presence of dodecanol and toluene as the mixed porogenic solvents. After polymerization, glycidyl groups were hydrolyzed with sulfuric acid to produce diol groups at the surface of the porous monolith. To investigate the extraction mechanism, several groups of model analytes (including neutral, acidic and basic) were selected to perform extractions. The resulting monolith showed high extraction selectivity towards polar compounds, which resulted from the enhancement of dipole-dipole and hydrogen bonding interactions relative to hydrophobic interactions. The equilibrium extraction time profiles were also monitored for those model compounds to assess the extraction capacity of the monolithic capillary. Moreover, the hydroxylated poly(GMA-co-EDMA) monolithic capillary exhibited satisfactory reproducibility and stability. Finally, the in-tube SPME-HPLC method, based on the developed monolithic capillary as the extraction media, was successfully applied to the determination of five polar organic contaminants in lake water.     

Preparation and characterization of polyethyleneimine modified ion-exchanger based on poly(methacrylate-co-ethylene dimethacrylate) monolith

A polyethyleneimine (PEI) modified ion-exchanger was prepared based on poly(methacrylate-co-ethylene dimethacrylate) monolith cast in 100 mm x 4.6 mm I.D. stainless steel tube with heptane as the porogenic solvent at 65 degrees C for 12 h. The pores larger than 500 nm presented 85% of total pore volume of PEI monolith and provided the better permeability for separation. Bovine serum albumin (BSA) binding capacity on the column was enhanced with increasing the molecular weight of PEI, indicated that the brush ligand emanated from the surface and captured more protein by multiple binding sites. Titration experiment as well as BSA retention versus the pH of mobile phase showed that the monolith exhibited weak ion-exchange property, and recovered BSA on the monolith reached 97% when NaCl content in mobile phase was higher than 0.5 M. Frontal analysis and gradient elution of BSA indicated that PEI monolith provided the rapid mass transfer in chromatographic procedure, which made the dynamic binding capacities as well as column efficiency keep as constants at high operating flow rate. Fast separation of three mode proteins mixture (lysozyme, hemoglobin and BSA) on the monolith was achieved within 3 min at velocity of 1445 cm/h. This demonstrated the potential of PEI monolith for the rapid analysis and separation of proteins.     

Preparation of a mixed-mode hydrophilic interaction/anion-exchange polymeric monolithic stationary phase for capillary liquid chromatography of polar analytes

A novel cationic hydrophilic interaction monolithic stationary phase based on the copolymerization of 2-(methacryloyloxy)ethyltrimethylammonium methyl sulfate (META) and pentaerythritol triacrylate (PETA) in a binary porogenic solvent consisting of cyclohexanol/ethylene glycol was designed for performing capillary liquid chromatography. While META functioned as both the ion-exchange sites and polar ligand provider, the PETA, a trivinyl monomer, was introduced as cross-linker. The monolithic stationary phases with different properties were easily prepared by adjusting the amount of META in the polymerization solution as well as the composition of the porogenic solvent. The hydrophilicity of the monolith increased with increasing content of META in the polymerization mixture. A typical hydrophilic interaction chromatography mechanism was observed when the content of acetonitrile in the mobile phase was higher than 20%. The poly(META-co-PETA) monolith showed very good selectivity for neutral, basic and acidic polar analytes. For polar-charged analytes, both hydrophilic interaction and electrostatic interaction contributed to their retention. Peak tailing of basic compounds was avoided and the efficient separation of benzoic acid derivatives was obtained.     

Rapid Separation of Proteins by Capillary HPLC on a Short Polymethacrylate-based Strong Cation-exchange Monolithic Column

A polymethacrylate‐based strong cation‐exchange capillary monolithic column was prepared by in‐situ copolymerization for the fast separation of proteins. Glycidyl methacrylate (GMA) was used as monomer, ethylenedimethacrylate (EDMA) as cross link agent and the mixture of 1‐propanol, 1,4‐butanediol and water as porogen solvent. The monolith was sulfonated using 1 mol/L Na₂SO₃ based on a ring opening of epoxides. The influences of the contents of the porogen solvent and GMA and the various concentration ratios of 1‐propanol to 1,4‐butanediol in the polymerization mixture on the morphology, porosity, globule size, stability and column efficiency were investigated. The morphology and pore size distribution of the monolith were characterized by SEM and mercury intrusion porosimetry, respectively. Using only 1.5 cm length of this monolithic capillary column, four kinds of proteins, trypsin, cytochrome C, lysozyme (egg white) and egg albumin, were successfully separated from each other in 5 min at a high flow rate of 110 mm/s.     

Novel Polymer Monolithic Column for Hydrophilic Compounds

A hydrophilic polymer-based monolithic column was prepared from water-soluble crosslinking agents for liquid chromatography. The column media were prepared with a diacrylate monomer, aqueous polyethylene glycol, methanol, and a water-soluble radical initiator. A fused silica capillary column, which was significantly modified by vinyl groups, was utilized as the outer column in order to control polymer shrinkage. We optimized the modification of the capillary column, composition of the monomer and porogen, and polymerization condition. The optimized column exhibited high hydrophilicity and achieved the baseline separation of nucleobases by using only a buffered solution as the mobile phase. Additionally, another column prepared from binary water-soluble crosslinking agents exhibited lower nonspecific adsorption of several proteins. Basically, we would show the possibility of a new type of polymer monolith prepared from water-soluble crosslinker and water-soluble porogenic solvent, and they can be used for chromatographic separation without non-specific hydrophobic interaction.     

Fast preparation of photopolymerized poly(benzyl methacrylate-co-bisphenol A dimethacrylate) monoliths for capillary electrochromatography

A novel porous polymer monolith was prepared in situ in a fused-silica capillary using photoinitiated polymerization. Bisphenol A dimethacrylate (BPADMA) was selected as a crosslinker, copolymerized with benzyl methacrylate (BMA) in the presence of a binary porogenic solvent consisting of cyclohexanol and 1-decanol in ≤10 min. The resulting poly(BMA-co-BPADMA) monoliths exhibited good permeability and mechanical stability. Mixtures of alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs) or phenolic compounds were successfully separated by CEC. A similar monolith was also prepared with ethylene dimethacrylate (EDMA) as the crosslinker instead of BPADMA to compare the separation ability of the resulting monoliths. The results indicated that poly(BMA-co-BPADMA) monoliths have better selectivity for aromatic analytes and greater chromatographic stability in higher aqueous mobile phase.     

Capillary electrochromatography with monolithic stationary phases: 1. Preparation of sulfonated stearyl acrylate monoliths and their electrochromatographic characterization with neutral and charged solutes

A novel monolithic stationary phase having long alkyl chain ligands (C17) was introduced and evaluated in capillary electrochromatography (CEC) of small neutral and charged species. The monolithic stationary phase was prepared by the in situ copolymerization of pentaerythritol diacrylate monostearate (PEDAS) and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a ternary porogenic solvent consisting of cyclohexanol/ethylene-glycol/water. While AMPS was meant to support the electroosmotic flow (EOF) necessary for transporting the mobile phase through the monolithic capillary, the PEDAS was introduced to provide the nonpolar sites for chromatographic retention. Monolithic columns at various EOF velocities were readily prepared by conveniently adjusting the amount of AMPS in the polymerization solution as well as the composition of the porogenic solvent. The monolithic stationary phases thus obtained exhibited reversed-phase chromatography behavior toward neutral solutes and yielded a relatively strong EOF. For charged solutes (e.g., dansyl amino acids), nonpolar as well as electrostatic interaction/repulsion with the monoliths were observed in addition to electrophoretic migration. Therefore, for charged solutes, selectivity and migration can be readily manipulated by changing various parameters including the nature of the monolith and the composition of the mobile phase (e.g., pH, ionic strength and organic modifier). Ultrafast separation on the time scale of seconds of 17 different charged and neutral pesticides and metabolites were performed using short capillary columns of 8.5 cm x 100 microm ID.     

Specific capture of phosphopeptides by Zr4+-modified monolithic capillary column

A method to prepare zirconium phosphate (ZrP)-modified monolithic capillary column for highly specific capture of phosphopeptides is presented. In this method, the phosphate monolithic capillary column was prepared by direct copolymerization of the functional monomer containing phosphate group (ethylene glycol methacrylate phosphate) and cross-linker (bis-acrylamide) in a ternary porogenic solvent. Copolymerization of cross-linker and functional monomer simplifies the procedure for the preparation of phosphate chromatographic media. After Zr4+ was immobilized, the ZrP-modified monolithic capillary column was evaluated by the analysis of standard phosphoproteins and the excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of beta-casein and BSA with molar ratio of 1:200.     

胃蛋白酶亲和有机聚合物毛细管整体柱的制备及性能考察

以热引发原位聚合方法制备了聚（甲基丙烯酸缩水甘油酯（glycidyl methacrylate,GMA）-乙二醇二甲基丙烯酸酯（ethyleneglycol dimethacrylate,EDMA））毛细管整体柱,对整体柱的性能进行了表征。结果表明,柱内部结构均匀、渗透性好;整体柱能够实现苯等中性小分子化合物的分离,具有反相色谱特征,重现性和稳定性良好。利用整体柱环氧基团的活性,采用间接法,以戊二醛为连接臂制备胃蛋白酶亲和手性整体柱。在毛细管电色谱模式下进行了柱分离性能研究,并对缓冲液pH值和运行电压等分离条件进行了考察。结果表明,亲和整体柱对4种碱性手性药物（奈福泮、氨氯地平、西酞普兰、扑尔敏）有拆分效果,奈福泮、氨氯地平、西酞普兰能达到基线分离。本文为蛋白质亲和毛细管电色谱整体柱的制备和应用提供了新的思路和方法。