Synthesis of Monodisperse Poly（glycidylmethacrylate-co-ethylene dimethacrylate） Beads and Their Application in Separation of Biopolymers

Introduction Since 19541 the polymeric separation media has attracted much attention due to their chemical stability over the entire pH range. The rigid, highly cross-linked styrene copolymers were first used for chromatography by Moore.2 The macroporous copolymers currently available are not only chemically stable but also more resistant to mechanical forces prevailing in a column and therefore are comparable to the traditional packings based on silica gel. Most polymer separation media are …     

Synthesis of monodisperse poly(chloromethylstyrene-co-divinylbenzene) beads and their application in separation of biopolymers

The monodisperse, porous poly(chloromethylstyrene-co-divinylbenzene) beads of 7.9 microm were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. Based on this media, a weak cation-exchange (WCX) stationary phase for HPLC was synthesized by a new chemically modified method. The prepared resin has advantages of biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery, and good resolution for proteins. The dynamic protein-loading capacity of the synthesized WCX packings was 18.2 mg/g. Five proteins were separated in 3.0 min using the synthesized WCX stationary phase. The experimental results show that the obtained WCX resin has very weak hydrophobicity. The WCX resin was also used for the rapid separation and purification of lysozyme from egg white in 5.0 min with only one step. The purity and specific bioactivity of the purified lysozyme were found to be more than 93% and 70 245 U/mg, respectively.     

Synthesis of non-porous poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their application in separation of biopolymers

The monodisperse, 5.0 μm non-porous poly(glycidylmethacrylate-co-ethylenedimethacrylate) (P_GMA/EDMA) beads were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. Based on this media, a weak cation exchange (WCX) stationary phase for high performance liquid chromatography (HPLC) was synthesized by a new chemical modification method. The prepared resin has advantages of biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery and good resolution for proteins. The measured bioactivity recovery for lysozyme was 97 ± 5%. The dynamic protein loading capacity of the synthesized WCX packings was 20.5 mg/g. Four proteins were completely separated in 3.0 min using the synthesized WCX stationary phase. The experimental results show that the obtained WCX resin has very weak hydrophobicity. The WCX resin was also used for the rapid separation and purification of lysozyme from egg white in 3.0 min with only one step. The purity and specific bioactivity of the purified lysozyme was found more than 95% and 70.264 IU/mg, respectively.     

Preparation of weak cation exchange packings based on monodisperse poly(glycidyl methacrylate-<Emphasis Type="Italic">co</Emphasis>-ethylene dimethacrylate) beads and their chromatographic properties

The monodisperse, macroporous poly(glycidyl methacrylate- co-ethylene dimethacrylate) beads were synthesized by a single-step swelling and polymerization method. Based on this media, a weak cation exchange (WCX) stationary phase for HPLC was synthesized by a new chemically modified method. The prepared resin has advantages for biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery, and good resolution for proteins. The measured bioactivity recovery for lysozyme was 98+/-5%. The dynamic protein loading capacity of the WCX packings was 17.3 mg g(-1). The experimental results show that the synthesized WCX resin has very weak hydrophobicity.     

单分散树脂基质的弱阳离子交换色谱固定相的制备及其在生物大分子分离中的应用研究

采用分散聚合法制备种子和“一步种子溶胀聚合法”制备了粒径为6~15 μm的单分散多孔氯甲基苯乙烯-二乙烯苯微球。该微球经化学改性后得到一种亲水性良好的新型高效弱阳离子交换色谱固定相。详细考察了该固定相的表面亲水性、对标准蛋白的分离性能和盐的种类对蛋白质保留行为的影响。考察结果表明该固定相是一种性能优异的弱阳离子交换色谱固定相。将其应用于鸡蛋清中溶菌酶的快速分离纯化,纯化后的溶菌酶纯度高于96%,比活高达71184 U/mg。     

Preparation of strong anion-exchange chromatographic packings based on monodisperse polymeric beads and their application in the separation of biopolymers

A new hydrophilic strong anion-exchange (SAX) stationary phase for HPLC has been synthesized by chemical modification of macroporous 8.0-m monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads (P_GMA/EDMA). The stationary phase was evaluated in detail to determine its ion-exchange properties, separability, reproducibility, hydrophilicity, and the effect of column loading and pH on the separation and retention of proteins. It was found to have an ion-exchange chromatographic (IEC) retention mechanism. The highest dynamic protein loading capacity of the synthesized SAX packing for BSA was 22.6 mg g^–1. Five proteins were separated within 6.0 min using the synthesized SAX resin. The SAX resin was also used for rapid separation and purification of recombinant human stem cell factor (rhSCF) from a crude extract solution in only one step. The purity of the purified of rhSCF was >92.4%.     

重组人干扰素-γ的制备与鉴定

用聚乙二醇200疏水相互作用色谱固定相(PEG200-STHIC)分别在色谱柱和色谱饼上完成了一步复性并同时纯化来源于大肠杆菌(E.coli)表达的重组人干扰素-γ(rhIFN-γ)。为了能使色谱分离方法用于不同来源的rhIFN-γ的纯化,对rhIFN-γ在反相色谱、离子交换色谱、固定化镍离子亲和色谱上的保留行为也进行了研究。色谱柱纯化的rhIFN-γ收集液经排阻色谱除盐和冷冻干燥得到rhIFN-γ干粉。用基质辅助激光解吸电离飞行时间质谱对rhIFN-γ干粉进行了测定,rhIFN-γ单体的相对分子质量为17184.0,二聚体的相对分子质量为34204.4。用细胞病变抑制法(CPEI)测定rhIFN-γ干粉的比活性为9.5×108 IU/mg。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定rhIFN-γ干粉的纯度高于95%。用色谱柱复性并同时纯化rhIFN-γ的质量回收率达到93.7%,纯度高于95%,比活性为4.3×107 IU/mg。结果表明,采用PEG200-STHIC色谱柱复性并同时纯化rhIFN-γ是一种十分高效的方法。     

Preparation of Medium Cation Exchange Stationary Phase of Polymeric Matrix and Their Chromatographic Properties

Based on the monodisperse poly（glycidyl methacrylate-co-ethylenedimethacrylate） beads （PGMA/EDMA） with macropore as a medium, a new hydrophilic medium cation exchange （MCX） stationary phase for HPLC was synthesized by a new chemically modified method. The stationary phase was evaluated with the property of ion exchange, separability, reproducibility, hydrophilicity, effect of salt concentration, salt types, column loading and pH on the separation and retention of proteins in detail. It was found that it follows ion exchange chromatographic （IEC） retention mechanism. The measured bioactivity recovery for lysozyme was （96 ± 5）%. The dynamic protein loading capacity of the synthesized MCX packings was 21.8 mg/g. Five proteins were almost completely separated within 6.0 min at a flow rate of 4 mL/min using the synthesized MCX resin. The MCX resin was also used for the rapid separation and purification of lysozyme from egg white with only one step. The purity and specific bioactivity of the purified lysozyme was found more than 95% and 70345 U/mg, respectively.     

肝素温度敏感色谱填料的制备与性能研究

利用自由基聚合法制备了含有聚(N-异丙基丙烯酰胺/N-丙烯酰氧琥珀酰亚胺)和肝素共聚物的温度敏感的亲和色谱填料; 通过热失重分析得到该填料表面聚合物的接枝率为9.45%; Elison-Morgan 法测定该填料表面的肝素含量为3.6mg/g. 将其用作HPLC 固定相, 分离苯和氢化可的松, 表明该色谱固定相具有温度敏感特性; 该色谱柱对凝血酶具有较强的亲和作用, 通过控制温度缩短了凝血酶的释放时间, 回收率为81.4%, 这为蛋白质的分离提供了一个快速有效的方法.     

连续棒状疏水色谱柱的合成及表征

以单体,致孔剂在不锈钢柱管中直接聚合的方法,合成了一种丙烯酰胺－甲基丙烯酸丁酯－Ｎ,Ｎ‘－亚甲基双丙烯酰胺聚合物连续棒状疏水色谱柱。考察了聚合物单体中甲基丙酸丁酯的含量对于标准蛋白质的分离效果和介质疏水性的影响。     

Preparation of High‐capacity,Monodisperse Polymeric Weak Cation Exchange Packings Using Surface‐initiated Atom Transfer Radical Polymerization and Its Chromatographic Properties

A novel stationary phase for weak cation exchange (WCX) chromatography was prepared by "grafting from" strategy. Surface initiated atom transfer radical polymerization (ATRP) of acrylic acid (AA) was conducted in toluene medium, starting from the macromolecule initiators of poly(4‐vinylbenzyl chloride‐co‐divinylbenzene) (P_CMS/DVB) beads. The amounts of poly(acrylic acid) grafted chains with different ATRP formulations were calculated based on the elemental analyses. The poly(acrylic acid) grafted beads obtained with different ATRP formulations were tried as chromatographic packings in the separation of proteins by ion‐exchange chromatography. The effect of the poly(acrylic acid) grafted chain lengths on P_CMS/DVB beads for the separation of proteins was investigated in details. Simultaneously, characterization of the column was investigated as ion chromatographic stationary phase for the separation of inorganic cations. The results show that poly(acrylic acid) grafted columns had excellent performance for separation of proteins and inorganic cations. The highest of the dynamic capacity of the column was 35.55 mg/mL. The columns were provided with high column efficiency.     

Preparation of a monolithic column for weak cation exchange chromatography and its application in the separation of biopolymers

A procedure for the preparation of a monolithic column for weak cation exchange chromatography was presented. The structure of the monolithic column was evaluated by mercury intrusion. The hydrodynamic and chromatographic properties of the monolithic column--such as back pressures at different flow rates, effects of pH on protein retention, dynamic loading capacity, recovery, and stability--were determined under conditions typical for ion-exchange chromatography. The prepared monolithic column might be used in a relatively broad pH range from 4.0 to 12.0 and exhibited an excellent separation to five proteins at the flow rates of both 1.0 and 8.0 mL/min, respectively. In addition, the prepared column was first used in the purification and simultaneous renaturation of recombinant human interferon gamma (rhIFN-gamma) in the extract solution with 7.0 mol/L guanidine hydrochloride. The purity and specific bioactivity of the purified rhIFN-gamma in only one chromatographic step were obtained to be 93% and 7.8 x 10(7) IU/mg, respectively.     

磷酰胆碱色谱固定相的制备及其在分离生物大分子中的应用

以三氯氧磷、氯化胆碱和甲基丙烯酸-2-羟乙酯(HEMA)为原料合成了含磷酰胆碱的单体2-甲基丙烯酰氧乙基磷酰胆碱(MPC). 在硅胶表面嫁接聚合MPC, 得到磷酰胆碱两性离子交换色谱填料. 研究了该填料对标准蛋白的分离性能及pH对蛋白质保留的影响. 结果表明, 该填料对溶菌酶和牛血清白蛋白的动态吸附容量分别为13.8和18.7 mg/g, 其基质磷酰胆碱色谱固定相可同时基线分离两种酸性和两种碱性蛋白.     

Preparation of poly(vinyltetrazole) chain-grafted poly(glycidymethacrylate-co-ethylenedimethacrylate) beads by surface-initiated atom transfer radical polymerization for the use in weak cation exchange and hydrophilic interaction chromatography

A novel stationary phase for weak cation exchange (WCX) and hydrophilic interaction chromatography (HILIC) was prepared with surface-initiated atom transfer radical polymerization (SI-ATRP). Vinyltetrazole was grafted onto the surface of the beads in water medium with the polyglycidylmethacrylate beads (P_GMA/EDMA) previously modified with 2-bromoisobutryl bromide as the macromolecule initiators and CuCl as catalyst. The poly(vinyltetrazole)-grafted beads obtained with different atom transfer radical polymerization (ATRP) formulations were tried as chromatographic packings in ion-exchange chromatography. The results showed that the prepared columns could separate the tested proteins with high efficiency and high capacity, and the retention time of protein had a positive relationship with increasing the chain lengths of the grafted poly(vinyltetrazole) (PVT). The prepared column was also found to be able to separate nucleosides by hydrophilic interaction chromatographic mode.     

原子转移自由基聚合法制备新型亲水作用色谱固定相及其性能评价

以自制的6.0μm单分散大孔交联聚氯甲基苯乙烯-二乙烯基苯(Poly(4-vinylbenzylchloride-co-divi-nylbenzene),PCMS/DVB)微球为基质和引发剂,CuCl和自行合成的三[(2-二甲基氨基)乙基]胺(Tris[2-(dimeth-ylamino)ethyl]amine,Me6TREN)组成混合催化体系,使4-乙烯基吡啶(4-Vinyl pyridine,4-VP)在甲苯中进行原子转移自由基聚合,制得4-乙烯基吡啶聚合物,单体4-乙烯基吡啶的接枝率为8.55%。将该聚合物与正溴丁烷反应制得新型亲水色谱固定相。在亲水作用色谱模式下,流速1 mL/min,乙腈-水为流动相可分离5种芳胺化合物和4种酚类化合物。在离子交换色谱模式下,6 mmol/L Na2CO3-5.5 mmol/L NaHCO3为淋洗液可分别分离5种无机阴离子和4种短链有机酸。结果表明,此固定相对极性化合物和无机阴离子具有良好的分离性能,是一种性能优异的亲水作用色谱固定相。     

原子转移自由基聚合技术用于以单分散树脂为基质的温敏色谱固定相的制备及性能评价

以α-溴异丁酰溴为引发剂,CuCl/CuCl2/2,2′-联二吡啶(Bpy)为催化体系,在室温条件下通过原子转移自由基聚合(ATRP)使N-异丙基丙烯酰胺(NIPAM)键合在单分散交联聚甲基丙烯酸环氧丙酯树脂（PGMA/EDMA 树脂）表面,制备了具有温敏性的聚合物色谱填料,并用元素分析、红外光谱等对其进行了表征;详细考察了该填料对芳香烃化合物的分离性能、温敏性能、稳定性和重现性。元素分析得出NIPAM单体的接枝率为10.4%;通过改变温度,可以有效地分离对羟基苯甲醛、邻甲酚和4-丁基苯胺3种混合物。结果表明,所合成的固定相具有很好的色谱性能和温敏性能,稳定性和重现性良好。     

原子转移自由基聚合技术制备L-苯丙氨酸手性配体交换色谱固定相及其对手性化合物的拆分

在室温条件下,以甲基丙烯酸环氧丙酯(GMA)为单体,溴异丁酰溴为引发剂,CuCl/2,2′-联吡啶(Bpy)为催化剂,通过原子转移自由基聚合(ATRP)反应,将甲基丙烯酸环氧丙酯聚合在硅胶表面。然后再将L-苯丙氨酸共价键合在硅胶表面的聚合物上,制备了新型手性配体交换色谱固定相,并用该固定相对DL-氨基酸进行分离。用元素分析对其进行了表征;详细考察了固定相的合成过程以及流动相pH值、流动相铜离子浓度、柱温等色谱条件对DL-氨基酸对映体拆分的影响。元素分析得出该固定相表面L-苯丙氨酸接枝密度达到4.32 mg/m2;在手性配体交换分离模式下,流动相为0.05 mol/L KH2PO4-0.1 mmol/L Cu(Ac)2水溶液、流速为1.0 mL/min、柱温为50 ℃和检测波长为223 nm条件下,该色谱固定相可以分离DL-天冬氨酸、DL-天冬酰胺等。同时,流动相pH值、铜离子浓度以及柱温对手性对映体的拆分有较大影响。与传统的在硅胶表面直接键合L-苯丙氨酸制得的固定相相比,所合成的固定相接枝密度高,分离效果好,对DL-天冬氨酸及DL-天冬酰胺实现了基线分离。结果表明,在手性配体交换分离模式下,固定相具有良好的拆分性能。     

氨基己酸为配基的新型弱阳离子交换/疏水双功能色谱固定相对蛋白质的分离纯化

以硅胶为基质、氨基己酸为配基制备了一种新型弱阳离子交换/疏水（WCX/HIC）双功能混合模式色谱固定相。该固定相配基具有一定的疏水性且含有羧基,在高盐浓度下表现为HIC的性质,可作为HIC固定相使用;在低盐浓度条件下表现为离子交换的性质,可作为WCX固定相使用。分别考察了该介质在WCX和HIC两种模式下对标准蛋白质的分离性能,并与商品柱进行比较。结果表明,所合成的WCX/HIC双功能固定相在WCX和HIC两种模式下对蛋白质均有较高的分离度和选择性,且分离能力与商品柱相当,两种模式下标准蛋白质的质量和活性回收率均大于93%,表明该柱具有“一柱二用”的功能,适于生物大分子的分离纯化。基于此双功能色谱柱构建的在线单柱二维液相色谱（2DLC-1C）可在60 min内实现8种蛋白质的快速分离。在70 min内完成了对蛋清中溶菌酶的二维纯化,纯度可达到98.3%。该技术中一根色谱柱可当作两根色谱柱使用,对蛋白质组学研究和重组蛋白药物的生产具有重要的应用价值。     

Immobilized Stationary Phases for Hydrophobic Interaction Chromatography of Proteins

Immobilized stationary phases for hydrophobic interaction chromatography (HIC) of proteins are prepared by coating macroporous silica Daisogel of different porosity with hydrophobized cellulose derivatives. The polymer adsorbed on the silica surface afterwards was cross-linked with bifunctional compounds. A uniform polymer nanocoating was indicated using the nitrogen gas adsorption method BET and scanning electron microscopy. The absence of non-specific protein sorption of the synthesized adsorbents shows that the developed polymeric coating isolates silica surface from contact with the sorbate. The retention data of bovine serum albumin (BSA) in the HIC mode on different synthesized adsorbents were evaluated. It was shown that sorption capacity of such phases may vary over a wide range and depends mainly on the substitution degree of the immobilized polymer. The dynamic sorption capacity of BSA was up to 63 mg mL⁻¹. The results proved that the new stationary phases have significant promise for the separation and purification of proteins in the HIC mode.

     

单分散大孔亲水弱阳离子交换填料的制备及其对鱼精蛋白的分离纯化

以自制的5.0 μm单分散大孔亲水交联聚甲基丙烯酸环氧丙酯(PGMA/EDMA)微球为基质,对其表面进行化学改性,合成弱阳离子交换色谱填料(WCX)。详细考察了该填料对标准蛋白质的分离性能、表面亲水性能、稳定性和重现性以及流速对蛋白保留的影响。实验结果表明,该色谱填料对蛋白的分离性能、重现性及稳定性良好;在流速为3 mL/min时,采用线性梯度洗脱,6 min内可分离4种标准碱性蛋白质,以溶菌酶测定的该填料的动力学吸附容量为29.86 mg/g。将其用于鱼精蛋白的分离纯化,经反相高效液相色谱测定纯化后鱼精蛋白的纯度为99.2%;与商品Shodex IEC SP-825强阳离子交换色谱柱比较,纯化结果几乎一样。