以单分散树脂为基质的中强阳离子交换色谱填料的合成及应用

将粒径为8.0μm多孔单分散交联氯甲基苯乙烯-二乙烯苯树脂的表面经化学方法改性,得到了一种亲水性良好的中强阳离子交换色谱填料,详细考察了该改性后树脂的表面亲水性、对标准蛋白的分离性能、盐种类及盐浓度对蛋白保留行为的影响。实验结果表明,当流动相流速为3 mL/min时,四种标准蛋白可在3min内基线快速分离,蛋白质的保留符合阳离子交换色谱规律。该填料应用于鸡蛋清中溶菌酶快速分离纯化,取得较好效果。     

无孔单分散亲水性中强阳离子交换树脂的制备及其在蛋白质快速分离中的应用研究

以3.0μm无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯树脂为基质,将其表面经新的化学方法改性后制备了一种新型的无孔中强阳离子交换色谱填料。详细考察了该无孔填料对标准蛋白分离性能,有机溶剂、pH、流动相盐种类和流速等对蛋白质保留的影响。实验结果表明,在流速为4 mL/min时,线性梯度时间在2.0 min内可快速分离4种标准蛋白,蛋白质的保留符合阳离子交换色谱规律。将其应用于快速纯化鸡蛋清中的溶菌酶,取得较好效果。     

原子转移自由基聚合法制备强阴离子交换色谱填料及其在蛋白质分离中的应用

在大孔硅胶表面引入原子转移自由基聚合的引发基团,通过该技术将甲基丙烯酰氧乙基三甲基氯化铵聚合于硅胶表面,制得了强阴离子交换色谱填料.详细考察了该填料对标准蛋白质的分离性能及流动相pH、流速、有机溶剂对蛋白质保留的影响.实验结果表明,在流速为0.75 mL/min时,采用线性梯度洗脱,20 min内可快速分离不同体系的三种标准蛋白;并将该填料用于鸡蛋中卵清蛋白的快速分离纯化,取得了很好的效果.     

快速弱阴离子交换无孔聚合物固定相的制及用于蛋白质的高效液相色谱分离

将粒径为3.0 μm无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯树脂(PGMA/EDMA)的表面经不同的化学方法改性,制备了两种不同配基的弱阴离子交换(WAX-Ⅰ和WAX-Ⅱ)色谱填料.在同等色谱条件下,比较了两种填料对蛋白质的分离性能,发现WAX-Ⅰ型填料分离性能优于WAX-Ⅱ.详细考察了WAX-Ⅰ型弱阴离子交换色谱填料流动相pH值、流速及有机溶剂等对蛋白质保留的影响.实验结果表明,当流动相流速为3mL/min时,4种标准蛋白可在2 min内基线快速分离,蛋白质的保留符合阴离子交换色谱规律.该填料可以用于生物工程产品的快速分离和纯化.     

高亲水性强阳离子交换色谱填料的制备及其在蛋白质分析中的应用

以具有双孔结构的聚甲基丙烯酸环氧丙酯(PGMA)微球为基质,以葡萄糖进行表面亲水改性,制备了强阳离子交换色谱填料,并将其用于复杂生命体系中生物大分子的快速而高效的分离、分析与纯化。葡萄糖亲水改性增进了填料的生物相容性,提高了蛋白质样品的回收率;双孔结构及较高的比表面积赋予填料良好的柱渗透性和样品负载量。以标准蛋白质为样品,考察了该填料对生物样品的分离性能。以100 mm×4.6 mm的色谱柱分离4种蛋白质,在6 min内实现了基线分离;以溶菌酶为样品,填料的吸附容量为39.5 g/L,在蛋白质快速分离纯化分析中显示了良好的应用前景。     

无孔单分散亲水性强阳离子交换固定相的制备及其在蛋白质快速分离中的应用研究

采用分散聚合法制备小颗粒种子及“一步种子溶胀聚合”法成功地制备了粒径为3.0 μm的无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯树脂,其表面经水解、环氧化、再水解后与氯磺酸反应,制备了一种新型的强阳离子交换色谱填料（SCX）。详细考察了该填料对标准蛋白质的分离性能及流动相中盐的种类、有机溶剂、流速等对蛋白质保留的影响。实验结果表明,在流速为4 mL/min时,采用线性梯度洗脱,1.0 min内可快速分离4种标准蛋白质,蛋白质的保留符合阳离子交换色谱规律。将SCX应用于快速纯化鸡蛋清中的溶菌酶和猪心中的细胞色素-C,取得了较好的效果。     

快速弱阴离子交换无孔聚合物固定相的制备及用于蛋白质的高效液相色谱分离

将粒径为3.0μm无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯树脂(PGMA/EDMA)的表面经不同的化学方法改性,制备了两种不同配基的弱阴离子交换(WAX-Ⅰ和WAX-Ⅱ)色谱填料。在同等色谱条件下,比较了两种填料对蛋白质的分离性能,发现WAX-Ⅰ型填料分离性能优于WAX-Ⅱ。详细考察了WAX-Ⅰ型弱阴离子交换色谱填料流动相pH值、流速及有机溶剂等对蛋白质保留的影响。实验结果表明,当流动相流速为3mL/min时,4种标准蛋白可在2min内基线快速分离,蛋白质的保留符合阴离子交换色谱规律。该填料可以用于生物工程产品的快速分离和纯化。     

连续棒状弱阳离子交换柱的合成及其对蛋白质保留行为

以甲基丙烯酸缩水甘油酯为单体,乙二醇二甲基丙烯酸酯为交联剂在空管柱内就地聚合制备了一种聚合物连续棒状色谱基质,并通过“在线”化学改性将其修饰为弱阳离子交换柱。考察了该色谱柱的孔结构特征、表面亲水性能、对标准蛋白的分离效果和ｐＨ值对保留行为的影响以及色谱柱的重现性。结果表明,该色谱柱对蛋白的分离性能及重现性良好,柱寿命不短于半年。对溶菌酶的活性回收率达９６．２％。流动相流速为８．０ｍＬ／ｍｉｎ条件下     

新型聚合物单分散温敏色谱固定相的制备及其在生物大分子分离中的应用

以2.5 μm的单分散聚苯乙烯为种子,乙二醇二甲基丙烯酸酯 (EDMA)为交联剂,甲苯和环己醇为致孔剂,采用"一步种子溶胀聚合法"制备了单分散微球; 再以过硫酸钾为引发剂将水溶性温敏单体N-异丙基丙烯酰胺(NIPAM)分子引发聚合到微球表面,制备了粒径为7.0 μm、分散系数为0.02的单分散交联温敏色谱填料,温敏单体NIPAM的接枝率为5.2%.考察了该填料对标准蛋白质的分离性能、温敏性能、稳定性和重现性以及动态吸附容量对蛋白保留的影响.实验结果表明,该色谱填料对蛋白的分离性能、温敏性能、稳定性及重现性良好,且对溶菌酶的动态吸附容量为32.3 mg/g.在疏水模式下,该填料不但可以同时基线分离5种标准蛋白,而且通过改变温度可以有效地将3种在低温下保留时间重叠的蛋白(细胞色素-C、β-乳球蛋白和核糖核酸酶)完全分离.     

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。     

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 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.     

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.     

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.     

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.     

Weak anion and cation exchange mixed-bed microcolumn for protein separation

To separate proteins with a wide distribution of pIs under the conditions compatible to online tryptic digestion (with preferable pH=8.0), weak anion and cation exchange chromatography (WAX/WCX) mixed‐bed microcolumn has been developed. With a mixture of five proteins with pIs ranging from 4.2 to 11.4, the effect of WAX/WCX ratio on the separation performance was investigated, and an optimum packing ratio of 1:1 w/w was obtained. Moreover, the undesirable hydrophobic interaction between the proteins and the stationary phase was suppressed with 10% ACN v/v added in the mobile phases. Under the optimized conditions compatible to tryptic digestion, basic and acidic proteins were resolved simultaneously, with RSDs of relative retention time on six columns less than 6%, indicating the good resolution and packing reproducibility. Furthermore, one RPLC fraction of proteins extracted from rat middle brain and the whole protein mixture extracted from rat liver were analyzed, respectively. The results demonstrated better separation performance on WAX/WCX microcolumns than that on both weak anion exchange chromatography and weak cation exchange chromatography at pH ～8. We anticipate that WAX/WCX microcolumns are promising for the integration of protein separation and tryptic digestion aiming at high‐throughput proteome study.     

在线单柱二维液相色谱法快速纯化牛胰腺中细胞色素C

用二维（弱阳,疏水）色谱柱首次完成了在线单柱二维液相色谱法快速纯化牛胰腺中的细胞色素C.在将牛胰腺粗提液进样到该二维色谱柱后,在弱阳离子交换模式下,以梯度洗脱方式进行一维色谱分离,并将分离得到的细胞色素C样品液收集到色谱仪的附加样品储液管内.然后将储液管中样品液全部排出,并二次进样到同一根二维色谱柱中,与此同时也完成了对该样品液的缓冲溶液交换,按疏水色谱（HIC）分离模式进行分离.最终对细胞色素C完成了第二维的HIC纯化.上述全部操作均为在线,在一具有正压的封闭体系中进行并可在52分钟内完成.细胞色素C的最终产品纯度高达94.7%（RSD=1.91%）,质量回收率为80.5%（RSD=2.20%）.预计此在线单柱二维液相色谱法也可能用于牛胰腺中其他功能蛋白的快速纯化,并可能将其放大到制备和生产规模.     

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%.     

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.