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。     

单分散亲水两性离子交换树脂的制备及其在生物大分子分离中的应用

采用分散聚合与溶胀聚合相结合的方法及高分子溶液致孔技术, 成功地制备了粒径为5.0 μm大孔和超大孔结构的单分散亲水性交联聚甲基丙烯酸环氧丙酯树脂, 并进行了结构表征. 将该树脂经胺化后再与1,3-丙磺酸内酯反应, 得到一种新型的两性离子交换(强阳-强阴型)高效液相色谱填料. 研究了该填料对标准蛋白分离性能及流动相中有机溶剂、 流速和pH值对蛋白保留的影响. 实验结果表明, 在流速为3 mL/min时, 采用线性梯度洗脱, 在4.0 min内可同时快速基线分离3种酸性和2种碱性蛋白.     

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。     

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。     

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。     

以单分散交联聚甲基丙烯酸环氧丙酯树脂为基质的亲水性排阻色谱固定相的合成及其在生物大分子分离中的应用

以单分散交联聚甲基丙烯酸环氧丙酯树脂为基质,将该树脂经化学改性后得到亲水性良好的新型尺寸排阻色谱固定相,其表面羟基含量高达6．1mmol／g。详细评价了该改性后树脂对蛋白的质量回收率、亲水性、耐压性能及化学稳定性。用三羟甲基胺基甲烷(Tris)缓冲溶液(pH=7)为流动相,对蛋白质混合样的分离遵循体积排阻色谱分离机理。     

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。     

单分散交联聚甲基丙烯酸环氧丙酯树脂的制备及亲合纯化伴刀豆球蛋白-A

采用一步种子溶胀聚合法制备了颗粒呈单分散的交联聚甲基丙烯酸环氧丙南树脂，对其环氧基的组成比进行了表征，并以氨基葡萄糖为配基，首次制备了纯化粗品伴刀豆球蛋白-A(Ⅲ）(Con-A，Ⅲ)的聚合物基质的高效亲合色谱柱。配基在树脂上的键合量为8．2mg／g，对纯化后的Con-A（Ⅲ）的吸附量为13．4mg／g。使用该亲合色谱介质成功地从粗品Con-A中快速纯化了Con-A，电泳分析显示为一个主要的谱带，纯度从15％提高到95％。     

活性蓝F3GA固载的无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯亲和色谱填料的制备与应用

以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为基质,与三嗪染料活性蓝F3GA(Cibacron Blue F3GA)反应,制备出 一种固定化染料聚合物高效亲和色谱填料。考察了应用该填料时流动相中的盐离子浓度、有机溶剂及流速等对牛血清白蛋白(BSA)和 溶菌酶(Lys)保留行为的影响。实验结果表明,该染料亲和填料具有良好的色谱性能。利用前沿色谱法测定了染料与溶菌酶之间的表观 解离常数为5.26×10-5 mol/L。使用该填料成功地从鸡蛋清和小牛血清中分别分离纯化了Lys和BSA,十二烷基硫酸钠-聚丙烯酰胺凝胶 电泳(SDS-PAGE)分析显示Lys和BSA的纯度分别为95%和92%。     

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 for chromatographic separation of proteins using "click chemistry'

"Click chemistry" is defined as a class of robust and selective chemical reactions affording high yields and is tolerant to a variety of solvents (including water), functional groups, and air. In this study, click chemistry was used as an effective strategy for coupling three alkyne-carboxylic acids onto the azide-silica to obtain three novel stationary phases of weak cation exchange chromatography, which were characterized with FTIR and elemental analysis. Six kinds of standard proteins, such as myoglobin, RNase A, RNase B, cytochrome C, α-chymotrypsin A, and lysozyme, were separated completely with the three novel weak cation exchange chromatography stationary phases. Compared with commercial weak cation exchange chromatography columns, the three kinds of novel weak cation exchange chromatography packings prepared by click chemistry approach have better resolution and selectivity. The mass recovery of more than 97% was obtained for all the tested proteins, and the bioactivity recovery of lysozyme on the prepared column was determined to be 96%. In addition, lysozyme was purified successfully from egg white with the novel weak cation exchange chromatography column by one step. The purity was more than 97% and a high specific activity was achieved to be 81 435 U/mg. The results illustrate the potential of click chemistry for preparing stationary phase for ion-exchange chromatography.     

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%.     

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.     

Preparation of weak cation exchange packings based on monodisperse poly(chloromethylstyrene‐co‐divinylbenzene) particles and its chromatographic properties

Monodisperse poly (chloromethylstyrene‐co‐divinylbenzene) particles were firstly prepared by a two‐step swelling method. Based on this media, one kind of weak cation ion exchange packings was prepared. It was demonstrated that the prepared packings have comparative advantages for biopolymer separation with high column efficiency, low interstitial volume and low column backpressure, and have good resolution to proteins. The effects of salt concentration and pH of mobile phase on protein retentions were investigated. The properties of the weak cation ion exchange packings were evaluated by the unified retention model for mixed‐mode interaction mechanism in ion exchange and hydrophobic interaction chromatography.     

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(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.     

强阳离子交换色谱分离多肽混合物的条件优化

以酵母全蛋白提取液的胰蛋白酶切产物为研究对象,对多维色谱分离中常用的强阳离子交换色谱的分离条件,包括上样量、盐的种类、调节缓冲液pH值的酸的种类及有机溶剂的比例进行了考察和比较。结果表明,在强阳离子交换色谱分离线性上样量范围内,在氯化铵溶液作为流动相,用磷酸调节流动相缓冲液pH值至2.7,且流动相中乙腈的体积分数为30%条件下进行梯度洗脱时,可获得最佳的分离结果。本结果可为采用二维色谱-质谱策略分析多肽混合物时的第一维强阳离子交换色谱分离条件的选择提供有益的参考。