阴离子交换整体柱对蛋白质的分离与纯化

分别用乙二胺、二乙胺、三乙胺将自制的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)为交联剂的整体柱修饰为弱、强阴离子交换整体柱。考察了该整体柱的性能,选择出分离蛋白质(牛血清白蛋白、溶菌酶和谷胱甘肽)的最佳实验条件,并在最佳分离条件下考察了这些蛋白质在整体柱上的色谱行为和该整体柱对纤维素降解酶的分离纯化情况。实验结果表明,该整体柱性能良好,可以实现对纤维素降解酶的快速分离与纯化。同时,实验也证明采用梯度洗脱可以实现对某些蛋白质的分离纯化。     

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。     

酯型十八烷基键合硅胶整体柱的制备、表征及性能评价

将硅烷偶联剂γ-(2,3-环氧丙氧)丙基三甲氧基硅烷与十八酸反应,再键合到硅胶整体柱上,得到了酯型十八烷基键合固定相,并用红外光谱、元素分析对其进行了表征。在以甲醇-水为流动相的反相色谱条件下分离了苯、联苯和蒽的混合样品,评价了该整体柱的色谱性能,考察了该整体柱适用的pH范围,以及柱压降、柱效与流速的关系。结果表明,该硅胶整体柱键合效果良好,具有较好的反相色谱性能,且在pH=2～8时稳定性好,柱压降、柱效受流速影响较小,可有效地用于化合物的快速分离分析。     

Electroosmotic pump‐supported molecularly imprinted monolithic column for capillary chromatographic separation of nitrophenol isomers

In this work, a novel molecularly imprinted polymer (MIP) monolithic column with integrated in‐column electroosmotic pump (EOP) was designed and successfully prepared to facilitate the capillary chromatography with MIP column. A silica‐based EOP was synthesized at the detection end of the MIP monolithic capillary column by so‐gel to provide the hydrodynamic driven force for the capillary chromatography. Because of large surface area and low fluidic resistance of the silica monolith,a strong and steady EOF was generated by silica‐based EOP, indicating that the EOP was quite compatible with MIP capillary column. With the sufficient EOF provided by EOP, the electro‐driven based capillary chromatographic separation of nitrophenol isomers was achieved in 4‐vinylpyridine‐based MIP monolithic capillary, which was originally proved infeasible because of the EOF shortage. No significant influence upon the specific recognition of the MIP was found due to the setting of EOP after the detection window of the column. The influence of experimental parameters on the EOF such as voltage and pH value of running buffer was investigated. The column was also evaluated by capillary liquid chromatographic mode to compare with EOP‐driven capillary chromatography. Higher column efficiency was obtained by EOP‐driven separation with improved peak shape. The results suggested that EOP‐supported technique would be a good way to solve the problem of weak EOF generation in electro‐driven capillary chromatography.     

以二甲亚砜/正十二醇为二元致孔剂的新型苯乙烯-二乙烯苯毛细管整体柱的制备与表征

首次以二甲亚砜/正十二醇为二元致孔剂,苯乙烯为单体,二乙烯苯为交联剂,过氧化苯甲酰为引发剂,通过原位聚合反应制备了苯乙烯-二乙烯苯聚合物型毛细管整体柱。结果表明,苯乙烯∶二乙烯苯∶二甲亚砜∶正十二醇的体积比为18.7∶15.3∶13.2∶52.8,即二甲亚砜占致孔剂的比例为20%,交联度为45%,致孔剂含量为66%为最优配比。所合成的整体柱实现了反相色谱模式下对小分子苯系物与生物大分子蛋白的快速分离。其中蛋白分离实验的流速达到104μL·min-1,线速度约为12 mm·s-1,而常规色谱柱的线速度为1~2 mm·s-1。该整体柱的渗透性好,可用于物质的高速分离,若对其进行化学修饰,有望用于其它色谱分离模式。     

Histidine‐modified organic‐silica hybrid monolithic column for mixed‐mode per aqueous and ion‐exchange capillary electrochromatography

A novel organic‐silica hybrid monolith was prepared through the binding of histidine onto the surface of monolithic matrix for mixed‐mode per aqueous and ion‐exchange capillary electrochromatography. The imidazolium and amino groups on the surface of the monolithic stationary phase were used to generate an anodic electro‐osmotic flow as well as to provide electrostatic interaction sites for the charged compounds at low pH. Typical per aqueous chromatographic behavior was observed in water‐rich mobile phases. Various polar and hydrophilic analytes were selected to evaluate the characteristics and chromatographic performance of the obtained monolith. Under per aqueous conditions, the mixed‐mode mechanism of hydrophobic and ion‐exchange interactions was observed and the resultant monolithic column proved to be very versatile for the efficient separations of these polar and hydrophilic compounds (including amides, nucleosides and nucleotide bases, benzoic acid derivatives, and amino acids) in highly aqueous mobile phases. The successful applications suggested that the histidine‐modified organic‐silica hybrid monolithic column could offer a wide range of retention behaviors and flexible selectivities toward polar and hydrophilic compounds.     

甲基丙烯酸酯毛细管整体柱分离微囊藻毒素的研究

以甲基丙烯酸丁酯为单体,乙二醇二甲基丙烯酸酯为交联剂,在致孔剂存在的条件下原位聚合制备了甲基丙烯酸丁酯毛细管整体柱(150 μm i.d.)。实验中优化了用此整体柱分离3种微囊藻毒素(MC-LR,-YR和-RR)的色谱条件(流动相种类、缓冲溶液浓度、pH、流动相流速),建立了微囊藻毒素的整体柱毛细管液相色谱分离方法,该法可以在9 min之内实现3种微囊藻毒素的基线分离。将该方法应用于实际水样中微囊藻毒素的分析,成功实现了培养水样和巢湖水样中微囊藻毒素的快速分离,两种样品中均检测到MC-LR。结果表明,所制备的甲基丙烯酸酯毛细管整体柱具有良好的重现性、渗透性,在微囊藻毒素的常规检测中具有很好的应用前景。     

整体柱高效液相色谱法测定草甘膦原药中甲醛含量

建立了衍生化-高效液相色谱法对草甘膦原药中痕量甲醛含量进行测定的方法。样品中残留甲醛经超声波水浴提取，与2，4-二硝基苯肼衍生反应，生成的2，4-二硝基苯腙用反相整体柱色谱进行快速分离，在360nm紫外波长下检测，外标法定量。该分析方法在2．0～200mg/L浓度范围呈良好线性，添加回收率在88％～105％之间，相对标准偏差小于5％。样品中甲醛的最小检测浓度为0．5mg/kg。比较了整体色谱柱和常规C18反相柱分离效果，表明整体色谱柱可在1．5min内实现衍生化产物的快速分离并进行定性定量，同时发现相对于常规柱，采用整体柱提高了检测灵敏度约10倍。     

Determination of DL-tetrahydropalmatine in Corydalis yanhusuo by L-tetrahydropalmatine imprinted monolithic column coupling with reversed-phase high performance liquid chromatography

A method for direct determination of DL-tetrahydropalmatine (DL-THP) in Corydalis yanhusuo, a traditional Chinese herb, by L-THP imprinted monolithic precolumn on-line/off-line coupling with reversed-phase high performance liquid chromatography (RP-HPLC) was developed. The L-THP imprinted monolithic column has been prepared by in situ polymerization using methacrylic acid (MAA) and ethylene dimethacrylate (EDMA) as functional monomer and cross-linker, respectively. With the optimization of chromatographic conditions, such as mobile phase composition, flow rate, column temperature and sample loading, for the separation of enantiomer, DL-THP was base-line separated on the MIP. The imprinted monolithic column was used as a precolumn for fractionation of the C. yanhusuo extract. Both the non-retained and retained fractions were separated by RP-HPLC. Meanwhile, the D-THP and L-THP can be detected in the non-retained and retained fractions, respectively. Additionally, direct determination of L-THP using molecularly imprinted monolith on-line coupling with a reversed-phase column was acquired.     

Simultaneous determination of a drug candidate and its metabolite in rat plasma samples using ultrafast monolithic column high-performance liquid chromatography/tandem mass spectrometry

An ultrafast bioanalytical method using monolithic column high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) was evaluated for the simultaneous determination of a drug discovery compound and its metabolite in plasma. Baseline separation of the two compounds was achieved with run times of 24 or 30 s under isocratic or gradient conditions, respectively. The monolithic column HPLC/MS/MS system offers shorter chromatographic run times by increasing flow rate without sacrificing separation power for the drug candidate and its biotransformation product (metabolite). In this work, the necessity for adequate chromatographic resolution was demonstrated because the quantitative determination of the drug-related metabolism product was otherwise hampered by interference from the dosed drug compound. The chromatographic performance of a monolithic silica rod column as a function of HPLC flow rates was investigated with a mixture of the drug component and its synthetic metabolite. The assay reliability of the monolithic column HPLC/MS/MS system was checked for matrix ionization suppression using the post-column infusion technique. The proposed methods were successfully applied to the analysis of study rat plasma samples for the simultaneous quantitation of both the dosed drug and its metabolite. The analytical results obtained by the proposed monolithic column methods and the 'standard' silica particle-packed HPLC column method were in good agreement, within 10% error.     

Chromatographic refolding of recombinant human interferon gamma by an immobilized sht GroEL191-345 column

Minichaperone sht GroEL191-345 was covalently coupled to NHS-activated Sepharose Fast Flow gel. Refolding of recombinant human interferon gamma (rhIFN-gamma) was carried out on a chromatographic column packed with immobilized minichaperone. The effects of salt concentration, urea concentration gradient, elution flow rate and protein loading on the refolding efficiency were investigated. The results indicated that immobilized sht GroEL191-345 chromatography was an effective protocol for the refolding of rhIFN-gamma. When loading 100 microl denatured rhIFN-gamma (17.8 mg/ml), the protein mass recovery and total activity obtained in this optimal process reached 74.25% and 6.74 x 10(6)IU/ml, respectively with the immobilized minichaperone column which was reused for 10 times with 25% decrease of renaturation capacity.     

全二维液相色谱(NPLC×RPLC)接口及其应用

用内径为0.53 mm的填充毛细管正相液相色谱为第一维, 用4.6 mm(i.d.)×50 mm RP-18e整体柱反相色谱为第二维, 建立了定量环-阀切换接口的全二维液相色谱系统(NPLC×RPLC). 第一维色谱分离洗脱出的组分交替存储在十通阀上的两个定量环中, 同时定量环中前一个组分被转移到第二维进行反相分离. 因为第一维的流动相流量仅是第二维的1/500, 自然解决了流动相兼容问题. 采用芳香族化合物的混合物和中药丹参正己烷提取液对该全二维液相系统的分离能力进行了评价.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

Preparation of hydrophobic interaction chromatographic packings based on monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their application

The monodisperse, poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads with macroporous in the range of 8.0-12.0 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. The pore size distribution of the beads was evaluated by gel permeation chromatography and mercury instrusion method. Based on this media, a hydrophobic interaction chromatographic (HIC) 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 dynamic protein loading capacity of the synthesized HIC packings was 40.0 mg/ml. Six proteins were fast separated in less than 8.0 min using the synthesized HIC stationary phase. The HIC resin was firstly used for the purification and simultaneous renaturation of recombinant human interferon-gamma (rhIFN-gamma) in the extract solution containing 7.0 mol/l guanidine hydrochloride with only one step. The purity and specific bioactivity of the purified of rhIFN-gamma was found more than 95% and 1.3 x 10(8) IU/mg, respectively.     

Fast fabrication of a hybrid monolithic column containing cyclic and aliphatic hydrophobic ligands via photo‐initiated thiol‐ene polymerization

Three monomers, octakis (3‐mercaptopropyl) octasilsesquioxane, 1,2,4‐trivinylcyclohexane and isophytol were employed to synthesize a novel monolithic stationary phase via photo‐initiated thiol‐ene click polymerization for reversed‐phase liquid chromatography. Several factors such as porogenic system, reaction time and the molar ratio of functional groups were investigated in detail. The resulting poly(POSS‐co‐TVCH‐co‐isophytol) monolithic column exhibited suitable permeability for fast separation and outstanding thermal stability. Five alkylbenzenes were employed to evaluate the ability of chromatographic separation of the resulting monolithic columns at different flow rates, and showed the highest column efficiencies of 90,200–93,100 N/m (corresponding to 10.4–10.6 μm of plate height) at a velocity of 0.41 mm/s. The baseline separations of five anilines and eight phenols further proved the applicability of poly(POSS‐co‐TVCH‐co‐isophytol) monolithic column in the separation of small molecules.     

正交试验法优化制备用于小分子物质分离的聚(甲基丙烯酸缩水甘油酯-二乙烯基苯)型整体柱

以甲基丙烯酸缩水甘油酯(GMA)为单体,二乙烯基苯(DVB)为交联剂,以环己醇和正十二醇为致孔剂,过氧化苯甲酰(BPO)为引发剂,直接以50 mm×4.6 mm色谱柱为模具,通过原位聚合制备聚(GMA-DVB)型整体柱。以GMA和DVB的体积比、环己醇和十二醇的体积比以及BPO占聚合物的质量分数为三因素,以分离苯和乙苯等小分子物质时的半峰宽分离度(R1/2)为考察指标,进行三因素三水平的正交试验,通过测定整体柱的比表面积、孔径和孔容分布对其进行表征。结果表明,制备整体柱的最优配方为V(GMA): V(DVB): V(环己醇): V(正十二醇)=0.825:0.825:1.32:2.03, BPO的质量分数为0.7%。应用所制备的整体柱分离苯和乙苯等小分子物质,理论塔板数达到37000塔板/m, R1/2值达到7.14,完全达到基线分离,分离时间小于10 min。该方法制备整体柱的重复性好,柱效较高,基本满足商品化要求。     

High-speed liquid chromatography/tandem mass spectrometry using a monolithic column for high-throughput bioanalysis

With the ever-increasing workload from a variety of in vitro and in vivo screening procedures, new analytical methodologies to perform bioanalysis in an accurate and high-throughput manner are in great demand. In this work, monolithic columns were used instead of conventional particulate HPLC columns to perform chromatographic separations. Because the pressure drop on a monolithic column was considerably lower than that on a particulate column, a high flow rate (6 mL/min) was used for a 4.6 x 50 mm monolithic column with a total backpressure of about 61 bar measured using acetonitrile/water (50:50). The capability of using a regular column length at high flow rates, combined with the extremely small dependency of separation efficiency on linear flow velocity, allowed for the generation of sufficient chromatographic resolving power in a significantly reduced runtime. As demonstrated in this work, a plasma extract of a mixture of tempazepam, tamoxifen, fenfluramine, and alprozolam were baseline separated within a total analysis time of one minute. An average peak width at half maximum of approximately one second was noted using a generic broad gradient. It was also found that the separation efficiency and signal/noise (S/N) ratios for this separation remained almost constant at flow rates of 1, 3, and 6 mL/min, respectively. The ruggedness of the separation was evaluated by injecting 600 plasma extracts containing the replicates of a standard curve of the above mixture during an overnight run. The chromatographic retention time, separation quality, peak response and sensitivity were highly reproducible throughout the run. This high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) system has been used routinely in the authors' laboratory to support drug discovery programs.     

整体柱离子相互作用色谱快速分析无机阴离子

研究了用硅胶整体柱和直接电导检测的离子相互作用色谱快速分析常见无机阴离子的方法。实验采用氢氧化四丁铵和邻苯二甲酸为淋洗液,讨论了包括淋洗液浓度、流速和pH对分离的影响。当以1.5 mmol/L氢氧化四丁铵和1.1 mmol/L邻苯二甲酸为淋洗液(pH 5.5),流速6 mL/min时,可以在1 min内分离Cl-、NO2-、Br-、NO3-、ClO3-、SO42-和I-7种阴离子。方法的检出限为0.3~1.9 mg/L,峰面积、峰高的相对标准偏差(RSD,n=5)分别为0.4%~2.2%和0.1%~1.5%。将该法用于测定矿泉水和地下水中的阴离子,加标回收率在97.9%~100.3%之间。     

Sample displacement chromatography of monoclonal antibody charge variants and aggregates

The rise of biosimilar monoclonal antibodies has renewed the interest in monoclonal antibody (mAb) charge variants composition and separation. The sample displacement chromatography (SDC) has the potential to overcome the low separation efficiency and productivity associated with bind-elute separation of mAb charge variants. SDC in combination with weak cation exchanging macroporous monolithic chromatographic column was successfully implemented for a separation of charge variants and aggregates of monoclonal IgG under overloading conditions. The charge variants composition was at-line monitored by a newly developed, simple and fast analytical method, based on weak cation exchange chromatography. It was proven that basic charge variants acted as displacers of IgG molecules with lower pI, when the loading was performed 1 to 1.5 pH unit below the pI of acidic charge variants. The efficiency of the SDC process is flow rate independent due to a convection-based mass transfer on the macroporous monolith. The productivity of the process at optimal conditions is 35 mg of purified IgG fraction per milliliters of monolithic support with 75–80% recovery. As such, an SDC approach surpasses the standard bind-elute separation in the productivity for a factor of 3, when performed on the same column. The applicability of the SDC approach was confirmed for porous particle-based column as well, but with 1.5 lower productivity compared to the monoliths.     

Hybrid organic-inorganic phenyl monolithic column for capillary electrochromatography

A novel hybrid organic-inorganic silica-based monolithic column possessing phenyl ligands for reversed-phase (RP) capillary electrochromatography (CEC) is described. The monolithic stationary phase was prepared by in situ co-condensation of tetraethoxysilane (TEOS) with phenyltriethoxysilane (PTES) via a two-step catalytic sol-gel procedure to introduce phenyl groups distributed throughout the silica matrix for chromatographic interaction. The hydrolysis and condensation reactions of precursors were chemically controlled through pH variation by adding hydrochloric acid and dodecylamine, respectively. The structural property of the monolithic column can be easily tailored through adjusting the composition of starting sol solution. The effect of PTES/TEOS ratios on the morphology of the created stationary phases was investigated. A variety of neutral and basic analytes were used to evaluate the column performance. The CEC columns exhibited typical RP chromatographic retention mechanism for neutral compounds and had improved peak shape for basic solutes.