基于微孔过滤板的蛋白吸附高通量筛选方法

针对色谱分离过程优化,建立了基于微孔过滤板的蛋白吸附高通量筛选方法,用于介质初筛、吸附性能考察、吸附等温线和吸附动力学测定、吸附和洗脱条件优化等。首先优化了96孔过滤板的操作参数,以2种离子交换介质和2种混合模式介质为典型代表,采用微孔过滤板方法考察了不同介质和液相条件下牛血清白蛋白的吸附,得到结合载量分布图,确定了合适的蛋白吸附和解吸条件。进一步测定了4种介质在特定吸附条件下的吸附等温线和吸附动力学曲线,获得吸附相关参数。最后,采用微孔过滤板进行了洗脱条件优化,并与填充柱色谱分离进行比较,验证了方法的可靠性。结果表明,基于微孔过滤板的蛋白吸附高通量筛选是切实可行的,可以快速筛选色谱介质和液相,优化蛋白分离条件,具有资源消耗小、实验通量大、研发周期短、适用性广、稳定性高的特点,是蛋白色谱分离过程优化的一种新方法。     

交变电场下离子交换色谱分离过程

以牛血清白蛋白在阴离子交换剂DEAE -SepharoseFastFlow上的吸附和解吸过程为例 ,考察了交变电场下离子交换色谱过程的吸附和洗脱过程及其传质特性 .结果表明 :在本文实验条件下 ,交变电场电流强度的变化对离子交换吸附平衡无显著影响 ,而外加电场在介质孔内所产生的电渗流可加速待分离组分进出固相介质的传递过程 ,显著提高色谱填充床的动态吸附容量 ,改善洗脱峰的特性     

逆向法测定酮洛芬对映体在Chiralpak AD柱上的吸附等温线

模拟移动床（SMB）色谱分离与纯化的操作过程复杂,对其进行设计与优化需要使用数值模拟方法,准确测定竞争型吸附等温线具有重要意义。采用逆向法确定了25℃下酮洛芬对映体在直链淀粉手性固定相（Chiralpak AD）上的竞争吸附等温线,先用高效液相色谱测得酮洛芬对映体在Chiralpak AD 柱上的过载流出曲线,再通过拟合实验测得的流出曲线确定吸附等温线模型及其参数。研究中采用平衡扩散模型描述酮洛芬对映体在色谱柱上流出的瞬态过程。参数拟合过程中,首先用非支配基因算法（NSGA-Ⅱ）在较广的参数空间内搜索吸附等温线模型参数,再以所得结果作为初值,使用Levenberg-Marquardt 算法（LMA）对参数进一步优化。比较了4种不同竞争吸附等温线模型对实验测得的流出曲线的拟合结果,其中五参数的Bi-Langmuir 模型拟合程度最好。测量了不同进料浓度和进料量条件下的流出曲线,并通过与模型预测结果的对照验证了所确定吸附等温线模型和参数。     

逆向法确定色氨酸对映体竞争型吸附等温线

引言 吸附等温线测定是研究色谱分离过程的关键,其准确性直接影响到色谱分离过程的准确程度,因此,选择适当的实验方法和数学模型来模拟吸附等温线是模拟色谱分离过程的一个重要环节.常用的几种方法包括:前沿分析法、特征点洗脱法、特征点前沿分析法以及扰动法[1-2].其中前沿分析法是最常用的方法,该方法简单,准确性高,可用于多组分竞争型吸附等温数据的测定,但其数据采集过程非常耗时,对原料的需求量大,且需要考察单组分吸附等温模型与吸附数据的拟合程度.     

H103大孔吸附树脂分离纯化苦参碱与氧化苦参碱的研究

通过对比苦参总碱在九种大孔树脂上的静态吸附,筛选出对苦参总碱吸附能力较强的H103树脂;研究了H103树脂对上述两种生物碱的吸附等温曲线及吸附动力学行为;考察了苦参总碱在H103树脂上的动态吸脱附过程,用薄层色谱法(TLC)对洗脱液跟踪分析,用高效液相色谱法(HPLC)检测苦参碱、氧化苦参碱含量,确定了动态吸脱附的过程参数。结果表明,H103树脂对苦参总碱的等温吸附可采用Freundlich方程描述;由吸附动力学曲线得H103树脂在100 min内达到平衡,具有较快吸附速度,吸附动力学规律可用二级速率方程表示;动态吸脱附过程的洗脱条件为：用30%乙醇-25%氨水（115:1,v/v）,80%乙醇梯度洗脱,氧化苦参碱先被洗脱,苦参碱随后,两种生物碱可达到很好的分离,经计算苦参碱收率为90.1%,氧化苦参碱收率为85.3%。     

疏水性电荷诱导色谱分离抗HER2单克隆抗体

采用疏水性电荷诱导色谱（HCIC）新型生物分离方法,从CHO细胞培养液中高效分离抗HER2单克隆抗体（单抗）。选用典型HCIC介质MEP HyperCel,考察了细胞培养液中单抗稳定性,比较了不同pH下MEP HyperCel介质对抗HER2单抗的吸附性能,发现pH6～9范围内吸附容量均较高,酸性条件下吸附容量显著下降。进一步考察了抗HER2单抗的动态吸附载量,优化了上样和洗脱条件,确定了合适的分离条件,实现了细胞培养液中高效分离单抗,纯度达到94.6%,收率约0.1 mg·（ml料液）^-1。结果表明,HCIC从哺乳动物细胞培养液中分离单抗是切实可行的,为单抗分离提供了新思路。     

羟基磷灰石电色谱中电渗流动与分离过程特性

在羟基磷灰石色谱上施加电场 ,发展了一种新的电色谱分离技术 .对蛋白质电泳和溶液电渗对分离特性的影响进行了实验研究 ,测量了羟基磷灰石电色谱装置中膜、膜与填充介质在不同浓度磷酸缓冲溶液和在不同电流下产生的电渗流 ,分别计算出由膜和羟基磷灰石电渗而产生的推动力 .实验结果表明电渗流量随缓冲溶液离子强度的降低和电流的增加而增加 .在施加电场的情况下 ,羟基磷灰石对牛血清白蛋白的吸附速度加快 ,动态吸附量增加 .分别考察了不同洗脱条件下电场对洗脱效果的影响 .在高浓度的洗脱溶液下 ,电渗的作用很小 ,电场的作用主要是电迁移的作用 ,可以通过选择适宜的电场方向来提高分离精度 ;在低浓度的洗脱溶液下 ,电渗的作用有效地促进了洗脱液与介质颗粒的传质 ,提高了洗脱效果 .在羟基磷灰石色谱上施加电场提高了该色谱分离过程的速率和效率 .     

H103大孔吸附树脂分离纯化苦参碱与氧化苦参碱

通过对比苦参总碱在9种大孔树脂上的静态吸附,筛选出对苦参总碱吸附能力较强的H103树脂;研究了H103树脂对上述两种生物碱的吸附等温曲线及吸附动力学行为;考察了苦参总碱在H103树脂上的动态吸脱附过程,用薄层色谱法(TLC)对洗脱液跟踪分析,用高效液相色谱法(HPLC)检测苦参碱、氧化苦参碱含量,确定了动态吸脱附的过程参数。结果表明,H103树脂对苦参总碱的等温吸附可采用Freundlich方程描述;由吸附动力学曲线得H103树脂在100 min内达到平衡,具有较快吸附速度,吸附动力学规律可用二级速率方程表示;动态吸脱附过程的洗脱条件为:用体积分数30%乙醇-体积分数25%氨水(体积比115∶1),体积分数80%乙醇梯度洗脱,氧化苦参碱先被洗脱,苦参碱随后,两种生物碱可达到很好的分离,经计算苦参碱收率为90.1%,氧化苦参碱收率为85.3%。     

逆向法测定氨氯地平在色谱柱Chiralpak OD-H上的竞争吸附等温线

使用逆向法测定氨氯地平在色谱柱Chiralpak OD-H上的竞争吸附等温线,先用泵进样的方式在高效液相色谱上测定氨氯地平的过载流出曲线,再选用5参数Bi-langmuir吸附等温线模型对过载流出曲线进行拟合,确定吸附等温线模型及模型参数,并验证参数准确性。     

大孔树脂法分离纯化阿维菌素工艺研究

研究了大孔树脂分离纯化阿维菌素的工艺。采用HPLC检测方法,从7种大孔树脂中筛选出吸附阿维菌素性能最好的树脂并优化其吸附和洗脱参数。结果表明,采用大孔树脂HZ816吸附阿维菌素的效果最佳,其动态吸附量为62mg·mL^-1,在吸附流速为1．5～2BV·h^-1、90％乙醇作为洗脱剂的优化条件下,解吸收率大于90％、阿维菌素中B1a含量大于91％、总收率大于65％。该阿维菌素分离纯化方法工艺简单。分离效果好,适于工业化生产。     

Volatile fatty acid adsorption on anion exchange resins: kinetics and selective recovery of acetic acid

ABSTRACT

The removal of volatile fatty acids was examined through adsorption on anion exchange resins in batch systems. During the initial screening step, granular activated carbon and 11 anion exchange resins were tested and the resins Amberlite IRA-67 and Dowex optipore L-493 were chosen for further investigation. The adsorption kinetics and diffusion mechanism and adsorption isotherms of the two resins for VFA were evaluated. Based on the selective adsorption capacity of the resins, a sequential batch process was tested to achieve separation of acetic acid from the VFA mixture and selective recoveries > 85% acetic acid and ~ 75% propionic acid was achieved.     

THE SEPARATION OF Au(III) AND Pd(II) IN HYDROCHLORIC ACID SOLUTIONS BY STRONG ANION TYPE II EXCHANGE RESINS: THE EFFECT OF COUNTER ION CONCENTRATION AND TEMPERATURE

ABSTRACT

The separation of gold and palladium ions in hydrochloric acid solutions by strong anion Type II exchange resins is studied in batch and column operations. Increases in the hydrochloric acid concentration and the temperature of the adsorption process have the effect of decreasing the adsorption capacity and the separation factor of both metals. A faster kinetics of the adsorption process of both ions results from temperature increase and this effect is higher for gold ions as a result of greater changes in its diffusivity. Although the adsorption capacity of palladium ions is lower at equilibrium conditions, the adsorption isotherms obtained suggest that they are more strongly retained by the polymers.

The high capacities of these resins for both metals permits their use in the preconcentration of gold and palladium but their application in quantitative separation is restricted by the co-elution of up to% of the interfering ion during the desorption process. The best method for ion desorption is sequential elution with% ammonia for palladium recovery followed by 0·5M thiourea in HCI 1M for the recovery of gold.     

Preparation of high purity squalene from soybean oil deodorizer distillate with the combination of macroporous resin and thin-film evaporation coupling distillation

ABSTRACT

In this study, the combination of macroporous adsorbent resins (MARs) and thin-film evaporation coupling distillation (TFECD) was studied systematically, with aim to obtain high value-added squalene from soybean oil deodorizer distillate (SODD). Five types of resins (X-5, D-101, D4020, DM301, and AB-8) were first used to evaluate the adsorption/desorption properties of squalene. D101 resin exhibited higher adsorption/desorption capacities and desorption ratios for squalene based on static adsorption results among the tested resins. We further investigated the adsorption kinetics, isotherms, and thermodynamics with D101 resin as adsorbent. The adsorption of squalene on D101 was best fitted to pseudo-second-order kinetic model and the Langmuir equation. The dynamic adsorption and desorption indicated that similar results were observed in the static adsorption test. The purity of squalene was increased from 7.5 to 82.5% with the recovery up to 88.5% after separation on D101 column. The resin-refined sample was directly subjected to TFECD purification. Under optimized process parameters, the final product with purity of 98.5% and recovery yield of 76.5% was confirmed by high-performance liquid chromatography (HPLC) analysis. The present study provided an effective method for large-scale production of high purity squalene.     

Importance of Surface Area and Pore Size Distribution of Resin for Organic Toxicants Adsorption

Adsorption of benzoic acid and reactive brilliant blue 4 (RBB4) by hypercrosslinked resins and macroporous resins was examined. Micropore areas were much higher in the hypercrosslinked resins than macroporous resins while the pore volume and average pore diameter of macroporous resins were superior to the hypercrosslinked resins. The investigation showed that the pore size distributions of the resins significantly influenced the adsorption capacity of adsorbents. Adsorption of benzoic acid was dominated by BET surface area and the micropore area while the adsorption of RBB4 shifted to a pore-size-dominant process. The maximum mol adsorption capacities of benzoic acid were all higher than those of RBB4 by the resins, showing the influence of the molecular dimension on adsorption. The adsorption thermodynamic parameters were calculated and it revealed that the adsorption was an exothermic, spontaneous, and more ordered process. Adsorption kinetics and elution processes were also investigated for establishing the appropriate adsorption conditions for purifying wastewater containing benzoic acid or RBB4.     

Preparative Purification of Solanesol from Tobacco Leaf Extracts by Macroporous Resins

In the present study, the preparative purification of solanesol by macroporous resins was studied. The adsorption and desorption properties of solanesol on macroporous resins including SP207, SP700, SP825, SP850 and HP20 were compared. SP850 resin offered the best adsorption and desorption capacities based on the research results. Both Langmuir and Freundlich isotherms were used to describe the interactions between the solutes and resin at different temperatures, and the experimental data fits best to the Freundlich isotherms. Adsorption and desorption kinetic curves were obtained for solanesol adsorption on SP850 resin in the temperature range of 298–303 K. The results showed that the mobility of solanesol adsorption increased and the proportion of adsorbed solanesol decreased with increasing temperature. The adsorption kinetic curve can be classified into three portions, i.e., instantaneous adsorption stage, intraparticle diffusion stage and the final equilibrium stage. Dynamic adsorption and desorption experiments were carried out with the column packed by SP850 resin to optimize the separation process. The isocratic and stepwise operations were designed for the separation of solanesol. The use of stepwise elution allowed a reduction in the process time and solvent consumption, but also led to some loss in purity. The corresponding optimal parameters for adsorption and desorption were determined. According to the research results, it can be concluded that SP850 resin possesses a good ability to separate solanesol and the results in this study may provide scientific references for large‐scale solanesol production from tobacco leaf extracts.     

Preparative Separation of Glabridin from Glycyrrhiza glabra L. Extracts with Macroporous Resins

Abstract

In the present study, the performance and adsorption characteristics of five macroporous resins for the separation of glabridin from Glycyrrhiza glabra L. have been evaluated. The adsorption and desorption properties of glabridin on macroporous resins including HPD100, HPD300, HPD800, NKA and H103 were compared. HPD100 resin offered the best adsorption and desorption capacities based on the research results. Both Langmuir and Freundlich isotherms were used to describe the interactions between the solutes and resins at different initial concentrations. Dynamic adsorption and desorption experiments on HPD100 resin packed column were conducted to optimize the separation process of glabridin from licorice extracts. After the treatment with stepwise elution on HPD100 resin, the content of glabridin in the product increased from 0.21% to 32.2% which is 153-fold higher than it in G. glabra L. roots and the recovery yield was 79.7%. The results indicated the good ability of HPD100 resin for separation glabridin and the study may provide scientific references for the large-scale glabridin production from G. glabra L. or other plants extracts.     

Penicillin G acylase purification with the aid of high-throughput screening approach

Penicillin G acylase (PGA) is one of the most important enzymes for the production of semi-synthetic β-lactam antibiotics and their key intermediates. Purification of penicillin G acylase from fermentation broth with the aid of high-throughput screening (HTS) process has been examined in this study. We used a microtiter-plate based on screening method to find appropriate purification conditions for the target protein. The screening method is based on a 96-well plate format, and different matrices and conditions (pH, salt concentration and type) were tested. Through analyses of all pooled fractions (flow-through and elution) we gained appropriate information to choose the best performing matrix and buffer conditions for upscaling. After an upscaled purification step the second unit operation is screened in the similar way and parameters for this operation can be chosen. The purification parameter of purified PGA at the small-screen and upscaling levels were measured, respectively. The results indicate that high-throughput progress based on a 96-well plate is a flexible and efficient paradigm for recombinant protein purification.     

Adsorption behaviors of avian immunoglobulins and purification of immunoglobulin Y from chicken serum with mixed-mode resins

The importance of immunoglobulin Y(IgY) as a specific antibody equivalent to mammalian immunoglobulin G(IgG) is well recognized. However, production of highly purified IgY is still difficult due to the lack of specific purification methods. In this study, adsorption behaviors of Ig Y on four mixed-mode resins with functional ligands of 4-mercatoethyl-pyridine(MEP), 2-mercapto-1-methyli-midazole(MMI), 5-aminobenzi-midazole(ABI) and tryptophan-5-aminobenzi-midazole(W-ABI) were evaluated. The results showed that high adsorption ratio were found at p H 6.0–7.0 with little adsorption under acidic conditions. The resin with ABI ligand was then used to separate IgY from immunized chicken serum. An efficient process with Ig Y purity of 95% and recovery of 90% was developed after optimization of loading and elution p H and injection volume. The biological activity of the purified Ig Y was fully maintained. These results indicated that mixed-mode chromatography with specially-designed ligands has great potential for the separation of Ig Y from crude feedstock.     

大孔树脂分离纯化双脱甲氧基姜黄素的研究

探讨了大孔树脂分离纯化双脱甲氧基姜黄素的最佳工艺。通过考察5种大孔树脂对双脱甲氧基姜黄素的洗脱及纯化性能，确定HZ801树脂为最佳树脂，并优化了该树脂分离纯化双脱甲氧基姜黄素的工艺条件。结果表明，在上样量为1g·100g^-1填料。依次用50％乙醇、80％乙醇、90％乙醇梯度洗脱，异丙醇为结晶溶荆时．可得到纯度大于90％、总收率大于60％的双脱甲氧基姜黄素。