用参数Z表征疏水色谱中脲浓度与蛋白质分子的构象变化

研究了５种标准蛋白在流动相中含有不同脲浓度条件下的疏水色谱保留行为。当脲浓度不变时，蛋白质的保留仍然服从计量置换保留模型，并可测定在该特定脲浓度条件下蛋白质的Ｚ值。计量置换参数Ｚ可作为疏水色谱中生物大分子的构象变化的表征。     

4，4'-（-3，3'二甲基）联苯二胺（o-Tolidine）作紫外探针试剂的间接光度色谱法研究(I)—o-Tolidine在反相色谱柱上的吸附性能

]用甲醇-水缓冲溶液作流动相,测定紫外探针试剂o-Tolidine在反相（ODS）柱上的吸附等温线。Freundlich吸附等温方程能解释探针试剂在ODS上的吸附。分别得到了在不同流动相条件下o-Tolidine的吸附等温方程。流动相的组成如流动相的PH值、甲醇含量以及有机添加剂的性质和浓度均影响o-Tolidine在柱上的吸附。o-Tolidine在反相色谱柱上的吸附量与间接光度色谱中的溶质保留和检测响应有一定的关系。     

强极性中药组分的亲水作用色谱保留行为

采用亲水作用色谱模式,以二醇基硅胶为固定相,水-有机溶剂为流动相,通过改变流动相中有机溶剂种类及浓度、缓冲盐、有机酸种类及其浓度、柱温等条件,研究了强极性中药组分在亲水作用色谱中的保留行为。结果表明:流动相中水的比例在0～100%(V/V)变化时,溶质保留呈U型曲线,属于亲水色谱和反相色谱的混合保留机理。水含量在0～50%(V/V)范围时,亲水作用控制溶质的保留。溶质保留随流动相中缓冲盐浓度的增大而增强。对于酸性溶质,其保留随有机酸三氟乙酸、甲酸、乙酸的酸性降低而增大。     

微柱高效液相色谱大体积进样技术

在色谱分离过程中,溶质在流动相中产生扩散效应,其最大峰浓度与柱效和溶质量呈正比,与柱子直径及柱长呈反比。所以使用微孔径柱可使峰浓度升高,提高分析的灵敏度。此外还可节省溶剂,操作条件选择方便,尤为适用于液相色谱和质谱的联机系统。目前使用的微孔径柱其直径大都为     

某些酚类的高温液相色谱行为

建立了高温液相色谱系统,在高温条件下,采用甲醇-水作为流动相,在Polymerx RP-1聚合物(PSDVB)色谱柱上考察了6种酚类样品的色谱行为.实验条件:温度40～160 ℃,流速0.2～5.5 mL/min,流动相中甲醇浓度范围40%～80%.考察了温度、流速和流动相组成对酚类样品的保留、分辨、柱效和系统压力的影响,探讨了酚类样品在聚合物柱上的热力学行为.温度升高2.35℃大约相当于流动相中甲醇浓度增加1%,可以通过改变色谱柱温度调节样品保留和改变选择性.柱温升高,降低了流动相的粘度,允许在高温条件下使用较高的流速实现快速分离.在160℃、V(甲醇):V(水)=40:60,为流动相和3 mL/min流速条件下,可于2.5 min内实现6种酚类的完全分离.     

用于蛋白同时复性及纯化的制备型装置中流动相用量的优化研究

 在生物大分子的高效液相色谱分离中 ,由计量置换保留模型可得出生物大分子在色谱柱上的保留行为取决于流动相中置换剂的浓度的结论。据此提出了用于蛋白同时复性及纯化的制备型装置 (USRPP)中最小流动相用量的估算公式 ,并进一步得出在保持最小流动相用量不变的条件下 ,改变流动相流速和线性梯度时间几乎不影响制备型USRPP分离蛋白的分离度和复性效率的结论。该结论与实验结果一致。     

低共熔溶剂——新型亲水作用色谱流动相改性剂

低共熔溶剂被用作亲水作用色谱流动相的新型改性剂。选用硅胶柱(150 mm×4.6 mm, 3 μm),以乙腈与低共熔溶剂(氯化胆碱-乙二醇(摩尔比为1:3))的混合溶液为流动相,考察了6个碱基与核苷的色谱分离效果,并讨论了低共熔溶剂在流动相中的比例及温度条件对分离的影响。结果表明,与传统的水相流动相条件相比,在加入低共熔溶剂改性后的流动相条件下,碱基与核苷分离效果得到明显的改善,尤其是胞嘧啶与胞苷能达到完全分离;同时,随着低共熔溶剂在乙腈中浓度的增加,6个碱基与核苷在色谱柱上的保留均有不同程度的减小,其中胞苷的保留减小最为显著;随着柱温的升高,碱基与核苷的保留同样有所减小。本文验证了低共熔溶剂作为亲水作用色谱流动相改性剂的可行性,可在一定程度上解决传统亲水作用色谱分离的困难。     

环孢素A在反相液相色谱中的吸附行为及分离纯化北大核心CSCD

环孢素A(cyclosporine A,CsA)是由11个氨基酸组成的中性环状多肽,是临床拮抗器官和组织移植后排异反应的首选药物。高效液相色谱法广泛应用于CsA的分离分析,开展CsA色谱行为的研究是使用制备高效液相色谱纯化CsA的关键。该文首先在C18固定相上比较了CsA在甲醇-水和乙腈-水两种流动相体系中的保留行为,结果表明其保留时间对有机相比例变化比较敏感。控制甲醇比例在84%~88%,或者乙腈比例在75%~85%,CsA的保留因子(k)在3~7范围内。考察了上样量对CsA峰形的影响。随着上样量增加,在两种流动相体系中,CsA的峰形都由对称开始变得拖尾,保留时间前移。因此在进行CsA纯化时,需要特别注意前杂的去除情况。然后采用吸附等温线描述CsA的保留行为,当流动相中CsA的质量浓度较低时,有机相比例对溶质在固定相上的吸附量影响并不明显。随着溶质的质量浓度增加至0.5 g/L以上,有机相比例降低有助于提高CsA在固定相上的吸附量。和甲醇-水体系相比,在乙腈-水体系中固定相对溶质有更大的吸附容量。用模型对CsA的等温吸附曲线拟合,在甲醇-水体系中符合Langmuir模型,在乙腈-水体系中为Moreau模型。由模型参数可知在两种体系下,CsA在C18固定相上均为单层吸附,区别在于乙腈-水体系中CsA会产生较大的分子间作用力。最后,实验采用0~60 min 65%~75%乙腈、60~80 min 75%乙腈的条件开展了环孢素A纯化的探索实验,可将CsA的杂质控制在0.2%以下。本研究结果对采用制备高效液相色谱纯化CsA具有指导意义。     

蛋白质在离子交换色谱上选择性和非吸附特性

对蛋白质在离子交换柱上选择民性和非吸附特性进行了研究。蛋白质在有机磷酸锆阳离子色谱柱上,其保留作用随流动相ｐＨ值在离子强度的增加而减小;蛋白质在强阳离子和强阴离子色谱柱上的保留作用,即是流动相中的ｐＨ值等于蛋白质的等当点,其净电荷为零。不册蛋白质仍有不同程度的保留,这主要是由于蛋白质的三维结构使电荷 密度的大小和分布的不均匀以及离子交换填料表面性质的影响。     

反相高效液相色谱中溶质保留过程的热力学研究4: 焓熵补偿

依据液相色谱中溶质计量置换保留模型, 对溶质在反相液相色谱(RPLC)保留过程及其吸附、解吸附过程中的焓熵补偿进行了研究, 证实了在RPLC中焓熵补偿确实存在。从焓熵补偿的定义出发, 从理论上证明了溶质在保留过程中的焓熵补偿温度本质上为溶质保留值的收敛温度, 其数值为Z对1/T线性作图的斜率与截距之比。与惯常计算焓熵补偿温度的方法相比, 本文的方法所得补偿温度更为合理且不受流动相中强溶剂浓度变化的影响。     

胶原在聚乳酸旋涂膜表面的吸附研究

采用石英晶体微天平(QCM)、原子力显微镜(AFM)研究了胶原在聚乳酸旋涂膜表面的吸附,考察了溶液浓度(0～9.25μg/mL)和温度(10～50℃)对胶原吸附的影响.实验结果表明,随着胶原浓度的增加,胶原在聚乳酸表面的吸附量和吸附初速率都相应增加.采用Langmuir模型和Freundlich模型拟合实验数据得到吸附等温线方程,分别为q=11690.99c1+0.99c和q=610c3.179.实验结果显示,Langmuir模型拟合效果要好于Freundlich模型.采用Lagergren拟一阶吸附速率方程和Lagergren拟二阶速率方程拟合不同浓度下的吸附动力学数据.在低浓度下Lagergren拟一阶速率方程拟合效果比较好,在高浓度下Lagergren拟二阶速率方程拟合效果比较好,说明在低浓度时扩散过程是胶原吸附的控速步骤,高浓度时胶原和聚乳酸表面的相互作用是吸附的控速步骤.原子力显微镜显示,吸附在聚乳酸表面的胶原形成网状结构.胶原的吸附受温度影响显著,说明胶原是一种对温度非常敏感的物质.实验结果表明随着温度的升高,在10～40℃范围内胶原的吸附量逐渐降低,在40～45℃范围内锐减,40℃是本实验条件下胶原的变性温度.     

疏水界面上标准蛋白质吸附等温线的研究

测定了几种具有代表性的标准蛋白质在疏水色谱填料上的吸附等温线.除溶菌酶和牛血清白蛋白的吸附近似呈线性外,细胞色素-c、肌红蛋白、胰岛素、α-淀粉酶和卵清蛋白均呈凸型吸附.用Langmuir、计量置换吸附模型(SDM-A)、BET和Jovanovic吸附模型对这些蛋白的吸附等温线拟合后发现,SDM-A关系式能够良好描述这7种蛋白的吸附,Langmuir关系仅能较好地描述后5种蛋白质的吸附.而Jovanovic和BET关系式对上述两类蛋白质吸附的描述欠妥.实验发现对于不同盐浓度条件下卵清蛋白的吸附,用SDM-A和Langmuir公式拟合所得的参数与盐浓度之间呈现出一定的规律性.     

STUDY OF THE ADSORPTION OF HUMAN SERUM ALBUMIN ON REVERSED-PHASE SUPPORTS.

This work examines the adsorption of Human Serum Albumin (HSA) on a Reversed-Phase High Performance Liquid Chromatographic (RPLC) support. The adsorption experiments were performed by frontal analysis. Adsorption isotherms were determined in pure buffer and in the presence of acetonitrile. Saturation is always reached, even at the lower protein concentrations. In view of the pore size of the particles (80 Å), it is assumed that HSA is adsorbed on the external surface of silica

In presence of acetonitrile, a variability in the amount of HSA adsorbed is found showing a maximum at 25% of acetonitrile. Slower adsorption kinetics are observed when the concentration of the organic modifier in the eluent is increased. The reversibility of HSA binding to the surface was investigated by desorbing the protein with 40% acetonitrile. The amount of HSA irreversibly adsorbed depends upon the experimental conditions used during the adsorption step. It is at a maximum when HSA is adsorbed with 25% acetonitrile. As the temperature is raised and only in the presence of acetonitrile, an important increase of the amount of HSA irreversibly adsorbed is observed.     

Comparison of Adsorption Isotherms of Methylxanthines on C18 Column

A comparison of the adsorption isotherms of caffeine, theophylline and theobromine and the competitive adsorption of the three compounds on a C 18 column were investigated. The experimental parameters of the equilibrium isotherms were estimated by linear and nonlinear regression analyses. The linear equation as a function of the adsorption concentration of the single compound in its solution and the competitive adsorption of a single compound in a mixed solution were then determined. The adsorption equilibrium data were then correlated to the linear, Langmuir, Freundlich, Langmuir-Freundlich and stoichiometric displacement theory for adsorption(SDT-A) isotherm models. The mixed compounds of the three compounds were competitively adsorbed on the C 18 particles. The expression of stoichiometric displacement theory for adsorption was found to be more suitable for adsorption of methylxanthines on a C 18 column.     

Adsorption of mercury(II) on macroreticular styrene-divinylbenzene copolymer beads

The adsorption characteristics of mercury(II) on several kinds of styrene-divinylbenzene copolymer beads having different surface properties were studied. It was found that the polymer beads selectively adsorbed mercury(II) from solutions over a wide range of pH with high efficiency. The amount of mercury(II) adsorbed increased with increase in specific surface area of the polymer beads and the adsorption behaviour was found to be of the Langmuir type. The presence of chloride strongly reduced the adsorption, but this interference was not observed with nitrate, sulphate, perchlorate, cadmium(II), cobalt(II), copper(II), nickel(II), silver(I) and zinc(II). More than 95% of the mercury(II) adsorbed on a column of polymer beads could be recovered with dilute hydrochloric acid.     

奥美拉唑对映体在自制手性柱上吸附等温线的测定

采用吸附-脱附法研究了奥美拉唑对映体在自制的纤维素三苯基氨基甲酸酯涂敷型手性柱上的吸附性能,并用Langmuir方程拟合出吸附平衡方程.当样品浓度降低时,Langmuir方程退化为直线关系,直线斜率即为吸附平衡常数.这一吸附平衡常数与从脉冲色谱流出曲线求得的吸附平衡常数相等,表明吸附-脱附法测得的吸附平衡关系准确,Langmuir方程能描述这一吸附平衡关系.     

A Novel Model for Protein Immobilization on Microspheres

The immobilization of proteins, especially receptor proteins commonly used in high through-put screening of drugs (HTS), have received great attention in recent years. There are many successful isothermal models for describing the adsorption of protein onto solid surface, such as Langmuir model, Bi-Langmuir model, Fowler model, Freundlich model, Freundlich-Langmuir model and Tekmin model etc. In all these models, Langmuir model was the most favorable one model accepted by many researchers, but the experimental results showed that it was not entirely fit to all adsorption behaviors. So new models were required for describing protein adsorption onto microspheres in different conditions.In our research, a novel isothermal model, including Langmuir and other adsorbing behaviors was presented basing on the holding degree of surface active sites and the interaction styles of protein immobilization. In Langmuir model, the adsorbing amount of protein was described as [PS] =Km[P]/1 + K[P], where [PS] was the concentration of adsorbed protein, [P] was the concentration of freeprotein at equilibrium state, and Km and K was constant. According to the interactions of protein and ligands, there were three patterns in the interactions of protein and ligands. On the similar assumption that the interaction of protein and microspheres were three styles, and based on the definition of the holding degree of surface active sites (Y), three adsorption behaviors could be described as Y K[ P ]φ/ K[P]φ+1 or ln K + φ ln[P] =ln(Y/1-Y) in which [P] was the concentration of free protein at equilibrium state, and φ and K was constant. Different scale of φ presented different adsorption behaviors, especially when φ was 1, the adsorption behavior was Langmuir adsorbing model. Figure I indicated the different adsorbing results in different adsorption behaviors (φ＞1, φ＜1,and φ=1).     

STUDY ON THE ADSORPTION OF PHENOL BY CHITOSAN FROM AQUEOUS SOLUTION

The effects of pH, initial concentration and temperature on the adsorption of phenol by chitosan were investigated in this paper. The isothermal data was applied to Langmuir linear and the Freundlich linear isotherm equation, and the thermodynamic parameters （AH, AG, AS） were calculated according to the values of binding Langmuir constant, KL. Results indicated that the adsorption between chitosan and phenol was significantly physical in nature, the negative ΔH constant at lower temperature confirmed that more phenol was adsorbed by chitosan at lower temperature. The kinetics of the sorption process of phenol on chitosan was investigated using the pseudo-first order and pseudo-second order kinetics, and results showed that the second order equation model provided the best correlation with the experimental results.     

BINARY COMPETITIVE AND COOPERATIVE ADSORPTION OF AROMATIC COMPOUNDS ON POLYMERIC RESINS

The adsorption behaviors of 2-naphthalenesulfonic acid and aniline on a conventional macroporous resin Amberlite XAD4 and the other two newly-developed hypercrosslinked resins NDA101 and NDA100 were investigated in a single or binary batch system at 293 K and 313 K,respectively.All the adsorption isotherms of 2-naphthalenesulfonic acid and aniline on the test resins in both systems can fit well with the Langmuir equation,indicating that the adsorption is a favorable process.At the identical equilibrium concentration,the amount of aniline adsorbed on polymeric resins in the single system is higher than that in the binary system because of the competitive adsorption between 2-naphthalenesulfonic acid and aniline on the resin surface.However,the uptake amount of 2-naphthalenesulfonic acid in the binary system is markedly larger than that in the single system,which is presumably due to the cooperative effect arisen from the electrostatic interaction between 2-naphthalenesulfonic acid and aniline adsorbed on the resin surface.The simultaneous adsorption system was proven to be helpful for the selective adsorption toward 2-naphthalenesulfonic acid due to its larger selective index.     

Selective foam separation of binary protein solution by SDS complexation method

A fundamental study about the selective foam separation of protein mixture was carried out. A solution containing two proteins, ovalbumin (OA) and lysozyme (LZ), and an anionic surfactant, sodium dodecyl sulfate (SDS), was adjusted to pH 6.0, which referred to an intermediate state between the isoelectric points of the proteins. The solution was processed by continuous foam separation. The results showed that a proper addition of SDS greatly improved the selective recovery of LZ to OA. The experimental data were well explained by a simple model that most of cationic protein molecules (LZ) are associated with SDS and the adsorption of all the species including LZ-SDS complexes are subjected to Langmuir adsorption isotherm. The results also showed that one of the Langmuir parameters, which means a kind of lyophillic property of adsorbed material, of LZ-SDS complexes was extremely large as compared with that of primary protein.