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101.
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. 相似文献
102.
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104.
A unified retention equation of proteins was proved to be valid for a mixed-mode interaction mechanism in ion exchange chromatography (IEC) and hydrophobia interaction chro-matography (HIC). The reason to form a "U" shape retention curve of proteins hi both HIC and IEC was explained and the concentration range of the strongest elution ability for the mobile phase was determined with this equation. The parameters in this equation could be used to characterize the difference for either HIC or IEC adsorbents and the changes in the molecular conformation of proteins. With the parameters in this equation, the contributions of salt and water in the mobile phase to the protein retention in HIC and IEC were discussed, respectively. In addition, the comparison between the unified equation and Melander' s three-parameter equation for mixed-mode interaction chromatography was also investigated and better results were obtained in former equation. 相似文献
105.
With insulin methanol-water,and the ion-pairing agent,hydrochloric acid and trifluroacetic acid(TEA),the character of the first plateau(FP)on the elution curve of frontal analysis in reversed phase liquid chromatography(RPLC)was investigated by on-line UV-spectrometry and identified with nuclear magnetic resonance(NMR) spectrometry and mass spectrometry.The profile of the FP is the same as that of a usual elution curve of methanol in frontal analysis(FA).When the insulin concentration was limited to a certain range,the height of the FP was found to be proportional to the insulin concentration in mobile phase and its length companying to shorten,The FP profile on the intersection of two tangents reflects the components of the microstructure in the depth direction of the bonded stationary phase layer and the desorption dynamics of the displaced components.The displaced methanol was quantitatively determined by NMR and on-line UV spectrometries.TFA with high UV absorbance can not be used as an ionpairing agent for the investigation of the FP in RPLC,but if can be used as a good marker to investigate the complicated transfer process of components in the stationary phase in RPLC.A stoichiometric displacement process between solute and solvent was proved to be valid in both usual and FA in RPLC.From the point of view of dynamics of mass transfer, the solutes can only contact to the surface of stationary phase in usual RPLC,while solute can penetrate into it in FA of RPLC.The solvation of insulin in methanol and water solution as an example indicating the usage of the FP in the FA was also investigated in this paper. 相似文献
106.
反相高效液相色谱中溶质保留过程的热力学研究 Ⅱ. 焓变及其分量, 吸附及解吸附焓变 总被引:2,自引:0,他引:2
依据液相色谱中溶质计量置换保留模型,从理论上分别推导出了在反相高效液相色谱(RP-HPLC)中lgI(与1mol溶质对固定相的亲合势有关的常数)和Z(对应于1mol溶质被固定相吸附时释放出置换剂的摩尔数)对绝对温度导数的线性关系式发现非极性与极性小分子溶质的lgI和Z均具有热力学平衡常数的特征.如在前报报告的吉布斯自由能变一样,也推导出了可将溶质保留过程中的总吸附焓变△H_(Pa)分成两个独立的分量,吸附焓变面△H_(I,a)和解吸附焓变△H_(Z,D)的表达式.可同时对在流动相中不同置换剂浓度条件下的△H_(Pa)和△H_(Z,D)进行估算,并与实验值进行了比较,偏差小于±10%并用面△H_(I,a)和△H_(Z.D)值对溶质在RP-HPLC的保留过程中热变化进行了解释. 相似文献
107.
线性参数logI对反相液相色谱中的非极性小分子溶质体系的表征 总被引:2,自引:0,他引:2
从理论上阐明了计量置换平衡常数的对数logKa对液相色谱中计量置换保留模型的线性参数logI起着主导作用。logI表示溶质对固定相的亲合势,且具有热力学平衡常数的性质。 相似文献
108.
通过对硅胶基质进行化学改性键合伴刀豆球蛋白(Con A),制备了对糖蛋白具有特异亲和作用的亲和色谱固定相;该固定相非特异性吸附弱,对于糖蛋白和糖肽的分离效果良好。对亲和色谱的分离条件进行了优化,以标准糖蛋白核糖核酸酶B(RNase B)为模型,对其进行了纯化;用糖苷酶切除糖链,并对切除糖链前后的RNase B用胰蛋白酶酶解;用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)对亲和色谱分离得到的糖蛋白、糖链及糖肽进行了分析,确定了RNase B的一级结构、糖含量、糖基化位点及糖连接方式。该方法快速准确,适于糖蛋白和糖肽的分离表征。将其应用于血清中糖蛋白及酶解后血清中糖肽的分离富集,取得了很好的效果。 相似文献
109.
With four kinds of mobile phases,methanol-water,ethanol-water,2-propanol and acetonitrile-water(all containing 0.1%rifluroacetic acid),the displacement between solute and solvent in RPLC was proved to be universal in frontal analysls(FA).Based on the measured Z value in usual RPLC to be a constant and the quantitative determination of methanol increment in mobile phase in FA,the stoichiometric displacement(SD)between insulin and methanol was directly proved by the experiment.The SD was also proved to occur only on about the one-fourth of the maximum amount of adsorbed methanol in the bonded phase layer(BPL)without any dynamic problem of mass transfer,while in FA,the SD firstly occurs on the surface of the BPL and then gradually sinks into the deeper sites companied with a dynamic problem.Although the displaces soplvent by the same solute is less in the former case,the SD is independent of how deep of the solute enters the BPL.In addition,the adsorbed amount of solute on and adsorbent not only depends on the numbers of the adsorbed layer on the adsorbent surface,but also on the extent of the complete removal of the displace able solvent in the BPL.The pyhsical fundamental of the SD and the methodoloby for investigation were also discussed. 相似文献
110.