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Optimization of affinity capillary electrophoresis for routine investigations of protein–metal ion interactions
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Hassan A. Alhazmi Sami El Deeb Markus Nachbar Sabine Redweik Hassan M. Albishri Deia Abd El‐Hady Hermann Wätzig 《Journal of separation science》2015,38(20):3629-3637
To facilitate the implementation of affinity capillary electrophoresis into routine binding screening studies of proteins with metal ions, method acceleration, transfer and precision improvement were investigated. Affinity capillary electrophoresis was accelerated by using shorter capillaries, employing lower sample concentrations and smaller injection volumes. Intra‐ and inter‐instrument method transfers were investigated considering the temperature setting of the capillary cooling system. For intra‐instrument method transfer, similar results were obtained when transferring a method from a long (62 cm) to a short (31 cm) capillary. The analysis time was reduced from 9 to 4 min. In case of inter‐instrument method transfer, interaction results showed small variation on the capillary electrophoresis instrument with inefficient capillary cooling system. Binding measurement precision was enhanced by slightly pushing the sample above the beginning of the capillary. Changing the buffer vials after each 30 runs and employing extra flushing after each 60 subsequent runs further enhanced the precision. The use of 0.1 molar ethylenediaminetetraacetic acid in the rinsing solution successfully desorbs the remaining metal ions from the capillary wall. Excellent precision for apparent mobility ratio measurements was achieved for different protein–metal ion interactions (relative standard deviation of 0.16–0.89%, 15 series, 12 runs for each). 相似文献
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The frontier orbital interactions of electron pushing and drawing substituents with ferrocenyl group were analyzed based on the electrochemical,UV visible spectral and spectroelectrochemical results of five ferrocene derivatives,R-Fc-A1(PⅠ),A1-Fc-A1(PⅡ),D-Fc-R (PⅢ),D-Kc-A1(PIV) and D-Fc-A2(PV)(R,CH2OH;A1 CHO;A2,CH=C(CN)2 and D,(C18H37)2N-C6H4-CH=CH) It was found that there are strong interactions of the LUMO (πA) of electron drawing substituents with le2g(dxy,dx2 y2)and e2u of the ferroeenyl group because the energy levels of πA and e2g,C2U of (Cp )2 are close,which lower not only the energy levels of bonded orbits,πA+ and dx2-y2+[πA] of PⅠ,PⅡ,PⅣ and PⅤobviously,but also those of their non-bonded orbu dxy For PⅢ,PⅣ and PⅤ,there are strong interactions of HOMO(πD) of the electron pushing substituent with le of the ferrocenyl group because the levels of πD and e of (Cp)2 are close,which result in the formation of anti-bonded orbit,πD- and bonded orbit 相似文献
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推拉电子取代基对二茂铁衍生物性质及电子结构的影响 总被引:4,自引:0,他引:4
根据前线轨道理论分析3种具有推拉电子取代基的二茂铁衍生物循环伏安曲线、电子吸收光谱及光谱电化学行为,并给出了分子轨道能级图,实验结果表明,PⅡ有两对可逆氧化还原峰,E1、E2分别为0.33、0.59V,第一氧化态PⅡ+ ̄(D—Fc ̄+—R)在613nm有强的LMCT(ligand-to-metal-charge-transfer)带,是一种良好的光学特性氧化还原开关.PⅡ在354nm处出现强的LMCT带,PⅢ在356nm和320nm处分别出现强的LMCT和π→πCT带,应具有较高的二阶分子极化系数,应当是较好的SHG材料. 相似文献
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