Schmidt B, Wandrey Ch, Freitag R
Department of Chemistry, Swiss Federal Institute of Technology Lausanne, Ecublens.
J Chromatogr A. 2002 Jan 25;944(1-2):149-59. doi: 10.1016/s0021-9673(01)01203-1.
A novel type of linear polyelectrolyte, namely poly-DADMAC [poly(diallyldimethylammonium chloride)], was prepared and studied as a displacer for cation-exchange displacement chromatography of proteins. In contrast to the commercially available polymers of that chemistry, the novel type of poly-DADMAC introduced here is characterized by a homogeneous linear structure, a narrow distribution of the (adjustable) molar mass as well as by a defined and homogeneous affinity for the stationary phase. Five poly-DADMACs of different size (17900 to 88000 g/mol) were prepared and compared with regard to their stationary phase affinity and protein separation potential, taking a mixture of basic proteins, namely lysozyme, cytochrome C, and ribonuclease A (from bovine pancreas), as an example. The steric mass action model was employed to aid method development. Under the chosen conditions (low ionic strength of the mobile phase guaranteeing strong binding of both the proteins and the displacer) the poly-DADMAC with the lowest molar masses proved to be the most efficient displacers for the basic proteins with a stationary phase affinity constant of 5.3 x 10(16) and a steric factor of 224. Using this substance as displacer, a sample mixture containing up to three proteins was separated and the proteins recovered at high yields (80-97%) and in high purity and concentration.
制备了一种新型线性聚电解质，即聚二烯丙基二甲基氯化铵（poly-DADMAC），并将其作为蛋白质阳离子交换置换色谱的置换剂进行了研究。与该化学类型的市售聚合物相比，本文介绍的新型聚二烯丙基二甲基氯化铵的特点是具有均匀的线性结构、（可调节的）摩尔质量分布窄以及对固定相具有明确且均匀的亲和力。制备了五种不同大小（17900至88000 g/mol）的聚二烯丙基二甲基氯化铵，并以碱性蛋白质混合物，即溶菌酶、细胞色素C和（来自牛胰腺的）核糖核酸酶A为例，比较了它们对固定相的亲和力和蛋白质分离潜力。采用空间质量作用模型辅助方法开发。在所选条件下（流动相的低离子强度保证了蛋白质和置换剂的强结合），摩尔质量最低的聚二烯丙基二甲基氯化铵被证明是碱性蛋白质最有效的置换剂，其固定相亲和常数为5.3×10¹⁶，空间因子为224。使用该物质作为置换剂，分离了含有多达三种蛋白质的样品混合物，蛋白质以高收率（80 - 97%）、高纯度和高浓度回收。
