Abstract This paper describes for the first time the automation of immobilized metal affinity chromatography by sequential injection analysis. A generic poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith was synthesized by thermal free radical polymerization inside a 2.1 mm inner diameter activated fused silica-lined stainless steel tubing and then modified with iminodiacetate (IDA) onto which Cu(II) was immobilized. The retention mechanism controlled by metal ion affinity was verified for proteins such as ovalbumin and myoglobin. These proteins were unretained in the monolith modified only with IDA using a mobile phase composed of 20 mM phosphate buffer (pH 7.0) and 0.50 M NaCl. On the other hand, ovalbumin and myoglobin were retained in the monolith to which Cu(II) was immobilized and elution was achieved only following the addition of imidazole to the mobile phase. The sequential injection strategy created a fast gradient by mutual dispersion of three mobile phases containing imidazole at concentrations of 10, 20, and 50 mM, which enabled the efficient separation of cytochrome C, ribonuclease A, and myoglobin. The proposed methodology may be useful to isolate proteins from complex matrices as demonstrated by the separation of ovalbumin and lysozyme from unretained compounds in an egg-white sample.

中文翻译：

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用于蛋白质分离的固定化金属亲和顺序进样色谱

摘要 本文首次介绍了连续进样分析固定化金属亲和层析的自动化。在 2.1 mm 内径活化的熔融石英衬里不锈钢管内通过热自由基聚合合成了一种通用的聚（甲基丙烯酸缩水甘油酯-共聚二甲基丙烯酸乙烯酯）整体料，然后用亚氨基二乙酸酯 (IDA) 进行改性，在其上固定有 Cu(II) . 对于卵清蛋白和肌红蛋白等蛋白质，验证了由金属离子亲和力控制的保留机制。这些蛋白质未保留在仅用 IDA 修饰的整体中，使用由 20 mM 磷酸盐缓冲液 (pH 7.0) 和 0.50 M NaCl 组成的流动相。另一方面，卵清蛋白和肌红蛋白保留在固定有 Cu(II) 的整体中，只有在向流动相中加入咪唑后才能洗脱。连续进样策略通过相互分散含有浓度为 10、20 和 50 mM 的咪唑的三种流动相产生快速梯度，从而实现细胞色素 C、核糖核酸酶 A 和肌红蛋白的有效分离。所提出的方法可用于从复杂基质中分离蛋白质，如从蛋清样品中未保留的化合物中分离卵清蛋白和溶菌酶所证明的那样。这使得细胞色素 C、核糖核酸酶 A 和肌红蛋白的有效分离成为可能。所提出的方法可用于从复杂基质中分离蛋白质，如从蛋清样品中未保留的化合物中分离卵清蛋白和溶菌酶所证明的那样。这使得细胞色素 C、核糖核酸酶 A 和肌红蛋白的有效分离成为可能。所提出的方法可用于从复杂基质中分离蛋白质，如从蛋清样品中未保留的化合物中分离卵清蛋白和溶菌酶所证明的那样。