el Rassi Z, De Ocampo L F, Bacolod M D
Department of Chemistry, Oklahoma State University, Stillwater 74078-0447.
J Chromatogr. 1990 Jan 19;499:141-52. doi: 10.1016/s0021-9673(00)96969-3.
Hydrophobic-interaction chromatography of mixtures of acidic and basic proteins having a wide range of molecular weights and hydrophobic character was carried out by using binary and ternary salt gradients. Chaotropic and antichaotropic salts as well as organic salts were incorporated in the eluents. The stationary phase consisted of macroporous silica with surface-bound polyether moieties. At constant eluent surface tension, gradient elution with two or three aqueous salt solutions was found to be superior to single-salt gradients in modulating hydrophobic-interaction chromatography retention and selectivity. The effect was attributed to the competitive salt-specific binding to the protein molecule and/or the stationary phase surface. Chaotropic/antichaotropic salt gradient systems exhibited vastly different selectivities upon changing the nature and concentrations of salts in the eluents. In general, the retention of basic proteins increased while that of acidic proteins either decreased or remained unchanged with the use of chaotropic salts. At the same surface tension of the eluent, KSCN and KC1O4 yielded different selectivities. The addition of organic salts, such as tetrabutylammonium bromide was found to be suitable for the separation of proteins having a wide range of isoelectric points.
采用二元和三元盐梯度对具有广泛分子量和疏水特性的酸性和碱性蛋白质混合物进行疏水相互作用色谱分析。离液盐和抗离液盐以及有机盐被纳入洗脱液中。固定相由具有表面结合聚醚部分的大孔二氧化硅组成。在洗脱液表面张力恒定的情况下，发现用两种或三种盐水溶液进行梯度洗脱在调节疏水相互作用色谱保留率和选择性方面优于单盐梯度。这种效应归因于盐特异性与蛋白质分子和/或固定相表面的竞争性结合。当改变洗脱液中盐的性质和浓度时，离液盐/抗离液盐梯度系统表现出截然不同的选择性。一般来说，使用离液盐时碱性蛋白质的保留率增加，而酸性蛋白质的保留率要么降低要么保持不变。在洗脱液表面张力相同的情况下，KSCN和KClO4产生不同的选择性。发现添加有机盐，如四丁基溴化铵适用于分离具有广泛等电点的蛋白质。
