Gritti Fabrice, Guiochon Georges
Department of Chemistry, University of Tennessee, Knoxville, TN 37996-1600, USA.
J Chromatogr A. 2014 Dec 29;1374:112-121. doi: 10.1016/j.chroma.2014.11.036. Epub 2014 Nov 20.
A new brand of BEH-C18 hybrid particles chemically bonded to a leash carrying an amine group permits the implementation of electrostatic repulsive interactions chromatography. Using columns packed with this material, the influence of the concentration of positive charges bonded to the BEH-C18 surface on the overloaded band profiles of a few positively charged peptides and proteins was investigated in the gradient elution mode. Three columns packed with endcapped BEH-C18 particles bonded with three different surface-charge densities (LOW, MEDIUM and HIGH) were used and compared with those provided by a column packed with non-doped, endcapped BEH-C18 particles. The surface concentrations of fixed charges in the LOW, MEDIUM and HIGH columns were estimated at 0.029, 0.050, and 0.064μmol/m(2), for example, about two orders of magnitude smaller than the surface density of bonded C18 chains (2.1μmol/m(2)). Three different mobile phase additives (0.1% v/v of trifluoro-acetic, phosphoric, and formic acid) were used to optimize the purification levels of proteins under different loading conditions. The weak ion-pairing ions (formate and phosphate) generate smaller retention but broader, more fronting band profiles than those eluted with a stronger ion-pairing ion (trifluoroactate). This effect is worse in the presence of fixed charges at the surface of the BEH-C18 particles. This was explained by an enhanced anti-Langmuirian adsorption behavior of the charged proteins in the presence of fixed surface charges. As the protein concentration increases in the bulk, so does the internal ionic strength, the electrostatic repulsive interactions weaken, and retention increases. Band fronting is mostly eliminated by replacing weak ion-pairing acids with TFA with which the adsorption isotherm remains weakly langmuirian. Faster but still complete gradient separation of insulin and myoglobin were achieved with the HIGH column than with the reference neutral column, despite a measurable loss in selectivity.
一种新品牌的BEH-C18杂化颗粒，化学键合到带有胺基的链上，可实现静电排斥相互作用色谱法。使用填充有这种材料的色谱柱，在梯度洗脱模式下研究了键合到BEH-C18表面的正电荷浓度对几种带正电荷的肽和蛋白质过载峰形的影响。使用了三根填充有封端的BEH-C18颗粒的色谱柱，其表面电荷密度分别为三种不同水平（低、中、高），并与填充有未掺杂、封端的BEH-C18颗粒的色谱柱进行比较。例如，低、中、高色谱柱中固定电荷的表面浓度估计分别为0.029、0.050和0.064μmol/m²，比键合C18链的表面密度（2.1μmol/m²）小约两个数量级。使用三种不同的流动相添加剂（0.1% v/v的三氟乙酸、磷酸和甲酸）来优化不同负载条件下蛋白质的纯化水平。弱离子对离子（甲酸根和磷酸根）产生的保留较小，但峰形比用较强离子对离子（三氟乙酸根）洗脱的峰形更宽、更前沿。在BEH-C18颗粒表面存在固定电荷的情况下，这种影响更严重。这可以通过在存在固定表面电荷的情况下带电蛋白质增强的反朗缪尔吸附行为来解释。随着本体中蛋白质浓度的增加，内部离子强度也增加，静电排斥相互作用减弱，保留增加。通过用TFA代替弱离子对酸，大部分峰前沿被消除，此时吸附等温线仍保持弱朗缪尔性质。尽管选择性有一定损失，但与参比中性色谱柱相比，高电荷密度色谱柱实现了胰岛素和肌红蛋白更快但仍完全的梯度分离。
