Abstract:
Ion-pair chromatography is the de facto standard for separating oligonucleotides and related impurities, particularly for analysis but also often for small-scale purification. Currently, there is limited understanding of the quantitative modeling of both analytical and overloaded elution profiles obtained during gradient elution in ion-pair chromatography. Here we will investigate a recently introduced gradient mode, the so-called ion-pairing reagent gradient mode, for both analytical and overloaded separations of oligonucleotides. The first part of the study demonstrates how the electrostatic theory of ion-pair chromatography can be applied for modeling gradient elution of oligonucleotides. When the ion-pair gradient mode is used in a region where the electrostatic surface potential can be linearized, a closed-form expression of retention time can be derived. A unified retention model was then derived, applicable for both ion-pair reagent gradient mode as well as co-solvent gradient mode. The model was verified for two different experimental systems and homo- and heteromeric oligonucleotides of different lengths. Quantitative modeling of overloaded chromatography using the ion-pairing reagent gradient mode was also investigated. Firstly, a unified adsorption isotherm model was developed for both gradient modes. Then, adsorption isotherms parameter of a model oligonucleotide and two major synthetic impurities were estimated using the inverse method. Secondly, the parameters of the adsorption isotherm were then used to investigate how the productivity of oligonucleotide varies with injection volume, gradient slope, and initial retention factor. Here, the productivity increased when using a shallow gradient slope combined with a low initial retention factor. Finally, experiments were conducted to confirming some of the model predictions. Comparison with the conventional co-solvent gradient mode showed that the ion-pairing reagent gradient leads to both higher yield and productivity while consuming less co-solvent.
摘要:
离子对色谱法是分离寡核苷酸和相关杂质的事实上的标准,特别是用于分析,但也经常用于小规模纯化。目前,对离子对色谱中梯度洗脱过程中获得的分析和过载洗脱曲线的定量建模的理解有限.在这里,我们将研究最近引入的梯度模式,所谓的离子对试剂梯度模式,用于分析和过载的寡核苷酸分离。研究的第一部分演示了离子对色谱的静电理论如何应用于寡核苷酸的梯度洗脱建模。当在可以线性化静电表面电势的区域中使用离子对梯度模式时,可以得出保留时间的闭式表达式。然后推导出一个统一的保留模型,适用于离子对试剂梯度模式以及共溶剂梯度模式。针对两个不同的实验系统以及不同长度的同聚和异聚寡核苷酸验证了该模型。还研究了使用离子配对试剂梯度模式的过载色谱的定量建模。首先,对两种梯度模式都建立了统一的吸附等温线模型。然后,模型寡核苷酸和两种主要合成杂质的吸附等温线参数使用反方法估计。其次,然后使用吸附等温线的参数来研究寡核苷酸的生产率如何随注射体积而变化,坡度,和初始保留因子。这里,当使用浅梯度斜率和低初始保留因子时,生产率提高。最后,进行了实验以证实一些模型预测。与常规共溶剂梯度模式的比较表明,离子-配对试剂梯度导致更高的产率和生产率,同时消耗更少的共溶剂。