Improving the LC Separation of Biomolecule Mixtures using Novel Mixed-Mode Gradient Stationary Phases

在化学系化学测量和成像项目的支持下，弗吉尼亚联邦大学的Maryanne Collinson教授和她的团队正在寻求通过探索分离科学的另一种范式来改进复杂生物样品的分离：使用混合模式梯度固定相。这些材料被装入不锈钢管中，固定相上化学相互作用位点的强度沿着管的长度变化。当与传统的液体流动相梯度相结合时，随着流动相溶剂强度的增加，分析物分子被顺序置换，这种材料有可能在分离大分子时提供显着改善的选择性。在实践中实现这种改进的选择性的一个重要障碍是在可控和可预测的方式下实验制造这些混合模式梯度固定相的能力。这一挑战将通过与色谱模拟及其预测能力领域的专家Sarah Rutan博士的持续合作，通过实验设计和模拟的结合，在目前的工作中解决。这个项目将为科学发现和学生的训练和学习提供机会。这项研究有望推进分离科学领域的知识，特别是在针对大的、生物相关分子的分析领域，包括蛋白质、肽，最终是对新型生物药物开发特别感兴趣的单克隆抗体。它探索了一个原始的概念，旨在提高分离的选择性，并最终在复杂蛋白质样品中化学相似分析物的分辨率。参与该项目的学生将获得有关色谱柱填充、硅烷化学修饰二氧化硅以及使用热重分析仪和其他光谱和微观工具对其进行详细表征的宝贵专业知识。同时，他们将成为LC和生物分子混合物分离的专家，使他们在生物制药领域具有独特的就业机会。在大多数化学分离中，梯度在流动相中。另一种范式将梯度置于固定相位。最近的模拟工作表明，当双固定相梯度与流动相梯度耦合时，可以在大分子的分离中“打开以前看不见的选择性”。在目前的工作中，将解决与制造和实现适合生物分子分离的这种混合模式固定相梯度相关的挑战。通过这项工作的过程中，将探索新的方法，以梯度方式战略性地用功能化的单氯硅烷（例如，苯基，C8, C4，…）修饰颗粒填充的二氧化硅柱。梯度的陡峭度将被改变和优化，以便当与流动相梯度耦合时，将在生物分子的分离中观察到协同选择性和/或带压缩。将开发模拟方法来预测色谱响应并优化柱制作条件。获得最佳分离所需的梯度长度和组成将通过模拟与实验相结合来获得，从而避免了材料开发中通常使用的试错方法。从长远来看，通过本研究开发的新色谱柱技术和伴随的模拟有可能影响蛋白质组学和脂质组学以及二维液相色谱分离领域。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Maryanne Collinson and her group at Virginia Commonwealth University are seeking to improve the separation of complex biological samples by exploring an alternate paradigm in separation science: the use of mixed-mode gradient stationary phases. These materials are packed into stainless steel tubes and the strength of the chemical interaction sites on the stationary phase is varied along the length of the tube. When coupled with a traditional liquid mobile phase gradient, which displaces the analyte molecules sequentially as the strength of mobile phase solvent is increased, such materials have the potential to provide significantly improved selectivity in the separation of large biomolecules. An important obstacle to realizing such improved selectivity in practice is the ability to experimentally fabricate these mixed-mode gradient stationary phases in a controlled and predictable fashion. This challenge will be addressed in the present work through a combination of experimental design and simulation via a continued collaboration with Dr. Sarah Rutan, an expert in the field of chromatographic simulations and their predictive power. This will project provide opportunities for both scientific discovery and student training and learning. This research is expected to advance knowledge in the field of separation science, particularly in areas that target the analysis of large, biologically relevant molecules including proteins, peptides, and ultimately monoclonal antibodies that are of particular interest in the development of novel biologic pharmaceuticals. It explores an original concept aimed to improve the selectivity of a separation and ultimately the resolution of chemically similar analytes within complex protein samples.Students involved in this project will obtain valuable expertise in the packing of chromatography columns, the modification of silica using silane chemistry, and its detailed characterization using TGA and other spectroscopic and microscopic tools. Simultaneously, they will become experts in LC and the separation of mixtures of biomolecules making them uniquely employable in the biopharmaceutical field. In most chemical separations, the gradient is in the mobile phase. An alternate paradigm puts the gradient on the stationary phase. Recent simulated work has shown that dual stationary phase gradients when coupled with a mobile phase gradient can “open up previously unseen selectivities” in the separation of large biomolecules. In the present work, the challenges associated with the fabrication and implementation of such mixed-mode stationary phase gradients suitable for biomolecule separations will be addressed. Through the course of this work, new approaches will be explored to strategically modify a particle-packed silica column with a functionalized monochlorosilane (e.g., phenyl, C8, C4,...) in a gradient fashion. The steepness of the gradients will be varied and optimized so that when coupled with a mobile phase gradient synergistic selectivity and/or band compression will be observed in the separation of biomolecules. Simulation methods will be developed to predict the chromatographic response and to optimize the conditions for column fabrication. The gradient lengths and compositions needed to obtain optimum separations will be obtained by coupling simulations with experiments, thus avoiding the trial-and-error approach usually used in materials development. Over the long term, the new column technology and accompanying simulations developed through this research have the potential to impact the fields of proteomics and lipidomics, as well as two-dimensional liquid chromatographic separations.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.