Waters Corporation, Instrument/Core Research/Fundamental, 34 Maple Street, Milford, MA, 01757, USA.
J Chromatogr A. 2020 Dec 20;1634:461673. doi: 10.1016/j.chroma.2020.461673. Epub 2020 Nov 2.
The fundamental relationships are derived for the retention, peak width, and peak capacity of non-retained polymers eluting from multiple standard size-exclusion chromatography (SEC) columns connected in series. The standard SEC columns may have different dimensions and are packed with particles having distinct average particle diameters (APDs) and average mesopore sizes (AMSs). The performances (peak capacity, local resolution power, and sensitivity) of three standard SEC columns connected in series (called a tri-SEC column) packed with bridged-ethylene-hybrid (BEH) fully porous particles (FPPs) having three different APDs (1.7, 2.5, and 3.5 μm) and AMSs (200, 450, and 900 Å, respectively) are calculated as a function of the applied flow rate and size of polystyrene standards. Irrespective of the APD and AMS, the present investigation assumes isomorphological materials relative to the mesopore space of the three different BEH particles. The advantage of a 15 cm long tri-SEC column over a single reference SEC column (APD=3.5 μm, AMS=900 Å), which generates the same back pressure and separation window as those of the tri-SEC column, is expected at flow rates larger than the optimum flow rate generating the maximum peak capacity. The calculations predict a significant relative increase of the peak capacity (from +25% to +85%), resolution of small molecules (from +75% to +225%), and of the detection limit of intermediate size (from +15% to +70%) and largest polymers (from +25 to +110%). This is explained by 1) the exclusion of the largest polymers from the internal volume of the particles having the smallest mesopores (restricted access media) and 2) the minimum dispersion along the columns packed with the smallest particle sizes in the tri-SEC column. The main benefit of multi-SEC columns is to easily adjust the desired pore size distribution by properly selecting the lengths of each individual SEC column. The user can then control the pore size distribution for any specific separation problem. A potential application is theoretically demonstrated for the fast purification of monoclonal antibodies from metabolites, host cell proteins, aggregated forms, and from virus-like particles.
推导出了非保留聚合物在串联连接的多个标准尺寸排阻色谱（SEC）柱中洗脱时的保留、峰宽和峰容量的基本关系。标准 SEC 柱可能具有不同的尺寸，并填充有具有不同平均粒径（APD）和平均中孔尺寸（AMS）的颗粒。串联连接的三个标准 SEC 柱（称为三 SEC 柱）的性能（峰容量、局部分辨率和灵敏度）用具有三种不同 APD（1.7、2.5 和 3.5μm）和 AMS（200、450 和 900Å，分别）的 bridged-ethylene-hybrid（BEH）全多孔颗粒（FPP）填充计算为施加的流速和聚苯乙烯标准的尺寸的函数。无论 APD 和 AMS 如何，本研究都假设相对于三种不同 BEH 颗粒的中孔空间是同构材料。与生成与三 SEC 柱相同的背压和分离窗口的单个参考 SEC 柱（APD=3.5μm，AMS=900Å）相比，15cm 长的三 SEC 柱的优势预计在大于最佳流速的流速下，最大峰容量产生。计算预测了峰容量（从+25%到+85%）、小分子分辨率（从+75%到+225%）以及中等大小（从+15%到+70%）和最大聚合物（从+25%到+110%）的检测限的显著相对增加。这可以通过以下两种方式来解释：1）将最大聚合物排除在具有最小中孔的颗粒的内部体积之外（受限进入介质）；2）在三 SEC 柱中用最小颗粒尺寸填充的柱中最小的分散。多 SEC 柱的主要优点是可以通过适当选择每个单独 SEC 柱的长度来轻松调整所需的孔径分布。用户可以根据特定的分离问题来控制孔径分布。从理论上演示了一种潜在的应用，用于从代谢物、宿主细胞蛋白、聚集形式和病毒样颗粒中快速纯化单克隆抗体。
