Department of Chemical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4L7.
Department of Materials Science and Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4L7.
Talanta. 2019 Jul 1;199:472-477. doi: 10.1016/j.talanta.2019.02.090. Epub 2019 Feb 27.
Columns packed with ultrafine particles (e.g. sub-2 µm porous particles) are suitable for high-resolution, high-speed analytical separation of proteins. However, they require very expensive chromatography systems to provide the ultra-high pressure required for carrying out separations using such columns. Also, frictional heating at high pressure could result in peak broadening and on-column protein degradation. In this paper, we discuss the use of nanoparticles, packed in a box-shaped or cuboid packed-bed device having 50 mm length, 5 mm width, and 3 mm bed-height for fast, high-resolution separation of proteins at low pressure. The low bed height allows the separation to be carried out at low pressure while the cuboid device reduces dispersion effects and thereby keeps the resolution high. Two different types of hydroxyapatite nanoparticles, i.e., needle-shaped (about 20 nm × 150 nm) and spherical (<200 nm) ones, were examined. The experimental results showed that while the needle-shaped nanoparticles were suitable for the separation of small proteins such as lysozyme, the spherical nanoparticles were better suited for separation of larger proteins such as bovine serum albumin and monoclonal antibody. The separation of the two proteins could be carried out in less than 2 min at a pressure lower than 0.8 MPa, using inexpensive chromatography devices a7nd systems, and without high pressure related problems such as frictional heating and on-column protein denaturation.
柱内填充超细微粒（例如亚 2μm 多孔颗粒）适用于蛋白质的高分辨率、高速分析分离。然而，它们需要非常昂贵的色谱系统来提供进行此类柱分离所需的超高压力。此外，高压下的摩擦加热可能导致峰展宽和柱上蛋白质降解。在本文中，我们讨论了使用纳米颗粒，填充在具有 50mm 长度、5mm 宽度和 3mm 床高的盒形或长方体填充床装置中，以在低压下快速、高分辨率地分离蛋白质。低床高允许在低压下进行分离，而长方体装置减少了分散效应，从而保持了高分辨率。我们检查了两种不同类型的羟磷灰石纳米颗粒，即针状（约 20nm×150nm）和球形（<200nm）纳米颗粒。实验结果表明，虽然针状纳米颗粒适用于分离小蛋白质（如溶菌酶），但球形纳米颗粒更适合分离大蛋白质（如牛血清白蛋白和单克隆抗体）。使用廉价的色谱设备和系统，在压力低于 0.8MPa 的情况下，不到 2 分钟即可完成两种蛋白质的分离，而不会出现与高压相关的问题，如摩擦加热和柱上蛋白质变性。
