Department of Chemistry, University of Tennessee, Knoxville, TN 37996-1600, USA.
J Chromatogr A. 2012 Mar 9;1228:2-19. doi: 10.1016/j.chroma.2011.07.014. Epub 2011 Jul 23.
This work revisits the exceptionally rapid evolution of the technology of chromatographic columns and the important progress in speed of analysis and resolution power that was achieved over the last ten years. Whereas columns packed with 10 and 5 μm fully porous particles dominated the field for nearly thirty years (1975-2000), it took barely six years to see the commercialization of monolithic silica rods (2000), their raise to fame and decay to oblivion, the development of finer fully porous particles with size down to 1.7 μm (2006), and of sub-3 μm superficially porous particles (2006). Analysis times and plate heights delivered by columns packed with these recent packing materials have then been improved by more than one order of magnitude in this short period of time. This progress has rendered practically obsolete the age-old design of LC instruments. For low molecular weight compounds, analysts can now achieve peak capacities of 40 peaks in about 15s with a hold-up time of the order of 1.5s , in gradient elution, by operating columns packed with sub-3 μm shell particles at elevated temperatures, provided that they use optimized high pressure liquid chromatographs. This is the ultimate limit allowed by modern instruments, which have an extra-column band broadening contribution of 7 μL² at 4.0 mL/min and data acquisition rate of 160 Hz. The best 2.1 mm × 50 mm narrow-bore columns packed with 1.7 μm silica core-shell particles provide peaks that have a variance of 2.1 μL² for k=1. Finally, this work discusses possible ways to accelerate separations and, in the same time perform these separations at the same level of efficiency as they have today. It seems possible to pack columns with smaller particles, probably down to 1 μm and operate them with current vHPLC equipments for separations of biochemicals. Analyses of low molecular weight compounds will require new micro-HPLC systems able to operate 1mm I.D. columns at pressures up to 5 kbar, which would eliminate the heat friction problems, and providing extra-column band broadening contributions smaller than 0.1 μL². Alternatively, a new generation of vHPLC systems with minimal extra-column contributions of less than 0.5 μL² could run 2.1mm I.D. columns if these latter were to be packed with high heat conductivity materials such as core-shell particles made with an alumina or gold core.
本文重新审视了色谱柱技术的快速发展，以及过去十年中在分析速度和分辨率方面取得的重要进展。1975 年至 2000 年近三十年的时间里，填充有 10μm 和 5μm 全多孔颗粒的色谱柱占据主导地位，而仅仅六年时间就实现了整体硅胶棒的商业化（2000 年），其从成名到被淘汰，再到更细的 1.7μm 全多孔颗粒（2006 年）和亚 3μm 表面多孔颗粒（2006 年）的发展。在这段时间内，使用这些最新填充材料填充的色谱柱的分析时间和塔板高度提高了一个数量级以上。这种进步使得 LC 仪器的古老设计几乎过时。对于低分子量化合物，分析人员现在可以在梯度洗脱中，在约 15s 内实现约 40 个峰的峰容量，保留时间约为 1.5s ，通过在高温下操作填充有亚 3μm 壳颗粒的色谱柱来实现，前提是他们使用优化的高压液相色谱仪。这是现代仪器允许的最终极限，在 4.0mL/min 的流速和 160Hz 的数据采集率下，仪器的柱外带宽展宽贡献为 7μL²。最好的 2.1mm×50mm 窄径柱填充 1.7μm 硅胶核壳颗粒，提供的峰方差为 2.1μL²，k=1。最后，本文讨论了加速分离的可能方法，并在相同的效率水平下进行这些分离。用更小的颗粒（可能小至 1μm）填充色谱柱，并使用当前的 vHPLC 设备进行操作，用于生化物质的分离似乎是可行的。低分子量化合物的分析将需要新的微 HPLC 系统，能够在高达 5kbar 的压力下运行 1mm ID 柱，这将消除热摩擦问题，并提供小于 0.1μL² 的柱外带宽展宽贡献。或者，如果新一代 vHPLC 系统的柱外贡献最小，小于 0.5μL²，则可以运行 2.1mm ID 柱，如果这些柱采用氧化铝或金核的核壳颗粒等高热导率材料填充。
