Analytical Research and Development, Bristol Myers Squibb Company, 1 Squibb Drive, New Brunswick, NJ 08903, USA.
J Chromatogr A. 2011 Aug 12;1218(32):5456-69. doi: 10.1016/j.chroma.2011.06.026. Epub 2011 Jun 17.
The theoretical increase in performance from the use of high efficiency columns with conventional HPLC equipment is generally not observed due to the design limitations of such equipment, particularly with respect to extra-column dispersion (ECD). This study examines the impact of ECD from a Waters Alliance 2695 system on the performance of 2.7 μm HALO C(18) Fused-Core superficially porous particle columns of various dimensions. The Alliance system was re-configured in different ways to reduce extra-column volume (ECV) and the ECD determined in each case as a function of flow rate up to a maximum of 2 mL/min. The results obtained showed a progressive decrease in ECD as the ECV was reduced, irrespective of the flow rate employed. However, this decrease in ECD was less than theoretically expected for the lower ECV configurations. The inability to reduce the actual extra-column dispersion further was attributed to additional dispersion associated with the design/volume of the auto-injector. This was confirmed by making sample injections with a low dispersion manual injection valve, instead of auto-injection, for the two lowest ECV configurations studied. In each case, the measured and predicted ECD values were in good agreement. The auto-injector module is an integral part of the Alliance 2695 instrument and cannot be easily modified. However, even with autosampler injection, for a 3mm ID × 100 mm Fused-Core column approximately 70% of the maximum plate count (∼84% of the resolution or more) could still be obtained in isocratic separations for solutes with k ≥ ∼4.5 when using the lowest ECV configuration. This study also highlights some of the problems inherent in trying to measure accurately the true extra-column dispersion of a chromatographic system and compares the results obtained to those theoretically predicted. Using this same lowest volume instrument configuration, two real-world pharmaceutical methods were scaled to separations that are ∼3-3.5-fold faster, while still maintaining comparable data quality (resolution and signal-to-noise ratios).
由于此类设备的设计限制，特别是柱外展宽（ECD），使用高效柱和传统 HPLC 设备通常无法观察到理论上的性能提高。本研究考察了 Waters Alliance 2695 系统的 ECD 对不同尺寸的 2.7μm HALO C(18) 熔融核表面多孔颗粒柱性能的影响。该 Alliance 系统以不同方式重新配置，以减小柱外体积（ECV），并在每种情况下确定 ECD 作为流速的函数，最高可达 2mL/min。结果表明，随着 ECV 的减小，ECD 逐渐减小，与所采用的流速无关。然而，对于较低的 ECV 配置，ECD 的减小量小于理论预期。无法进一步减小实际柱外展宽归因于与自动进样器的设计/体积相关的附加展宽。通过对于研究的两个最低 ECV 配置，使用低分散手动进样阀而不是自动进样来进行样品进样，从而证实了这一点。在每种情况下，测量和预测的 ECD 值非常吻合。自动进样器模块是 Alliance 2695 仪器的一个组成部分，不易修改。然而，即使使用自动进样器，对于 3mm ID×100mm Fused-Core 柱，当使用最低 ECV 配置时，对于 k≥∼4.5 的溶质，在等度分离中仍然可以获得大约 70%的最大板数（∼84%的分辨率或更高）。本研究还强调了在尝试准确测量色谱系统的真实柱外展宽时存在的一些问题，并将获得的结果与理论预测进行了比较。使用相同的最低体积仪器配置，将两种实际的药物方法扩展到分离速度提高了约 3-3.5 倍，同时仍保持可比的数据质量（分辨率和信噪比）。
