De Pauw Ruben, Swier Tim, Degreef Bart, Desmet Gert, Broeckhoven Ken
Vrije Universiteit Brussel, Department of Chemical Engineering (CHIS-IR), Pleinlaan 2, 1050 Brussels, Belgium.
Vrije Universiteit Brussel, Department of Chemical Engineering (CHIS-IR), Pleinlaan 2, 1050 Brussels, Belgium.
J Chromatogr A. 2016 Nov 18;1473:48-55. doi: 10.1016/j.chroma.2016.10.008. Epub 2016 Oct 6.
The limits in operating pressures are extended for narrow-bore columns in gradient elution up to 2000bar. As the required pumps for these pressures are incompatible with common chromatographic solvents and are not suitable to apply a mobile phase composition gradient, a mobile phase delivery and injection system is described and experimentally validated which allows to use any possible chromatographic solvent in isocratic and gradient elution. The mobile phase delivery and injection system also allows to perform multiple separations without the need to depressurize the column. This system consists out of 5 dual on/off valves and two large volume loops in which the gradient and equilibration volume of initial mobile phase are loaded by a commercial liquid chromatography pump. The loops are then flushed toward the column at extreme pressures. The mobile phase delivery and injection system is first evaluated in isocratic elution and shows a comparable performance to a state-of-the-art commercial flow-through-needle injector but with twice the pressure rating. Distortion of the loaded gradient by dispersion in the gradient storage loop is studied. The effect of the most important parameters (such as flow rate, pressure and gradient steepness) is experimentally investigated. Different gradient steepnesses and volumes can be applied at different flow rates and operating pressures with a good repeatability. Due to the isobaric operation of the pumps, the gradient is monitored in real-time by a mass flow meter installed at the detector outlet. The chromatograms are then converted from time to volume-base. A separation of a 19-compound sample is performed on a 300×2.1mm column at 1000bar and on a 600×2.1mm column at 2000bar. The peak capacity was found to increase from 141 to 199 and thus scales with L as is predicted by theory. This allows to conclude that the inlet pressure for narrow-bore columns in gradient elution can be increased up to 2000bar without fundamental pressure-induced limitations.
对于窄径柱，梯度洗脱时的操作压力上限可扩展至2000巴。由于达到这些压力所需的泵与常用的色谱溶剂不相容，且不适用于施加流动相组成梯度，因此描述并通过实验验证了一种流动相输送和进样系统，该系统允许在等度洗脱和梯度洗脱中使用任何可能的色谱溶剂。该流动相输送和进样系统还允许进行多次分离而无需对柱进行减压。该系统由5个双通开关阀和两个大体积定量环组成，初始流动相的梯度和平衡体积由商用液相色谱泵加载到定量环中。然后在极高压力下将定量环中的流动相冲洗至柱中。首先对等度洗脱中的流动相输送和进样系统进行了评估，结果表明其性能与最先进的商用流通针进样器相当，但耐压等级是其两倍。研究了梯度存储环中分散对加载梯度的影响。通过实验研究了最重要参数（如流速、压力和梯度陡度）的影响。不同的梯度陡度和体积可以在不同的流速和操作压力下应用，且具有良好的重复性。由于泵的等压操作，梯度由安装在检测器出口的质量流量计实时监测。然后将色谱图从时间基准转换为体积基准。在1000巴下于300×2.1毫米柱上以及在2000巴下于600×2.1毫米柱上对一个19种化合物的样品进行了分离。发现峰容量从141增加到199，因此正如理论预测的那样与柱长成正比。由此可以得出结论，梯度洗脱中窄径柱的入口压力可提高至2000巴，而不会受到基本的压力诱导限制。
