Abstract：
Due to the decoupling of the first (1D) and second (2D) dimension in pulsed elution-LC × LC (PE-LC × LC), method development is more flexible and straightforward compared to fast comprehensive LC × LC where the dependencies of key parameters between the two dimensions limits its flexibility. In this study we present a method for pulse generation, which is based on a switching valve alternating between one pump that delivers the gradient and a second pump that delivers low eluotrophic strength for the pause state. Consequently, the dwell volume of the system was circumvented and 7.5, and 3.75 times shorter pulse widths could be generated at flow rates of 0.2, and 0.4 mL/min with satisfactory accuracies between programmed and observed mobile phase composition (relative deviation of 6.0 %). We investigated how key parameters including pulse width and step height, 2D gradient time and flow rate affected the peak capacity in PE-LC × LC. The conditions yielding the highest peak capacity for the PE-LC × LC- high-resolution mass spectrometry (HRMS) system were applied to a wastewater effluent sample. The results were compared to a one dimensional (1D)-LC-HRMS chromatogram. The peak capacity increased with a factor 34 from 112 for the 1D-LC run to 3770 for PE-LC × LC-HRMS after correction for undersampling. The analysis time for PE-LC × LC-HRMS was 12.1 h compared to 67.5 min for the 1D-LC-HRMS run. The purity of the mass spectra improved for PE-LC × LC-HRMS by a factor 2.6 (p-value 3.3 × 10-6) and 2.0 (p-value 2.5 × 10-3) for the low and high collision energy trace compared to the 1D-LC-HRMS analysis. Furthermore, the signal-to-noise ratio (S/N) was 4.2 times higher (range: 0.06-56.7, p-value 3.8 × 10-2) compared to the 1D-LC-HRMS separation based on 42 identified compounds. The improvements in S/N were explained by the lower peak volume obtained in the PE-LC × LC-HRMS.
摘要：
由于脉冲洗脱-LC×LC（PE-LC×LC）中第一（1D）和第二（2D）维度的解耦，与快速综合LC×LC相比，方法开发更加灵活和直接，其中两个维度之间的关键参数的依赖性限制了其灵活性。在这项研究中，我们提出了一种脉冲生成方法，这是基于一个切换阀，在一个泵之间交替输送梯度和第二泵，提供低洗脱强度的暂停状态。因此,在流速为0.2和0.4mL/min的情况下，可以规避系统的停留时间，并在编程和观察到的流动相组成之间具有令人满意的精度（相对偏差为6.0％），可以产生7.5和3.75倍的短脉冲宽度。我们研究了包括脉冲宽度和步长在内的关键参数，2D梯度时间和流速影响PE-LC×LC中的峰值容量。将产生PE-LC×LC-高分辨率质谱（HRMS）系统的最高峰容量的条件应用于废水流出物样品。将结果与一维(1D)-LC-HRMS色谱图进行比较。在校正欠采样后，峰值容量从1D-LC运行的112增加到PE-LC×LC-HRMS的3770。PE-LC×LC-HRMS的分析时间为12.1小时,而1D-LC-HRMS的分析时间为67.5分钟。对于低和高碰撞能量迹线，PE-LC×LC-HRMS的质谱纯度提高了2.6倍（p值3.3×10-6）和2.0倍（p值2.5×10-3）与1D-LC-HRMS分析相比。此外，与基于42种鉴定化合物的1D-LC-HRMS分离相比，信噪比（S/N）高4.2倍（范围：0.06-56.7，p值3.8×10-2）。在PE-LC×LC-HRMS中获得的较低的峰体积解释了S/N的改善。
