Modulation optimization when using a splitter pump after the first dimension in comprehensive two- dimensional liquid chromatography.

The rapid growth in the use of two dimensional liquid chromatography (2D-LC) applied to the analysis of moderately to highly complex mixtures, has been fueled by continuous improvements in performance and robustness of the instrument components, as well as the ease-of-use of software necessary for controlling the 2D-LC instrument hardware, and analysis of the large data files that result from this type of work. This work has focused on the evaluation of the performance of an online full comprehensive mode (LC×LC), when an active modulation is implemented using a flow splitter pump placed after the D effluent. Two different types of splitting pumps were evaluated: a binary ultra-high pressure liquid chromatography (UHPLC) pump and a high precision syringe pump. We analyzed the performance (reproducibility in peak area and retention times and the D peak dispersion) as a function of the location of the active pump Before or After the modulation valve, and the influence of connecting tubes (based on internal diameter and length) necessary between the interface, waste, and the splitting pump. The effect on the flow direction on the filling and flushing of the injection loops at the modulation valve was also analyzed for each pump. In this study, we demonstrate that flow-splitting LCxLC assembly can be performed using either a UHPLC binary pump or a simple syringe pump. Flow splitting after the first dimension is a straightforward strategy to: (i) independently select the D column and flow rates with respect to the second dimension; (ii) consciously dilute the eluate according to the solvent characteristics of the second dimension, thereby avoiding D peak distortions; and (iii) adapt the injected amount to the second column according to the relative concentration of the components in a complex sample. However, careful consideration of the system setup is necessary. It is demonstrated how experimental results can be significantly affected in terms of peak broadening and reproducibility if optimization of the interface is not taken into account. In addition, under the optimized conditions, the reproducibility in peak area and dispersion in the D dimension were evaluated as a function of the amount of sample transferred in terms of percentage of filled loop.