Separations of complex peptide mixtures have been a common target application for two-dimensional liquid chromatography over the last few decades. These separations have most frequently been carried out at the capillary scale, with columns on the order of 75 µm i.d. and flow rates on the order of 500 nL/min. Recently, however, several groups have worked to optimize comprehensive 2D-LC (LC × LC) separations of peptides at the analytical scale (i.e., 2 mm i.d. columns, and ca. 1 mL/min flow rates) and demonstrated peak capacities on the order of 5000 in analysis times of a few hours, using reversed-phase separations in both dimensions. In this paper we aim to advance the performance of such separations in two primary ways. First, we demonstrate that active solvent modulation (ASM) can be used to improve the 2D peak capacity by both enabling use of long and highly efficient first dimension (1D) columns, and by mitigating the deleterious effects of injecting large fractions of 1D effluent into the small columns that are required for fast and highly sensitive second dimension (2D) separations. Taken together these two benefits enable the realization of a peak capacity of 10,000 in an analysis time of four hours. This comes at the cost of increased instrument complexity compared to 1D-LC separations, but the 2D-LC approach is unquestionably the most efficient way to improve upon the resolving power of existing 1D-LC. Second, we have systematically studied the compromise between the peak capacity of each 2D separation and the operating pressure required to achieve that peak capacity. Understanding this compromise will be important to the development of LC × LC methods that both produce high peak capacities, and are sufficiently robust to operate for days at a time without significant losses in separation performance. Based on the results of this study we chose conditions for subsequent separations that required less than 400 bar operating pressure in the second dimension, but yielded a 2D peak capacity of about 3500 in 2 h. After 160 h of continuous operation of the LC × LC separation under these conditions (and about 20,000 injections into the 2D column) the 2D column had only lost about 18% of its initial isocratic efficiency. These results should motivate further development and implementation of such high performing and robust separations for the identification and quantification of peptides in a variety of application areas, including digests of therapeutic proteins such as monoclonal antibodies.
Keywords: Active solvent modulation; Column lifetime; Focusing; Optimization; Peak capacity; Peptides; Two-dimensional liquid chromatography.
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