Comprehensive Multidimensional Separations of Peptides Using Nano-Liquid Chromatography Coupled with Micro Free Flow Electrophoresis

The throughput of existing liquid phase two-dimensional separations is generally limited by the peak capacity lost due to under sampling by the second dimension separation as peaks elute off the first dimension separation. In the current manuscript, a first dimension nanoliquid chromatography (nLC) separation is coupled directly with a second dimension micro free flow electrophoresis (mu FFE) separation. Since mu FFE performs continuous separations, no complicated injection or modulation is necessary to couple the two techniques. Analyte peaks are further separated in mu FFE as they elute off the nLC column. A side-on interface was designed to minimize dead volume in the nLC x mu FFE interface, eliminating this as a source of band broadening. A Chromeo P503 labeled tryptic digest of BSA was used as a complex mixture to assess peak capacity. 2D nLC X mu FFE peak capacities as high as 2,352 could be obtained in a 10 min separation window when determined according to the product of the first and second dimension peak capacities. After considering the orthogonality of the two separation modes and the fraction of separation space occupied by peaks, the usable peak capacity generated was determined to be 776. The 105 peaks/min generated using 2D nLC x mu FFE was nearly double the previously reported maximum peak capacity production rate achieved using online LC x LC.