Practical considerations in comprehensive two-dimensional liquid chromatography systems (LCxLC) with reversed-phases in both dimensions

Online comprehensive two-dimensional liquid chromatography (online LCxLC) presents high peak capacity compared with one-dimensional liquid chromatography, and reasonable operation time compared with an offline or stop-flow mode. Among various combinations, coupling of two reversed-phase (RPxRP) columns generates a high peak capacity product rate. Its wide applicability made RPxRP a promising technique in separation of complex samples. This review discusses the practical considerations in development and application of an RPxRP system, including systematic investigation of column stationary phase chemistry, column combinations, mobile phase system, interface, gradient types, and achieving fast analysis in LCxLC. In addition, many efforts are given to methods that increase the fraction coverage because this is the main obstacle caused by the correlated separation mechanism in RPxRP.