Effect of Sampling Rate on Resolution in Comprehensive Two-Dimensional Liquid Chromatography Robert E. Murphy,* ,²,‡ Mark R. Schure, § and Joe P. Foley ‡ Analytical Research and Theoretical Separation Science Laboratory, Rohm and Haas Company, 727 Norristown Road, Spring House, Pennsylvania 19477, and Department of Chemistry, Villanova University, Villanova, Pennsylvania 19085 In “comprehensive” two-dimensional liquid chromatog- raphy, the column effluent from the first separation system (the first dimension) is sequentially sampled by the second dimension separation system. The total analysis time is largely determined by the speed of the second dimension separation system; the most retained compo- nent must elute before the least retained component of the next second dimension separation. Optimization of multidimensional separation systems requires that one understand the relationship between system resolution and the number of second dimension samples across a first dimension peak. In this paper, we study the theo- retical and experimental aspects of this sampling process. To obtain high two-dimensional resolution, each peak in the first dimension should be sampled at least three times into the second dimension when the sampling is in-phase. If the sampling is maximally out of phase, there should be at least four samples per peak for high-fidelity separa- tion. The sensitivity of the resolution with respect to the sampling phase is discussed in detail and shown to be insignificant when four or greater samples are taken across the first dimension peak width. These results suggest optimal criteria for method development with multidimensional chromatography. Two-dimensional liquid chromatographic systems (2DLC) have been used for many years to characterize and separate biomol- ecules, polymers, and other complex mixtures. 1-3 Natural prod- ucts and polymer samples are extremely complex, containing a large number of components of various composition and size. Two- dimensional liquid chromatography has the peak capacity to separate plant extracts 1 and the selectivity and resolution to separate copolymers 2 by their size and composition. The most common form of two-dimensional separation is heart-cutting, where one discrete zone is collected from the first dimension column and reinjected into the second dimension separation system. 4-7 The resulting data are two individual one-dimensional data sets and are useful for the higher resolution analysis of a single fused peak from the first dimension. In so-called “com- prehensive” automated systems, sequential aliquots from the first dimension effluent are sampled by the second dimension separa- tion system. 8 The resulting data is a matrix, usually represented as a contour plot with each chromatographic separation along an axis. This technique is very useful for the higher resolution analysis of multiple fused peaks from the first dimension column and resolved in the second dimension separation system when orthogonal separation systems can be found. There are many combinations of separation techniques and methods of coupling these techniques currently employed in multidimensional separation systems. Giddings 9 discussed a number of the possible combinations of techniques that can be coupled together to form two-dimensional systems. We will restrict our discussion to only two-dimensional systems here although many of the principles will apply to more than two coupled separation systems. The two-dimensional techniques can be categorized by the number of fractions and amount of eluent sampled into the second dimension (Table 1). We will focus exclusively on the comprehensive mode, with the entire first dimension effluent sampled by the second dimension separation system in this paper. A host of methods exist for coupling the various separation systems. An eight-port valve with matching sample loops is typically used for the coupling and repetitive sampling of the first dimension separation system when the comprehensive mode of operation is utilized. 8,10 A six-port valve is generally used for the partial sampling of the first dimension separation with repetitive sampling. 11-13 A direct interface is also possible which utilizes † Analytical Research, Rohm and Haas Co. ‡ Villanova University. § Theoretical Separation Science Laboratory, Rohm and Haas Co. (1) Erni, F.; Frei, R. W. J. Chromatogr. 1978 , 149, 561-569. (2) Balke, S. T.; Patel, R. D. J. Polym. Sci., Polym. Lett. Ed. 1980 , 18, 453-456. (3) Majors, R. E. J. Chromatogr. Sci. 1980 , 18, 571-579. (4) Augenstein, M.; Strickler, M. Makromol. Chem. 1990 , 415-428. (5) Pasch, H.; Brinkmann, C.; Much, H.; Just, U. J. Chromatogr. 1992 , 623, 315-322. (6) Van Doremaele, G. H. J.; Geerts, F. H. J. 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