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The different operating conditions of an online two-dimensional liquid chromatographic separation (2D-LC), such as the length of the column, the linear velocity and the composition of the mobile phase used in the second dimension, its initial organic content if this separation is carried out in gradient elution, the number of fractions of the first column eluent collected, and the analysis time of the first dimension all affect the achievable separation power of 2D-LC online systems. The influences of these factors on the separation performance were investigated, and an equation was derived for the calculation of the achievable peak capacity in online 2D-LC assuming (1) that the option of undersampling the first-dimension separation is acceptable, (2) that the solutes follow linear-solvent-strength behavior, and (3) that all the separations are made in gradient elution. This theoretical discussion shows that (1) highly efficient separations made with online 2D-LC require the second-dimension peaks to be very narrow, (2) the separation power of 2D-LC systems is maximum for an optimum number of fractions collected in the first dimension, (3) higher peak capacities can be achieved by using shorter second-dimension columns and collecting a relatively large number of fractions, (4) the achievable 2D peak capacity is maximum for a certain eluent flow rate and column length of the second-dimension column, and (5) the maximum achievable peak capacity increases with decreasing velocity and initial organic content of the second-dimension eluent. As a consequence, due to the time restriction of the second-dimension gradient time, online 2D-LC schemes cannot realistically afford peak capacities exceeding 10,000, even if they are implemented with exceptionally efficient columns and if long analysis times are accepted.

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