A protocol for designing comprehensive two-dimensional liquid chromatography separation systems

In this paper a protocol is proposed for establishing suitable column dimensions (length and diameters), particle sizes, flow rates, and second-dimension injection volumes (i.e. loop sizes) in comprehensive two-dimensional liquid chromatography (LC × LC). The chromatographer should select the maximum allowable first-dimension retention time, which is approximately equal to the overall analysis time. Also, (s)he should define the maximum allowable pressure in both dimensions and the (minimum) diameter of the first-dimension column. The proposed protocol provides design parameters corresponding to the ideal (theoretically optimal) conditions or to realistic practical conditions. The protocol also allowed us to study the implications of contemporary developments in LC, such as the use of high temperatures (implying reduced viscosities and increased diffusion coefficients), monolithic columns (implying smaller flow-resistance factors), and ultra-high-pressure LC. The combination of (reversed-phase or normal-phase) liquid chromatography with size-exclusion chromatography (LC × SEC) is frequently employed for analysing complex polymers. The proposed protocol is used to design a suitable LC × SEC system under realistic conditions. The results resemble the systems that have been designed and implemented by expert chromatographers, but they also indicate how current practice can be improved.