Optimizing Gradient Separations for Speed

At this point, the stationary phase particle diameter is extremely important for the kinetic optimization of separations. A smaller particle diameter reduces the distance for the necessary radial diffusion of analyte molecules on the one hand, but increases the geometrical radial concentration gradient that drives the diffusion. Both effects are synergistic for an efficient analyte transport and this is the physicochemical foundation for the decrease of the C-term with the squared particle diameter (dp ). This will be used effectively in the speed optimization strategy. 

Others have examined the necessary parameters that should be optimized to make the two-dimensional separation operate within the context of the columns that are chosen for the unique separation applications that are being developed. This is true for most of the applications shown in this book. However, one of the common themes here is that it is often necessary to slow down the first-dimension separation system in a 2DLC system. If one does not slow down the first dimension, another approach is to speed up the second dimension so that the whole analysis is not gated by the time of the second dimension. Recently, this has been the motivation behind the very fast second-dimension systems, such as Carr and coworker s fast gradient reversed-phase liquid chromatography (RPLC) second dimension systems, which operate at elevated temperatures (Stoll et al., 2006, 2007). Having a fast second dimension makes CE an attractive technique, especially with fast gating methods, which are discussed in Chapter 5. However, these are specialized for specific applications and may require method development techniques specific to CE. 

MD-HPLC for natural products requires thoughtful selection of orthogonal and complementary separation modes, of the order of their utilization and independent optimization with respect to the chromatographic goals (speed, resolution, capacity, and recovery). Furthermore, besides the mobile phase composition of the employed chromatographic modes, the elution mode (isocratic, step, or gradient elution), flow rates, and mobile phase temperatures need to be considered. 

Gradient development in TLC is a technique that allows one to improve the resolution of a given pair of adjacent bands, to accelerate a separation, to concentrate the sample band and lower the detection limit, and to speed up the search for an optimal chromatographic system. 

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