Separation efficiency parameters peak capacity

In the development and optimization of a comprehensive LCxLC method, many parameters have to be taken in acconnt in order to accomplish snccessfnl separations. First of all, selectivity of the columns used in the two dimensions must be different to get maximum gain in peak capacity of the 2D system. For the experimental setup, column dimensions and stationary phases, particle sizes, mobile-phase compositions, flow rates, and second-dimension injection volumes should be carefully selected. The main challenges are related to the efficient coupling of columns and the preservation of mobile phase/column compatibility. 

It is obvious that n independent molecular properties require -dimensional methods for accurate (independent) characterization of all parameters. Additionally, the separation efficiency of any single separation method is limited by the efficiency and selectivity of this separation mode (i.e., the plate count N of the column and the phase system selected). Adding more columns will not overcome the need to identify more components in a complex sample, due to the limitation of peak capacities, n. The corresponding peak capacity... 

Column design involves the application of a number of specific equations (most of which have been previously derived and/or discussed) to determine the column parameters and operating conditions that will provide the analytical specifications necessary to achieve a specific separation. The characteristics of the separation will be defined by the reduced chromatogram of the particular sample of interest. First, it is necessary to calculate the efficiency required to separate the critical pair of the reduced chromatogram of the sample. This requires a knowledge of the capacity ratio of the first eluted peak of the critical pair and their separation ratio. Employing the Purnell equation (chapter 6, equation (16)). 

FIGURE 3.13 Dependence on the resolution of two adjacent peaks from the separation selectivity, column efficiency, and capacity factors of peaks. Curves were calculated by keeping values of two parameters constant at the starting value and varying the third parameter. 

Several important parameters to characterize the separation and the efficiency can be directly deduced from the chromatogram. The retention time of the component i is the residence time of the component i and can be measured directly from the chromatogram as the time distance between the sample injection and the top of the peak due to component i (see Fig. 1). From this, a number of other parameters can be calculated (Table 4). The capacity ratio or mass distribution ratio hfi (Eq. 1) is defined as the ratio of the amount of i in the stationary and the mobile phase, respectively. The capacity ratio is the product of the distribution coefficient and the phase ratio. It follows that the analyte molecules spend an average time fraction of 1 / ( -I- 1) in the mobile phase and of ( + 1) in the stationary phase. Analytes with different retention times spend different periods of time in the stationary phase. The separation factor a , of the phase system for the components i and j (Eq. 2) is another important parameter characterizing the separation. 

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