Gradient time optimization

In this study, chromatographic experiments were 10 times faster with the monolithic column and results were equivalent to those obtained with the silica-based columns. This approach could be further optimized with faster gradient since flow rate should be increased by a factor 3 or 7 compared to conventional Cig supports [61, 62] and gradient time reduced by the same factor [63] to fully exploit the potential of monolithic supports. 

Dolan, J.W., Snyder, L.R., Djordjevic, N.M., Hill, D.W., Waeghe, T.J. (1999). Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time I. Peak capacity limitations. J. Chromatogr. A 857, 1-20. 

By replacing conventional 3.5 or 5 jtm columns with sub-2-micron columns, gradient time can be reduced dramatically. The flow rate must be increased for optimal conditions as well but solvent consumption will be less than the amount used by the original method. To use the full power of these columns, an LC instrument must be thoroughly optimized toward lowest extra-column dispersion. The smaller the column (small ID and short length), the more sensitive the performance is to dispersion. With smaller internal diameter columns, the injection volumes and internal diameters of the capillaries should be reduced. 

Modem technologies provide many techniques for expanding the throughput of an analytical laboratory. The task that needs to be accomplished and the possible drawbacks should be carefully considered. Optimized LC equipment can utilize columns packed with much smaller stationary phase particles to achieve significant reductions in gradient time while still achieving the same or even better peak capacities than conventional methods. 

FIGURE 5 Optimization of gradient time (to) resolution map and chromatogram. 

As the gradient time and other parameters (temperature, pH, or initial concentration of solvent B) often show synergistic effects on separation, simultaneous optimization of two or more parameters... 

Suppose that you have optimized a gradient on a 0.46 X 25 cm column and you want to transfer it to a 0.21 X 10 cm column. The quotient V2/V is ( nr2L)2/( nr1L), where r is column radius and L is column length. For these columns, V2/Vj = 0.083. Equation 25-8 tells us to decrease the volume flow rate, the sample mass, and the delay time to 0.083 times the values used for the large column. The gradient time should not be changed. 

Figure 8-47. Resolution map for all critical pairs. Color scale on the left indicates the minimum resolution that is predicted for a particular color in the resolution map. The a axis is the gradient time and the y axis is the temperature. The crosshair can be moved to obtained the predicted conditions for optimal resolution of all critical pairs. See color plate.

Hie typical output from method optimization software is a resolution map, as shown in Figure 10-1. The map shows resolution of the critical pair (two closest eluting peaks) as a function of the parameter(s). The example shows resolution as a function of gradient time (slope of the gradient). The resolution map has several advantages as an experimental display tool It forms a concise summary of experiments performed, it allows the chromatographer to select areas of interest and communicate the expected result, and it facilitates the viewing of data that would allow for a more robust separation. 

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