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Initial efforts centered on chromatographic separation of benzo[a]pyrene-1,6/3,6 quinone benzo[a]pyrene-1,6/3,6 quinoneution and retention time.
Differences less than 6.8% between the predicted and the experimental values in terms of resolution and retention time indeed confirmed that the proposed approach is practical.
A Box-Behnken experimental design was used to build the mathematical models and then to choose the significant parameters for the optimisation by simultaneously taking both resolution and retention time as the responses.
Twenty experiments, taking the minimum resolution and retention time of the last eluted peak as the responses with three important factors, mobile phase composition, flow rate and column temperature, were used to design a mathematical model.
It was found that: (1) column and % acetonitrile affected significantly resolution and retention time, (2) column, % acetonitrile, column temperature, flow rate and time constant affected significantly the plate number of sennoside A, and (3) column and time constant affected significantly the tailing factor.
The influence of different concentrations of SDS (0.05 0.175 M) on the selectivity, resolution and retention times of the studied analytes was investigated.
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In case of a complex mixture with unknown number of components, number of peaks, sum of resolutions and retention time of ultimate peak were considered as output variables.
Instead of using an objective response function, combined models were built for elementary chromatographic criteria (retention factors, resolution and relative retention) of each solute or pair of solutes and, after their validation, the global separation was accomplished by means of Derringer's desirability functions.
Newly designed chromatographic response functions based on a combination of resolution RS and retention time of the last component eluted tRL were employed to evaluate the resolution with regard to quality and quantity.
An excellent feature of this approach is that, although fluorescence intensities decreased gradually, the resolution, efficiency, and retention time remained virtually unchanged (see Figure S2 in the SI for details).
Apparatus and protocol for characterizing online DNA and YOYO-1 interesolution refficiency efficiency, and retention time varying with number of runs; preparation of chip injector; capillary surface modification for capillary PCR; capillary PCR; incompatibility of YOYO-1 with PCR; and calculation of the three multiplex products length are provided as Supporting Information.
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