Response surface examination of the relationship between experimental conditions and product distribution in electrophoretically mediated microanalysis

This work presents the first known use of response surface methodology (RSM) in electrophoretically mediated microanalysis. This concept is demonstrated by examining the optimization of reaction conditions for the conversion of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced form by glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) in the conversion of glucose-6-phosphate to 6-phosphogluconate. Experimental factors including voltage, enzyme concentration, and mixing time of reaction at the applied voltage were selected at three levels and tested in a Box-Behnken response surface design. Upon migration in a capillary under CE conditions, plugs of substrate and enzyme are injected separately in buffer and allowed to react at variable conditions. Extent of reaction and product ratios were subsequently determined by CE. The model predicted results are shown to be in good agreement (7.1% discrepancy difference) with experimental data. The use of chemometric RSM provides a direct relationship between electrophoretic conditions and product distribution of microscale reactions using CE, thereby offering a new and versatile approach to optimizing enzymatic experimental conditions.