Extra column band broadening is a key concern when implementing chip-based microfluidics in a capillary LC system. Band broadening negatively influences the sensitivity for bioactivity detection, as the sensitivity is dependent on the height of the inhibitor peaks in the product trace(s). Both the design of the microreactors and the connections to the LC column and mass spectrometer are crucial. The band broadening was investigated by flow-injection of the inhibitor E-64 at various flow rates and injection volumes by calculating the peak width at half height (FWHM). Data were obtained by experiments using an autosampler connected to a UV detector in the absence and presence of the chip.
As expected, the lower flow rates and injection volumes resulted in broader peaks when using the microfluidic system. At an injection volume of 0.1 mL, 85% of the band broadening can be contributed to the microfluidic chip, independent of the flow rate. The reason is that the connections and channels of the chip increased the extra column volume and thus the sample dilution. At larger injection volumes (1 mL), the percentage of band broadening that can be contributed to the chip was less (60%). A reason for this difference could be that diffusion at the borders of the sample plug is relatively more problematic for smaller volumes.
The band broadening and the analyte dilution resulting from extracolumn band broadening were compared between the microfluidic chip system and the conventional macro-scale system. For a proper comparison, we calculated the analyte concentration at the peak maximum of the bioactive peaks (Cmax) from E-64 injections in both systems. It turned out that the dilution factor when comparing the concentration at peak maximum with the injected concentration was only 10% higher for the microfluidic chip system in comparison to the conventional macro-scale system.
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