Contact conductivity detection in poly(methyl methacrylate)-based microfluidic devices for analysis of mono- and polyanionic molecules

An on-column contact conductivity detector was developed for the analysis of various mono- and polyanionic compounds separated by electrophoresis chips fabricated in poly(methyl methacrylate) (PMMA) using hot embossing techniques from Ni electroforms. The detector consisted of a pair of Pt wires (127 microm diameter) with an end-to-end spacing of approximately 20 microm and situated within the fluidic channel. The waveform applied to the electrode pair was a bipolar pulse with a frequency of 5.0 kHz and was used to reduce the charging current from measurement so that the current recorded at the end of one pulse is more representative of the solution conductivity. Using the detector, separations of amino acids, peptides, proteins, and oligonucleotides were demonstrated. For the amino acids and peptides, free-solution zone electrophoresis was performed. A calibration plot for the amino acid alanine was found to be linear from approximately 10 to 100 nM in a carrier electrolyte consisting of 10 mM triethylamonium acetate. The concentration detection limit was found to be 8.0 nM, with the corresponding mass detection limit equal to 3.4 amol (injection volume = 425 pL). The protein separations with conductivity detection were performed using MEKC, in which the carrier electrolyte contained the anionic surfactant sodium dodecyl sulfate (SDS) above its cmc. Near baseline resolution was achieved in the PMMA microchip for a solution containing 8 different proteins. In the case of the DNA fragments, capillary electrochromatography was used with a C18-modified PMMA chip and a carrier electrolyte containing an ion-pairing agent.