| Two microfluidics devices for chemical analyses are fabricated and tested. In the first, bead-based strong cation exchange capillary electrochromatography (SCX CEC) of a series of poly-lysines is described, using laser-induced fluorescence detection. Conditions for efficient analyte separation are tested. The effect of buffer ionic strength and separation voltage on plate height (H) numbers is studied. Under optimal conditions, efficiencies equal to 196,000 plates/meter for dilysine, 400,000 plates/meter for trilysine and 238,000 plates/meter for tetralysine are obtained. A two-column CEC microsystem, using strong cation exchange followed by reverse-phase columns, is discussed: its use for two-column sequential analysis of a mixture of dyes and dilysine under laser-induced fluorescence detection is described. The second microfluidics device consists of a hybrid silicon-glass system for single- and multi-channel protein analysis by pressure-driven electrospray ionization mass spectrometry (ESI MS). Microchannels are defined in a glass substrate these are connected to silicon beds through a thin (< 20 mum) or thick (> 600 mum) polydimethylsiloxane (PDMS) layer. The silicon beds are octadecylsilylderivatized and therefore act as solid phase extraction (SPE) beds. For efficient elution of analytes off the silicon SPE beds in the multichannel device, uniform bed-to-bed porosity is shown to be a critical factor. Because the silicon SPE beds are micromachined to be porous using well-known lithographic techniques, all are uniform and of equal porosity. Microdevice fabrication is discussed including glass, silicon and PDMS processing. A novel PDMS processing technique utilizing reactive ion etching (RIE) is described. Single channel microdevices are used for on-chip protein sorption and on-bed trypsin digestion for cytochrome c, myoglobin and bovine serum albumin. In the multichannel microdevices built using the thin PDMS approach, defects in the PDMS layer prevent sequential elution off the silicon SPE beds. Laser-induced fluorescence microscopy shows sequential elution off the silicon SPE beds in the multichannel microdevice built using the thick PDMS. However, cytochrome c analysis under ESI MS detection showed that baseline-resolved peak elution is not possible due to the large dead volume introduced by the thick PDMS layer. Theoretical bandbroadening analysis describes conditions for resolved cytochrome c peak analysis by pressure-driven ESI MS. |
