On-chip separation and sensing systems for hydrodynamic chromatography

The feasibility of on-chip analytical separations using planar hydrodynamic chromatography (HDC) in Pyrex-silicon and fused silica chips has been demonstrated. In order to sketch the analytical separations area in which the HDC chip has to operate, an introduction was given of important macro-scale separation techniques and their microfabricated counterparts. Furthermore, an impression of separation techniques that are enabled specifically by microtechnology was presented. One of these techniques is on-chip planar hydrodynamic chromatography. Other techniques are mainly aimed at DNA analysis using differences in transport of DNA through various constraining (microfabricated) geometries. Two types of HDC chips have been fabricated. The first type employs a Pyrex top wafer and a silicon bottom wafer, while the second type is fabricated using fused silica for both top and bottom wafers. Separation and injection channels were defined in the bottom wafer, whereas the top wafer provided through-holes for external connections. One particular part of the fabrication technology, fusion bonding of Pyrex to silicon and of fused silica to fused silica, was thoroughly investigated both theoretically and experimentally. Bonding of Pyrex to silicon induces thermal stresses, which cause a slight deformation of the channel geometry. This deforms the sample zones thus decreasing the device performance. The thermal deformation was reduced by using fused silica. This has the additional advantage of a good transparency down to the deep-UV. Fabrication of the fused silica HDC chip required additional processing development, especially aimed at Reactive Ion Etching of deeper structures. The HDC chip designs were made using a combination of computational fluid dynamics and analytical expressions. Investigation of the chromatographic and technological constraints lead to a first Pyrex-silicon HDC chip design, incorporating a 1 μm deep and 0.5 or 1 mm wide separation channel and a deeper (20 μm) integrated injection configuration. Apart from an enhanced injection structure, the fused silica HDC chip also incorporated an optical detection cell, located directly after the separation channel. This detection cell had roughly the same cross-sectional area as the separation channel, but was 30 μm deep, thereby giving a 30-fold increase in the path length for external optical detection. All characterization steps were performed optically. For the Pyrex-silicon chip fluorescence imaging directly inside the 1 μm deep separation channel was used. The correct operation of the injection structure for the Pyrex-silicon and fused silica chips was demonstrated by visualization of the concentration distribution of a fluorescent dye. Subsequently, the separation capabilities in both chip types were demonstrated by separation of various polystyrene nanoparticles. In the fused silica chip detection of the particles was performed using UV absorption detection through the integrated optical detection cell. In the Pyrex-silicon chip separation of large biomolecules was shown as well. Additionally, it was shown that relatively small molecules could be separated, not by using the hydrodynamic separation effect, but by interaction with the relatively large channel wall surface area (adsorption chromatography). The retention data for the particle separations showed that the influence of electrostatic and hydrodynamic particle-wall interactions on the particle retention behavior is not yet well understood and needs further research. The particle separations, detected using UV absorption through the detection cell, enabled a quantitative estimate of the peak broadening. Plate numbers ranging from 10.000 for small analytes to more than 100.000 for large fluorescent and non-fluorescent polystyrene nanoparticles were obtained. Qualitative comparison of (fluorescent) zone shapes showed that thermal deformation of the Pyrex-silicon separation channel, resulting in a higher dispersion, is less pronounced in narrower channels. Narrower channels however exhibit a relatively large influence of the side-walls on the total peak broadening, which leads to smaller plate numbers. Improvement of the separation resolution must therefore be aimed at reduction of the side-wall induced dispersion and of the thermal deformation. This could be realized by using wider channels, defined in fused silica. For integration with the Pyrex-silicon HDC chip a prototype stand-alone viscosity detector was developed using a technologically compatible fabrication process. The functionality of the differential detector was demonstrated by measuring the viscosity change caused by an ethanol plug in deionized water. A viscosity sensing resolution of Δηsp = 3*10-3 was estimated from those measurements. For the HDC chip a resolution of 10-4 is required. The difference can be explained by the extremely low volume flows and sample volumes required in on-chip HDC. Upscaling of the viscosity detector for higher volume flows theoretically improves the detection limits. Upscaling could thus create a functional viscosity detector with sample volumes that are much smaller than required for commercial viscodetectors. This could enable application of the detector for miniaturized (not necessarily micromachined) liquid chromatography systems for polymer analysis.