The objective of this research was to develop efficient, non-invasive separation systems for various biological and non-biological substances. One of the major technological pushes in modern bioanalysis instrumentation development is the realization of efficient, miniaturized bioanalysis systems. In this work, three sizes of micromachined electrical field-flow fractionation (m-ElFFF) systems, with complementary on-column electrical and optical detection modalities were fabricated to achieve this objective. Field-flow fractionation (FFF) technology is capable of fractionating (or separating) a wide variety of materials and is capable of hundreds of consecutive analysis runs using a single system. A highly promising sub-technique, particularly for the analysis of biological / biochemical materials, is electrical field-flow fractionation (ElFFF). In this work, microfabrication technologies were used to fabricate m-ElFFF systems that have smaller system volumes, require smaller sample volumes and have shorter run times than their macro-scale counterparts. Direct, on-column detection within the miniaturized separation device improved the resolution, decreased the band broadening, lowered the plate height, and shortened the overall analysis time. Also, the information obtained from these detection systems can be used to elucidate information on the electrical and physical characteristics of a sample. Therefore, complimentary on-column detection systems, were designed, fabricated and characterized. Additionally, the data from the two detection systems was compared and a quantitative correlation was performed, enabling the independent use of each detection system.

The use of spectrofluorometers to examine nanomaterials is quite popular using either fluorescence or synchronous measurements. However, understanding how a material’s optical properties can influence spectral acquisition are of great importance to accurately characterize nanomaterials. This dissertation presents a series of computational and experimental studies aimed at enhancing the quantitative understanding of nanoparticle interactions with matter and photons. This allows for more reliable spectrofluorometer based acquisition of nanoparticle containing solutions. Chapter I presents a background overview of the works described in this dissertation. Correction of the gold nanoparticle (AuNP) inner filter effect (IFE) on fluorophore fluorescence using PEGylated AuNPs as an external reference method is demonstrated in Chapter II. The AuNP IFE is corrected to quantify tryptophan fluorescence for surface adsorbed proteins. We demonstrate that protein adsorption onto AuNPs will only induce ~ 20% tryptophan fluorescence reduction instead of the commonly assumed 100% reduction. Using water Raman intensities to determine the effective path lengths of a spectrofluorometer for correction of fluorophore fluorescence is discussed in Chapter III. Using Ni(NO3)2 and K2Cr2O7 as Raman IFE references, the excitation and emission path lengths are found to exhibit chromophore and fluorophore independence, however path lengths are spectrofluorometer dependent. Finally, ratiometric resonance synchronous spectroscopy (R2S2) is discussed in Chapter IV. Using a combination of UV-vis and R2S2 spectroscopy, the optical cross sections of a wide range of nanomaterials were determined. Also on-resonance fluorescence in solution is demonstrated for the first time. The nanoparticles discussed range from photon absorbers, scatterers, simultaneous photon absorbers and scatterers, all the way to simultaneous photon absorbers, scatterers, and emitters.