A method of determining the diffusion coefficient of one or more components in a polydisperse sample, the method comprising the following steps in the following order: introducing an auxiliary fluid flow into a fractionation channel; introducing the polydisperse sample comprising one or more components into the fractionation channel; allowing the sample fluid and the auxiliary fluid to create a combined flow; fractionating the combined flow into two or more fractions by diffusive sizing; adding into each fraction solutes or solvents selected from: affinity probes or immunoprobes for labelling; ions or salts to perform pre-concentration; additives to reduce risk of sample adhesion to the wall of the channel of the microfluidic device; additives to modify the amount of the electro-osmotic flow within an electrophoresis separation channel; additives to enhance the quantum yield or lifetime of a fluorophore; separating two or more components from each fraction by creating a distribution of the components within a separation channel; processing the components in one or more of the fractions to separate and detect components added before or during the method, wherein said added components include: a blank or auxiliary fluid; any additive such as immunoprobes or affinity probes; ions or salts to perform pre-concentration; additives to modify the amount of the electro-osmotic flow within an electrophoresis separation channel; additives to enhance the quantum yield or lifetime of a fluorophore; detecting a characteristic of each of the two or more components in each fraction; and comparing the characteristic of each component in each fraction in order to determine the diffusion coefficient of each of the one or more components in the polydisperse sample introducing an auxiliary fluid flow into a fractionation channel; introducing the polydisperse sample comprising one or more components into the fractionation channel; allowing the sample fluid and the auxiliary fluid to create a combined flow; fractionating the combined flow into two or more fractions by diffusive sizing; adding into each fraction solutes or solvents selected from: affinity probes or immunoprobes for labelling; ions or salts to perform pre-concentration; additives to reduce risk of sample adhesion to the wall of the channel of the microfluidic device; additives to modify the amount of the electro-osmotic flow within an electrophoresis separation channel; additives to enhance the quantum yield or lifetime of a fluorophore; affinity probes or immunoprobes for labelling; ions or salts to perform pre-concentration; additives to reduce risk of sample adhesion to the wall of the channel of the microfluidic device; additives to modify the amount of the electro-osmotic flow within an electrophoresis separation channel; additives to enhance the quantum yield or lifetime of a fluorophore; separating two or more components from each fraction by creating a distribution of the components within a separation channel; processing the components in one or more of the fractions to separate and detect components added before or during the method, wherein said added components include: a blank or auxiliary fluid; any additive such as immunoprobes or affinity probes; ions or salts to perform pre-concentration; additives to modify the amount of the electro-osmotic flow within an electrophoresis separation channel; additives to enhance the quantum yield or lifetime of a fluorophore; a blank or auxiliary fluid; any additive such as immunoprobes or affinity probes; ions or salts to perform pre-concentration; additives to modify the amount of the electro-osmotic flow within an electrophoresis separation channel; additives to enhance the quantum yield or lifetime of a fluorophore; detecting a characteristic of each of the two or more components in each fraction; and comparing the characteristic of each component in each fraction in order to determine the diffusion coefficient of each of the one or more components in the polydisperse sample