This is a copy of the slides presented at the meeting but not formally written up for the volume.

Nanoparticles (NPs) are showing great promise in their utility towards biomedical applications in areas such as drug delivery, diagnostics and image contrast enhancement. The standardization of physical characterization protocols for NPs is critical for their eventual approval and use in clinical settings, and for the development of reliable nanosize reference materials. Field-flow fractionation (FFF) is emerging as a powerful tool to obtain information on the composition, size, and molecular weight of fractionated NP solutions. FFF is classified into several sub-techniques based on the applied "field", with the most common and broadly applicable being asymmetric-flow (A-FFF). A-FFF separates constituents according to their hydrodynamic size, and can be coupled with various detectors, such as UV-Vis, multi-angle light scattering (MALS), differential refractive index (DRI), photon correlation spectroscopy (PCS), fluorescence, and, more recently, inductively coupled plasma-mass spectrometry (ICP-MS). Depending on the different detector systems employed, further information such as the number and distribution of ligands or drug molecules attached to a multifunctional nanomaterial and the frequency of dimer, trimer and higher order aggregates can be obtained.In the present work we have employed commercial A-FFF systems customized with various detectors, including MALS, PCS, DRI and UV-Vis, to establish fundamental protocols for the characterization of gold nanoparticles and their bio- or dendridic-conjugates. These protocols are being applied in the development of new gold-based nanosize reference materials intended for the cancer research community. We optimized the experimental conditions by controlling various parameters, such carrier composition, membrane material, and ratios of channel-to-cross flow rates. Also, samples were separated by collection mode from A-FFF, to coincide with the MALS signal, and subsequently investigated for purity and accuracy by further supportive analysis of off-line UV-Vis and PCS measurements. We report on the results of these studies and their implications for biomedical research.NCL is funded by NCI Contract N01-CO-12400.