Separation of equivalently sized polystyrene, n=1.59, poly(methylmethacrylate), n=1.49, and silica, n=1.43, beads has been accomplished using optical chromatography. The optical separations were performed using a glass flowcell that permits the optical trapping laser to be lightly focused into the fluid pathway against the fluid flow. Separation occurs due to the balance of fluid and optical forces; particles come to rest when the force due to the fluid flow equals the radiation pressure force. The ability to optically separate particles based upon their refractive index opens avenues for the characterization of colloidal samples based upon chemical characteristics, in addition to size. © 2003 American Institute of Physics.