In this paper, we report a range of factors which affect the retention of colloidal particles in thermal field-flow fractionation (ThFFF). These results are observed among different sizes of polystyrene (PS) latex particles suspended in both aqueous and nonaqueous liquid carriers and very low density lipoproteins in a phosphate buffer. These factors include particle size and chemical composition, field strength, cold-wall temperature of the channel and the nature of the suspension medium. These results show that ThFFF can be used to fractionate colloidal particles according to size and that for an unknown colloidal sample material, a calibration curve must be obtained using particles of similar composition. This is necessary because the degree of retention of the colloidal material is dependent on chemical composition of sample material as evidenced by the chemical composition study. The potential of using ThFFF for physico-chemical characterization of colloidal material is illustrated through the evaluation of thermal diffusion coefficient of PS particles as a function of size, cold-wall temperature, and carrier solution composition. The tunabiliy of the extent of retention of the colloidal particles in a ThFFF channel is illustrated by results of the influence of field strength on retention.