Purpose. Conversion of animal collagen to commercial gelatin yields a heterogeneous proteinaceous preparation with a broad range of molecular weights. By characterizing the time-dependant effect of temperature, pH and desolvating agent on the molecular weight profiles (MWP), a process for precipitation and separation of gelatin nanoparticles was optimized. Methods. Aqueous B225 gelatin (Sigma) solutions (0.2% w/v) were incubated under various conditions of time, temperature, pH and ethanol concentration, before analysis by size exclusion HPLC. The HPLC chromatograms were divided into molecular weight fractions. Nanoparticles were prepared by dissolving gelatin in water, adjusting to pH 7 and diluting with anhydrous ethanol to a final concentration of 0.2% w/w gelatin in 70% w/w ethanol. The system was further diluted 1:30 in 70% w/w ethanol and stored at 37°C while reacting with 1% w/w glutaraldehyde for 2 hours. Excess glutaraldehyde was neutralized with sodium metabisulfite and unreacted gelatin and ethanol removed by ultrafiltration. The particles were sized using a Malvern Zetasizer and by scanning electron microscopy. Results. The system denatured when the temperature was increased above 37°C, while renaturation was optimized over a temperature range of 7°C to 20°C, at a pH of 5.0, equivalent to the isoelectric point (IEP). The MWP remained unchanged at a temperature of 37°C over the pH range 5 to 7. The higher molecular weight fractions (>700 kDa) were observed to precipitate at lower percentage ethanol concentrations than the lower molecular weight materials, with maximum precipitation occurring close to the IEP. The nanoparticles obtained under these conditions had a mean size of 234 nm (polydispersity 0.068). Conclusions. The MWP of gelatin was critically dependent on the pH and temperature of the solution. This affected the phase behavior of gelatin in hydroalcoholic solutions which, in turn, influenced nanoparticle formation.