We analyzed the kinetics of the reaction cycle by HPLC and 31 P-NMR and refitted our kinetic model to the obtained data (Fig. and Fig. ). From the kinetic model, we can derive that the droplets dissolved when the total concentration of phosphorylated species fell below 262 µM. We can also conclude that the droplets, after roughly 30 hours, can only comprise 3-pHis as a phosphorylated species because 1-pHis has already been hydrolyzed. Next, we tested how the chemical fuel can affect the macroscopic behavior of the active droplets using optical density at 500 nm as a readout. To produce sufficient droplet material for a reproducible increase in optical density, we added 2.5% PEG8000 as a crowding agent. We added various amounts of MAP as fuel. Above 10 mM of MAP, we found evidence of the first droplets. We then determined the time needed for the optical density to pass a threshold which we define as the lag time (Fig. and). With increasing amounts of fuel, the lag time decreased from 8.6 ± 1.17 hours with 10 mM MAP to 2.55 ± 0.13 hours with 50 mM of fuel. Notably, the decrease in the lag time tended to level off beyond 40 mM of fuel. With increasing fuel, the maximum turbidity reached, as a measure for the maximum amount of droplet material, increased too (Fig. and).