In both steps 2 and 3, the diffusion through chloroform is essential. We thus determined if the transport of molecules is limited by diffusion within the chloroform by testing different stirring speeds, interface cross-sections, and lengths of the chloroform phase. First, we tested the influence of the stirring speed. We found that increasing the stirring from 1000 to 1600 rpm does not significantly influence the transport efficiency (Fig. , Fig. ), indicating the chloroform phase is well mixed. It also indicates that hydrolysis in the chloroform does not play a major role, i.e., a faster transfer from sender to receiver by faster mixing did not result in the loss of fewer molecules, affecting the efficiency. We found that increasing the interface area from 79 mm 2 to 113 mm 2 did not significantly influence the transport efficiency (Fig. , Fig. ). Furthermore, we found that increasing the length of the chloroform phase from 25 mm to 35 mm does not significantly influence the transport efficiency (Fig. , Fig. ), corroborating our finding that the chloroform phase is well mixed. To further understand the details and efficiencies of the transport in each step, we devised a simple 1D model that describes the timedependent transport of Cbz-D across the hydrophobic barrier in response to the fuel (see Supporting Notes). The model considers the fuel-driven activation and spontaneous hydrolysis of activated molecules and the partitioning of the activated molecules inside the hydrophobic domain (chloroform). It also describes the diffusion in the sender and receiver phases and the transfer across the senderchloroform and chloroform-receiver interfaces. Notably, when we applied least-squares fitting for the diffusion constants, the rate of transfer across the interfaces, and the excess chemical-potential differences of Cbz-D and its anhydride, the model predicted the experimental data on the evolution of transporter molecules for various initial fuel concentrations (Fig. ). Interestingly, the transport efficiency increases drastically when the fuel concentration goes to zero, which is caused by passive transport, i.e., the diffusion of nonactivated Cbz-D molecules through the chloroform phase, which does not require EDC.