Phase-Separation Pathway Selection in PNIPAM through End-Group Ionisation and NaCl Salting-Out

Nile Red fluorescence revealed three distinct phase-separation pathways in heterotelechelic HS–PNIPAM–COOH: multistage, monotonic, and suppressed transitions. Aqueous HS–PNIPAM–COOH (Mn ≈ 6 kDa) was investigated over pH 3–10 and at NaCl concentrations from 0 to 1.0 M using fluorescence, turbidimetry, dynamic light scattering, and tempera-ture-controlled single-particle tracking. Multistage transitions, comprising an initial fluo-rescence rise, an intermediate plateau, and a second rise, occurred under weakly ionised, high-salt conditions. Intermediate conditions produced a monotonic fluorescence increase, whereas strongly ionised, weakly screened conditions suppressed phase separation. The multistage sample, citrate containing 1.0 M NaCl, formed stable, micrometre-scale PNIPAM-rich droplets close to their final size that showed no further growth across the flu-orescence plateau despite increasing turbidity. Growth resumed only above the plateau. By contrast, the representative monotonic samples (citrate without added NaCl and borate con-taining 1.0 M NaCl) showed continuous droplet growth during the temperature holds. Dur-ing cooling, multistage droplets remained nearly unchanged in size on cooling from 60 to 40 °C before undergoing delayed, rapid, collective dissolution at lower temperature, where-as monotonic droplets shrank progressively before later rapid dissolution. These results show that pH and NaCl concentration control both the phase-separation pathway and meso-globule persistence, producing different high-temperature states that rehydrate differently on cooling.