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

Nile Red fluorescence reveals three distinct phase-separation pathways in heterotelechelic HS–PNIPAM–COOH: multistage, monotonic, and suppressed transitions. Aqueous HS–PNIPAM–COOH (Mn ≈ 6 kDa) is investigated over pH 3–10 and at NaCl concentrations from 0 to 1.0 M using fluorescence, turbidimetry, dynamic light scattering, and temperature-controlled single-particle tracking. Multistage transitions, comprising an initial fluorescence rise, an intermediate plateau, and a second rise, occur under weakly ionised, high-salt conditions. Intermediate conditions produce a monotonic fluorescence increase, whereas strongly ionised, weakly screened conditions suppress detectable phase separation. The multistage sample, citrate buffer containing 1.0 M NaCl, forms stable, micrometre-scale PNIPAM-rich droplets with no further growth across the fluorescence plateau despite increasing turbidity. Growth resumes only above the plateau. By contrast, the representative monotonic samples (citrate buffer without added NaCl and borate buffer containing 1.0 M NaCl) show continuous droplet growth during the temperature holds. On cooling from 60 to 40 °C, multistage droplets remain nearly unchanged in size before undergoing delayed, rapid, collective dissolution at lower temperature, whereas monotonic droplets shrink progressively before later rapid dissolution. These results show that pH and NaCl concentration control both the phase-separation pathway and mesoglobule persistence, producing different high-temperature states that rehydrate differently on cooling.