How Osmolyte and Denaturant Affect Water at the Air–Water Interface and in Bulk: A Heterodyne-Detected Vibrational Sum Frequency Generation (HD-VSFG) and Hydration Shell Spectroscopic Study

Preferential orientation and expulsion/accumulation of trimethylamine N-oxide (TMAO; a protecting osmolyte) and tert-butyl alcohol (TBA; a denaturant) have been investigated at the hydrophobic air–water interface by phase-sensitive heterodyne-detected vibrational sum frequency generation (HD-VSFG) spectroscopy. The imaginary χ(2)spectrum (Imχ(2); χ(2)is the second order electric susceptibility), which is directly obtainable from the HD-VSFG measurement, provides the accurate absorption characteristics of interfacial molecules, and the sign of Imχ(2)reveals the net orientation of these molecules at the interface. For the aqueous TMAO and TBA solutions, the Imχ(2)spectra in the CH-stretch region show a negative sign, which demonstrates that both TMAO and TBA orient in the same manner at the air–water interface, by pointing their methyls away from the aqueous phase (“methyl-up” orientation). Nevertheless, they affect the interfacial water quite differently: TMAO increases the H-bond strength and preferential H-down orientation of interfacial water, while the dangling OH remains almost unperturbed. TBA, on the other hand, does not affect the H-bond strength and preferential orientation of interfacial water, but reduces the propensity of the dangling OH at the air–water interface. The preferential orientation of TMAO and TBA and their distinct effect on the interfacial water have been correlated with their hydration characteristics in bulk water by retrieving the vibrational spectrum of water in their respective hydration shells, using Raman multivariate curve resolution (Raman-MCR) spectroscopy. The MCR-retrieved hydration water spectra clearly show that the water around TBA has strong water–water interaction (hydrophobic hydration) and that around TMAO has a hydrophobic hydration around the N-methyl ((CH3)3N+−) group and a hydrophilic hydration around the N-oxide group (strong H-bonding of water with the N-oxide group). The different hydration characteristic of the N-methyl and N-oxide groups orients the TMAO molecules as “methyl-up” at the air–water interface. Moreover, the strong hydration of the N-oxide group leads to a depletion of TMAO from the hydrophobic water surface, such that the preferentially oriented TMAO molecules are located beneath the topmost water layer at the air–water interface. As a result, the topmost water molecules are largely unaware of the presence of TMAO at the interface, even at very high bulk concentration of TMAO (5.0 mol dm–3). In the case of TBA, the hydrophobic hydration leads to an accumulation of TBA at the water surface, mainly affecting the topmost water molecules.