The standard free energy of hydrophobic interaction between alkyl chains has been measured in aqueous solutions of t-butanol, dioxan, dimethyl sulphoxide and dimethyl formamide.

The results make it clear that hydrophobic interaction (in the sense of a pair-wise interaction between hydrophobic groups otherwise surrounded by water) is not simply a partial reversal of solution of the non-polar molecules in water. Hydrophobic interaction should therefore be distinguished from phenomena, such as the formation of detergent micelles or phospholipid bilayers, in which non-polar molecules are completely removed from water. Only in the latter case is “reversal of solution” a correct description.

The experimental method was measurement of conductance of long chain alkyltrimethylammonium carboxylate solutions. Ion pair association constants, and hence free energies of ion pair formation were calculated from these data. Then by systematically varying the alkyl chain length it was possible to separate the free energy of hydrophobic interaction from the electrostatic interaction between the charged groups.

Moderate concentrations of t-butanol, dimethyl sulphoxide and dimethyl formamide strengthened hydrophobic interactions. All concentrations of dioxan weakened hydrophobic interactions. Maximum hydrophobic interaction occurred with those solute concentrations which maximised the three dimensional hydrogen bonded structure of water.

The results explain some of the odd effects of alcohols and other structure making solutes on the stability of proteins.