Recently, a theoretical model was proposed to study the existence of pockets of acetonitrile (ACN) called clusters in a hydroorganic mixture. The proposal used ACN as an interaction organic modifier between D,L-dansyl amino acids and their binding site in human serum albumin at site II. This solute binding is governed by primary and secondary interactions. The primary interactions are under the dependence of the solute solvation by ACN clusters and electrostatic interactions. Following this first step, the solute engages strong short-range interactions with the residues of site II. Using a biochromatographic approach, the solute binding, i.e., the solute retention, was divided into two dielectric constant (epsilon) ranges. In the first range, epsilon > epsilon c (epsilon c is the critical dielectric constant); the primary and secondary nonstereoselective electrostatic interactions were the major contributions to the variation in the solute binding with the ACN fraction in the mixture. In the second range, epsilon < epsilon c, the solute retention variation with the ACN fraction was governed by its solvation by the ACN clusters and also by the secondary hydrophobic stereoselective interaction. The mathematical model developed provided the determination of the surface charge density of site II as well as the cluster number that solvates each solute.