**3. Hydrophobic Interaction Chromatography process**

The HIC process consists of injecting a macromolecule solution in a column packed with a stationary phase specifically designed to promote hydrophobic interaction with macromolecules such as proteins (solute). Usually retention is accomplished under high salt concentration conditions. Elution is achieved by decreasing the ionic strength in the mobile phase, building a decreasing salt gradient. At a microscopic level, the macromolecule enters in contact with the hydrophobic ligands at the pores surface of the resin, suffering a spatial reorientation. The hydrophobic ligands of the stationary phase interact with the hydrophobic zones of the macromolecule exposed to the solvent (usually aqueous solution), and thus the protein is reversibly attached to the resin.

Figure 4 shows a schematic representation of a HIC process. Here, A and B represent the vessels that contain the buffers used to manage the chemical environment in order to promote adsorption and desorption of the macromolecules present in the sample. The solution in A corresponds to a buffer with a low concentration of a neutral salt (usually 0.1 M), aiming to stabilize the macromolecular three-dimensional structure. The solution in B corresponds to buffer "A" added with a high salt concentration (usually higher than 1 M). Adsorption is promoted by using buffer "B", while desorption is induced by mixing both A and B forming a decreasing gradient salt concentration.

Fig. 4. Schematic representation of the HIC process. The HIC process consists of injecting a protein sample in a hydrophobic column under high salt concentration conditions such that hydrophobic interaction between the protein and the resin is promoted. Elution is achieved by decreasing the ionic strength in the mobile phase, building a decreasing salt gradient. In a microscopic level, the hydrophobic patches on the protein surface interact with the hydrophobic ligands of the resin, being reversibly attached to it. The protein concentration in the outlet is recorded as a function of time, and then a chromatogram is obtained.

The macromolecule concentration in the outlet solution is continuously determined through absorbance at 280 nm, and finally the elution curve or "chromatogram" is obtained. The chromatographic behavior in HIC can be characterized by several parameters, including the elution curve (most commonly by using the theoretical plate theory), the retention time or volume, or other parameters based on the preceding ones. To predict the behavior of proteins in HIC, the preferred parameter is the "Dimensionless Retention Time" (DRT), given by equation (9), where tR is the time corresponding to the peak maximum, t0 is the time at the beginning of the elution gradient, and tf the time at the end of the gradient. In

solution in A corresponds to a buffer with a low concentration of a neutral salt (usually 0.1 M), aiming to stabilize the macromolecular three-dimensional structure. The solution in B corresponds to buffer "A" added with a high salt concentration (usually higher than 1 M). Adsorption is promoted by using buffer "B", while desorption is induced by mixing both A

Fig. 4. Schematic representation of the HIC process. The HIC process consists of injecting a protein sample in a hydrophobic column under high salt concentration conditions such that hydrophobic interaction between the protein and the resin is promoted. Elution is achieved by decreasing the ionic strength in the mobile phase, building a decreasing salt gradient. In a microscopic level, the hydrophobic patches on the protein surface interact with the hydrophobic ligands of the resin, being reversibly attached to it. The protein concentration in the outlet is recorded as a function of time, and then a chromatogram is obtained.

The macromolecule concentration in the outlet solution is continuously determined through absorbance at 280 nm, and finally the elution curve or "chromatogram" is obtained. The chromatographic behavior in HIC can be characterized by several parameters, including the elution curve (most commonly by using the theoretical plate theory), the retention time or volume, or other parameters based on the preceding ones. To predict the behavior of proteins in HIC, the preferred parameter is the "Dimensionless Retention Time" (DRT), given by equation (9), where tR is the time corresponding to the peak maximum, t0 is the time at the beginning of the elution gradient, and tf the time at the end of the gradient. In

and B forming a decreasing gradient salt concentration.

HIC, the exploited property is hydrophobicity (Eriksson, 1998), and accordingly retention time is highly influenced by this property. Therefore, knowing macromolecule hydrophobicity allows predicting its behavior in HIC. Currently there is no universally agreed definition of protein hydrophobicity, but there is consensus in that it is determined by the hydrophobic contribution of the amino acids that compose the protein (Tanford, 1962).

$$DRT = \frac{t\_R - t\_0}{t\_f - t\_0} \tag{9}$$

On the other hand, protein retention in HIC is significantly affected by the operating conditions, which influence the resolution and selectivity of purification processes that include a HIC step (Ladiwala et al., 2006). From a process point of view, it is essential to count on methodologies and mathematical models to describe and to predict a protein behavior in HIC, ideally under varying operating conditions. Many efforts have been carried out to develop theories to explain this behavior based on protein properties, mainly protein hydrophobicity. At this point, controversial approaches have been proposed to theoretically estimate or experimentally determine protein hydrophobicity. These approaches include different amino acid hydrophobicity scales as well as diverse methodologies to perform calculations that use some scale to describe and predict protein retention time in HIC.
