**2. Theoretical principles underlying macromolecule retention in Hydrophobic Interaction Chromatography**

### **2.1 Thermodynamics fundamentals**

Hydrophobicity can be defined, in general terms, as the repulsion between a non-polar molecule and a polar environment, such as that conferred by water, methanol, and other polar solvents. Two hydrophobic molecules (non-polar) located in a polar environment will show a trend to minimize the contact with the polar solvent. This is accomplished by coming in contact with each other thus minimizing the molecular surface exposed to the solvent. This phenomenon is known as "hydrophobic interaction". Hydrophobic interaction is the most common macromolecular interaction in biological systems. It is also the driving force of several biological and physicochemical processes, such as protein folding, antigen-antibody recognition, stabilization of enzyme-substrate complexes, among others.

From a thermodynamic point of view, the interaction between hydrophobic molecules is an entropy-driven process, based on the second law of Thermodynamics and considering that temperature (T) and pressure (P) remain constant during the process, in this case, the hydrophobic interaction between two biological molecules. Considering equation (1), when a non-polar molecule enters in contact with a polar solvent (usually water), an increase in the degree of order of the solvent molecules that surround the hydrophobic molecule is observed, producing a decrease in entropy (ΔS < 0). Given that enthalpy (ΔH) does not suffer a significant increase in this kind of processes (constant temperature) in comparison with TΔS, an overall positive change in the Gibbs energy (ΔG > 0) is produced. Hence, the dissolution of a non-polar molecule in a polar solvent does not occur spontaneously, since it is thermodynamically unfavorable.

$$
\Delta G = \Delta H - T\Delta S \tag{1}
$$

The thermodynamics situation changes when two or more non-polar molecules are located in a polar environment. In this case, the hydrophobic molecules spontaneously aggregate because of hydrophobic interaction, and in this way the hydrophobic surfaces of the macromolecules become hidden from the polar surrounding. Entropy increases (ΔS > 0) owing to a displacement of the highly structured solvent molecules surrounding the exposed surface of the hydrophobic molecules towards the solvent bulk consisting of less structured molecules. As a consequence, the Gibbs energy decreases (ΔG < 0), and therefore, hydrophobic interaction becomes a thermodynamically favorable process. In conclusion, the hydrophobic interaction between two or more non-polar molecules in a polar solvent solution is a spontaneous process governed by a change in entropy. Accordingly, hydrophobic interactions can be weakened by raising temperature or by modifying the solvent polarity through the addition of another solute.

### **2.2 Retention mechanisms in Hydrophobic Interaction Chromatography**

Macromolecule retention in HIC occurs due to hydrophobic interactions between the hydrophobic ligands immobilized on a stationary phase and the hydrophobic moieties on the macromolecule surface (Queiroz et al., 2001). There is a variety of stationary phases used in HIC, corresponding to organic polymers or silica. Their main characteristics are being chemically modifiable, highly porous, and of high moisturizing power. Among them, the most commonly used are polyacrylamide (BiogelPTM), cellulose (CellulafineTM), dextran (SephadexTM), agarose (SepharoseTM), and others. These supports are further modified by linking hydrophobic ligands that become a sort of "active group" that allows hydrophobic interaction with the macromolecule to be separated from a solution. The ligand is linked to the support through a spacer arm (usually glycidyl ether), so that there is no steric impediment for macromolecule-ligand interaction, and avoiding hydrophobic interaction between the ligands. Figure 1 depicts the retention of a protein to a HIC stationary phase.

solvent. This phenomenon is known as "hydrophobic interaction". Hydrophobic interaction is the most common macromolecular interaction in biological systems. It is also the driving force of several biological and physicochemical processes, such as protein folding, antigen-antibody recognition, stabilization of enzyme-substrate complexes,

From a thermodynamic point of view, the interaction between hydrophobic molecules is an entropy-driven process, based on the second law of Thermodynamics and considering that temperature (T) and pressure (P) remain constant during the process, in this case, the hydrophobic interaction between two biological molecules. Considering equation (1), when a non-polar molecule enters in contact with a polar solvent (usually water), an increase in the degree of order of the solvent molecules that surround the hydrophobic molecule is observed, producing a decrease in entropy (ΔS < 0). Given that enthalpy (ΔH) does not suffer a significant increase in this kind of processes (constant temperature) in comparison with TΔS, an overall positive change in the Gibbs energy (ΔG > 0) is produced. Hence, the dissolution of a non-polar molecule in a polar solvent does not occur spontaneously, since it

The thermodynamics situation changes when two or more non-polar molecules are located in a polar environment. In this case, the hydrophobic molecules spontaneously aggregate because of hydrophobic interaction, and in this way the hydrophobic surfaces of the macromolecules become hidden from the polar surrounding. Entropy increases (ΔS > 0) owing to a displacement of the highly structured solvent molecules surrounding the exposed surface of the hydrophobic molecules towards the solvent bulk consisting of less structured molecules. As a consequence, the Gibbs energy decreases (ΔG < 0), and therefore, hydrophobic interaction becomes a thermodynamically favorable process. In conclusion, the hydrophobic interaction between two or more non-polar molecules in a polar solvent solution is a spontaneous process governed by a change in entropy. Accordingly, hydrophobic interactions can be weakened by raising temperature or by modifying the

Δ =Δ − Δ *G H TS* (1)

among others.

stationary phase.

is thermodynamically unfavorable.

solvent polarity through the addition of another solute.

**2.2 Retention mechanisms in Hydrophobic Interaction Chromatography** 

Macromolecule retention in HIC occurs due to hydrophobic interactions between the hydrophobic ligands immobilized on a stationary phase and the hydrophobic moieties on the macromolecule surface (Queiroz et al., 2001). There is a variety of stationary phases used in HIC, corresponding to organic polymers or silica. Their main characteristics are being chemically modifiable, highly porous, and of high moisturizing power. Among them, the most commonly used are polyacrylamide (BiogelPTM), cellulose (CellulafineTM), dextran (SephadexTM), agarose (SepharoseTM), and others. These supports are further modified by linking hydrophobic ligands that become a sort of "active group" that allows hydrophobic interaction with the macromolecule to be separated from a solution. The ligand is linked to the support through a spacer arm (usually glycidyl ether), so that there is no steric impediment for macromolecule-ligand interaction, and avoiding hydrophobic interaction between the ligands. Figure 1 depicts the retention of a protein to a HIC

Fig. 1. Protein retention mechanism in HIC. (A) The basic structure of a HIC resin is depicted, and a protein is schematized highlighting the hydrophobic zones on the protein surface. (B) The protein gets in contact with the hydrophobic ligands of the resin, suffering a spatial reorientation. The hydrophobic ligands of the matrix interact with the exposed hydrophobic zones of the protein, and thus the protein is reversibly attached to the resin.

The most common hydrophobic ligands are alkyl or aryl groups of 4 to 10 carbons (Jennissen, 2000). The length of the carbon chain usually does not exceed 10 units in order to avoid self-folding. The nature of the hydrophobic ligand determines the performance of a HIC process. Figure 2 shows a scheme of stationary phases used in HIC and the chemical structure of the most commonly used alkyl and aryl groups, such as butyl (four carbons), octyl (eigth carbons) and phenyl (aromatic ring that promotes π-π interactions with the aromatic residues on a proteins surface). The hydrophobic interaction is directly proportional to the length of the alkyl chain. The most commonly used ligands in HIC resins are butyl, octyl and phenyl, in the following order in terms of relative interaction strength:

### Phenyl > Octyl > Butyl

In the HIC process, retention is reinforced by the presence of a neutral salt. When a neutral salt is added to a solution consisting of a polar solvent, i.e. water, and a non-polar macromolecule, such as a protein, a competition for the water molecules that hydrate the macromolecule is observed, being more favorable to the salt. As a consequence, high salt concentration will reduce the number of solvent molecules that surround the macromolecules, thus favoring the hydrophobic interaction between them. Furthermore, if such solution comes in contact with a HIC resin, the interaction between the macromolecule and the hydrophobic ligand on the resin surface will be promoted, resulting in the adsorption of the macromolecule to the HIC stationary phase. From a process point of view, it is essential to choose the right type of salt and a concentration that minimizes macromolecule precipitation due to solubility decrease in the presence of high salt concentration ("salting-out").

Fig. 2. Schematic representation of stationary phases used in HIC. Butyl is the shortest carbon chain used as HIC ligand and therefore the less hydrophobic one; octyl exhibits an intermediate hydrophobicity, and phenyl offers the strongest hydrophobic interaction.

The effect of different types of salt on macromolecule retention in HIC follows the Hofmeister (or lyotropic) series according to their positive influence in increasing the molal surface tension of water (Melander & Horvath, 1977). Besides, anions and cations exhibit cosmotropic or chaotropic properties. The salts at the beginning of the series are known as "cosmotropic" or "antichaotropic", since they promote hydrophobic interactions (as well as protein precipitation due to the "salting-out" effect) because of their water structuring ability. On the other hand, the salts at the end of the series, called "chaotropic", tend to randomize the structure of water and therefore they disfavor hydrophobic interactions. The salts ammonium sulfate and sodium chloride are most preferred in HIC.

Once the macromolecule of interest is attached to the stationary phase, it is necessary to detach it in order to recover it as a bio-product. Desorption is most commonly accomplished by reducing the ionic strength in the mobile phase, by building a decreasing gradient of salt concentration (Fausnaugh et al., 1984). In this stage, the hydrophobic interaction between the macromolecule and the ligand is weakened as salt concentration diminishes in the mobile phase. As a consequence, the macromolecule is desorbed when a specific salt concentration is reached. This salt concentration, or ionic strength, depends on the physicochemical properties of the macromolecule. In this way, HIC can be used to selectively detach different macromolecules in a solution, thus becoming a powerful separation process.

Protein retention in HIC has been interpreted in the light of the underlying thermodynamic phenomena, by considering the effect of salt. Melander et al. (1989) proposed a thermodynamic model that describes protein retention in terms of electrostatic and hydrophobic interactions. This model describes protein retention due to only electrostatic interactions (case of ion Exchange Chromatography), only hydrophobic interactions (case of HIC), and both types of interactions (case of a weakly hydrophobic support or a chromatographic support bearing both hydrophobic and charged ligands). Simplifications of this model have been used to develop methodologies to predict protein retention in HIC. This model is described below.

Fig. 2. Schematic representation of stationary phases used in HIC. Butyl is the shortest carbon chain used as HIC ligand and therefore the less hydrophobic one; octyl exhibits an intermediate hydrophobicity, and phenyl offers the strongest hydrophobic interaction.

salts ammonium sulfate and sodium chloride are most preferred in HIC.

separation process.

This model is described below.

The effect of different types of salt on macromolecule retention in HIC follows the Hofmeister (or lyotropic) series according to their positive influence in increasing the molal surface tension of water (Melander & Horvath, 1977). Besides, anions and cations exhibit cosmotropic or chaotropic properties. The salts at the beginning of the series are known as "cosmotropic" or "antichaotropic", since they promote hydrophobic interactions (as well as protein precipitation due to the "salting-out" effect) because of their water structuring ability. On the other hand, the salts at the end of the series, called "chaotropic", tend to randomize the structure of water and therefore they disfavor hydrophobic interactions. The

Once the macromolecule of interest is attached to the stationary phase, it is necessary to detach it in order to recover it as a bio-product. Desorption is most commonly accomplished by reducing the ionic strength in the mobile phase, by building a decreasing gradient of salt concentration (Fausnaugh et al., 1984). In this stage, the hydrophobic interaction between the macromolecule and the ligand is weakened as salt concentration diminishes in the mobile phase. As a consequence, the macromolecule is desorbed when a specific salt concentration is reached. This salt concentration, or ionic strength, depends on the physicochemical properties of the macromolecule. In this way, HIC can be used to selectively detach different macromolecules in a solution, thus becoming a powerful

Protein retention in HIC has been interpreted in the light of the underlying thermodynamic phenomena, by considering the effect of salt. Melander et al. (1989) proposed a thermodynamic model that describes protein retention in terms of electrostatic and hydrophobic interactions. This model describes protein retention due to only electrostatic interactions (case of ion Exchange Chromatography), only hydrophobic interactions (case of HIC), and both types of interactions (case of a weakly hydrophobic support or a chromatographic support bearing both hydrophobic and charged ligands). Simplifications of this model have been used to develop methodologies to predict protein retention in HIC.
