Hydrophobic Interaction Chromatography

Performance of a HIC medium (selectivity, resolution and binding capacity) is influenced by many parameters:

• Protein properties
• Ligand type
• Degree of ligand substitution (ligand density)
• Concentration and type of salt used during sample application
• Presence of detergents

and to a lesser extent::

• Temperature
• pH
• Type of matrix

Dynamic binding capacity of a HIC medium depends on factors such as protein properties, salt type and concentration, flow rate, temperature, and pH, but also on the properties of the medium.

Binding capacity of HIC media increases with increased degree of ligand substitution. Simultaneously, the strength of the interaction increases, which might lead to difficulties in elution of bound components.

Selecting a medium substituted with the same ligand, but at a lower degree of ligand substitution can solve the problem.

Selectivity is the most important parameter when choosing a HIC medium. Different ligands and differences in ligand density influence selectivity of a HIC medium:

Resolution is influenced by factors such as binding and elution conditions, column packing, and flow rate, but also by the properties of the base matrix. A small bead size will give a higher resolution, while a larger bead size allows higher flow rates.

Choose medium according to the purpose of the separation: capture, intermediate purification, or polishing. The choice of matrix will also often dependent of the availability of a medium with the correct selectivity.

Sepharose Fast Flow media are based on a matrix of particles (mean size 90 µm) made from 4% or 6% agarose that is highly cross-linked for chemical and physical stability. The matrices are substituted with hydrophobic ligands (phenyl, butyl or octyl) coupled via a chemically stable O-ether or S-ether linkage. Two different degrees of substitution of the phenyl ligand provide a further chance to optimize binding capacity and elution conditions.

Sepharose High Performance are high-resolution, high-capacity HIC media based on highly cross-linked, 34 μm agarose beads modified with phenyl or butyl groups via uncharged, chemically stable ether linkages. These media are truly hydrophobic, displaying a minimum of ionic interactions. In addition, the media tolerate detergents, polar organic solvents, and chaotropic agents, as well as agents for cleaning in place and sanitization.

SOURCE media are based on a hydrophilic matrix made from monodispersed, rigid, polystyrene/divinyl benzene and substituted with hydrophobic ligands: phenyl, ether, or isopropyl. The media demonstrate extreme chemical and physical stability. The small particle sizes allow fast binding and dissociation to facilitate high resolution while the uniformity and stability of the particles ensures high flow rates at low back pressure.