Introduction to Multimodal or Mixed-Mode Chromatography

Multimodal or mixed-mode protein chromatography is based on media supports that have been functionalized with ligands capable of multiple modes of interaction: ion exchange, hydroxyapatite, affinity, size exclusion, and hydrophobic interactions. The ability to combine these separation methods can enhance selectivity in a protein purification process. This section provides an overview of multimodal chromatography, discusses some general considerations for mixed-mode chromatography, and describes hydrophobic ion-exchange and mixed-mode media and ligands.

To find out whether multimodal chromatography is the most suitable approach for your application and to learn about other chromatographic methods that may be applicable for your purification needs, visit our Chromatography page.

Related Topics: Affinity Chromatography, Size Exclusion Chromatography, Ion Exchange Chromatography, Hydrophobic Interaction Chromatography, Low Pressure Chromatography Systems and Medium Pressure Chromatography Systems.

Page Contents
General Considerations

Unlike affinity chromatography where a specific site on the protein is targeted, with mixed-mode ligands there is no known specificity. Accordingly, screening mixed-mode media becomes a search for sites on the target protein that will provide useful affinity and selectivity.

Mixed-mode chromatography interactions are not independent of one another. For example, when using a mixed-mode ligand containing both hydrophobic and ionic elements, increasing ionic strength will disrupt ionic bonds, however, increasing salt concentration will promote hydrophobic interactions.

Mixed-mode media effectively combine complementary chromatography methods within a single media and can reduce the total number of column steps needed in a purification process. Because these mixed-mode elements are present in a single ligand, it contributes affinity-like binding and selectivity.

Binding and elution are controlled and optimized by the parameters relevant to each mode — salt for hydrophobic interactions and ionic strength for ionic interactions.

Mixed-Mode Media

There are a number of commercially available mixed-mode media combining different chromatographic elements:

  • Hydroxyapatite (CHT/CFT/Bio-Gel HT/HTP) — electrostatic and calcium coordination complexes
  • Hydrophobic ion exchange ligands
Hydroxyapatite Chromatography

Hydroxyapatite, Ca10(PO4)6(OH)2, is a form of calcium phosphate used in the chromatographic separation of biomolecules. Sets of five calcium doublets (C-sites) and pairs of –OH containing phosphate triplets (P-sites) are arranged in a repeating geometric pattern. Space-filling models and repeat structure from Raman spectroscopy have also been constructed. Hydroxyapatite has unique separation properties and unparalleled selectivity and resolution. It often separates proteins shown to be homogeneous by electrophoretic and other chromatographic techniques.

Applications of hydroxyapatite chromatography include the purification of:

  • Different subclasses of monoclonal and polyclonal antibodies
  • Antibodies that differ in light chain composition
  • Antibody fragments
  • Recombinant proteins
  • Viral particles
  • Vaccines
  • Isozymes
  • Supercoiled DNA from linear duplexes
  • Single-stranded from double-stranded DNA

CHT™ ceramic hydroxyapatite is a spherical, macroporous form of hydroxyapatite. It has been sintered at high temperatures to modify it from a nanocrystalline to a ceramic form. The ceramic material retains the unique separation properties of crystalline hydroxyapatite, and lot-to-lot control assures reproducibility in large-scale production columns. Unlike most other chromatography adsorbents, CHT is both the ligand and the support matrix. Separation protocols originally developed on crystalline hydroxyapatite can often be transferred directly to the ceramic material with only minor modifications.

Two types of CHT ceramic hydroxyapatite, Type I and Type II, are available in three particle sizes, 20, 40, and 80 μm. Although both types have elution characteristics similar to crystalline hydroxyapatite, there are also some important differences. CHT Type I has a higher protein binding capacity and better capacity for acidic proteins. CHT Type II has a lower protein binding capacity but has better resolution of nucleic acids and certain proteins. The Type II material also has a very low affinity for albumin and is especially suitable for the purification of many species and classes of immunoglobulins.

Protein binding to hydroxyapatite. A is a basic protein. B is an acidic protein. Double parentheses indicate repulsion. Dotted lines indicate ionic bonds. Triangular linkages indicate coordination bonds.

Hydrophobic Ion-Exchange Ligands

These ligands incorporate hydrophobic and ionic elements. For ion exchange binding and elution, parameters such as salt, pH, ionic strength, and buffer apply. With hydrophobic interactions, salt type, concentration, and additives apply. As mentioned above, it is important to remember that binding and elution factors are not independent and can work counter to one another. For example, increasing the ionic strength for ion-exchange elution will drive hydrophobic binding. Due to the dependency of these interactions, buffer conditions must be optimized for binding, washing, and elution to determine the optimal balance for a highly selective purification scheme.

Mixed-Mode Ligands
  • Hydrophobic, Anionic Ligand with Hydrogen Bonding — this ligand features hydrogen bonding, a quaternary amine, and a phenyl group
  • Mixed-Mode Cationic Ligand with Hydrophobic Binding — this ligand contains a secondary amine and is cationic over a wide pH range, therefore, it behaves as both a hydrophobic interaction resin and an anionic exchange media. At low ionic strength, it can bind acidic proteins as well as proteins with moderately high isoelectric points. However, hydrophobic interactions predominate, as binding capacity increases with temperature and salt
  • Mixed-Mode pH-Controllable Sorbents — this ligand contains a 4-mercaptoethylpyridine (MEP) ligand. The pyridine ring is uncharged at neutral and basic pH. As the pH decreases, the pyridine nitrogen becomes positively charged, turning the resin into a mixed-mode media. MEP becomes a pH-controlled mixed-mode ligand, a property that has been termed "hydrophobic charge induction chromatography" (Burton and Harding, 1998)

Example of mixed-mode ligands. A is a hydrophobic, anionic ligand with hydrogen bonding. B is a mixed-mode pH-controllable sorbent. C is a mixed-mode cationic ligand with hydrophobic binding.




Mixed Mode Chromatography Media Selection Guide
Media Type Packaging
Hydroxyapatite and Fluoroapatite
Type I
B, C, MPC ++++ ++++ ++++ Antibody purification (higher capacity than Type II); virus purification/removal; DNA purification/removal; aggregate and endotoxin removal; Fab purification
Type II
B, C ++++ ++++ ++++ Antibody purification; removal of albumin from feedstream; Fab purification
Types I and II
B, C ++++ ++++ ++++ Similar properties to CHT but exhibits greater stability in the lower pH range (5.5); suitable for Fab purification
Bio-Gel® HT B ++++ +++   Purification of proteins, nucleic acids and other biomolecules; crystalline hydroxyapatite not as mechanically stable as CHT (ceramic hydroxyapatite)
Bio-Gel® HTP B ++++ +++   Similar to Bio-Gel HT but in powder form
DNA grade Bio-Gel® HTP B ++++ +++   Similar to Bio-Gel HTP with smaller particle size; selectivity for dsDNA; separation of ss- and dsDNA

* B, bottle; C, cartridge (1 ml or 5 ml); GC, gravity column; SC, spin column; HPLC, high-pressure column; MPC, medium-pressure column.

** +, low suitability; ++, moderate suitability; +++, suitable; ++++, high suitability.


Burton SC and Harding DR (1998). Hydrophobic charge induction chromatography: Salt independent protein adsorption and facile elution with aqueous buffers. J Chromatogr A 814, 71–81.


Number Description Options
Protein and Peptide Purification Applications, Rev B
Purification of Horse IgG T Using Macro-Prep DEAE and CHT Ceramic Hydroxyapatite Type I Supports, Rev C
Purification of Transgenic Antibody From Corn Seed Using UNOsphere S and CHT Ceramic Hydroxyapatite Supports, Rev A
Evaluation of Different Approaches for the Chromatographic Purification of Monoclonal Antibodies
Chimeric IgG Monoclonal Antibody Purification: A Comparative Study Using CHT Ceramic Hydroxyapatite and CFT Ceramic Fluoroapatite Chromatographic Media, Rev A