How Does CHT Ceramic Hydroxyapatite
Media Work?
™

CHT Chemical Interactions
CHT Structure
Each molecule of CHT consists of:
5 positively charged calcium pairs (C-sites)
2 hydroxyl residues
2 phosphate triplets (P-sites), each with
6 negatively charged oxygen atoms

Ca+

■■

Ca+

■■

OH

■■

CHT

CHT Ceramic Hydroxyapatite, Ca10(PO4)6(OH)2, is a mixed-mode chromatography media
used for the purification of monoclonal and polyclonal antibodies, antibody fragments,enzymes, nucleic acids, and membrane proteins. It is formed from the chemicalcombination of calcium and phosphate salts. Biomolecules can interact with CHT
through calcium affinity interactions and/or cation exchange interactions. This wall
chart highlights the different ways in which these chemical interactions can occur.

PPPOOOOOO-CHT Chemical Interaction Mantra■■

Carboxyl groups on biomolecules are attracted to C-sites and repelled 		by P-sites on CHT■■

Amino groups on biomolecules are attracted to P-sites and repelledby C-sites on CHTCHT Metal Affinity Interactions
ElutionBinding
Interaction of calcium groups on CHT with carboxyl groups onproteins.

Interaction of calcium groups on CHT with phosphoryl groups onnucleic acids.

Feed loading

Desorption from metal affinity sites due to increasing phosphate gradient.
Elution buffer

Elution buffer

Feed loading

DNA

DNA
Ca+Ca+-OOC-OOC

Stationary phase

Ca+

Protein

Ca+

OH

CHTOH

P

OO-

P

OO-

P

OO-

BufferUnbound biomoleculesin flowthrough-OP-O-O
P-O-O
P-O-O
P-O

P

OO-

P

OO-

P

OO-

OOOP
OOP O
OP
OP-OOC-OOC

Ca+Ca+Protein

OHOH

P

OO-

P

OO-

P

OO-

P

OO-

P

OO-

POO-

BufferUnbound biomoleculesin flowthroughCHT typically interacts with acidic proteins, antibodies, nucleic acids, endotoxins, and enveloped viruses through metal
affinity interactions.

CHT Cation Exchange Interactions-OP-O-O
P-O-O
P-O-O
P-O

Ca+Ca+

Moderate phosphateconcentrations forprotein elutionOOOPOOP OOPOP

High phosphateconcentrations fornucleic acid elution

Phosphate gradients are typically used to elute biomolecules bound to CHT by metal affinity interaction. However, NaCl also
affects their retention time, indicating a minor contribution by cation exchange.1

CHT Mixed-Mode Interactions

Binding

ElutionBinding

ElutionInteraction of phosphate groups on CHT with amino groups onproteins.

Desorption from cation exchange sites due to increasing salt
gradients or increasing pH.

Binding of proteins to CHT through a combination of affinity
and cation exchange interactions.

Desorption from metal affinity and cation exchange sites dueto increasing NaCl concentrations in the presence of low levels(5 –25 mM) of phosphates.2

Elution buffer

Feed loading
Ca+

Ca+

Ca+

Ca+

Ca+OHOHOH

CHT

Stationary phase

Ca+POO-POO-

P

OO-

+H3N

Protein

+H3N

PP

OO-

P

OO-

BufferUnbound biomoleculesin flowthroughOO-+H3N+H3N

Elution buffer

Feed loading

Ca+-OOC
-OOC

-OOC
-OOC

Ca+Protein

ProteinOH

N+H 3Protein

POO-

POO-

P

Elution of protein

CHT typically interacts with basic proteins and antibodies through cation exchange interactions. Salt or pH gradients are usuallyused to elute these bound biomolecules.

OO-

N+H 3

N+H 3

BufferUnbound biomoleculesin flowthrough

P

OO-

P

OO-

POO-

N+H 3

Elution of protein

CHT typically interacts with monoclonal antibodies through mixed-mode interactions. A combination of phosphate and NaClis usually required to elute these bound biomolecules.

CHT metal affinity interactions are 15 to 60 times stronger than ionic interactions and are not affected by increasing ionic strength
when using typical elution ions.

Tips and Tricks
Load Preparation: Initial load should be free of agents such as citrate or EDTA that could degrade CHT via chelation.
Purification Process: Keep pH >6.5 during purification. For increased CHT stability, phosphate levels should be at least 5 mM in process solutions. Use hydrated buffer salts.
Do not use anhydrous sodium phosphate or dodecahydrates. These two salt types can cause irreproducible results.
12NaCl at 1.0 M typically reduces retention time by approximately 10% in the presence of phosphate gradients, indicating a minor contribution by cation exchange.
Unless a minimal concentration of phosphate is present, most IgGs remain bound to CHT even in saturated NaCl. Retention on CHT is progressively reduced with increased phosphate concentration.

Visit bio-rad.com/web/CHTwallchart to get detailed information about CHT Ceramic Hydroxyapatiteand bio-rad.com/web/ProcessApplications to learn about its various applications.

Bulletin 6902 Ver A 16-11800317

How Does CHT Ceramic Hydroxyapatite
Media Work?
™

How Does CHT

Ceramic Hydroxyapatite

Media Work?

™