Bio-Rad Bio-Scale™ CHT™ Type I Columns User Manual

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Section 1
Characteristics of the Bio-Scale CHT2, CHT5,
CHT10, CHT20 Ceramic Hydroxyapatite, Type I
Columns

1.1 Introduction

Bio-Scale prepacked hydroxyapatite columns meet the needs of the bio-chromatograph-

er for rapid and reproducible high resolution separations of biomolecules including proteins,
peptides and polynucleotides. The availability of four column sizes (bed volumes of 2, 5,
10, and 20 ml) provides unrivaled flexibility for the economical and predictable scale up
of separation and purification protocols without sacrificing resolution due to overloading.

1.2 The CHT-I Separation Medium

Each column contains a newly developed, chemically pure form of hydroxyapatite. The

spherical, ceramic nature of the CHT-I support overcomes the physical and chemical instability
that limited the use of traditional crystalline forms of hydroxyapatite for high resolution chro-
matography. The 10 µm particle size and narrow particle size distribution produce excellent
resolution of biomolecules at high flow rates and with very low back-pressures.

The CHT-I support demonstrates high affinity for basic proteins of relatively high pI and

lower affinity for proteins of relatively low pI. The hydroxyapatite structure (Ca

ions at the

positively-charged centers and PO

ions at the negatively-charged centers) results in essen-

tially a mixed-mode ion exchange separation. Separation of biomolecules using hydroxyap-
atite is a very useful complement to other separation techniques such as ion-exchange and
hydrophobic interaction chromatography particularly in the later stages of a purification
scheme. It will often resolve components that other techniques fail to separate. The support
binds proteins under mild conditions using neutral phosphate buffer systems. Selective elution
is accomplished with higher concentrations of phosphate and/or a pH gradient. Recovery of
protein/enzymatic activity is normally high due to the gentle elution conditions employed.

Stability of the CHT-I Support

The columns are stable over the pH range 5.5-14, allowing for easy cleaning and regen-

eration. The support is compatible with aqueous solutions of 6 M guanidine-HCl and 8 M
urea. Detergents such as Triton

(reduced form), CHAPS, CTAB and 1% SDS and organic

solvents such as methanol, ethanol and acetonitrile may also be used.

Buffers containing calcium chelators such as EDTA and EGTA should be avoided. If

chelating agents must be present in the buffers, the addition of some calcium chloride may be
necessary.

1.3 Connection to FPLC and HPLC Systems

Each Bio-Scale column is supplied with 0.02 inch ID (1/16 inch OD) Tefzel tubing,

1/4x28 flangeless fittings and end caps for connection to the BioLogic system. For connec-
tion to HPLC systems, 10-32 fingertight fittings and M6fittings for FPLC systems.

Instructions for assembling the fittings are included with the column.