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

Bio-Scale Ceramic

Hydroxyapatite,

Type I Columns

Instruction Manual

Catalog Numbers

751-0021, 751-0023,

751-0025, 751-0027

For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)

Table of Contents

Page

Section 1 Characteristics of the Bio-Scale CHT2, CHT5, CHT10, CHT20

Ceramic Hydroxyapatite, Type I Columns...............................................1

1.1 Introduction ................................................................................................................1

1.2 The CHT-I Separation Medium.................................................................................1

1.3 Connection to the BioLogic, FPLC®and HPLC Systems ........................................1

Section 2 Use of the Bio-Scale CHT-I Columns.........................................................2

2.1 Preparation for Initial Use..........................................................................................2

2.2 Sample Preparation ....................................................................................................3

2.3 Elution Conditions......................................................................................................3

2.4 Use of Detergents.......................................................................................................4

2.5 Effect of Calcium Chloride........................................................................................4

2.6 Chromatography Pre-Runs.........................................................................................4

Section 3 Care of the Bio-Scale CHT-I Columns ......................................................5

3.1 Column Hygiene ........................................................................................................5

3.2 Bed Height Adjustment..............................................................................................5

3.3 Frit Removal...............................................................................................................5

3.4 Top-Off Resin.............................................................................................................5

3.5 Storage Conditions.....................................................................................................5

3.6 References ..................................................................................................................8

Section 4 Product Information....................................................................................8

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-chromatograph­er 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 chro­matography. 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 (Ca2+ions at the
positively-charged centers and PO

4

3-

ions at the negatively-charged centers) results in essen­tially a mixed-mode ion exchange separation. Separation of biomolecules using hydroxyap­atite 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 regen­eration. 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 connec­tion to HPLC systems, 10-32 fingertight fittings and M6fittings for FPLC systems.

Instructions for assembling the fittings are included with the column.

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2

Table 1. Column Characteristics CHT2-I

CHT2-I CHT5-I CHT10-I CHT20-I

Column volume (ml) 2 5 10 20
Recommended max.

protein loading (mg) 20 50 100 200
Recommended flow

arates (ml/min) 0.5 to 3.0 0.5 to 5.0 0.5 to 7.0 0.5 to 10.0
Dynamic protein binding

capacity (mg Lysozyme)/
column 30 75 150 300

Static DNA binding
capacity (mg calf thymus
DNA) 0.20 0.5 1.0 2.0

Average particle size (µm) 10 ± 3.0 10 ± 3.0 10 ±3.0 10 ± 3.0
Column dimensions (mm) 7 x 52 10 x 64 12 x 88 15 x 113
Maximum operating

pressure (psi/bar) 1,000/67 750/50 600/40 500/34

Note: Bio-Rad Laboratories does not recommend nor warranty the use of the Bio-Scale
columns with solvent delivery systems containing stainless steel parts and used with corrosive
eluents containing e.g. halide salts. We recommend the use of inert, biocompatible (ceramic,
PEEK, titanium) solvent delivery systems for maximum column life and recovery of sample
biological activity.

Section 2
Use of the Bio-Scale CHT-I Columns

2.1 Preparation for Initial Use

The columns are supplied in a storage solution of 20% ethanol in 5 mM phosphate buffer,
pH 6.8. Prior to initial use and after extended storage periods, each column should be condi­tioned as described below (steps 1-4). Always use HPLC grade reagents, and filter and degas
buffers). During this operation do not exceed more than 25% of the recommended maximum
flow rates (see Table 1).

1. Wash with 5 column volumes of water.

2. Wash with 5 column volumes of low ionic strength equilibration buffer. Typically 5-10

mM sodium phosphate pH 6.8

3. Wash with 5 column volumes of high ionic strength limit buffer. Typically 500 mM sodium

phosphate pH 6.8.

4. Wash with 5 column volumes of low ionic strength equilibration buffer.

The column may now be further equilibrated in the starting buffer at the desired flow
rate.

Always use buffer components of the highest available purity as UV absorbing impurities
may cause baseline disturbances that interfere with the detection of protein peaks.

2.2 Sample Preparation

Proper adjustment of the sample pH and ionic strength is critical for consistent and repro­ducible chromatography. For best results, the sample should be exchanged into the start buffer