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.
Table of Contents Page Section 1 Characteristics of the Bio-Scale CHT2, CHT5, CHT10, CHT20 Ceramic Hydroxyapatite, Type I Columns ...............................................1 1.1 1.2 1.3 Introduction ................................................................................................................1 The CHT-I Separation Medium.................................................................................1 Connection to the BioLogic, FPLC® and HPLC Systems .............................
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.
Table 1. Column Characteristics CHT2-I Column volume (ml) Recommended max. protein loading (mg) Recommended flow arates (ml/min) Dynamic protein binding capacity (mg Lysozyme)/ column Static DNA binding capacity (mg calf thymus DNA) Average particle size (µm) Column dimensions (mm) Maximum operating pressure (psi/bar) CHT2-I 2 CHT5-I 5 CHT10-I 10 CHT20-I 20 20 50 100 200 0.5 to 3.0 0.5 to 5.0 0.5 to 7.0 0.5 to 10.0 30 75 150 300 0.20 10 ± 3.0 7 x 52 0.5 10 ± 3.0 10 x 64 1.0 10 ± 3.
or diluted to the start buffer’s concentration. Buffer exchange can be accomplished using Bio-Spin® 6 or Bio-Spin 30 columns, Econo-Pac® 10DG desalting columns, Bio-Gel® P6DG size exclusion gel or the Econo-Pac P6 cartridge. The choice of product depends on sample volume. Always centrifuge or filter the sample (0.2-0.45 mm filter) to remove particulates. Application of turbid or lipid-containing samples may reduce the column lifetime. See Ref. 1 for further information about sample preparation.
1 Scale-up [Buffer B] M Recommended gradient volumes: 0.5 CHT2-I 20 to 40 ml CHT5-I 50 to 100 ml CHT10-I 100 to 200 ml CHT20-I 200 to 400 ml load sample 0 0 6 12 18 24 30 36 42 48 Volume (ml) Fig. 1. Schematic gradient for separation on Bio-Scale CHT2-I column. 2.4 Use of Detergents Most of the common detergents used for protein isolation may be used with the Bio-Scale CHT-I support. We recommend the use of the reduced form of Triton® X-100 to minimize UV absorption artifacts.
sharp, UV-absorbing peaks during the gradient, complicating subsequent analysis of the chromatogram. Section 3 Care of the Bio-Scale CHT-I Columns 3.1 Column Hygiene Careful preparation (especially filtration) of the sample and the buffers will maintain the column performance and lifetime. Normally, washing with 0.5 M phosphate will remove most bound components.
3.4 Top-Off Resin If the top of the resin bed becomes fouled and the above hygiene steps do not restore performance, then a few mm of the bed should be removed and replaced with fresh resin obtainable from Bio-Rad. See page 6 for ordering information. 3.5 Storage Conditions Prior to long-term storage, the column should be cleaned as previously described and then washed with 3 column volumes of 20% ethanol. This will prevent microbial growth. Store the column in a safe place at room temperature.
Upper end fitting Upper lock nut Top end cap Protective shield Upper bed support Distribution screen Top frit FLOW Glass column Bottom end cap Lower end fitting Fig. 2. Instructions for removal of top frit. 1. Remove the upper and lower end fittings from the column. 2. Remove the upper lock nut from the top end cap. 3. Unscrew the bottom end cap from the protective shield and slide out the glass column. 4. Pull out the upper bed support from the glass column. 5.
3.6 References 1. 2. 3. 4. 5. Guide to Sample Preparation. Bio-Rad Laboratories Technical Bulletin # 1322. Hydroxylapatite. T. L. Brooks (Calbiochem Biochemicals). HPLC of Proteins on an Hydroxyapatite Column. T. Kadoya et al, (1988), J. Liquid Chromatography, 11, No 14, 2951-2967. Ceramic Hydroxyapatite HPLC of Complexes Membrane Protein and Sodium Dodecyl Sulfate. T. Horigome et. al., (1989), European Journal of Biochemistry, 186, 63-69.
Bio-Rad Laboratories Life Science Group 2000 Alfred Nobel Drive Hercules, California 94547 Telephone (510) 741-1000 Fax: (510) 741-5800 Australia, Bio-Rad Laboratories Pty Limited, Block Y Unit 1, Regents Park Industrial Estate, 391 Park Road, Regents Park, NSW 2143 • Phone 02-9414-2800 • Fax 02-9914-2888 Austria, Bio-Rad Laboratories Ges.m.b.H., Auhofstrasse 78D, 1130 Wien • Phone (1) 877 89 01 • Fax (1) 876 56 29 Belgium, Bio-Rad Laboratories S.A./N.V.
