4006078A 10/1/98 10:02 AM Page CVR1 UNO™ Q&S Polishing Column for Continuous Bed Ion Exchange Chromatography Instruction Manual Catalog Numbers 720-0009 720-0029 For Technical Service Call Your Local Bio-Rad Office or in the U.S.
4006078A 10/1/98 10:02 AM Page 1 UNO Q & S Polishing Ion Exchange Column Introduction UNO prepacked ion-exchange columns are designed to meet the needs of the bio-chromatographer for rapid and reproducible high-resolution separations of biomolecules including proteins, peptides and polynucleotides. The UNO Polishing column is designed specifically as a late-stage purification tool to obtain the highest resolution and recovery from a small sample load (up to 2 mg).
4006078A 10/1/98 10:02 AM Page 2 use of inert, biocompatible (ceramic, PEEK, titanium) solvent delivery systems for maximum column life and recovery of sample biological activity. Table 1. Column Characteristics Q Polishing Column Column volume (ml) 0.16 Recommended max. protein loading (mg) 2 Recommended flow-rates (ml/min) 0.1 to 1 Column Dimensions (mm) 4.6 x 10 Max. operating pressure (psi/MPa/Bar) 200/1.3/14 S Polishing Column 0.16 2 0.1 to 1 4.6 x 10 200/1.
006078A 10/1/98 10:02 AM Page 3 while maintaining maximum buffering capacity. In any case, a buffer concentration of 20 mM is recommended. As can be seen in Table 2 and 3, the pKa and hence the pH of the buffer, changes with temperature. Therefore the pH of the buffer must be adjusted at the working temperature. Table 2. Buffers for Anion-Exchange Chromatography pH range 5.0 - 6.0 5.5 - 6.0 5.8 - 7.2 6.4 - 7.3 7.3 - 8.3 7.6 - 8.6 8.4 - 8.8 9.0 - 9.9 9.8 - 10.
4006078A 10/1/98 10:02 AM Page 4 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 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® P-6DG size exclusion gel or the Econo-Pac P6 cartridge.
10/1/98 10:02 AM Page 5 Gradient Volumes & Salt Concentrations As a starting point for developing a separation, we recommend using the UNO Q Polishing column with a simple gradient profile over 2–6 ml. Protocol: Use a flow-rate of 0.5 ml/min. Following sample application, wash unbound proteins from the column with 2 ml of Start Buffer A. For elution, use a gradient volume of 6 ml to a Cl- concentration of 0.5 M (50% B). Follow this segment of the gradient by raising the salt concentration to 1.
4006078A 10/1/98 10:02 AM Page 6 concentration (CMC). As the salt concentration (i.e. the counter-ion concentration) increases, the CMC drops and eventually micelles will form. This may cause a sudden increase in the UV baseline as the micelles themselves scatter light. We recommend using a concentration of detergent above the CMC during gradient elution. If subsequent chromatographic steps (e.g.
4006078A 10/1/98 10:02 AM Page 7 Storage Conditions Prior to long-term storage, the column should be cleaned as previously described and then washed with 5 ml of 20% ethanol. This will prevent microbial growth. Store the column in a safe place at room temperature. NEVER allow the column to freeze. References 1. 2. W. Kopaciewicz, M. A. Rounds, J. Fausnaugh and F. E. Regnier (1983) Retention Model for High-performance Ion-Exchange Chromatography. J. Chromatography, 266, 3-21.
4006078A 10/1/98 10:02 AM Page 8 Catalog Number Product Description 751-0091 Bio-Scale 2 Replacement Part Kit, includes 5 frits, 5 distribution screens, 2 o-rings, 1 frit remover. Use this kit for the UNO S-1 or Q-1 Column. 751-0095 Bio-Scale 10 Replacement Part Kit, includes 5 frits, 5 distribution screens, 2 o-rings, 1 frit remover. Use this kit for the UNO S-6 or Q-6 Column. 751-0097 Bio-Scale 20 Replacement Part Kit, includes 5 frits, 5 distribution screens, 2 o-rings, 1 frit remover.
4006078A 10/1/98 10:02 AM Page CVR2 Bio-Rad Laboratories Life Science Group U.S.
