UNO™ Q&S Continuous Bed Ion Exchange Column Instruction Manual Catalog Numbers 720-0001, 720-0003, 720-0005, 720-0021, 720-0023 and 720-0025 For Technical Service Call Your Local Bio-Rad Office or in the U.S.
UNO Anion and Cation Exchange Columns 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 availability of three column sizes (bed volumes of 1.3, 6, and 12 ml) provides unrivaled flexibility for the economical and predictable scale up of separation and purification protocols without sacrificing resolution due to overloading.
Table 2. UNO S Column Characteristics Column volume (ml) Recommended max. protein loading (mg) Recommended flow-rates (ml/min) Column Dimensions (mm) Maximum operating pressure (psi/MPa/bar) S-1 1.3 20 0.5 to 6.0 7 x 35 700/4.5/48 S-6 6 80 0.5 to 8.0 12 x 53 700/4.5/48 S-12 12 160 0.5 to 10.0 15 x 68 700/4.5/48 Preparation for Initial Use The columns are supplied in a storage buffer of 0.1 M NaCl + 20% ethanol. The counter ion for the Q column is Cl- and Na+ for the S column.
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 - 10.3 Buffer Piperazine L-Histidine Bis-Tris Bis-Tris Propane Triethanolamine Tris Diethanolamine Ethanolamine 1,3-diamino-propane Mwt 86.1 155.2 209.2 282.3 149.2 121.1 105.1 61.1 74.1 pKa @25 °C 5.7 6.15 6.5 6.8, 9.0 7.8 8.1 8.9 9.5 10.5 Counter-ion ∆ pKa/°C Cl- /HCOO-0.015 ClCl-0.017 ClCl- /CH3COO-0.020 Cl-0.031 Cl -0.025 Cl-0.029 Cl-0.026 Table 3.
Elution Conditions Separations by ion-exchange chromatography are typically accomplished by increasing the salt concentration of the eluent either as a “step” or as a “continuous” gradient. For many separations, varying the pH of the elution buffer in addition to its salt concentration may be advantageous. Generally, it is best to choose initial pH and ionic strength conditions such that the protein of interest elutes early in the gradient.
1 Scale-up [Buffer] M 0.9 0.8 Recommended gradient volumes: 0.7 S-1 or Q-1 10 to 30 ml 0.6 S-6 or Q-6 60 to 120 ml 0.5 S-12 or Q-12 120 to 240 ml 0.4 Load Sample 0.3 Load Sample 0.2 0.1 0 0 6 12 18 24 30 36 42 48 Volume (ml) Fig. 1. Schematic gradient for separation on UNO S-1 or UNO Q-1 column. Use of Detergents Cationic or non-ionic detergents may be used with the UNO Q support. Anionic or nonionic detergents may be used with the UNO S support.
ing protocol as described below (steps 1–7) should be used. Always reverse the flow during this procedure so tightly-bound substances at the top of the column are quickly removed. During this operation do not exceed more than the following maximum flow-rates. 1. 2. 3. 4. 5. 6. 7. S-1 or Q-1 1 ml/min S-6 or Q-6 2 ml/min S-12 or Q-12 3 ml/min Wash with 2 column volumes of deionized water. Elevated backpressures may occur when washing with deionized water. Do not exceed 700 psi.
Frit Removal The top frit and screen may need to be replaced after extensive column use or if increasing back-pressures are noticed. Each column is supplied with a Frit Removal Tool, Polyethylene Frit and Distribution Screen. The screen assists in uniformly distributing the sample over the entire column surface and also acts as a pre-filter for the frit. It should be replaced every time the frit is changed.
Catalog Number Product Description 720-0029 UNO S Polishing Column 720-0031 UNO S-1 R Column, replacement column 720-0033 UNO S-6 R Column, replacement column 720-0035 UNO S-12 R Column, replacement column 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.
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.
