Ge Appliances Ion Exchange Column Mono Q 5 50 Gl Users Manual [PDF] マニュアル 5/50 GL, S
Mono Q 550 GL to the manual 39da0379-eced-4523-86f8-6463f6ec5131
2015-01-23
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GE Healthcare First-time use Sample recommendations Equilibrate the column for first-time use or after long-term storage as follows: Instructions 71-5017-88 AC a) Ion Exchange Columns Mono Q 5/50 GL and Mono S 5/50 GL 5 column volumes (CV) distilled water at 1 ml/min at room temperature. b) 5 CV start buffer at 2 ml/min at room temperature. c) 5 CV elution buffer at 2 ml/min at room temperature. d) 5 CV start buffer at 2 ml/min at room temperature. Try these conditions first Elution buffer (Mono Q)*: 20 mM Tris-HCl + 1.0 M NaCl, pH 8.0 TM Users of ÄKTA design system may select one of the buffer recipes recommended for anion exchange chromatography at pH 8 or cation exchange chromatography at pH 6. Flow: 2 ml/min at room temperature 1. Equilibrate column with 5–10 column volumes (CV) of start buffer or until baseline, eluent pH and conductivity are stable. 2. Adjust the sample to the chosen starting pH and ionic strength and apply to the column (see sample recommendations). The columns are supplied with two union M6 female/1/16” male for connection TM to FPLC System, two fingertight connector 1/16” for connecting 1/16” tubing to column and ÄKTA, two stop plugs 1/16” male to seal the column (attached to column when delivered) and instruction. 3. Wash with 5–10 CV of start buffer or until the baseline, the eluent pH and the conductivity are stable i.e. when all unbound material has washed through the column. 4. Begin elution using a gradient volume of 10–20 CV and an increasing ionic strengt up to 0.5 M NaCl (50% elution buffer). Column data 5. Wash with 2–5 CV of 1 M NaCl (100% elution buffer) to elute any remaining ionically-bound material. TM TM Mono Q™ 5/50 GL and Mono S 5/50 GL are Tricorn high performance columns. The columns are pre-packed glass columns for high performance ion exchange chromatography of proteins, peptides, polynucleotides and other biomolecules. Matrix Polystyrene/divinyl benzene Bead form Rigid, spherical, porous monodisperse Particle size 10 μm Column dimensions 5 × 50 mm Bed volume 1 ml Average loading capacity 50 mg 6. 2–12 1–14 ▼ To avoid local disturbances in pH caused by buffering ions participating in the ion exchange process, select an eluent with buffering ions of the same charge as the substituent groups on the ion exchanger. Choose the start buffer pH so that substances to be bound to the ion exchanger are charged, e.g. at least 1 pH unit above the isoelectric point for anion exchangers and at least 1 pH unit below the isoelectric point for cation exchangers. Figure 2 and Figure 3 list a selection of standard aqueous buffers. pH 4 6 5 7 8 N-methyl piperazine Piperazine bis-Tris Requilibrate with at least 5–10 CV of start buff er or until eluent pH and conductivity reach the required values. All commonly used aqueous buffers, pH 2–12 Urea, up to 8 M Guanidine hydrochloride, up to 6 M Acetonitrile, up to 30% in aqueous buffers Non-ionic detergents Cationic detergents (Mono Q) Anionic detergents (Mono S) Flow rate (water at room temperature) recommended 0.5–3.0 ml/min maximum 3 ml/min Pressure over column maximum 4 MPa, 40 bar, 580 psi Type of exchanger Mono Q Strong anion + Charged group -CH2-N (CH3)3 Ionic capacity 0.27–0.37 mmol Cl /ml medium Recommended to have an on-line filter upstream of the injection valve. Buffers and solvents with increased viscosity will affect the back-pressure and flow rate. De-gas and filter all solutions through a 0.22 μm filter. Daily use 4 to 40 ºC - Choice of eluent Buffers and solvent resistance Temperature operating Dissolve the sample in start buffer, filter through a 0.22 μm filter or centrifuge at 10 000 × g for 10 min Read the section ”Optimization” for information about how to optimize a separation. (will vary depending on sample and loading conditions) pH stability regular use cleaning Preparation The column is delivered in degassed 20% ethanol sealed with two stop plugs to prevent the column from drying out. For column storage, wash with 5 column volumes of distilled water followed by 5 column volumes of 20% ethanol. Degas the ethanol/water mixture thoroughly and apply at a low flow rate to avoid overpressuring the column. Store at room temperature or, for long periods, store at +4° C to +8 °C. Ensure that the column is sealed well to avoid drying out. Do not freeze. Separation by gradient elution Quick information ≤ 45 mg Delivery/storage 20 mM 2-[N-morpholino] ethanesulphonic acid (MES), pH 6.0 Elution buffer (Mono S)*: 20 mM MES + 1.0 M NaCl, pH 6.0 * negative (Mono Q), positive (Mono S) Recommended initial sample load In-depth information Start buffer (Mono Q)*: 20 mM Tris-HCl, pH 8.0 Start buffer (Mono S)*: Net charge of target molecule Mono S Strong cation -CH2-SO3 - Cleaning 0.12–0.15 mmol + H /ml medium Acetonitrile, up to 100% Sodium hydroxide, up to 2 M Ethanol, up to 100% Methanol, up to 100% Acetic acid, up to 75% Isopropanol, up to 100% Hydrochloric acid, up to 1 M 1% Trifluoroacetic acid Note: Before connecting the column to a chromatography system, start the pump and remove all air and debris in the system, particularly in the tubing and valves. Avoid: Oxidizing agents Anionic detergents (Mono Q) Cationic detergents (Mono S) Fig 1. Illustration of how to lock the upper adapter. The locking ring (black) must be in down position to prevent uncontrolled adjustment of the column’s bed height. Tricorn ™ 9 10 pKa (25 ˚C) 11 4.75 5.33 6.48 6.65; 9.10 bis-Trispropane 7.76 Triethhanolamine 8.07 Tris 8.52 N-methyldiethanolamine 8.88 Propane-1,3-diamino Ethanolamine 9.50 Piperazine 9.73 Propane-1,3-diamino 10.55 Piperidine 11.12 Fig 2. Recommended buffers for anion exchange chromatography. ▼ ▼ pH 2.5 3 4 5 6 7 8 9 Citric acid pKa (25 °C) 3.13 Lactic acid Butanedioic acid Acetic acid 3.86 4.21 4.75 5.76 Methyl Malonic acid MES Phosphate 6.27 7.20 7.56 HEPES BICINE 8.33 Fig 3. Recommended buffers for cation exchange chromatography. Table 1 lists suggested volatile buffers that can be used in cases where the purified substance has to be freeze-dried. Table 1. Volatile buffer systems. pH Substance 3.3–4.3; 4.8–5.8 Pyridine/formic acid 3.3–4.3; 9.3–10.3 Trimethylamine/formic acid 4.3–5.8 Pyridine/acetic acid 3.3–4.3; 8.8–9.8 Ammonia/formic acid 4.3–5.3; 8.8–9.8 Ammonia/acetic acid 5.9–6.9; 9.3–10.3 Trimethylamine/carbonate 5.9–6.9; 8.8–9.8 Ammonium carbonate/ammonia 4.3–5.3; 7.2–8.2 N-ethylmorpholine/acetate Optimization Troubleshooting Perform a first run as described in the section “Try these conditions first”. If the results obtained are unsatisfactory, consider the following: Symptom Remedy Increased back-pressure over the column Reverse the flow direction and pump 5 ml elution buffer at a flow rate of 0.5 ml/min through the column. Return to normal flow direction and run for 5 minutes at 1 ml/min through the column. If high backpressure persists, clean the column. Action Effect Change pH/buffer salt (see Figure 1 and Figure 2 for buffers) Changes selectivity, gives weaker/stronger binding. Change salt, counter ions and/or co-ions Changes selectivity. Function test of Mono S 5/50 GL Clean the column according to the procedure described in the section “More rigorous cleaning”. mAU (280 nm) Reverse the flow direction and pump 10 ml well degassed start buffer through the column at a flow rate of 0.5 ml/min. 200 Decrease the sample load Improves resolution. Decrease the flow rate Improves resolution. Air in the column Change gradient slope Shallower gradients improve selectivity but broaden peaks (decrease efficiency). A steeper gradient will sharpen peaks, but move them closer together. Sample: Regular cleaning Sample volume: Gradient: Start buffer: Elution buffer: Flow rate: Flow: 0.5 ml/min at room temperature 3. Wash with at least 2 CV of 2 M NaCl 4. Rinse with at least 2CV of distilled water until the UV-baseline and the eluent pH are stable. 4. Wash with at least 4 CV of start buffer or storage buffer until pH and conductivity values have reached the required values. 100 50 Function test of Mono Q 5/50 GL It is recommended to reverse the direction of flow during column cleaning so that contaminants do not need to pass through the entire length of the column. Wash with 2 column volumes (CV) of 2 M NaCl. 1. Conalbumin (3 mg/ml) 2. α-lactalbumin, bovine milk (4 mg/ml) 3. Soybean trypsin inhibitor (6 mg/ml) 200 μl 0–100% elution buffer in 20 CV 20 mM Tris-HCl, pH 7.0 20 mM Tris-HCl + 0.25 M NaCl, pH 7.0 1.0 ml/min (room temperature) mAU (280 nm) 0 Depending on the nature of contaminant cleaning solution in the section ”Buffers and solvent resistance” may be appropriate. After cleaning the column wash with at least 2 CV of distilled water and 4 CV of start buffer or storage buffer. For more information on how to clean your column, please refer to the handbook ”Ion exchange chromatography & Chromatofocusing, Principles & Methods”. As an alternative to more rigorous cleaning or if column performance still not restored change the filter at the top of the column. (Since contaminants are introduced with the liquid flow, many of them are caught by the filter.) Instructions for changing the filter are supplied with the Filter Kit. Clean the column after filter change according to regular cleaning. 5.0 10.0 15.0 20.0 ml Fig 5. Typical chromatograms from a function test of Mono S 5/50 GL. Ordering information % Elution Buffer 100 Product 200 No. per pack Code No. Mono Q 5/50 GL 1 17-5166-01 Mono S 5/50 GL 1 17-5168-01 No. per pack Code No. Related products 150 ▼ 0 0.0 More rigorous cleaning Remove strongly hydrophobically bound proteins, lipoproteins and lipids by washing with 4 column volumes (CV) of 30% isopropanol or 70% ethanol at 0.25 ml/min. Remove precipitated proteins with 1 CV of 1 mg/ml pepsin in 0.5 M NaCl, 0.1 M acetic acid (leave overnight) or wash with 2 CV of 6 M Guanidine hydrochloride at 0.25 ml/min. % Elution Buffer 100 150 Check the performance of the column when new by running the separation described in Figures 4 and 5. Figures 4 and 5 shows a typical chromatogram run on an optimized system. Since the system can profoundly affect the resolution, it is meaningful to compare runs done on the same system. Check the column at regular intervals and whenever you suspect a problem. Cleaning Wash with 4 CV of 1 M NaOH 1. Ribonuclease A (1.5 mg/ml) 2. Cytochrome C (0.4 mg/ml) 3. Lysozyme (0.4 mg/ml) 100 μl 0–100% elution buffer in 20 CV 20 mM sodium phosphate, pH 6.8 20 mM sodium phosphate + 0.4 M NaCl, pH 6.8 1.0 ml/min (room temperature) Column performance control For more information, please refer to the handbook “Ion exchange chromatography, Principles & Methods”, which can be ordered from GE Healthcare or downloaded from our web site. 2. Sample volume: Gradient: Start buffer: Elution buffer: Flow rate: Loss of resolution and/or decreased sample recovery 1. Sample: Product ▼ 100 Mono Q 10/100 GL 1 17-5167-01 Mono Q 4.6/100 PE 1 17-5179-01 Mono S 10/100 GL 1 17-5169-01 Mono S 4.6/100 PE 50 TM HiTrap Desalting 1 17-5180-01 5 × 5 ml 17-1408-01 ▼ Accessories 0 0 0.0 5.0 10.0 15.0 20.0 ml Fig 4. Typical chromatograms from a function test of Mono Q 5/50 GL. Product No. per pack Code No. Tubing connectors: Fingertight connector 1/16” male Tricorn 5 filter kit* Filter Tool Union M6 female/1/16” male On-line filter (1/16”) 10 1 1 8 1 18-1112-55 18-1153-02 18-1153-20 18-1112-58 18-1118-01 Handbook: Ion Exchange Chromatography & Chromatofocusing, Principles and Methods 1 11-0004-21 www.gehealthcare.com/protein-purification www.gehealthcare.com GE Healthcare Europe GmbH Munzinger Strasse 5, D-79111 Freiburg, Germany Tricorn, Mono Q, Mono S, HiTrap, ÄKTA and Drop Design are trademarks of GE Healthcare companies. GE and GE Monogram are trademarks of General Electric Company. GE Healthcare UK Ltd Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK The Tricorn column and components are protected by US design patents USD500856, USD506261, USD500555, USD495060 and their equivalents in other countries. GE Healthcare Bio-Sciences AB Björkgatan 30 751 84 Uppsala Sweden GE Healthcare Bio-Sciences Corp 800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327, USA All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. General Electric Company reserves the right, subject to any regulatory and contractual approval, if required, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation. GE Healthcare Bio-Sciences KK Sanken Bldg, 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan © 2006 General Electric Company – All rights reserved. GE Healthcare Bio-Sciences AB, a General Electric Company. 71-5017-88 AC 03/2006 Elanders Östervåla 2006 * includes top and bottom filters and O-rings, 5 of each.
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