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GE Healthcare
Instructions 71-7149-00 AN HiTrap ion exchange columns
HiTrap SP HP, 1 ml and 5 ml
HiTrap Q HP, 1 ml and 5 ml
™
HiTrap SP HP and HiTrap Q HP are prepacked, ready to use cation and anion
exchange columns for method scouting, group separations, sample concentration
and sample clean-up of charged biomolecules. HiTrap SP HP and HiTrap Q HP provide
fast, reproducible, and easy separations in a convenient format.
The columns can be operated with a syringe, peristaltic pump or liquid
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Code No. Designation No. supplied 17-1151-01 HiTrap SP HP 5 x 1 ml 17-1152-01 HiTrap SP HP 5 x 5 ml 17-1153-01 HiTrap Q HP 5 x 1 ml 17-1154-01 HiTrap Q HP 5 x 5 ml Connectorkit Connectors supplied Usage No. supplied 1/16” male/luer female Connection of syringe to top of HiTrap column 1 Tubing connector Connection of tubing (e.g. Peristaltic flangeless/M6 female Pump P1) to bottom of HiTrap column* 1 Tubing connector Connection of tubing (e.g. Peristaltic flangeless/M6 male Pump P1) to top
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1. Description Media properties ™ SP Sepharose High Performance and Q Sepharose High Performance are strong cation and strong anion exchangers respectively. Both remain charged and maintain high capacity over broad pH ranges. The functional groups are coupled to the matrix via chemically stable ether linkages. Characteristics of HiTrap SP HP and HiTrap Q HP, 1 and 5 ml columns are listed in Table 1. Column HiTrap Q HP and HiTrap SP HP are 1 ml and 5 ml columns made of polypropylene, which
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Table 1. HiTrap SP HP and HiTrap Q HP columns characteristics Column volumes 1 ml or 5 ml Column dimensions 0.7 × 2.5 cm (1 ml) and 1.6 × 2.5 cm (5 ml) – Total ionic capacity 0.14–0.20 mmol (Cl )/ml medium (Q) + 0.15–0.20 mmol (H )/ml medium (SP) Dynamic binding capacity SP: approx. 55 mg ribonuclease/ml medium (0.1 M sodium acetate, pH 6.0 at 1 ml/min) Q: approx. 50 mg HSA/ml medium (20 mM Tris-HCl, pH 8.2 at 1 ml/min) Mean particle size 34 μm Bead structure 6% highly cross-linked spherical
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2. Selection of ion exchanger and conditions Ion exchange chromatography is based on adsorption and reversible binding of charged sample molecules to oppositely charged groups attached to an insoluble matrix. The pH value at which a biomolecule carries no net charge is called the isoelectric point (pI). When exposed to a pH below its pI, the biomolecule will carry a positive charge and will bind to a cation exchanger (SP). At pH’s above its pI the protein will carry a negative charge and w
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Strategy 1. Binding and elution of all sample components Binding is achieved by choosing a start buffer with a low pH for HiTrap SP HP or a high pH for HiTrap Q HP. The ionic strenght should be kept as low as possible to allow all components to bind to the ionic exchange (<5 mS/cm). This results in a concentration of the target substance and a complete picture of the whole sample. The drawback of this strategy is that the binding capacity of the ion exchanger for the target substance is dep
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The buffering ion should carry the same charge as the ion exchange group and should have a pKa within 0.5 pH units of the pH used in the separation. Buffering ions of opposite charge may take part in the ion exchange process and cause local disturbances in pH. Starting pH Cation exchangers (SP): At least 1 pH unit below the pI of substance to be bound. Anion exchangers (Q): At least 1 pH unit above the pI of substance to be bound. Table 2. Buffers for cation exchange chromatography. 1 pH in
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Table 3. Buffers for anion exchange chromatography. 1 pH interval Substance Conc. (mM) Counter-ion pKa (25 °C) - 4.3–5.3 N-Methylpiperazine 20 Cl 4.75 - - 4.8–5.8 Piperazine 20 Cl or HCOO 5.33 - 5.5–6.5 L-Histidine 20 Cl 6.04 - 6.0–7.0 bis-Tris 20 Cl 6.48 - 6.2–7.2; 8.6–9.6 bis-Tris propane 20 Cl 6.65; 9.10 - - 7.3–8.3 Triethanolamine 20 Cl or CH COO 7.76 3 - 7.6–8.6 Tris 20 Cl 8.07 2- 8.0–9.0 N-Methyldiethanolamine 20 SO 8.52 4 - - 8.0–9.0 N-Methyldiethanolamine 50 Cl or CH COO 8.52 3 - 8.4–9.
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Fig 3. Recommended buffer substances for anion exchange chromatography. The columns can be operated with a syringe, peristaltic pump or a chromatography system. 3. Operation Buff er preparation Water and chemicals used for buffer preparation should be of high purity. It is recommended to filter the buffers by passing them through a 0.22 μm filter immediately before use. See Tables 2 and 3, Figs 2 and 3 for recommended buffers. Sample preparation The sample should be adjusted to the composition
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p. 10 Table 4. Prepacked columns for desalting and buffer exchange. Code No Column Loading volume Elution volume Comments Application 17-1408-01 HiTrap 0.1–1.5 ml 1.3–4.0 ml Prepacked with For desalting and buffer Desalting Sephadex™ G-25 exchange of protein Superfine. Requires extracts (M >5000). r a syringe or pump to run. 17-5087-01 HiPrep Up to 15–20 ml Prepacked with For desalting and 26/10 15 ml Sephadex G-25 buffer exchange of Desalting Fine. Req
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Purifi cation 1. Fill the syringe or pump tubing with start buffer (low ionic strength). Remove the stopper and connect the column to the syringe (with the provided adaptor), “drop to drop” to avoid introducting air into the column. 2. Remove the snap-off end at the column outlet. 3. Wash out the preservatives with 5 column volumes of start buffer, at 1 ml/min for the 1 ml column and 5 ml/min for the 5 ml column. 4. Wash with 5 column volumes of elution buffer (start buffer with 1 M NaCl).
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Note: If a P1-pump is used a max flow rate of 1–3 ml/min can be run on a HiTrap 1 ml column packed with Sepharose High Performance media. 4. Optimization If sample composition is unknown, a simple screening test with the aid of a syringe or pump can be performed to optimize starting pH and ionic strength. 1. Set up a series of buffers with different pH´s, in the range 4–8 (SP) or 5–9 (Q), with 0.5–1 pH unit intervals between each buffer. Make one series with 1 M NaCl included in the buffer
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Further optimization The recommendations given above will give a sound basis for developing an efficient purification step. Details of how flow rate, sample loading, particle size and elution scheme may be optimized to meet the special needs can be found in the handbook, Ion Exchange Chromatography & Chromato- foucsing, Principles and Methods, Code No. 11-0004-21. GE Healthcare supplies a wide range of ion exchange chromatography media for purification of biomolecules at all scales. See Orde
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Elution with stepwise ionic strength gradients Stepwise elution is the sequential use of the same buffer at different ionic strengths. It is technically simple and fast, and is suitable for syringe operation. It is often used for sample concentration and sample clean-up. Stepwise elution gives small peak volumes and the resolution depends on the difference in elution power between each step. 1. Choose starting conditions as outlined under Optimizing starting conditions. 2. Equilibrate the c
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– A peristaltic pump and a gradient mixer e.g. pump P-1, gradient mixer GM-1. ™ – A one pump system, e.g. ÄKTAprime plus. – A two pump system, e.g FPLC or ÄKTA. 1. Choose starting conditions as outlined under Optimizing starting conditions. 2. Equilibrate the column, see Purification. 3. Adjust the sample to the chosen starting pH and ionic strength, see Sample preparation. 4. Apply the sample at 1 or 5 ml/min for the HiTrap 1 or 5 ml column respectively. Collect eluate. 5. Wash with 5–10 co
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Fig 4. Binding capacity of human IgG, HSA and human transferrin at different pH´s on HiTrap Q HP, 1 ml. 4. Apply the sample solution to the column with a pump or a syringe, at a flow rate equal to the flow rate to be used in the purification method. Collect fractions and continue sample application until the column is saturated. 5. Wash the column with 5–10 column volumes start buffer or until no material appears in the effluent. 6. Elute bound proteins with 3–5 column volumes of elution bu
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7. Scaling up For quick scale-up of purifications (back pressure will increase), two or three HiTrap ion exchange columns of the same type can be connected in series. For further scale-up SP Sepharose High Performance and Q Sepharose High ™ Performance are available in prepacked HiLoad columns or bulk media packs. See Ordering Information. 8. Storage HiTrap SP HP: Rinse with water and then wash with 5 column volumes of 20% ethanol, 0.2 M sodium acetate. HiTrap Q HP: Rinse with water and th
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Related products No. Supplied Code No. HiTrap IEX Selection Kit 7 x 1 ml 17-6002-33 HiTrap Desalting 5 x 5 ml 17-1408-01 HiTrap Desalting 100 x 5 ml* 11-0003-29 HiPrep 26/10 Desalting 1 x 53 ml 17-5087-01 HiPrep 26/10 Desalting 4 x 53 ml 17-5087-02 PD-10 Desalting column 30 17-0851-01 HiLoad 16/10 SP Sepharose High Performance 1 x 20 ml 17-1137-01 HiLoad 26/10 SP Sepharose High Performance 1 x 53 ml 17-1138-01 HiLoad 16/10 Q Sepharose High Performance 1 x 20 ml 17-1064-01 HiLoad 26/10 Q Sepharos
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Realated literature Ion Exchange Chromatography & Chromatofocusing Handbook, Principles and Methods 1 11-0004-21 Ion Exchange Columns and Media, Selection Guide 1 18-1127-31 Convenient Protein Purification, HiTrap Column Guide 1 18-1129-81 p. 19
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GE Healthcare Europe GmbH www.gehealthcare.com/hitrap Munzinger Strasse 5 www.gehealthcare.com D-79111 Freiburg Germany GE Healthcare Bio-Sciences AB GE Healthcare UK Ltd Björkgatan 30 Amersham Place Little Chalfont 751 84 Uppsala Buckinghamshire, HP7 9NA Sweden UK GE Healthcare Bio-Sciences Corp 800 Centennial Avenue P.O. Box 1327 Piscataway, NJ 08855-1327 USA GE Healthcare Bio-Sciences KK Sanken Bldg. 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073 Japan HiTrap, Sepharose, Sephadex, FPLC, ÄK
