CIM® IDA-8f mL Tube Monolithic Column
About CIM® IDA-8f Tube Monolithic Columns
The CIM® IDA-8f Tube Monolithic Column consists of a unique polymeric monolith encased in a specially designed housing. These affinity columns are used for the fast and highly efficient purification of histidine containing or (His)-tagged proteins which rely on histidine's affinity for immobilized transition metals. The design of these columns allows the mobile phase to run in a radial direction from the outer to the inner surface. In this way, resolution is preserved during scale-up while maintaining the short separation layer length. This short separation layer length and the specially engineered highly porous structure allow operations at elevated flow rates with low-pressure drops (only a few bar).
The following information is being provided to ensure proper product care and maximal product life. All other information can be found in the Instruction Manual that accompanied the product.
| Catalogue number: | 437.3010 |
| Tube chemistry: | affinity, iminodiacetic acid (IDA) as chelating ligand (uncharged) |
| Ligand density: | 0.22 ± 0.02 mmol/g dry support |
| Support matrix: | poly (glycidyl methacrylate- co-ethylene dimethacrylate) |
| Tube dimensions: | outer diameter: 15.0 mm; inner diameter: 6.5 mm; length: 56.0 mm; bed volume: 8.0 mL |
| Metal ion capacity: |
25 ± 10 μmol M2+/mL of support (metal ion dependent) |
| Dynamic binding capacity: | ≥ 3 mg Conalbumin/mL wet support (Conditions: Conalbumin 0.5 mg/mL, 50 mM phosphate buffer with 1.5 M ammonium sulphate, pH 7.0, flow rate 2 mL/min) |
| Working flow rates: | up to 400 mL/min (uav = 1340 cm/h) |
| Working system pressure: | up to 20 bar (2 MPa); WARNING: Do not exceed the maximum allowed pressure as this might seriously damage your column! |
| Temperature stability: | 4°C (39°F) to 50°C (122°F); WARNING: Avoid prolonged use at elevated temperatures! |
| Recommended pH: | pH>3 |
Caring for the CIM® IDA-8f Tube Monolithic Columns
Charging
When the CIM® IDA-8f Tube Monolithic Column is to be charged for the first time, the following procedure should be performed:- Wash with 20 column volumes of deionized water
- Charge with 10 column volumes of 30 mM metal salt solution (e. g. CuSO4, NiSO4)
- Wash with 20 column volumes of deionized water
- Re-equilibrate with 20 column volumes of the working mobile phase (buffer) at the working flow rate.
Regeneration
When the CIM® IDA-8f Tube Monolithic Column is to be recharged with metal ions, the following procedure should be performed:- Wash with 20 column volumes of deionized water
- Wash with 10 column volumes of 1 M HCl to strip the metal ions
- Wash with 20 column volumes of 0.5 M phosphate buffer/ 1MNaCl, pH 7.4
- Wash with 20 column volumes of deionized water
- Recharge with 10 column volumes of 30 mM metal salt solution (e. g. CuSO4, NiSO4)
- Wash with 20 column volumes of deionized water
- Re-equilibrate with 20 column volumes of the working mobile phase (buffer) at the working flow rate.
Cleaning In Place (CIP)
The CIM® Tube Monolithic Column does not have to be cleaned between purifications if the same protein is to be purified. It is sufficient to clean the tube monolithic column after 3–5 purifications, depending on the cell lysate, target proteins, conditions used, etc.
However, if an increase in back pressure is seen then the CIM® Tube Monolithic Column must be cleaned. The following procedures can be applied (WARNING: The columns must be regenerated after these CIP procedures are performed):
1. Removal of precipitated proteins
- Wash with 20 column volumes of deionized water
- Wash with 20 column volumes of 1 M NaOH
- Incubate with 1 column volume of NaOH for 1 hour
- Wash with 20 column volumes of 0.5 M phosphate buffer/ 1 M NaCl, pH 7.4
- Wash with 20 column volumes of deionized water at one-half of the working flow rate.
- Wash with 20 column volumes of a 30% 2-propanol at one-half of the working flow rate.
- Wash with 20 column volumes of distilled water at the working flow rate.
- Re-equilibrate with 20 column volumes of the working mobile phase (buffer) at the working flow rate.
Reducing nonspecific binding
- High concentrations of NaCl (1–2 M) are recommended in the binding, washing and elution buffer.
- Low concentrations of imidazole (1–20 mM) in the lysis or binding and washing buffer are recommended.
- Detergent (Tween 20 or Triton X) up to 2% can be used.
- Use of deoxyribonuclease (DNase) and ribonuclease (RNase) in the cell lysis procedure is recommended.
Storage of CIM® IDA-8f Tube Monolithic Columns
CIM® IDA-8f Tube Monolithic Columns should be stored at a temperature of +4°C to +8°C in the presence of a suitable bacteriostatic agent, e. g. 20% ethanol. |
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