CIM® EDA-8f mL Tube Monolithic Column for Ligand Immobillization
About CIM® EDA-8f Tube Monolithic Columns
The CIM® EDA-8f Tube Monolithic Column consists of a unique polymeric monolith encased in a specially designed housing. These columns are amine activated monolithic supports obtained by reacting the native epoxy groups with Ethylene Diamine. The CIM® EDA Tube Monolithic Columns can be used for affinity chromatography or bioconversion by coupling proteins, peptides and other ligands through a crosslinking reaction with a suitable bifunctional reagent, e. g. glutaric dialdehyde. 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: | 430.5116 |
| Disk chemistry: | ethylene diamino |
| Ligand density: | 3.2 ± 0.2 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 |
| 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: | working range 2–13 cleaning-in-place 1–14* (e. g. 1 M NaOH) * Valid for the matrix. Note: After immobilization, the working and CIP range depend on the stability of the coupled ligand! |
Caring for the CIM® EDA-8f Tube Monolithic Columns
The correct choice of a coupling method to the amino activated CIM® EDA-8f Tube Monolithic column depends on the substance to be immobilized (the chemistry of its active groups, pH and temperature stability, reactivity, etc.). An example of a coupling method is presented below. This procedure needs to be optimized on a case to case basis. Activation of the matrix with glutaric dialdehyde (generation of aldehyde groups).- Wash the CIM® EDA-8f Tube Monolithic Column with 10 column volumes of a 50 mM phosphate buffer, pH 7.5 at the working flow rate.
- Activate the amino groups by pumping at least 8 mL of a 50 mM phosphate buffer containing 1% glutaric dialdehyde through the CIM® EDA-8f Tube Monolithic Column at the flow rate of 4 mL/min to 8 mL/min. Remove the tube monolithic column from the LC/HPLC system, seal it at both ends and incubate overnight at room temperature.
Note: for all handling procedures of the tube monolithic columns, please refer to the Instruction Manual. - Remove the excess glutaric dialdehyde from the CIM® EDA-8f Tube Monolithic Column by washing it with 10 column volumes of a 50 mM phosphate buffer, pH 7.5 at the working flow rate.
Coupling the Ligand
• Prepare your ligand solution by dissolving your ligand in a suitable coupling buffer, to obtain a final concentration of at least 2 mg/mL.• Pump at least 8 mL of the ligand solution through the aldehyde activated CIM® EDA-8f Tube Monolithic Column to completely fill the monolith pores.
• Remove the CIM® Tube Monolithic Column from the LC/HPLC system (see above) and seal it at both ends with suitable column end stoppers.
• Incubate the CIM® Tube Monolithic Column for at least 20 hours at room temperature or 4 °C.
Note: The selection of the coupling temperature depends on your ligand's stability.
Washing the CIM® Affinity Tube Monolithic Column
• To remove the excess ligand entrapped within the pores of the CIM® Tube Monolithic Column, wash the column thoroughly with at least 10 column volumes of a suitable buffer (e. g. coupling buffer) at one-half of the working flow rate.• To remove any unspecifically bound ligand wash the affinity tube monolithic column with at least 10 column volumes of a high ionic strength buffer (e. g. coupling buffer with 2 M urea or 2 M NaCl) at one half of the working flow rate.
• Finally, wash the affinity column with 5 column volumes of a working buffer at the working flow rate.
Influence of the Excess Active Groups on Non-specific Adsorption
The CIM® EDA-8f Tube Monolithic Column contains charged ethylene diamino groups and, therefore, acts as a weak anion-exchanger. The effect of these charged groups can be overcome by using a relatively high salt concentration (e. g. 0.5 M NaCl) in the binding buffer for affinity chromatography.Storage of CIM® EDA-8f Tube Monolithic Columns
CIM® EDA-8f Tube Monolithic Columns should be stored at a temperature of +4 °C to +8 °C in the presence of a suitable bacteriostatic agent. The choice of buffer solution and bacteriostatic agent depends on the properties of the coupled ligand. |
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