CIM® Epoxy-8f mL Tube Monolithic Column
About CIM® Epoxy-8f Tube Monolithic Columns
The CIM® EPOXY-8f high throughput Tube Monolithic Column consist of a unique polymeric monolith encased in a specially designed housing. This column is used for direct coupling of proteins, peptides and other ligands containing amino or thiol groups. The design of the column 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 monolithic length and the specially engineered highly porous structure allow operations at elevated flow rates with low-pressure drops (only few bars).
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.
| Catalog number: | 433.7175 |
| Tube chemistry: | epoxy |
| Ligand density: | 4.22 ± 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 |
| 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 (coupling) range 1–14 WARNING: The working range is only valid for the matrix itself. After coupling, it depends on the stability of the coupled ligand! |
Storage of CIM® Epoxy-8f Tube Monolithic Column
CIM® Epoxy-8f Tube Monolithic Column should be stored at +4 °C (39 °F) to +8 °C (46 °F) in the presence of a suitable bacteriostatic agent, e.g. 20 % ethanol.
Coupling on CIM® Epoxy-8f Tube Monolithic Column
Coupling of the ligand
Coupling is performed at a higher pH where the epoxy groups are more reactive. The upper pH value is limited by the ligand stability. Hence, it should be thoroughly checked and optimized from case to case. Coupling at pH 8 is a good compromise.
Temperature dependence
The coupling of the ligand to the CIM® Epoxy-8f Tube Monolithic Column is obtained by a covalent reaction between the epoxy groups on the support and the amino or thiol groups of the ligand. Therefore, the coupling rate increases exponentially with temperature according to the Arrhenius equation. From this point of view, an elevated temperature would be preferred for shortening the coupling time. On the other hand, some ligands may become unstable at higher temperatures and tend to agglomerate or even to loose their biological activity. The coupling temperature depends on the stability of your ligand and should be optimized from case to case. For most ligands (especially those with high molecularmasses) the coupling is best performed for several days at 4 °C (39 °F). If, however, the ligand is stable, room or even higher temperature is recommended.
Buffer composition
The buffer type does not significantly influence the coupling efficiency. Of course, in some cases, the selection of the buffer is of utmost importance, especialy when the ligand stability depends on the environment. Because of this, physiologic buffers are prefferred. Tris(hydroxymethyl)aminomethane, glycine or other nucleophilic compounds should be avoided as they may compete with the ligand for the epoxy groups, thus lowering the coupling efficiency. For coupling, the ligand should be dissolved in a coupling buffer. In some cases, it is necessary to add organic solvents to dissolve the ligand. The polymeric backbone of the CIM® monolith is hydrophobic. Because of this, coupling can be enhanced by using buffers with higher ionic strengths (e. g. 0.5 M). In this way, the ligand comes closer to the surface
facilitating coupling. Higher ionicmay not be appropriate as some ligands can start to agglomerate and hinder coupling.
Blocking the excess epoxy groups
The remaining epoxy groups should be blocked after the coupling in order to avoid unspecific interactions during use. The epoxy groups can be blocked with various reagents like e. g. 1 M ethanolamine, overnight at 25 °C (77 °F) to 35 °C (95 °F), 0.1 M mercaptoethanol, pH 8, overnight at 25 (77 °F) to 35 °C (95 °F), or 0.5 M sulfuric acid, for about 3 hours at 50 °C (122 °F).WARNING: The selection of a suitable reagent for blocking of the excess epoxy groups depends on the coupled ligand's stability!
Storage of CIM® Epoxy-8f Tube Monolithic Columns
The CIM® Tube Monolithic Column should be stored at +4 °C (39 °F) to +8 °C (46 °F) in the presence of a suitable bacteriostatic agent, e.g. 20 % ethanol. |
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