PRODUCTS > CIM® Laboratory Columns

CIM® CDI Disk Monolithic Column

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About CIM® CDI Disk Monolithic Columns*

Thank-you for purchasing your CIM® CDI Disk Monolithic Column. CIM® Monolithic Columns are composed of a rigid highly cross-linked monolithic glycidylmethacrylate- co-ethyleneglycol- dimethacrylate polymer and an appropriate housing. They can be used to immobilize ligands to perform specific separations of large and small biomolecules. The CIM® CDI Disk Monolithic Column operates at high flow rates, low back pressures, and are extremely easy to use.
*Please also review the Product Specific Information Sheet(s) which accompanied these instructions for additional information (PSIS-CDID-0407)

Before you begin

IMPORTANT: Use the product according to guidelines in this Instruction Manual. Improper use may result in malfunction, personal injury or damage of the product or material. Follow safety instructions, wear gloves, safety glasses and a lab coat during operation.

Before you begin the immobilization procedure, please read the following sections in the Instruction Manual (# IMD010703) that accompanied the CIM® Disk Monolithic Column that you purchased:
  • Before you Begin
  • Preparation of the CIM® Monolithic Columns for use

Due to the nature of the monolithic structure, some of the conditions needed for immobilization may be different then your experience with existing chromatographic supports. Please consider the following when preparing your specific immobilization procedure:

  • Determine the pH stability of the ligand
    CDI groups are more reactive at a higher pH so it is recommended to perform the immobilization at the highest pH that the ligand can withstand. In general, a pH of 8.0 is a good compromise.
  • Determine the thermal stability of the ligand
    Ligand immobilization occurs via a covalent reaction between the CDI groups and the amino or thiol groups on the ligand. Therefore, the reaction rate increases exponentially with increases in temperature resulting in a shortening of the overall immobilization time. Since most ligands are stable at 4°C, the immobilization can be performed in the refrigerator and left to proceed for several days. If possible, it is best to perform the immobilization at the highest possible temperature that the ligand can withstand.
  • Buffer Composition
    The type of buffer selected does not significantly influence the efficiency of the immobilization. So, the buffer that is most suitable for the particular ligand should be chosen. Note: The use of buffers with amino groups, like Tris, should be avoided as they may compete with the ligand for the binding sites.
    To facilitate ligand coupling to the CDI groups, a buffer with a high ionic strength should be selected. A 0.5 M buffer has, in our experience, performed well. Note: At higher ionic strengths, some ligands might start to agglomerate.
  • Immobilization Protocol
    CIM® monolithic supports consist of a solid piece of material. To facilitate binding, the ligand solution needs to be pushed through the column in regular time intervals for a specified period of time.
    Note: Placing the disk in a beaker and mixing with a magnetic stirrer should be avoided when preparing the disk affinity column as this might damage the disk-shaped monolith and has no influence on ligand attachment inside the pores.

     

General Immobilization Procedure for CIM® CDI Disk Monolithic Columns


  1. Assemble the CIM® CDI Disk Monolithic Column according to the Instruction Manual (# IMD010703) that accompanied this product.
  2. Connect a syringe filled with deionized water to one side of the CIM® CDI Disk Monolithic Column and wash the column by pushing through at least 5 column volumes (CV) of water to remove the 96 % ethanol solution that the disk was stored in.
  3. Fill the syringe with i.e. 0.5 M Na-Phosphate Buffer, pH 8.0* and equilibrate the CIM® CDI Disk Monolithic Column by pushing through at least 5 CV of buffer (∼2 mL).
  4. Prepare 3 mL of your ligand solution by dissolving your ligand in a 0.5 M Na-Phosphate Buffer, pH 8.0* to obtain a final concentration of 2–3 mg/mL and fill the syringe with it.
  5. Connect the filled syringe to the CIM® CDI Disk Monolithic Column from one side and an empty syringe to the other.
  6. Push the ligand solution through the CIM® CDI Disk Monolithic Column by pressing the filled syringe while leaving the empty syringe free to collect the ligand solution passing through the CIM® CDI Disk Monolithic Column. Keep repeating the procedure for up two hours in regular time intervals of 15 minutes.
  7. After the immobilization is completed, disconnect one syringe and remove the residual ligand by washing the CIM® CDI Disk Monolithic Column with 10 CV of 0.5 M Na-Phosphate Buffer, pH 8.0* containing 1 M NaCl, and finally with another 5 CV of deionized water.
  8. Equilibrate the CIM® CDI Disk Monolithic Column by washing with 5 column volumes of a 0.5 M Na-Phosphate Buffer, pH 8.0 at a flow rate of 0.5–1.0 CV/minute.
  9. The immobilized or affinity CIM® CDI Disk Monolithic Column is now ready for use.
    Note: Blocking excessive reactive groups is not necessary since the groups left are hydrolyzed into OH groups.

* The most appropriate buffer for your ligand should be chosen.

Operating Conditions

  1. CIM® Disk Housings: Please refer to Operating Conditions in the Accompanying Instruction Manual (# IMD010703).
  2. Affinity CIM® Disk Monolithic Columns: this is dependent on the particular ligand immobilized.

Caring for the CIM® CDI Disk Monolithic Columns

To extend the life of your CIM® CDI Disk Monolithic Columns, please observe the following guidelines:

  • Always use freshly prepared mobile phases (buffers).
  • Always filter your mobile phases (buffers) and samples through a 0.45 μm or 0.22 μm filter.
  • Protect the monolithic column by using an appropriate high-pressure in-line filters (e. g. PEEK filter, 231.0851).

Cleaning and Regeneration of the CIM® CDI Disk Monolithic Columns

The cleaning and regeneration procedure for an immobilized ligand must be customized based upon the properties and tolerances of the molecule of interest.

Storing the CIM® CDI Disk

Up to 2 days of planned storage after column use:

  • Seal the CIM® CDI Disk Monolithic Columns tightly on both sides using the column blind fittings.
  • Store the column in the refrigerator.
    WARNING: Do not store the disk(s) or completely assembled column below 0 °C (32 °F)!
3 or more days of planned storage after column use:
  • Remove the CIM® CDI Disk from the housing and store in a labeled jar with an appropriate bacteriostatic agent eg. 96 % Ethanol or 0.02 % Sodium azide.
    WARNING: Take care that your ligand is stable in the used preservative! Sodium azide is toxic (follow the appropriate safety precautions and decontamination procedures)!
  • Store the CIM® CDI Disk in the refrigerator.
    WARNING: Do not store the disk(s) below 0 °C (32 °F)!

WARNING: Never let the disk(s) dry out! Always keep the CIM® CDI Monolithic Columns wetted with the buffer or storage solution!

Troubleshooting

  1. Quantity of immobilized ligand is small:
    • Optimize immobilization procedure by considering the following parameters: coupling buffer, pH, time, temperature and ligand concentration.
    • Make sure that the monolithic column has been thoroughly washed and equilibrated with the coupling buffer.
  2. Quantity of immobilized ligand is as expected, but affinity/ capacity is bad:
    • Check if the ligand already shows low affinity before immobilization (old, degraded, unstable).
    • The chromatographic binding and eluting buffers may not be optimal. Just one assay with an inadequate solution can damage the column.
    • Fouling of the column by non-specific binding, or blockage of pores due to residual particles in the sample, may have occurred. Try developing a general procedure to sanitize (gently!) the column or a CIP procedure.
    • Immobilization of the ligand may have blocked a lysine essential to the affinity/activity of the ligand. If a lysine is known to be in the binding site or close to it, consider performing a different immobilization strategy.
  3. Loss of binding/capacity with time
    • Affinity columns have a limited lifetime, especially if not used regularly.
    • Eluting conditions may not be optimal, i.e. either not strong enough to elute the target molecule completely, or too strong and therefore destroying or modifying the immobilized ligand. Both effects lead to a loss in binding capacity.

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