PRODUCTS > CIM® Laboratory Columns

CIM® Epoxy Disk Monolithic Column

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About the CIM® EPOXY Monolithic Columns*

Thank-you for purchasing your CIM® Epoxy Disk or Tube Monolithic Column. CIM® Monolithic Columns are composed of a rigid highly cross-linked monolithic glycidylmethacrylate-co-ethyleneglycoldimethacrylate polymer and an appropriate housing. They can be used to immobilize ligands to perform specific separations of large and small biomolecules. The Epoxy CIM® Disk or Tube Monolithic Column operate 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-EpoxyD-0202, or PSIS-Epoxy8-0202, or PSIS-Epoxy80-0202).

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 (# IMD010701, or # IM8T010901, or # IM80T051201) that accompanied the CIM® Disk or Tube 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 exisiting chromatographic supports. Please consider the following when preparing your specific immobilization procedure:

  • Determine the pH stability of the ligand
    Epoxy 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 epoxy 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 epoxy binding sites.
    To facilitate coupling of the ligand to the epoxy 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 or pumped into the monolith's flow through pores. Once the monolith pores are filled with the ligand, the immobilization can be left to proceed.
    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® Disk Monolithic Columns

  1. Assemble the CIM® Disk Monolithic Column according to the Instruction Manual (# IMD010701) that accompanied this product.
  2. Equilibrate the CIM® 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 1–3 mL/min.
  3. Prepare your ligand solution by dissolving your ligand in a 0.5 M Na-Phosphate Buffer, pH 8.0 to obtain a final concentration of 3.0 mg/mL.
  4. Push or pump at least 1mL of the ligand solution through the CIM® Disk Monolithic Column to completely fill the monolith pores and collect the flow through in a beaker.
  5. Remove the CIM® Disk according to the Instruction Manual (# IMD010701) in the section – Exchanging the Column Chemistry.
  6. Place the disk in the beaker with the flow through solution (see step 4). You may also view an Instructional Video for this product on the product CIM® Tube Monolithic Columns consist of a CIM® monolith in an inert polyacetal (Ertacetal®) housing (Figure 1). The chemistry of the monolith and the flow direction are marked on the label.
  7. Cover the beaker with parafilm and let it stand for 24 hours at room temperature or 4 °C for 2–3 days.
    Note: Allow the immobilization to occur at the highest possible temperature that the ligand can withstand.
  8. Reinsert the CIM® Disk according to the Instruction Manual (# IMD010701) in the section – Exchanging the Column Chemistry.
  9. Reconnect the CIM® Disk Monolithic Column to the HPLC according to the Instruction Manual (# IMD010701).
  10. Wash the CIM ® 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 1–3 mL/min.
  11. Block the excess Epoxy groups according to the procedure listed in the Product Specific Information Sheet (# PSIS-EpoxyD.0202).
    WARNING: Take care that your particular ligand is stable in the reagent that will be used for blocking epoxy groups!
  12. Wash the CIM® Disk Monolithic Column by washing with 5 column volumes of a 0.5 M Na-Phosphate Buffer, pH 8.0 with 1 M NaCl added at a flow rate of 1–3 mL/min.
  13. Equilibrate the CIM® Disk Monolithic Column by washing with 5 column volumes of a 0. 5M Na-Phosphate Buffer, pH 8.0 at a flow rate of 1–3 mL/min.
  14. The immobilized or affinity CIM® Disk Monolithic Column is now ready for use.
    * The most appropriate buffer for your ligand should be chosen.

General immobilization procedure for CIM® Tube Monolithic Columns

  1. Attach the CIM® Tube Monolithic Column to the LC/HPLC as per the Instruction Manual (# IM8T010901, or # IM80T051201) that accompanied this product.
  2. Equilibrate the CIM® Tube Monolithic Column by washing with about 5 column volumes of a 0.5 M Na-Phosphate Buffer, pH 8.0* at a flow rate of 0.5–1.0 CV (column volume)/minute.
  3. Prepare your ligand solution by dissolving your ligand in a 0.5M Na-Phosphate Buffer, pH 8.0 to obtain a final concentration of 3.0 mg/mL.
    Note: The total amount of ligand solution should be equal to at least 1CV in order to completely fill the monolith flow through pores.
  4. Pump the entire ligand solution through the CIM® Tube Monolithic Column at a flow rate of 0.5–1.0 CV/min.
  5. When all of the solution is pumped into the column, stop the pumps so that the ligand solution will become entrapped in the flow through pores of the monolith.
  6. Disconnect the CIM® Tube Monolithic Column from the LC/HPLC.
  7. Close the CIM ® Tube Monolithic Column on both sides with the end fittings.
  8. Store the CIM® Tube Monolithic Column at room temperature for 24 hours or 4 °C for 2–3 days.
    Note: Allow the immobilization to occur at the highest possible temperature that the ligand can withstand.
  9. Reconnect the CIM ® Tube Monolithic Column to the HPLC according to the Instruction Manual (# IM8T010901, or # IM80T051201) that accompanied this product.
  10. Wash the CIM® Tube 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.
  11. Block the excess Epoxy groups according to the procedures listed in the Product Specific Information Sheet (# PSIS-Epoxy 8-0202, or # PSIS-Epoxy80-0202).
    WARNING: take care that your particular ligand is stable in the reagent that will be used for blocking epoxy groups!
  12. Wash the CIM® Tube Monolithic Column by washing with 5 column volumes of a 0.5 M Na-Phosphate Buffer, pH 8.0 with 1 M NaCl added at a flow rate of 0.5–1.0 CV/minute.
  13. Equilbrate the CIM® Tube 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.
  14. The immobilized or affinity CIM® Tube Monolithic Column is now ready for use.
    * The most appropriate buffer for your ligand should be chosen.

Operating Conditions

1. CIM® Disk and Tube Housings: Please refer to Operating Conditions in the Accompanying Instruction Manual (# IMD010701, or # IM8T010901, or # IM80T051201).
2. Affinity CIM ® Disk and Tube Monolithic Columns: this is dependent on particular ligand immobilized.

Cleaning and Regeneration of the CIM® Tube Monolithic Column

To extend the life of your CIM® Disk and Tube 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® Disk and Tube 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® Disk and Tube Monolithic Columns

Up to 2 days of planned storage after column use:

  • Seal the CIM® Disk or Tube 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:
Disk

  • Remove the CIM® Disk from the housing and store in a labeled jar with an appropriate bacteriostatic agent eg. 20% 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® Disk in the refrigerator.
    WARNING: Do not store the disk(s) below 0 °C (32 °F)!
Tube
  • Fill the CIM® Tube Monolithic Column with at least 1 CV of a 20 % 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)!
  • Seal the CIM® Tube Monolithic Columns tightly on both sides using the column blind fittings.
  • Store the CIM® Tube Monolithic Columns in the refrigerator.
    WARNING: Do not store the columns(s) below 0 °C (32 °F)!
  • WARNING: Never let the disk(s) or tube(s) dry out! Always keep the CIM® Monolithic Columns wetted with the buffer or storage solution!

    Troubleshooting

    1. Quantity of immobilized ligand is small:
      • Optimize immobilization procedure by considering the parameters like: coupling buffer, pH, time, temperature and ligand concentration.
      • Assure yourself that the monolithic column has been thoroughly washed and equilibrated with the coupling buffer.
      • Make a new column and compare. Sometimes immobilization does not work without any good explanation.
    2. Quantity of immobilized ligand is as expected, but affinity/ capacity is bad:
      • Check if 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, can have occurred. Try developing a general procedure to sanitize (gently!) the column or a CIP procedure.
      • Immobilization of the ligand can 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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