PRODUCTS > CIM® Laboratory Columns

CIM® r-Protein L Disk Monolithic Column

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About the CIM® Disk Monolithic Columns*

Thank-you for purchasing your CIM® Disk Monolithic Columns. CIM® Disk Monolithic Columns are composed of a rigid, highly cross-linked monolithic polymer disk, and an appropriate housing. The monolithic columns are innovative chromatographic and bioconversion supports that offer new ways of performing chromatography. They can be used for the separation of large molecules such as proteins or DNA and also for smaller molecules such as peptides and oligonucleotides. CIM® Disk Monolithic Columns operate at high flow rates, low back pressures, and are extremely easy to use. The advantageous features of the CIM® Disk Monolithic Columns include:
  • Flow-unaffected resolution
  • High dynamic binding capacity unaffected by flow rate
  • Extremely fast separations
  • Low back pressure
  • User-exchangeable monolithic support
  • User-adjustable column length
  • Easy to use
*Please also review the Product Specific Information Sheet which accompanied these instructions for additional information.

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.

Unpacking

Carefully unpack the CIM® Disk Monolithic Column, CIM® disk(s) and housing(s), and inspect them for any damage that may have occurred during shipping. Report any such damage to the Transport Company and to your BIA Separations distributor within 30 days. CIM® Disks are shipped in a 20 % ethanol solution (Note: Reversed Phase-SDVB Disks are shipped in a 50 % ethanol solution) and need to be stored in the refrigerator at +4 to +8 °C (39 to 46 °F ) until ready to use.
WARNING: Do not store the CIM® Disk(s) below 0 °C (32 °F).

Optimization of the HPLC System for Efficient Separations

The set-up of the HPLC system is one of the crucial factors in achieving optimal performance from CIM® Disk Monolithic Columns. Since CIM® Disk Monolithic Columns are commonly used for gradient separations, the HPLC system should include at least two HPLC pumps connected via capillaries and a mixing tee to the injection valve or autosampler, a filling loop for the sample injection, a column, a detector, and a recorder or data acquisition system. Each of these components has a direct influence on the separation quality when fast analyses are required.
Capillaries: The inner diameter of the capillaries strongly affects the peak shape.
Using smaller diameters results in sharper peaks, increases the quality of the separation, and raises the back pressure of the LC system. Logically, small diameter capillaries also have a smaller volume, thus reducing the total system dead volume.
We recommend using capillaries with an inner diameter of 0.5 mm or less.
Back pressure: Check the back pressure of the system at a flow rate that is up to 2 mL/min higher than the flow rate that will be used during the actual work. It is advisable that the back pressure of the system alone is below 20 bar (2 MPa).
Mixing tee: A low dead volume mixing tee, e. g. 10 μL, should be used for fast gradient separations. This will improve the peak quality and resolution.
Detector: To obtain an optimal performance from CIM® Disk Monolithic Column(s), the detector response time should be set to the lowest possible value – for most UV detectors this is 0.1 s.
Acquisition rate: The acquisition rate depends on the analysis time. Typical analysis times in the case of CIM® Disk Monolithic Columns are just a few minutes. To obtain optimal resolution for separations of less than 1 minute, the acquisition rate should be 5 to 10 Hz.
Flow Rate: up to 10 mL/min (Note: typical flow rate is 3–6 mL/min).
Your HPLC system is now ready for fast analyses!

Preparation of the CIM® Disk Monolithic Column for Use

You may also view an Instructional Video for this product on the product instruction section.
The CIM® Disk Monolithic Column consists of a CIM® Disk (Figure 1), a housing (Figure 2) and the disk extractor (Figure 3). The chemistry of the disk is determined by the ring color as shown in the Ordering Information Section of this manual.

Assembly of the CIM® Disk Monolithic Column

(Refer to Figure 2)
  1. Unscrew one of the screw caps (e) from the housing.
  2. Remove one of the retaining fittings (d).
  3. Remove a CIM® Disk(s) from the labeled jar with a pincette and place it gently into the housing (hollow cylinder) (c).
    Note: Up to 4 CIM® Disks of the same or different chemistries (e. g., ion-exchange and affinity) can be inserted into one housing depending on the application.
  4. Reinsert the retaining fitting (d) to fix the disk approximately in the middle of the housing cylinder (c).
  5. Tighten both of the screw caps (e) by hand.
  6. Connect the CIM® Disk Monolithic Column to the HPLC system preferably using PEEK fingertight fittings (1/16" OD UNF 10–32). (Due to the symmetrical design of the column the flow direction is not important).
  7. We also recommend using high-pressure in-line filters between the column outlet and the detector.

Equilibrating the CIM® Disk Monolithic Column

Before you begin working with the CIM® Disk Monolithic Column the product should be equilibrated and the flow characteristics and backpressure checked in accordance with the following procedure:
  1. Wash the CIM® Disk Monolithic Column with at least 5 column volumes of the binding mobile phase (e. g. for ion-exchange chromatography a low ionic strength buffer) at one-half of the working flow rate.
    Note: Allow 2 column volumes to flow into a waste container. This will remove any small particles or air bubbles that may effect the detector cell.
  2. Wash the CIM® Disk Monolithic Column with at least 5 column volumes of the eluting mobile phase (e. g. for ion-exchange chromatography a buffer containing 1–2 M NaCl) at one-half of the working flow rate in order to elute any bound components and impurities.
  3. Finally, wash the CIM® Disk Monolithic Column again with at least 5 column volumes of the binding mobile phase at a working flow rate.
  4. Your CIM® Disk Monolithic Column is now ready for use.

Exchanging the Column Chemistry

One of the unique features of CIM® Disk Monolithic Columns is the fast and easy exchange of the monolith stationary phase. This is done as follows:
  1. Stop the pumps and disconnect the column from the HPLC/FPLC system.
  2. Remove both of the screw caps (e) and retaining fittings (d).
  3. Extract the disk from the housing cylinder (c) using the disk extractor (Figure 3).
  4. Place the CIM® Disk(s) in the labeled jar with 20 % ethanol.
    Note: Reversed Phase-SDVB Disks should be stored in at least 50 % Ethanol.
  5. The new CIM® Disk(s) is inserted according to the Assembly procedure (See above).

Operating Conditions

  1. Maximum Number of Disks per housing: 4
  2. Operating Flow Rates: up to 10 mL/min
  3. Operating BackPressure: up to 50 bar (5 MPa)
    WARNING:     Exceeding 5 MPa can seriously damage your column!
  4. Mobile Phases and Organic Solvents Used in Housings: pH, salt concentration, use of organic solvents
    • Columns using the Blue (made of HD-PE) and White Housing (made of Polyacetal) (No. 222.0830)
      1. Primarily for separation of biomolecules.
      2. Water-based phases within the pH range of 2–12 and at various ionic strengths (e. g., 6 M urea).
      3. Organic solvents, such as acetonitrile, should be avoided since they can seriously damage the column housing.
    • Columns using the PEEK housing (No. 222.0850)
      1. For separation of various molecules.
      2. Water-based phases within the pH range of 1–13 and at various ionic strengths (e. g., 6 M urea).
      3. Concentrated organic solvents (e.g., in reversed phase chromatography), such as acetonitrile, can be used without any limitations.
  5. Operating Temperatures
    • Polyacetal (Blue and White) (No. 222.0830): up to 50 °C (122 °F)
      WARNING: Exceeding 50 °C (122 °F) can seriously damage your column!
    • PEEK (No. 222.0850): up to 50 °C (122 °F)
      Note: PEEK housings can be thermally sterilized in the autoclave.

Caring for the CIM® Disk Monolithic Column

To extend the life of your CIM® Disk Monolithic Column, please observe the following guidelines:
  • Always use freshly prepared mobile phases (buffers).
  • Always filter your mobile phases (buffers) and samples through a 0.22 μm filter.
  • Protect the CIM® Disk Monolithic Column by using high-pressure in-line filters (e. g. PEEK filter, 231.0851).

Cleaning and Regeneration of the CIM® Disk Monolithic Column

Please refer to the Product Specific Information Sheet which accompanied these instructions.

Storing the CIM® Disk Monolithic Columns

Up to 2 days of planned storage after column use:
  • Wash the CIM® Disk Monolithic Columns with at least 5 column volumes of the eluting mobile phase (e. g. for ion-exchange chromatography a buffer containing 1–2 M NaCl).
  • Wash the CIM® Disk Monolithic Columns with at least 5 column volumes of the starting (binding) mobile phase.
  • Seal it tightly on both sides using the column blind fittings (Figure 2, f).
  • Store the column in the refrigerator.
    WARNING: Do not store the CIM® Disk(s) or the completely assembled column below 0 °C (32 °F).
3 or more days of planned storage after column use:
  • Wash the CIM® Disk Monolithic Columns with at least 5 column volumes of the eluting mobile phase (e. g. for ion-exchange chromatography a buffer containing 1–2 M NaCl).
  • Wash the CIM® Disk Monolithic Columns with at least 5 column volumes of the starting (binding) mobile phase.
  • Wash the CIM® Disk Monolithic Columns with at least 10 column volumes of 20 % ethanol.
  • Remove the disk (s) from the housing and store in a labeled jar with 20 % ethanol.
    Note: Reversed Phase-SDVB Disks should be stored in at least 50 % Ethanol.
  • Store CIM® Disk(s) in the refrigerator.
    WARNING: Do not store the disk(s) below 0 °C (32 °F).
WARNING: Never let the disk(s) dry out! Do not expose the CIM® Monolithic Disk(s) to dry air for more than 1 hour.

Troubleshooting

Problem

Possible source

Action
Increased back pressure
 Blocked disk, retaining fitting, mixing tee, capillaries or detector cell.
  • Exchange the disk, capillary or tee, wash the retaining fitting with distilled water under sonication.
  • Clear out the detector cell.
Leakage from the housing
 Screw caps not tightened enough.
O-ring damaged: Use of strong organic solvents.
  • Carefully tighten screw caps.
  • Exchange the O-rings.
Leakage at the inlet to or at the outlet from the housing
 Capillary fitting not tightened enough.
Damaged threads on the retaining fitting.
  • Carefully tighten the fitting.
  • Change the retaining fitting.
O-ring falls out  O-ring swelling from using strong organic solvents. 
  • Try to reinsert the O-rings into the retaining fittings. If the O-ring will not fit, then replace the O-rings.
Disk changes colors  Precipitate from the sample. 
  • Follow CIP Procedure in the Product Specific Information Sheet.
Poor resolution (i.e. wider peaks)  Various.
  • Perform regeneration and/or CIP procedure in the product specific information sheet.
No apparent separation  Various.
  • Check the pH and the composition of the buffer.
  • Verfiy that the disk chemistry is appropriate for your application.
Poor or no baseline separation  Various. 
  • Check the pH and the composition of the buffer.
  • Check the HPLC System (too much dead volume in the mixing chamber or capillaries).