CNBr Immobilization Procedure

Prepared by
Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111

List of Reagents and Instruments

A. Equipment

B. Reagents

Enzyme Immobilization Procedures

  1. Immerse glass fiber in DI water.
    Picture Figure: Immerse glass fiber in water. (Movie 238K)
  2. Add 5g CNBr per 50 mL of water.
    Picture Figure: Add CNBr. (Movie 297K)
  3. Maintain pH at 10-11 by adding NaOH, reflux overnight, and rinse with water.
    Picture Figure: Add NaOH. (Movie 304K)
  4. Immerse glass fiber/beads in 1g/L urease solution, and refrigerate for several days until use.
  5. Rinse with DI water several times, and check the glass fiber for urease activity before packing into a column.
  6. If enzyme is successfully immobilized on the glass surface, as indicated by positive activity, pack the glass fiber/beads into a column.
  7. Pump urea substrate to the bottom of the reaction column.
    Picture Figure: Pump substrate. (Movie 216K)
  8. As the substrate travels through the column, it is converted into product and opposing gradients of substrate and product concentrations develop within the column.
    Picture Figure: Reaction column. (Movie 236K)
  9. Reaction product exists the column into a collection jar.
    Picture Figure: Collection jar. (Movie 336K)

Data Acquisition Procedures

  1. Calibrate the spectrophotometer (Ocean Optics) for dark (i.e., 0% transmittance) by blocking the light path.
    Picture Figure: 0% transmittance. (Movie 575K)
  2. Calibrate the spectrophotometer for light (i.e., 100% transmittance) by placing a cuvet containing the reference sample, which is usually either water or the unreacted substrate solution, in the spectrophotometer.
    Picture Figure: 100% transmittance. (Movie 332K)
  3. Collect sample. Place it in the spectrophotometer.
    Picture Figure: Collect sample. (Movie 275K)
  4. Record absorbance at 558nm and save the sample spectrum.
    Picture Figure: Save sample spectrum. (Movie 424K)


  1. Adjust substrate flow rate with the speed knob on the pump. You can correlate the pump speed setting with the flow rate, but you should measure the actual flow rate with a graduated cylinder and a stop watch. You can visually inspect the flow (if you can see through the tubing) by momentarily lifting the tubing from the reservoir and introduce an air bubble. Of course, this does disrupt the flow pattern in the column a bit.
    Picture Figure: Watch the bubble move through the system. (Movie 424K)

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CNBr Immobilization Procedure
Forward comments to:
Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111
301-405-1910 (voice)
301-314-9126 (FAX)
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