1. Immerse glass fiber in DI water.
   Figure: Immerse glass fiber in water. (Movie 238K)
   
2. Add 5g CNBr per 50 mL of water.
   Figure: Add CNBr. (Movie 297K)
   
3. Maintain pH at 10-11 by adding NaOH, reflux overnight, and rinse with water.
   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.
   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.
   Figure: Reaction column. (Movie 236K)
   
9. Reaction product exists the column into a collection jar.
   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.
   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.
   Figure: 100% transmittance. (Movie 332K)
   
3. Collect sample. Place it in the spectrophotometer.
   Figure: Collect sample. (Movie 275K)
   
4. Record absorbance at 558nm and save the sample spectrum.
   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.
   Figure: Watch the bubble move through the system. (Movie 424K)
   

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CNBr Immobilization Procedure
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Nam Sun Wang Department of Chemical & Biomolecular Engineering University of Maryland College Park, MD 20742-2111 301-405-1910 (voice) 301-314-9126 (FAX) e-mail: nsw@umd.edu