Add 5g CNBr per 50 mL of water.
Figure: Add CNBr. (Movie 297K)
Maintain pH at 10-11 by adding NaOH, reflux overnight, and rinse with water.
Figure: Add NaOH. (Movie 304K)
Immerse glass fiber/beads in 1g/L urease solution, and
refrigerate for several days until use.
Rinse with DI water several times, and check the glass fiber for
urease activity before packing into a column.
If enzyme is successfully immobilized on the glass surface, as
indicated by positive activity, pack the glass fiber/beads into a
Pump urea substrate to the bottom of the reaction column.
Figure: Pump substrate. (Movie 216K)
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)
Reaction product exists the column into a collection jar.
Figure: Collection jar. (Movie 336K)
Data Acquisition Procedures
Calibrate the spectrophotometer (Ocean Optics) for dark (i.e., 0%
transmittance) by blocking the light path.
Figure: 0% transmittance. (Movie 575K)
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)
Collect sample. Place it in the spectrophotometer.
Figure: Collect sample. (Movie 275K)
Record absorbance at 558nm and save the sample spectrum.
Figure: Save sample spectrum. (Movie 424K)