CBL Interfacing
Photosynthesis In Isolated Chloroplasts
Procedure
A. Wrap four cuvettes in aluminum foil.
B. Using the appropriate pipette and pump, add phosphate buffer,
distilled water, DPIP and chloroplast suspension according to
the flow chart shown below. Your teacher will have test tubes
with boiled and unboiled chloroplast suspensions which are in
an enclosed test tube which is stored in an ice water bath. Maintain
your wrapped cuvettes, with their appropriate solutions, in ice
water baths also. Cover each cuvette with a numbered lid (1,2,3
or 4). Invert to thoroughly mix the cuvette. Now follow the instructions
below to properly set up the TI-82 and CBL and colorimeter.
C.
Setting Up The TI-82, CBL and Barometer
Connect the CBL unit to the TI-82 graphics calculator using the
unit-to-unit link cable and the I/O port located at the bottom
edge of each unit.
Connect a Vernier Colorimeter to the Channel 1 port located at
the top of the CBL unit.
B.
Entering the Program
Press ON to turn on your CBL.
Press ON to turn on your TI-82 calculator.
Press PRGM.
Choose SPECTRO.
prgm SPECTRO will appear on your screen. Press ENTER.
VERNIER SOFTWARE, SPECTROPHOTOMETER WITH CBL will appear on your
screen. Press ENTER.
C.
Setting up the Probe
A menu will appear. Choose SET UP PROBE.
CONNECT SPECTROPHOTOMETER TO CHANNEL 1 will appear. Press ENTER.
SET TRANSMITTANCE TO 0 PERCENT.....will appear. Set spectrophotometer
to 0 %T, allow voltage to stabilize and press TRIGGER on the CBL.
SET TRANSMITTANCE T 100 PERCENT...will appear. Set spectrophotometer
to Red (635 nm), insert the blank (test tube #1) , allow
voltage to stabilize and press TRIGGER on the CBL. Remove the
blank.
A menu will appear. Choose COLLECT DATA.
A menu will appear.
D. Precise timing is crucial to the remainder of the experiment.
Slide test tube #2, Dark out of its aluminum foil sleeve, wipe
it dry and clean with a tissue and place it in the colorimeter.
E. From the menu on the TI-82 choose MONITOR INPUT.
F. The spectrophotometer provides two types of information, the
% transmittance, shown on the CBL and absorption, shown on the
calculator. Record the absorbance and the precise time on your
data table (see Data Sheet).
G. Place cuvette 2, Dark back into its aluminum foil sleeve and
position it in the specimen position as is directed by the diagram
below. You will need to repeat your readings after 5, 10 and 15
minutes. Record all data below. (See Data Sheet).
H. Repeat procedural steps 1-4 for cuvette 3, unboiled-light,
but
DO NOT
return the cuvette to its aluminum foil sleeve. Be sure to plan
your timing so that you will not have to read more than one cuvette
within about one half of a minute of another cuvette. Record all
data below. (See Data Sheet)
I. Repeat procedural steps 1-4 for cuvette 4, boiled-light, but
DO NOT
return the cuvette to its aluminum foil sleeve. Be sure to plan
your timing so that you will not have to read more than one cuvette
within about one half of a minute of another cuvette. Record all
data below. (See Data Sheet)
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