Spectrophotometer Lab
For this lab it was necessary to bring a watch with a second hand, as well as personal protective gear: lab coat, safety goggles, and safety gloves. It was best to work in pairs and one partner needed to be a timekeeper while the other one would record the data. The timekeeper then would announce every 5 second interval, beginning from when the enzyme was added to the tube. On the other hand, the recorder would read and record the absorbance from the spectrometer at the 5 second intervals. This was done for all four experiments so the absorbance could be recorded every 5 seconds for 60 seconds. It was highly important to keep in mind that all the tubes that would enter the spectrophotometer are clean and dry before the substrate and buffer was added.
In the first experiment, optimal temperature for enzymatic activity was tested. Five clean spectrophotometer tubes wereare necessary with the different temperatures labeled on them using a wax pencil (Blank, Room Temp, 35 degrees Celsius, 45 degrees Celsius, and 55 degrees Celsius). Then 1mL of …show more content…
substrate mixture and 1 mL of pH 6 buffer was added to each tube. After that, one tube was placed in each of the three temperature baths (35 degrees Celsius, 45 degrees Celsius, and 55 degrees Celsius). Only tube 1 and 2 would remain at room temperature (25 degrees Celsius). While waiting 20 minutes for the tubes to equilibrate, it is important to add 0.1 mL of pH 6 buffer to the blank test tube. After the wait, one spectrophotometer tube was removed and 0.1 mL of enzyme from vial E1 ( 1x10^-7 M peroxidase) was added then shaken with a piece of parafilm covering the top. The test tube was placed quickly in the spectrophotometer and the absorbance was recorded every 5 seconds for 60 seconds. This was repeated every time the enzyme was added to a tube at a time, and the data was recorded.
As for the second experiment, the enzymatic activity was tested in various pH levels to find the optimal pH.
Again, five clean spectrophotometer tubes were necessary with the different pH numbers labeled on them using a wax pencil (3, 4, 5, 6, and 8), and 1 mL of the buffers with those pH levels was added. Each tube needed 1 mL of substrate mixture which was added. Then the spectrophotometer to 470nm and the first spectrophotometer tube was wiped to be used to blank the spectrophotometer. Then 0.1 mL of enzyme from vial E1 ( 1x10^-7 M peroxidase) was added, then it was shaken with parafilm covering the top. The tube was quickly placed into the spectrophotometer, and it was necessary to be sure not to spill any of the reaction mixture or this experiment would have to be repeated. The absorbance was recorded every 5 seconds for 60 seconds and this process was repeated for each spectrophotometer tube that went in one at a
time.
Following that experiment was experiment three that tested the varying substrate concentration to prove an optimal concentration for high enzymatic activity. For this one, six clean spectrophotometer tubes were necessary with the different guaiacol measurements labeled on them using a wax pencil (1.25 mM, 2.5 mM, 5 mM, 7 mM, 10 mM, and 20 mM). Each tube would begin with 1 mL of pH 6 buffer. Then one tube at a time, the respective guaiacol measurements were added and the tube was shaken with a parafilm covering the top. After that is was quickly placed in the spectrophotometer at 470 nm. The first spectrophotometer tube was tested as the blank. Continue the experiment for each tube and record the absorbance every 5 seconds for 60 seconds.
The final experiment was a test for the optimal enzyme concentration that would have high enzymatic activity. Five clean spectrophotometer tubes were necessary with the different peroxidase amounts labeled on them using a wax pencil ( E2 - 2.5x10^-8 M, E3 - 5x10^-8 M, E4 - 1x10^-7 M, E5 - 2x10^-7 M, and E6 - 4x10^-7 M). Each tube began with 1 mL of substrate mixture and 1 mL of pH 6 buffer. Then with one tube at a time, the respective peroxidase measurements were added, and it was shaken with a parafilm covering the top. It was then placed in the spectrophotometer at 470 nm. This first spectrophotometer tube was tested the blank to blank the spectrophotometer. After the first test tube, the experiment was continued for each tube and the absorbance was recorded every 5 seconds for 60 seconds.
In the first experiment, optimal temperature for enzymatic activity was tested. Five clean spectrophotometer tubes wereare necessary with the different temperatures labeled on them using a wax pencil (Blank, Room Temp, 35 degrees Celsius, 45 degrees Celsius, and 55 degrees Celsius). Then 1mL of …show more content…
substrate mixture and 1 mL of pH 6 buffer was added to each tube. After that, one tube was placed in each of the three temperature baths (35 degrees Celsius, 45 degrees Celsius, and 55 degrees Celsius). Only tube 1 and 2 would remain at room temperature (25 degrees Celsius). While waiting 20 minutes for the tubes to equilibrate, it is important to add 0.1 mL of pH 6 buffer to the blank test tube. After the wait, one spectrophotometer tube was removed and 0.1 mL of enzyme from vial E1 ( 1x10^-7 M peroxidase) was added then shaken with a piece of parafilm covering the top. The test tube was placed quickly in the spectrophotometer and the absorbance was recorded every 5 seconds for 60 seconds. This was repeated every time the enzyme was added to a tube at a time, and the data was recorded.
As for the second experiment, the enzymatic activity was tested in various pH levels to find the optimal pH.
Again, five clean spectrophotometer tubes were necessary with the different pH numbers labeled on them using a wax pencil (3, 4, 5, 6, and 8), and 1 mL of the buffers with those pH levels was added. Each tube needed 1 mL of substrate mixture which was added. Then the spectrophotometer to 470nm and the first spectrophotometer tube was wiped to be used to blank the spectrophotometer. Then 0.1 mL of enzyme from vial E1 ( 1x10^-7 M peroxidase) was added, then it was shaken with parafilm covering the top. The tube was quickly placed into the spectrophotometer, and it was necessary to be sure not to spill any of the reaction mixture or this experiment would have to be repeated. The absorbance was recorded every 5 seconds for 60 seconds and this process was repeated for each spectrophotometer tube that went in one at a
time.
Following that experiment was experiment three that tested the varying substrate concentration to prove an optimal concentration for high enzymatic activity. For this one, six clean spectrophotometer tubes were necessary with the different guaiacol measurements labeled on them using a wax pencil (1.25 mM, 2.5 mM, 5 mM, 7 mM, 10 mM, and 20 mM). Each tube would begin with 1 mL of pH 6 buffer. Then one tube at a time, the respective guaiacol measurements were added and the tube was shaken with a parafilm covering the top. After that is was quickly placed in the spectrophotometer at 470 nm. The first spectrophotometer tube was tested as the blank. Continue the experiment for each tube and record the absorbance every 5 seconds for 60 seconds.
The final experiment was a test for the optimal enzyme concentration that would have high enzymatic activity. Five clean spectrophotometer tubes were necessary with the different peroxidase amounts labeled on them using a wax pencil ( E2 - 2.5x10^-8 M, E3 - 5x10^-8 M, E4 - 1x10^-7 M, E5 - 2x10^-7 M, and E6 - 4x10^-7 M). Each tube began with 1 mL of substrate mixture and 1 mL of pH 6 buffer. Then with one tube at a time, the respective peroxidase measurements were added, and it was shaken with a parafilm covering the top. It was then placed in the spectrophotometer at 470 nm. This first spectrophotometer tube was tested the blank to blank the spectrophotometer. After the first test tube, the experiment was continued for each tube and the absorbance was recorded every 5 seconds for 60 seconds.