lab report #1
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Part 1: Use of Micropipettes
1.
Test Tube 1
0.2587 A.U. Test Tube 2
0.2787 A.U.
Test Tube 3
0.2683 A.U.
Absorbance values of p-nitrophenol and glycine buffer at 410nm of cuvette prepared with three tubes of the same concentrations to test pipetting competency. 2.
0.2ml p-nitrophenol (Test tube 1)
0.5116 A.U.
0.2ml p-nitrophenol (Test tube 2)
0.5417 A.U.
0.4ml p-nitrophenol (Test tube 3)
1.0238 A.U.
0.6ml p-nitrophenol (Test tube 4)
1.3954 A.U.
Absorbance values of increasing volumes of p-nitrophenol and glycine buffer were recorded by a
spectrometer at 410nm and zeroed with water as a reference. There is a proportional increasing positive relationship between the amount of p-
nitrophenol added and the absorbance. The increase is not exactly linear as the average of the first tubes (0.2 ml of p-nitrophenol) is 0.5267 which would give the expected linear amount of tube #3 to be 1.0533, but this value is not far away from the absorbance value obtained of 1.0238. Tube #4 is expected to have an absorbance if directly proportional to 1.5801, but the obtained value from the experiment is lower. These values should be directly proportional though due to the assumption in The Beer-Lambert law. In this law, absorbance should be directly proportional to the concentration as the amount of light intensity projected at the cuvette is measured to see the amount of intensity lost on the other side of the cuvette (Clark, 2016). The
amount of intensity lost in this process is the absorption by the sample, which should increase with the increasing amount of concentration as more molecules interact with the light (Clark,
2016). These lower amounts could be due to only completing one trial for the 0.4ml and 0.6ml of p-nitrophenol or improper pipette techniques resulting in lower concentrations. 3.
Test tubes 1 and 2 do not have the same numbers, but the two numbers fall within a similar range. They should have the same value as both received the same amount of p-
nitrophenol in the test tubes. Although test tube 1 and test tube 2 used two different micropipettes with differing ranges, both are able to achieve a similar absorbance value result as both allocated the same amount of solution. Part 2 Kinetics of hydrolysis of p-nitrophenylacetate
1.
Calculations to find the dilutions were found using the formula M
1
V
1
=M
2
V
2 and it was rearranged to V
1
=(M
2
V
2
)/M
1
This was used to find the Test tube 1 by inserting the known value
M
2
-0.05mM
V
2
-5 ml
M
1
-10mM
V
1
=(5ml x 0.05mM)/ 10mM =0.025ml of stock solution of 10mM p-nitrophenylacetate and 4.975ml of water
It is possible to make a serial dilution by following specific dilution factors for the preparation of
each sample. For this procedure to be successful the first test tube prepared should be #5 (1.5mM) and then subsequent dilutions should take place using this sample. The 1.5mM solution
could be diluted to prepare the #4 0.5mM sample by using 1.67ml of solution in 3.33ml of distilled water to make a ⅓ dilution. The subsequent test tube #3 0.25mM could be prepared via a ½ dilution using 2.5 ml of #4 0.5mM and 2.5 ml of distilled water. The next dilution for #2 0.10mM would require 2ml of #3 0.25mM and 3ml of distilled water. Finally, the #1 test tube could be prepared using a ½ dilution using 2.5 ml of #2 0.1mM and 2.5ml of water. This technique is typically used to make very diluted solutions from once concentrated solution, but if
your dilutions were kept track of this method could work for this procedure. 2
Diagram depicting serial dilution preparation for the differing concentrations of p-
nitrophylacetate. 2.
0.05mM p-nitrophenylacetate
0.0005092293
0.10mM p-nitrophenylacetate
0.01942366
0.25mM p-nitrophenylacetate
0.03258989
0.5 p-nitrophenylacetate
0.05606691
1.5 mM p-nitrophenylacetate
0.216025
Data points for the recorded slope of absorbance (A.U) vs time (secs) for differing p-
nitrophenylacetate concentrations in a glycine buffer. These data points were used to form Figure 1. 3
#4 0.5mM
#3 0.25mM
#2 0.1mM
#1 0.05mM
#5 1.5mM
2.5ml distilled water
2ml distilled water
2.5ml distilled water
3.33ml distilled water
1.67ml
2.5ml
3ml
2.5ml
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Figure 1. A spectrophotometer measured the Hydrolysis of differing p-nitrophenylacetate concentrations in a glycine buffer. The slope (m=0.1497 M/sec) indicates the rate of hydrolysis of the p-nitrophenylacetate in the buffer. Prelab and Raw Data: Scanned and attached at the end of the report. Sample Calculations:
Conversion of ml to ul:
0.1ml of p-nitrophenol x 1000
ul
1
ml
=
100
ul
Citations:
Clark, J. (2016, May). THE BEER-LAMBERT LAW
. Absorption spectra - the beer-lambert law. https://www.chemguide.co.uk/analysis/uvvisible/beerlambert.html Powloski, J., Kornblatt, M. J.,
Joyce, P., Sahlman, L., Turnbull, J., & Ciortea, M. (2023). Chemistry 271 Laboratory and Tutorial Manual (Vol. January 2023). Concordia University. 4
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