Lab 1

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271

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Chemistry

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Apr 3, 2024

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DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY CHEM 271 LABORATORY SECTION 03 MONDAY AFTERNOON LABORATORY 1: PIPETTING AND SPECTROPHOTOMETER PRACTICE, BUFFER PREPARATION PART 1 Sujay Patel 40283272 Date of Experiment: January 29, 2024 Date of Report Submission: February 12, 2024

Experimental Report 1 Part 1: Use of Micropipettors 1. What are your absorbance values at 410 nm for your triplicate solutions from the measurements? Please refer to Table 1: Data of Triplicate Solutions Test in section Results. 2. What absorbance values did you get using increasing volumes of p -nitrophenol? Do you get a proportional increase in absorbance? (Is the absorbance of tube #4 three times that of tube #2? Is tube #3 two times that of tube #2?) To find the absorbance values for each tube, please refer to Table 2: Absorbance of Tubes Using Increasing Volumes of P -Nitrophenol. According to the values obtained using increasing volumes of p- nitrophenol in each tube, there is a proportional increase in absorbance. The absorbance of tube #4 is approximately 3 times ( 1.4749 0.4865 = 3.032 ) that of tube #2 and tube #3 is approximately 2 times ( 0.9752 0.4865 = 2.005 ) that of tube #2.

3. Do tube #1 and tube #2 have the same absorbance? Should they have the same absorbance? According to the data of Table 2: Absorbance of Tubes Using Increasing Volumes of P -Nitrophenol , The absorbance value of both tubes are different with a difference of 0.0849 Au ( 0.5714 − 0.4865 = 0.0849 ). However, since they both are using the same volumes of buffer and p -nitrophenol, they should have the same values even if they were made using different pipettor. Part 2: Kinetics of Hydrolysis of p -nitrophenylacetate 1. Show your calculations to prepare all the p -nitrophenylacetate solutions starting from the stock solutions. Can you use serial dilution? Explain how. Please refer to section Calculations to find the sample calculations used to prepare the p - nitrophenylacetate solutions. Unfortunately, for this part of the experiment, serial dilution is not an optimal method to use when preparing the solutions because, in this method, the dilution factor must remain constant for each solution whereas, in this experiment, the dilution factors are not the same.

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2. Plot the rate of the reaction vs. [p-nitrophenylacetate]; you should get a straight line. Do not forget to include a descriptive title and the units. This is a reaction that is first-order in substrate-the rate is a linear function of the substrate concentration. Later in the course, you will learn about Michaelis-Menten kinetics. At that time, look at this data again and see the difference between first-order kinetics and Michaelis-Menten kinetics. Please refer to section Results to find Plot 1: Reaction Rate of Hydrolysis compared to the volume of p -Nitrophenylacetate .

Calculations Preparation of p -nitrophenylacetate solutions (Sample Calculations): Solution 1 (0.05 nM): Final [ p − nitrophenylacetate ] = C 2 = 0.05 mM Initial [ p − nitrophenylacetate ] = C 1 = 10 mM Final volume of solution = V 2 = 5 mL InitialVolumeof solution = V 1 = ? Formula = C 1 V 1 = C 2 V 2 C 2 V 2 C 1 = V 1 0.05 nM ∗ 5 mL 10 mM = 0.025 mL Volumeneeded for 0.05 nM of p − nitrophenylacetateis 0.025 mL ¿ theinitalsolution . Volumeof water needed = 5 mL − 0.025 mL = 4.975 mL

Solution 2 (0.10 mM): V 1 = 0.05 mL Volumeof water = 4.95 mL Solution 3 (0.25 mM): V 1 = 0.125 mL Volumeof water = 4.875 mL Solution 4 (0.5 mM): V 1 = 0.25 mL Volumeof water = 4.75 mL Solution 5 (1.5 mM): V 1 = 0.75 mL Volumeof water = 4.25 mL

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Results Table 1: Data of Triplicate Solutions Test Tube Absorbance (Au) 1 0.2309 2 0.2200 3 0.2184 Average 0.2231 Table 2: Absorbance of Tubes Using Increasing Volumes of P -Nitrophenol Tube Absorbance Value (Au) 1 0.5717 2 0.4865 3 0.9752 4 1.4749 Table 3: Hydrolysis Rate of Reaction Compared to the Concentration of P - Nitrophenylacetate Solutions [ P- Nitrophenylacetate] (mM) Hydrolysis Rate 1 0.05 0.007294724 2 0.10 0.0141073 3 0.25 0.0576928

4 0.05 0.466747 5 1.5 0.4290549 Table 4: Hydrolysis Rate of Reaction Compared to the Concentration of P - Nitrophenylacetate (Without Aberration) Solutions [ P- Nitrophenylacetate] (mM) Hydrolysis Rate 1 0.05 0.007294724 2 0.10 0.0141073 3 0.25 0.0576928 5 1.5 0.4290549 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 f(x) = 0.19 x + 0.12 R² = 0.27 Plot 1: Hydrolysis Rate of Reaction Compared to the Concentration of P-Nitrophenylacetate in mM [P-Nitrophenylacetate] (mM) Reaction Rate

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 f(x) = 0.29 x − 0.01 R² = 1 Plot 2: Hydrolysis Rate of Reaction Compared to the Concentration of P-Nitrophenylacetate in mM (Without Aberration) [P-Nitrophenylacetate] (mM) Reaction Rate Due to Solution 4 being an aberration, the result would have changed significantly and false therefore with the permission of the Lab technician, Solution 4 has been removed from the data.

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References - Powloski, J.; Kornblatt, M.J.; Joyce, P.; Sahlman, L.; Turbull, J.; Ciortea M. Biochemistry I – Laboratory Manual CHEM 271 ; Concordia University , Montreal, QC; p53-57