Onuska 8

A UV-visible Spectroscopic Study of the Kinetics of Imidazole Catalyzed Hydrolysis of p-Nitrophenyl Butyrate

Nicholas Onuska

Analytical Chemistry Laboratory II

02/13/15

Abstract:

A study of the affects of various concentrations of imidazole on the rate of imidazole-catalyzed hydrolysis of p-nitrophenyl butyrate is presented. UV-visible spectroscopy was used to quantify changes in the concentration of the product of the reaction with respect to time. The rate constant obtained, assuming pseudo-first-order kinetics, is less than literature values for analogous ester of a shorter chain length, indicating that steric hindrance of the longer alkyl chain retards the rate of the reaction.

1.) Introduction

UV (ultraviolet)-Vis (visible) absorption spectroscopy is a versatile and deceptively simple method for analysis within a variety of experimental designs. For substrates with absorbance within the ultraviolet-visible range (~ 100 1100 nm), UV-Vis spectroscopy measurements can be used to quantify reaction kinetics, chemical equilibrium and concentration, and identify molecules based on their absorbance fingerprint. UV-Vis spectrometers are inexpensive compared to many other instruments and are therefore readily available in many labs. Additionally, analysis by UV-Vis spectroscopy takes place in real time with little delay due to signal processing. In the case of this experiment, UV-Vis spectroscopy was used to measure the rate constant for the hydrolysis of p-nitrophenyl butyrate in the presence of imidazole (Figure 1).

Figure 1: Imidazole mediated hydrolysis of p-nitrophenyl Butyrate4-Nitrophenol (1) posses a bright yellow coloration, while all the other reactants and products are colorless, making the reaction very easy to monitor using absorption spectroscopy. The process is thought to occur by either a base catalyst process (Figure 2) or nucleophilic catalysis process[1],[3] (Figure 3).

Figure 2: Generalized transition state for base catalysis process[2]

Figure 3: Generalized mechanism for nucleophilic catalytic process[2]In both processes, imidazole is present in the transition state/rate determining step, and will therefore be present in the corresponding rate law[2]. In a system where the concentration of a catalyst is much greater than the concentration of the primary reactant, and assuming first order behavior with respect to the catalyst, the observed rate constant (kobs) is equal to the rate constant of the uncatalyzed reaction (k0) plus the rate constant of the catalyzed reaction (kcat) multiplied by the concentration of the catalyst ([C]) (Equation 1). (1)While experiments on nitrophenyl esters have been documented using 4-nitrophenylacetate, this study focuses on 4-nitrophenyl butyrate and prompts a comparison of the relative rates of hydrolysis of the two esters and potential causes of any differences in reaction rate.

2.) ExperimentalAll spectra were collected on a Varian Cary 50 UV-Vis spectrometer using a quartz cuvette. Solutions of imidazole (imidazole obtained from Acros Organics) were prepared using water as a solvent in 4 concentrations. A table of dilutions is given below (Table 1).

Table 1: Prepared solutions of imidazole in water

Mass of Imidazole (g)Moles of ImidazoleFinal Volume (mL)Concentration (mol/L)

0.2060.003031000.0303

0.3450.005071000.0507

0.490.007201000.0720

0.6240.009171000.0917

Using a 2M solution of hydrochloric acid, the pH of each prepared solution of imidazole was adjusted to be between 6 and 8. pH was monitored using a digital pH meter. The final pH of each solution is given below (Table 2).

Table 2: pH of prepared imidazole solutions after adjustment.Imidazole concentration (mol/L)pH

0.03037.63

0.05077.60

0.07207.68

0.09177.79

A 6.21-mM solution of p-nitrophenyl butyrate (NPB) was prepared by dissolving NPB (0.065 g) in acetonitrile (50 mL). Before recording any spectra, the UV-Vis instrument was turned on and allowed to warm up for half of an hour. In order to select the wavelength best suited to monitoring the formation of the product, 4-nitrophenol, a full absorption spectrum of the NPB solution as well as a solution of the product was taken. To obtain a sample of products, 2.5 mL of the 0.0917 M imidazole solution was mixed with 50 L of the 6.21 mM NPB solution, stirred and allowed to sit for 10 minutes before taking the absorbance spectrum. This gave sufficient time for the reaction to progress to completion. To eliminate matrix effects produced by the absorbance of the solvents, the spectrometer was zeroed using a solution composed of 2.5 mL H2O and 50 L acetonitrile. A full absorption spectrum, ranging from 190 1100 nm was taken of each sample. 400 nm was visually identified as a wavelength that possessed low absorbance in the starting material, and high absorbance in the product, making it an ideal wavelength at which to monitor changes in the concentration of the product. The full absorption spectrum for each sample is given below (Graph 1 and Graph 2).

Graph 1: Full absorbance spectra of products of ester hydrolysis reaction

Graph 2: Full absorbance spectra of 6.21 mM NPB solution

With 400 nm selected as the wavelength for the kinetics runs, samples were run varying the concentration of imidazole. A general procedure is given below, and was repeated for each of the four concentrations of imidazole.

General procedure:The UV-vis spectrometer was first zeroed using matrix-matching corresponding to the reaction solvents: 50 L acetonitrile and 2.5 mL of water. In order to obtain a reading for the initial absorbance of the imidazole solution, 2.5 mL of the imidazole solution was added to the cuvette. Initial absorbance (A0) was recorded with the parameters presented below (Table 3).

Time ScaleRun Time Cycle TimeWavelengthRate Calculation

Seconds30 seconds5 seconds400 nmNone

Table 3: UV-vis software parameters for the determination of initial absorbance

After the initial absorbance spectrum of the sample was collected, the software parameters were changed in order to increase run time (Table 4). This ensured that the reaction would reach completion before the spectral recording process ended. The absorbance at any time, t, during the reaction was determined from this spectrum. This absorbance is referred to henceforth as At.

Time ScaleRun Time Cycle TimeWavelengthRate Calculation

Seconds400 seconds5 seconds400 nmNone

Table 4: UV-vis software parameters for the determination of At

As soon as the kinetics run was started, the cover of the spectrometer was opened, and 50 L of the 6.21 mM NPB solution was injected into the cuvette using a micropipette (while the cuvette was still secured in the holder within the spectrometer) and the reaction mixture was stirred using the tip of the micropipette. After stirring for several seconds, the cover was closed and the full run time was allowed to pass. The absorbance after 6 minutes was assumed to be the absorbance of the reaction after it had reached completion. This value of absorbance is referred to henceforth as A. This general procedure was repeated for all concentrations of imidazole prepared, using the 6.21 mM NPB solution each time.

After the absorption versus time graphs were collected, the data was exported to Microsoft Excel, which was used for all manipulations of data and generations of graphs. Assuming pseudo-first-order reaction behavior, the rate constant of each reaction was calculated the slope of a linear best fit line fit to the graph of ln[(A-A0)/(A-At)] versus (t-t0) where t is the time corresponding to absorbance reading At and t0 is the so called dead time of the reaction, corresponding to the period of time before the NPB solution was injected into the reaction cuvette. These calculations and linear fitting were repeated for each concentration of imidazole. The calculated rate constants (kobs) were plotted versus the total concentration of imidazole in each sample. In this case, kcat, the rate constant for the catalyzed reaction, was determined as the slope of a line fitted to this curve. The rate constant for the uncatalyzed reaction, k0, was calculated from the y-intercept of the line fitted to the kobs versus [imidazole] graph.

3.) Results and DiscussionThe plot of ln[(A-A0)/(A-At)] versus (t-t0) for each concentration of imidazole is given below (Graph 3). Graph 3: Graphs of ln[(A-A0)/(A-At)] versus (t-t0) for all imidazole concentrations

The equations of the linear best-fit lines for each plot are given in the below table (Table 5).

Concentration of ImidazoleEquation of best fit lineSlope of best fit line (Kobs)

0.0303 My = 0.0131x - 0.37260.0131

0.0507 My = 0.0157x + 0.34370.0157

0.0720 My = 0.0181x-0.24080.0181

0.0917 My = 0.0187x + 0.63930.0187

Table 5: Information obtained from best fit lines of ln[(A-A0)/(A-At)] versus (t-t0) curvesAs the concentration of imidazole increased, the calculated rate constant (Kobs) increased as well. Plotting Kobs versus imidazole concentration gave a curve, which upon fitting with a linear regression, yielded an R2 value of 0.9474 (Graph 4).

Graph 4: Observed rate constants versus imidazole concentration, NPB concentration = 6.21 MThe linear relationship between kobs and imidazole concentration showed that the reaction was indeed first order with respect to imidazole, matching previously reported results[1],[2],[3].By examining the slope of the best-fit line for the graph of observed rate constants versus imidazole concentrations, the value of kcat was found to be 0.0937 s-1 and the value of k0 (the rate constant of the uncatalyzed reaction) was found to be 0.0107 s-1. This shows that the addition of a catalyst (hydrolysis) increases the reaction of the reaction by a factor of more than 9. A reported value[1] of kcat for hydrolysis of p-nitrophenylacetate (NPA) by imidazole (Figure 4) is 0.130 s-1.

Figure 4: Structure of p-nitrophenyl acetate (NPA)According to the observed value of kcat for the hydrolysis of NPB by imidazole, the catalytic hydrolysis of NPB occurs more slowly than the hydrolysis of NPA. This is possibly due to differences in steric crowding of the two substrates. NPA possesses a smaller (alkyl chain of a shorter length) carbonyl-containing group and therefore nucleophilic addition to the carbonyl is more energetically favored compared to nucleophilic addition to the more crowded butyrate carbonyl carbon. Due to these steric effects, the hydrolysis of NPA occurs more quickly than the hydrolysis of NPB. While the ln[(A-A0)/(A-At)] versus (t-t0) for the 0.0303 M imidazole solution showed the greatest linearity (R2 = 0.985) all other solutions shows some form of deviation from a linear plot as the reaction progressed. One problem encountered during the experiment was an inadequate mixing of the reactants, resulting in a solution that had the appearance of biphasicity. This caused part of the reaction mixture to be yellow, while another part remained clear. This separation may have causing deviations from a linear response of absorption as the interface of the two colored layers passed in front of the beam of the spectrometer. Additionally, previous studies [2] have shown that solvent identity can affect the value of the rate constant. In order to better understand these affects, the rate constant must be studied in other solvent mixtures. Mixtures of dioxane and water were used in previous kinetics studies[3].

4.) Conclusions

In conclusion, at room temperature in a mixture of acetonitrile and water, the observed rate constant (kobs) for the ester hydrolysis reaction of NPB and imidazole was found to increase linearly with changes in concentration of imidazole, indicating the reaction is pseudo-first-order with respect to imidazole. From the slope of a linear regression fit to the plot of kobs versus imidazole concentration, the catalytic rate constant was determined to be 0.0937 s-1 and the rate constant of the uncatalyzed reaction was found to be 0.0107 sec-1. For an identical solvent system and reaction temperature, the hydrolysis of NPB occurred more slowly than the hydrolysis of a shorter chain ester, NPA by a difference of 11%. This shows that the hydrolysis of shorter chain esters is expedited by a lack of steric crowing from a longer alkyl chain.

5.) References(1) Lombardo, A. J. Chem. Educ. 1982, 59, 887888.(2) Bruice, T. C.; Schmir, G. L. J. Am. Chem. Soc. 1957, 79, 16631667.(3) Bender, M. L.; Turnquest, B. W. J. Amer. Chem. Soc. 1957, 79, 16521655.

6.) Appendix

Figure A.1: Absorption versus time graph for the reaction of 0.0303 M imidazole with 6.21 mM NPB, 400 nm, 400 second run time, 5 second cycle time

Figure A.2: Absorption versus time graph for the reaction of 0.0507 M imidazole with 6.21 mM NPB, 400 nm, 400 second run time, 5 second cycle time

Figure A.3: Absorption versus time graph for the reaction of 0.0720 M imidazole with 6.21 mM NPB, 400 nm, 400 second run time, 5 second cycle time

Figure A.4: Absorption versus time graph for the reaction of 0.0917 M imidazole with 6.21 mM NPB, 400 nm, 400 second run time, 5 second cycle time