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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

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Abstract:
A study of the affects of various concentrations of imidazole on the rate of
imidazole-catalyzed hydrolysis of p-nitrophenyl butyrate is presented. UVvisible 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.

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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 Butyrate

4-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).

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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 (k obs) is equal to the
rate constant of the uncatalyzed reaction (k 0) plus the rate constant of the
catalyzed reaction (kcat) multiplied by the concentration of the catalyst ([C])
(Equation 1).

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k obs =k 0 + [ C ] k cat
(1)
While experiments on nitrophenyl esters have been documented using 4nitrophenylacetate, 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.

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2.) Experimental
All 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

Moles
Mass of
of
Imidazole
Imidaz
(g)
ole

Final
Volu
me
(mL)

Concentra
tion
(mol/L)

100

0.0303

100

0.0507

100

0.0720

100

0.0917

0.0030
0.206
3
0.0050
0.345
7
0.0072
0.49
0
0.0091
0.624
7
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).

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Table 2: pH of prepared imidazole solutions after adjustment.

Imidazole
concentrat
ion (mol/L)
0.0303
0.0507
0.0720
0.0917

pH
7.63
7.60
7.68
7.79

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

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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

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12
10
8
6
Absorbance
4
2
0
0

200

400

600

800

-2
Wavelength (nm)

1000

1200

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4
3.5
3
2.5
Absorbance

2
1.5
1
0.5
0
-0.5

200

400

600

800

1000

1200

Wavelength (nm)

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).

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Time
Scale
Secon
ds

Run
Time
30
secon
ds

Cycle
Time
5
secon
ds

Wavelen
gth

Rate
Calculati
on

400 nm

None

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
Scale
Secon
ds

Run
Time
400
secon
ds

Cycle
Time
5
secon
ds

Wavelen
gth

Rate
Calculati
on

400 nm

None

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

Onuska 12
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

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the uncatalyzed reaction, k0, was calculated from the y-intercept of the line
fitted to the kobs versus [imidazole] graph.

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3.) Results and Discussion
The 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
10

6
ln[(A-A0)/(A-At)]

0.0917 M

0.0303 M
0.0507 M
0.0720 M

0
0

50 100 150 200 250 300 350 400 450

-2
(t-t0) (s)

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

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Concentration
Imidazole
0.0303 M
0.0507 M
0.0720 M
0.0917 M

of Equation of best fit Slope of best fit line

line
y = 0.0131x - 0.3726
y = 0.0157x + 0.3437
y = 0.0181x-0.2408
y = 0.0187x + 0.6393

(Kobs)
0.0131
0.0157
0.0181
0.0187

Table 5: Information obtained from best fit lines of ln[(A -A0)/(A-At)]

versus (t-t0) curves
As 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 R 2 value of
0.9474 (Graph 4).

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0.02
0.02
0.02

f(x) = 0.09x + 0.01

R = 0.95

0.01
0.01
Kobs

0.01
Kobs

0.01

Linear (Kobs)

0.01
0
0
0
0.02

0.04

0.06

0.08

0.1

Imidazole concentration (mol/L)

Graph 4: Observed rate constants versus imidazole

concentration, NPB concentration = 6.21 M
The 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 k cat was found to be
0.0937 s-1 and the value of k 0 (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.

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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

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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].

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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 k obs 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.

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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, 1652

1655.

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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

1.4
1.2
1
0.8
Absorption at 400 nm

0.6
0.0303 M
0.4
0.2
0
0

50 100 150 200 250 300 350 400 450

-0.2
Time (s)

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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

1.2

0.8

Absorption at 400 nm

0.6
0.0507 M
0.4

0.2

0
0

50 100 150 200 250 300 350 400 450

Time (s)

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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

2.5

1.5

Absorption at 400 nm

1
0.0720 M
0.5

0
0

50 100 150 200 250 300 350 400 450

-0.5
Time (s)

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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

2.5

1.5

Absorption at 400 nm

1
0.0917 M
0.5

0
0

50 100 150 200 250 300 350 400 450

-0.5
Time (s)