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NMR Kinetics: Study of a Reversible Hydrolysis Reaction

Renato Sousa and Ricardo Avila Physical Chemistry II Lab April 17, 2013

I. ABSTRACT Using a simple equation relating the width of NMR spectrum signals to the rate of a reaction in thermodynamic equilibrium, we were able to determine the forward and reverse rates of the hydrolysis reaction of pyruvic acid to 2,2-dihydroxypropanoic acid, as well as the thermodynamic equilibrium constant. Furthermore, we were able to calculate the exact dependence of the rate constant on the concentration of hydronium ion used as a catalyst, and show that increasing the concentration of acid increases the rate of the reaction. The results of our experiment were compared with literature values of the kinetic constants for this reaction and show a reasonable match, taking into account the accuracy limits of our experiment. The experiment is based on one described in the book Introduction to Molecular Spectroscopy; Theory and Experiment (1970).1 II. INTRODUCTION Nuclear magnetic resonance (NMR) is a chemical analysis technique in which nuclear spins, which are usually randomly oriented, are aligned parallel to a strong magnetic field, and then showered with electromagnetic radiation in the radio wave frequency, which excites the nuclei and causes their spins to realign in an antiparallel conformation. Then, the radiation is turned off, and the spins are allowed to return to their low energy state, a process called relaxation, which causes a resonance in the magnetic field. This fluctuation is detected and displayed as peaks in an NMR spectrum.2 There are several types of NMR, which differ on the type of nuclei being analyzed. This particular experiment involves 1H NMR, which focuses on the excitation of the hydrogen nucleus. The resonance frequency of a nucleus depends on several factors, including the strength of the instruments magnetic field, but most importantly, on the chemical environment in which it resides. For example, a hydrogen atom that is found close to an electronegative atom loses electron density. An electron-deprived nucleus experiences a stronger external magnetic field, and a stronger radiation frequency is required to reverse the spin of the nucleus, thus the NMR signal appears farther to the left (downfield) in the spectrum. Because the strength of the magnetic field can vary from instrument to instrument, NMR resonance signals are often measured in the dimensionless quantity of ppm, which equals the frequency of the signal in Hz divided by the frequency of the instrument. This technique can be used to determine the structures of molecules, as well as determine kinetics of reactions. A researcher can often extrapolate the ratio of a mixture by integrating the curve for each proton, which reflects the abundance of the individual protons. This can lead to quantify the ratio of the mixture, and in the case of a reaction in thermodynamic equilibrium, it yields the equilibrium constant, which can be computed using Equation 1, where [B] is the concentration of the products, and [A] is the concentration of the reactants.

(1)

Using reaction progress NMR, the reaction rates can be found by measuring the change of concentration of the products over time. When dealing with reactions that are in thermodynamic equilibrium, as is the case in our experiment, another method called chemical exchange NMR can be used to find the rate constants. This method is based on the fact that in a system where a nucleus is switching back and forth between two or more environments, the widths of the signals will be proportional to the speed at which the nucleus is shifting. The system can involve multiple molecules, or it can also be intramolecular, but the general principle is the same and the idea is quite intuitive: as exchange rate increases, the peaks become less and less sharp, as the proton spends more time in between both of its states, and if the rate is sufficiently fast, both signals merge, or coalesce into one signal, which becomes sharp once more and resembles a single species. The chemical exchange system is described by the Blotch-McConnell equations, which can be fairly complex, however, for slow exchange rates where the peaks do not coalesce, they become simplified. The equations can be summarized as follows,

(2)

(3)

Where Equation 1 describes the forward reaction, and Equation 2 is its equivalent for the reverse reaction. The values of W are the widths of the NMR signals at half their maximum value, kH is the rate constant that varies with catalyst concentration (H+), and C is a constant that consists of the sum of two other constants: k0, or the rate constant independent of catalyst, and 1/T2, which is the spin-spin relaxation time (the longer the spin takes to return to its original energy, the wider the signal becomes). The complete rate constants of the reaction, kf and kr, are a sum of the catalyst-dependent, and catalyst-independent rate constants. In order to solve this equation, we make the assumption that the spin-spin relaxation time is approximately the same for both, the forward and reverse reaction, (this is reasonable, as both species under study are chemically similar) and call this constant C0, for simplicity. Then,

(4)

(5)

Which allows us to solve for the The reaction under study is the hydrolysis of pyruvic acid to 2,2-dihydroxypropanoic acid, which can proceed by a natural, and an acid-catalyzed path (Figure 1). It is considered a pseudo-first-order reaction-a reaction that proceeds at a rate that depends linearly only on one reactant concentration, because it behaves very much like one. There is an excess of pyruvic acid, so the reaction progresses toward the formation of 2,2-dihydroxypropanoic acid.

Figure 1: Pathways of the reversible reaction of pyruvic acid hydrolysis

III. EXPERIEMENTAL METHOD Materials and Instruments Used: 300 MHz NMR spectrometer, test tubes, NMR tubes, pyruvic acid, acetone, HCl, H2O, D2O, Adobe Photoshop CS6. All equipment was carefully cleaned with DI water and acetone beforehand to prevent any contamination. Six solutions of pyruvic acid in water and deuterium oxide, with an increasing concentration of hydrochloric acid were systematically prepared in test tubes; the ratios for these solutions are displayed in Table 1. A small sample of each of these solutions was transferred to an NMR tube with a pipette, making sure that the sample column was about three finger lengths in height.

Solution 1 2 3 4 5 6

Pyruvic Acid (mL) 1.0 1.0 1.0 1.0 1.0 1.0

HCl (mL) 0.00 0.10 0.20 0.30 0.40 0.50

H2O (mL)

D2O (mL)

1.10 1.00 0.90 0.80 0.70 0.60

0.10 0.10 0.10 0.10 0.10 0.10

Table 1: Ratios for the preparation of the experimental samples.

These samples were loaded in the NMR 300, the deuterium solvent was specified, and the sample was shimmed. The peaks in the resulting spectra were integrated using the machines software, and their location in the spectrum was labeled. The printouts of the resulting spectra where then scanned and the width of the peaks at half their maximum value (FWHM) was measured using the ruler tool in Adobe Photoshop CS6. Utilizing the equations described in the introduction, we where able to determine certain values of interest such as kHf, kHr, CA, CB, Keq, k0f, k0r, 1/T2A and 1/T2B. IV. RESULTS The NMR spectra obtained are displayed superimposed in Figure 2(appended at the end of the report), where signal A corresponds to pyruvic acid, the starting material, and signal B corresponds to 2, 2-dihydroxypropanoic acid. The table below shows the calculation of the equilibrium constant from the ratio of the integrated NMR Signals. Area Area under under peak B peak A 1.00 1.74 0.48 1.00 0.46 1.00 1.00 1.31 0.24 1.00 0.22 1.00 Average for all solutions:

Solution 1 2 3 4 5 6

Keq 1.740 2.083 2.174 1.310 4.167 4.545 2.670

Table 2: Integrated areas of peaks: A (reactant) and B (product) for each solution. The equilibrium constant was calculated by dividing the area of B over A for each solution.

The FWHM (Wa and Wb) in ppm of each peak was measured (Table 1) and then, after converting to hertz and multiplying by a factor of , was plotted as a function of the hydronium ion concentration (Plots 1 & 2). The linear fit of this plot was used to derive the kinetic rate constants from Equations (2) and (3). These rate constants were then used to solve for the equilibrium constant (Eqn.1), thus providing a benchmark for comparison with the data from Table 1. Solution 1 2 3 4 5 6 [H+](mol/L) 0.00 1.47 2.94 4.41 5.88 7.36 Wa (ppm) 0.0136 0.0144 0.0405 0.0269 0.0482 0.0545 *Wa (Hz) 12.818 13.609 38.170 25.353 45.427 51.365 Wb (ppm) 0.0109 0.0125 0.0246 0.0210 0.0313 0.0286 *Wb (Hz) 10.273 11.781 23.185 19.792 29.500 26.955

Table 3: Peak widths at half-maximum (Wa), in parts per million, and their corresponding *Wa values for each of the six solutions with different hydronium ion concentrations.

60.0 50.0 40.0

[H+] vs FWHM in the Forward (A->B) Reaction

*Wa (Hz)

30.0 20.0 10.0 0.0 0.00 2.00 4.00 y = 5.3482x + 11.454 R = 0.80895 6.00 8.00

[H+] (mol/L) Plot 1: Linear relationship between H+ concentration and the width of the absorption peak. The slope of the plot is equivalent to the rate constant for the forward reaction.

35.00 30.00 25.00 20.00 15.00 10.00 5.00 0.00 0.00

[H+] vs FWHM in the Reverse (B->A) Reaction

*Wa (Hz)

y = 2.5864x + 10.735 R = 0.8157

2.00 [H+]

4.00 (mol/L)

6.00

8.00

Plot 2: Linear relationship between H+ concentration and the width of the absorption peak. The slope of the plot is equivalent to the rate constant for the reverse reaction.

Applying the equations discussed in the Introduction to these linear fit functions the following values were obtained Constant kHf CA 1/T2A k0f kf Keq Value 5.348 11.454 10.065 1.384 3.964 2.069 Constant kHr CB 1/T2B k0r kr Value 2.586 10.735 10.065 0.669 1.917

Table 4: Experimental values for each of the constants in Equation 1.

V. DISCUSSION Knowing that the reaction under study progresses toward an equilibrium that favors the products, the calculation of the thermodynamic equilibrium constant from the ratio of the integrated areas of the peaks allowed us to determine that the peak that appears farthest upfield in the NMR spectrum is produced by the reactant, and the peak farthest downfield is produced by the product, as this ratio yields a value of Keq > 1. Although the equilibrium constant values obtained from this method were not perfectly consistent, there were no outliers. The mean value for Keq was 2.670, the median was 2.083 and the standard deviation was 1.346. Plotting the concentration of hydronium ions versus the FWHM values allowed us to determine the rate constants from the slopes of these two lines. The rate constants thus obtained are consistent with the observations described above, as the rate constant for the forward reaction (5.348) was larger than the rate constant for the reverse reaction (2.586), meaning that the reaction progresses in favor of the products. The equilibrium constant was recalculated from the ratio of the forward to the reverse rate constants, yielding a value of 2.069. Comparing this value with with the average value from the integration method yields a percent error of 22.5%. If the median is used as a standard for comparison, the percent error is much smaller, at 0.67%. VI. CONCLUSION A large source of error in the data is the accuracy of the NMR spectra readings, as due to a lack of time some of the spectra were improperly shimmed, and not all of the peaks are perfectly symmetric. This produces inaccuracies when measuring the FWHM of the peaks, and consequently interferes with the calculation of the rate constants. The paper Kinetic Study by NMR published by the West Ham College of Technology describes a very similar kinetics experiment involving the exact same reaction, and therefore allowed us to compare the values that we obtained in our rate constant to the average rate constants found under their results. The average values that they found for cwas 5.554, and the average value for khr was (7.866). Our calculated value for khf was 5.348, and for khr was 2.586 which give a percent difference of (3.70%) and (67%) respectfully. So this literature seems to support out value for khf , but not for khr . Although the overly large difference, coupled with the fact that their values indicate a reaction favoring pyruvic acid instead of 2,2-dihydroxypropanoic acid, casts some doubt on the validity of this comparison.

VII. REFERENCES
1. Brittain, Edward F. H., W. O. George, and C. H. J. Wells. "Exp. 21: NMR Study of a Reversible Hydrolysis Reaction." Introduction to Molecular Spectroscopy; Theory and Experiment. London: Academic, 1970. 26371. Print. Nerz-Stormes, M. The Basics Nuclear Magnetic Resonance Spectroscopy. http://www.brynmawr.edu/chemistry/ Chem/mnerzsto/The_Basics_Nuclear_Magnetic_Resonance%20_Spectroscopy_2.htm (accessed April 17, 2013).

2.

Figure 2: Compilation of the NMR spectra obtained, showing shifts in the location of the peaks. For each spectrum, the peak located farthest upfield is the absorption of the reactants, and the peak farthest downfield is the absorption of the products. The third, middle peak found in most spectra is believed to be a contaminant.