LABORATORY EXPERIMENT pubs.acs.org/jchemeduc
Self-Association of N-Methylacetamide Examined by Infrared and NMR Spectroscopies Heather L. Schenck* and KaWai Hui Department of Chemistry, University of Wisconsin—La Crosse, La Crosse, Wisconsin 54601, United States
bS Supporting Information ABSTRACT: These spectroscopic experiments investigate polarity and concentration effects on self-association behavior in N-methylacetamide. Inquiry can be limited to the concentration dependence of hydrogen bonding and estimation of dimerization constant (NMR studies) or to the effect of solvent polarity on extent of hydrogen bonding (IR studies). The combined experiments highlight differences between spectral timescales such as signal averaging seen in NMR spectra. Students learn how to obtain physical information about extent of aggregation from spectroscopic data. The experiments are best suited to an advanced undergraduate course. Undergraduate students are introduced to techniques that are relatively unusual in most college laboratory courses, including solution-state IR with solvent subtraction. The NMR experiments require students to use external referencing and to adjust collection parameters to accommodate a range of analyte concentrations spanning three orders of magnitude. KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Physical Chemistry, Equilibrium, Hydrogen Bonding, IR Spectroscopy, NMR Spectroscopy, Proteins/Peptides, Quantitative Analysis, Solutions/Solvents
H
predict a slightly lower Kdimer value for NMA in an aromatic solvent than in a nonaromatic solvent of comparable polarity. In the NMR study, students can predict the approximate Kdimer value of NMA in toluene based on toluene’s dielectric constant2,4 and on the data in Table 1.8 The potential effect of weak hydrogen bonds to the solvent (as proposed for dioxane) can be discussed, with regard to what impact such bonding would have on Kdimer. Students then measure extents of hydrogen bonding at various concentrations in toluene using the amide NH chemical shift. Extrapolation to zero concentration, where dimerization is most favored, permits estimation of Kdimer and comparison to the predicted value.1,2 In the IR study, students can predict the effect on solute hydrogen bonding of moving to a more or less polar solvent than toluene. Students then examine the effect of polarity on hydrogen bonding by studying equimolar solutions of NMA in these different solvents (we used toluene-d8 and methylene chloride). Students can do both IR and NMR or use either study in isolation, although the latter approach renders the IR work strictly qualitative. The combined experiments illustrate how IR and NMR spectra show hydrogen bonds differently (signal resolution versus signal averaging). The experiments also highlight the types of information that can (and cannot) be inferred from each type of spectrum. Each experiment can be completed in 2 h or less if students work in pairs. Larger groups are possible and (for the NMR work) should still permit each student to gain hands-on experience. We run this experiment as one of three different concurrent experiments because we have only one NMR; this has worked well with the advanced undergraduates who take this course.
ydrogen bonding is a key structural feature of many biological macromolecules. However, measurement of hydrogen bonding can present surprising challenges. In this experiment, upper-level undergraduate students study hydrogen bonding using NMR and IR spectroscopies and learn how a carefully designed inference can permit estimation of a quantity that cannot be directly measured. Students also have the opportunity to predict and test a new data point in an existing system used to model hydrogen bonds. N-Methylacetamide (NMA) is a small secondary amide with good solubility in many solvents. Dimerization of NMA by hydrogen bonding has been used as a model for hydrogen bonding within proteins.16 NMA also forms larger aggregates (e.g., trimer, tetramer, etc.), which are more favored at higher concentrations.1,2 Moreover, the association constant for dimerization is lower than the association constants for higher-order aggregates.1,3,5 This cooperative stabilization of higher aggregates renders direct measurement of the first association constant, Kdimer, impossible. Solvent polarity influences amide self-association; hydrogen bonds are most stable in nonpolar environments.1,2 Others have studied the association of NMA in CCl4, CHCl3, CD2Cl2, 9:1 CCl4/C6D6, 1,4-dioxane, and water and have reported Kdimer values (Table 1).1,2,4,6 The results illustrate the inverse relationship between solvent polarity and hydrogen bonding. The apparent incongruity of dioxane’s value has been attributed to this nonpolar solvent’s ability to participate in hydrogen bonding.1 No purely aromatic solvent systems have been examined. This opens the question of whether aromatic rings will affect NMA’s association behavior in a manner not predicted by overall polarity. The ability of aromatic rings to engage in weakly polar interactions, and to form weak hydrogen bonds to water via π-electrons, has been discussed.7 In this context, one might Copyright r 2011 American Chemical Society and Division of Chemical Education, Inc.
Published: June 02, 2011 1158
dx.doi.org/10.1021/ed100057s | J. Chem. Educ. 2011, 88, 1158–1161
Journal of Chemical Education
LABORATORY EXPERIMENT
This is the first hands-on experience most students have with the NMR spectrometer. The lecture group of up to 12 students is split into two laboratory sections of six students to accommodate this issue. If needed, instructor involvement with students can usually be limited to the first few NMR data collections (which typically occur about an hour after the start of laboratory). Assuming good procedure instructions are provided, minimal instructor involvement is needed for the IR experiment. Working in pairs or groups also decreases the cost of the NMR experiment, which requires 3 mL of toluene-d8 (99.5% atom-d available from Cambridge Isotope Laboratories in January 2011 for around $6/mL).
’ NMR EXPERIMENT Hydrogen bonding increases with solute concentration. When the NH chemical shift becomes invariant despite increasing concentration, self-association can be inferred to be complete.2 For this experiment, the solvent dielectric constant had to be low enough to cause complete association of NMA by about 6 M (the highest concentration accessible by NMR). Of the available lowdielectric and aromatic NMR solvents, toluene-d8 was selected on the basis of acceptable cost and risk profile. Students prepare seven NMR samples of NMA in toluene-d8 by serial dilution of a 6 M solution down to 0.006 M. Tetramethylsilane (TMS) is added as an internal reference to only the 0.6 M sample. This step maintains the other NMR samples as two-component mixtures for analysis by IR spectroscopy. Having Table 1. Reported Kdimer Values for NMA in Various Solvents Solvent Dielectrica
Solvent Water
0.005b
80
CH2Cl2 CHCl3
8.9 4.8
0.8c 2.72.8d
CCl4
2.2
4.7b,c, 24d,e
2.2
0.52b
1,4-dioxane a
Kdimer
b
c
d
TMS in only one sample introduces students to external referencing, a common NMR technique. Alternately, students can use residual solvent signals as an internal reference for the dilute samples. Solvent referencing is possible if the solvent is not 100% atom-d and assumes that solvent chemical shifts are not affected by NMA concentration. The relatively slow NMR timescale means that an NH proton samples both bound and unbound states during data collection. The observed chemical shift is a weighted average (based on time spent in each state) of the chemical shifts of the two pure states (so-called “signal averaging”). This phenomenon may be new to many advanced undergraduates. Exemplary student NH chemical shift data are shown in Figure 1. NMA appears essentially unbound at 0.006 M (NH chemical shift does not decrease further at lower concentrations). At 6 M, NMA appears fully hydrogen bonded (limiting high field NH chemical shift).2 The chemical shift range spans over 4 ppm, providing good dynamic range. Chemical shift data are used to calculate the concentrations of bound and free NH in each specimen. The chemical shift at 0.006 M, δ0.006M, is assumed to represent 100% free, and the chemical shift at 6 M, δ6M, is assumed to represent 100% bound. These chemical shifts should be the “bookends” of the data set. Formula 1 calculates the fraction of NH's that are bound (R) in a specimen of total concentration n; the corresponding fraction of unbound NH's would be 1 R. Formula 2 converts the fraction R into a concentration of hydrogen-bonded NH's (Cb). The concentration of free NH's (Cf) in each specimen is determined by subtracting Cb from the total concentration n. Source data for Figure 1 and the derived free and bound concentrations are shown in Table 2.
e
Ref 8. Ref 1. Ref 2. Ref 4. Ref 6.
Figure 1. Student data for amide NH chemical shift as a function of log(concentration).
R ¼ ðδn δ0:006M Þ=ðδ6M δ0:006M Þ
ð1Þ
Cb ¼ nR
ð2Þ
The final step of data analysis is estimation of Kdimer by the approach of Klotz and Franzen.1 These authors used the quantities of free and bound NH’s, which are measurable, to derive an approximation of Kdimer, which cannot be measured directly. Because hydrogen bonding decreases with concentration, R approaches zero as total concentration decreases. Equation 3 shows the resulting relationship between the fractions of free and bound NH’s and the concentration of free NH's. Kdimer is estimated as the y intercept of a graph of R/[(1 R)Cf] versus R. Instructors are referred to the work of Klotz and Franzen for additional detail on the derivation of eq 3.1 lim R 1 Cf s ð3Þ ¼ Kdimer f0 1 R Cf The cooperativity of aggregation at higher concentrations generates a strong positive curve, as noted by Klotz and Franzen.1
Table 2. Chemical Shift and Concentrations of Free and Bound NH for NMA in Toluene by NMR Concentration of NMA/M Parameter
6.0
2.0
0.60
0.20
Log concentration Chemical shift of NH/ppm
0.78 8.34
0.30 8.00
0.22 7.27
0.070 5.91
Concentration of hydrogen-bonded NH/M
6.0
1.8
0.45
0.089
0.0094
0.0011
0
Concentration of free NH/M
0
0.15
0.15
0.11
0.051
0.019
0.0060
1159
0.060 1.2 4.64
0.020 1.7 4.20
0.0060 2.2 3.95
dx.doi.org/10.1021/ed100057s |J. Chem. Educ. 2011, 88, 1158–1161
Journal of Chemical Education As a result, the curve with all concentrations included is poorly fit with exponential functions available in Excel software. Because higher-concentration samples represent regions where dimerization is least favorable, the four lowest concentration samples are used to approximate Kdimer. Figure 2 shows this derivation from data of Table 2; extrapolation to x = 0 yields Kdimer = 2.5. The average Kdimer from six student data sets was 2.09 (standard deviation 0.58). This result is in overall accord with literature values (Table 1; solvents of low dielectric promoting Kdimer values at or above 1). Students may have predicted a higher value based on CCl4’s reported Kdimer values. The lower experimental result may suggest that the presence of an aromatic ring can influence hydrogen bonding in a manner not predicted simply by solvent polarity. Alternatively, because the fit of the Excel-applied exponential is clearly not exact, this artifact may be a different possible explanation for toluene’s relatively low value compared to CCl4.
’ IR EXPERIMENT IR spectroscopy has also been used to investigate equilibrium behavior of NMA.1,2,9 This experiment uses solution-state IR spectroscopy to explore the effect of solvent polarity on NMA association at a single concentration. Using the idea that increasing solvent polarity will reduce the driving force for amide selfassociation, students can predict and then test the extent of hydrogen bonding in a solvent other than toluene. The experiment uses the 0.2 M NMR sample as one specimen. The other specimen is a solution of 0.2 M NMA in a solvent with a different dielectric constant, such as CH2Cl2 (dielectric constant = 8.9). Students collect spectra of pure solvent and of 0.2 M NMA for both toluene-d8 and CH2Cl2 in a solution cell with a path length of 0.2 mm. The rapid time scale of IR means that each NH proton is either bound or unbound during data collection. Because free and hydrogen-bonded NH's absorb at different wavenumbers,
LABORATORY EXPERIMENT
resolved peaks are obtained for these two states. After solvent subtraction and baseline correction (which may be new to students, but are necessary for quantification), students record absorbance values and wave numbers of free and bound NH peaks. Exemplary student IR data are shown in Figure 3. Students can qualitatively compare relative amounts of free and hydrogen-bonded NH's in the two solvents from absorbance values and spectral appearance. This analysis confirms the inverse relationship between solvent polarity and hydrogen bonding in NMA. However, because molar absorptivities of free and hydrogen-bonded NH stretches can vary, absorbance values cannot be converted to concentrations with confidence. Whereas NMR cannot resolve individual populations of free and hydrogenbonded NH's, these data can nevertheless aid the interpretation of the toluene IR spectrum at this point. Because NMR provided the concentrations of free and bound NH's at 0.2 M in toluene, students can calculate the respective molar absorptivities of these states in toluene using the BeerLambert law. Molar absorptivities of free and hydrogen-bonded NMA in toluene from Figure 3 are shown in Table 3. Students can rationalize the difference in values by recalling that hydrogen bonding often causes increased molar absorptivity for the XH stretching frequency of the hydrogen bond donor.10 It is not advisable to impute the same molar absorptivity values to the NH stretches in CH2Cl2; therefore, the exact concentrations of free and bound NH in that solvent are not determined in this experiment.
’ HAZARDS Hazards include toxicity, volatility, and flammability of all reagents and solvents. Measurements, transfers, and cleaning should be performed in a fume hood. Dichloromethane is a central nervous system toxin; is irritating to skin, eyes, and lungs; and is a possible carcinogen. Toluene is irritating to the skin and eyes; is readily absorbed through skin; and is a possible teratogen. N-Methylacetamide is irritating to all tissues; may be absorbed through skin; is a liver toxin; and a possible teratogen. Tetramethylsilane is extremely flammable and its vapors explosive. Students should use good laboratory hygiene and appropriate Table 3. Molar Absorptivities (IR) of Free and Bound NH for NMA in Toluene Molar Conc/ Wavenumber/ Parameter Free NH
Absorptivity/
M
cm1
0.11
3438
0.367
170
3322
0.378
210
Hydrogen-Bonded NH 0.089
Figure 2. Extrapolation to zero concentration. Y intercept is 2.5.
Absorption
Maximum (L mol1 cm1)
Figure 3. NH stretch regions of (A) 0.2 M NMA in CH2Cl2 and (B) 0.2 M NMA in toluene-d8. 1160
dx.doi.org/10.1021/ed100057s |J. Chem. Educ. 2011, 88, 1158–1161
Journal of Chemical Education
LABORATORY EXPERIMENT
personal protective equipment, including splash-proof goggles and gloves. NMA is difficult to measure, because it is a sticky solid that must be chopped apart. Students should understand the hazards of the spectrometers and be instructed in gentle handling of NMR tubes and IR windows.
’ CONCLUSIONS The experimental design allows students to explore a chemical question using a relatively sophisticated, indirect approach. Each experiment introduces techniques and concepts that many students will not have seen. The spectroscopic techniques used represent useful tools in the armamentarium of the well-schooled chemist. The concepts highlight caveats of spectral interpretation: the assumptions that an NMR signal possesses a single, true chemical shift, that a spectrum always represents a monomeric sample, and that a single functional group will exhibit uniform energy absorption regardless of state. ’ ASSOCIATED CONTENT
bS
Supporting Information Student instructions for NMR and IR experiments; student postlaboratory questions; spreadsheet (Excel 2004) for data analysis; information on reagent and solvent hazards; instructor notes This material is available via the Internet at http:// pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT Helpful discussions with S. H. Gellman and with J. L. Kirsch are gratefully acknowledged. ’ REFERENCES (1) Klotz, I. M.; Franzen, J. S. J. Am. Chem. Soc. 1962, 84, 3461–3466. (2) Gellman, S. H.; Dado, G. P.; Liang, G. B.; Adams, B. R. J. Am. Chem. Soc. 1991, 113, 1164–1173. (3) Albers, R. J.; Swanson, A. B.; Kresheck, G. C. J. Am. Chem. Soc. 1971, 93, 7075–7078 and references therein. (4) Krikorian, S. E. J. Phys. Chem. 1982, 86, 1875–1881. (5) Howlett, G. J.; Nichol, L. W.; Andrews, P. R. J. Phys. Chem. 1973, 77, 2907–2912. (6) Spencer, J. N.; Garrett, R. C.; Mayer, F. J.; Merkle, J. E.; Powell, C. R.; Tran, M. T.; Berger, S. K. Can. J. Chem. 1980, 58, 1372–1375. (7) Newcomb, L. F.; Haque, T. S.; Gellman, S. H. J. Am. Chem. Soc. 1995, 117, 6509–6519 and references therein. (8) CRC Handbook of Chemistry and Physics, 90th ed.; Lide, D.R; Haynes, W.M., Eds.; CRC Press: Boca Raton, FL, 2009. (9) Frohlich, H. J. Chem. Educ. 1993, 70, A3. (10) Silverstein, R. M.; Webster, F. X.; Kiemle, D. J. Spectrometric Identification of Organic Compounds, 7th ed.; John Wiley & Sons: Hoboken, NJ, 2005; pp 7678.
1161
dx.doi.org/10.1021/ed100057s |J. Chem. Educ. 2011, 88, 1158–1161
