Laboratory Experiment pubs.acs.org/jchemeduc
Characterizing Carbonyls with Infrared Spectroscopy: An Introductory Chemistry Experiment in a Molecular Bioscience Program James P. McEvoy* School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, United Kingdom S Supporting Information *
ABSTRACT: The organic chemistry of life is, to a great extent, the chemistry of the carbonyl group, and it is important that bioscience students should appreciate the physical effects that determine this chemistry. An experiment is described that uses attenuated total reflectance−Fourier transform infrared (ATR−FTIR) spectroscopy to teach first-year undergraduate bioscience students the significance of electronic effects in carbonyl structure and reactivity. Students first predict and then measure the carbonyl stretching frequencies of a set of compounds in a process designed to emphasize resonance and hydrogen bonding effects, both of which are frequently overlooked by inexperienced students in favor of inductive effects. Students then use their enhanced understanding of electronic effects to rationalize the reactivities of different types of carbonyl compounds to nucleophilic attack, which is an important theme in biological chemistry. KEYWORDS: First-Year Undergraduate/General, Biochemistry, Laboratory Instruction, Organic Chemistry, Hands-On Learning/Manipulatives, Misconceptions/Discrepant Events, Bioorganic Chemistry, IR Spectroscopy, Lewis Structures, Resonance Theory
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This experiment hinges on the following relationships between the carbonyl environment and its stretching frequency, which is mostly determined by the carbonyl stretching force constant. Although exact rationalizations of these dependences are beyond the scope of an introductory chemistry course, simple and satisfying electronic explanations are available to students. 1) More electronegative substituents tend to increase the carbonyl stretching frequency (Figure 1A). Inductively
lectronic effects have a central role in determining the reactivity of organic compounds. Students in introductory chemistry classes can usually understand and predict inductive effects, but they find it harder to understand and predict the importance of resonance effects1 and noncovalent interactions, such as hydrogen bonds.2 As part of an introductory chemistry course for molecular bioscience students and inspired by previous pedagogical uses of infrared spectroscopy,3−7 a laboratory experiment was designed using attenuated total reflectance−Fourier transform infrared (ATR−FTIR) spectroscopy to demonstrate to bioscience students the significance of inductive, resonance, and hydrogen bonding effects in determining the electronic structures of various aromatic carbonyl compounds. These compounds were chosen both for their ready availability and for their suitable physical properties, being either solids or fairly nonvolatile liquids. Students perform the exercise after several weeks of studying biochemical carbonyl reactions in class and after a lecture on biochemical spectroscopy, during which time they have read and answered quiz questions on these topics. Carbonyl compounds have three characteristics to recommend them in this exercise. First, they are exceptionally important in biological chemistry, and so bioscience students can appreciate the wider significance of their results. Second, the stretching frequencies of carbonyl groups are given by prominent peaks in ATR−FTIR spectra, so data of sufficient quality are easily obtained by spectroscopic novices. Third, these frequencies reflect the electronic environments of the carbonyl groups in a predictable and additive fashion, allowing students to draw meaningful conclusions from their data. © 2014 American Chemical Society and Division of Chemical Education, Inc.
Figure 1. Inductive (A), resonance (B), and hydrogen bonding effects (C) in carbonyl groups, where X represents any substituent.
withdrawing electron density from the carbonyl carbon gives it a greater partial positive charge and makes the CO σ bond more polar. This makes the carbonyl bond shorter and stronger and, therefore, gives it a higher stretching frequency.8,9 2) Substituents able to donate π electron density into the carbonyl group tend to lower the carbonyl stretching Published: April 10, 2014 726
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relationships outlined above, in order to accomplish three things. First, students identify the moiety the compounds have in common (i.e., the benzoyl group) and concentrate their attention on the other, variable parts. This systematic comparison is a fundamental cognitive process and underlies much scientific reasoning, making this a valuable exercise in itself.12 Second, a “discrepant event” is set up: students are confronted on the day of the experiment by their prior misconceptions, commonly an underestimation of resonance and hydrogen-bonding effects relative to inductive effects. Third, the minds of the students are focused on the connection between their measurements and the underlying theory in order to make them feel that they have an intellectual stake in their experimental data. There is some evidence to suggest that asking students to make predictions before they run their experiments is an effective strategy in science education.13 On the day of the laboratory experiment, students, working in pairs, measure and print the ATR−FTIR spectra of the five compounds assigned to them, making sure in each case that they have a strong carbonyl peak between 1600−1800 cm−1 and, thus, a reliable peak position to report. They then obtain the spectra of the remaining compounds from other students and fill in a table, ranking all ten compounds in order of decreasing carbonyl stretching frequency according to the experimental data that they and their colleagues obtain. In their lab reports, students justify theoretically the ranking of each compound, with the benefit of experimental hindsight. Students also obtain the ATR−FTIR spectrum of the skin of one of their fingers (their “IR fingerprint”) and consider this spectrum in the light of their other results. They obtain this spectrum by pressing their bare finger onto the diamond crystal of the ATR accessory and holding it there while the spectrum is recorded. This spectrum is found to resemble that of keratin,14 a protein found abundantly in the keratinocytes of the epidermis15 and which clearly exhibits the two amide bands expected of a protein. The details of the experiment are in the Supporting Information along with a student handout and a prelab quiz.
frequency. (Figure 1B). Mesomerically donated electron density is partially localized on the carbonyl oxygen atom, an effect represented by resonance structure II. This polarization of the π bond makes the carbonyl bond longer and weaker and, therefore, gives it a lower stretching frequency.8,9 3) Hydrogen bonding to a carbonyl oxygen tends to lower the carbonyl stretching frequency. (Figure 1C). The carbonyl oxygen forms hydrogen bonds through its sp2-hybridized lone pairs, which are part of its σ system. The hydrogen bond, therefore, draws σ electron density away from the carbonyl oxygen, depolarizing the CO σ bond and, in compensation, polarizing the CO π bond. These combined effects make the carbonyl bond longer and weaker and, therefore, give it a lower stretching frequency.10,11 The pedagogical goals of this experiment are that students should be able to predict the effect of these three electronic effects on carbonyl stretching frequencies and to understand their connection to carbonyl reactivities. The student learning objectives are that students should learn how to (1) predict relative carbonyl stretching frequencies, (2) obtain ATR−FTIR spectra to measure these frequencies, (3) compare their predictions with experimental results, and (4) relate these data to reactivity trends.
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EXPERIMENTAL OVERVIEW Students are given the laboratory procedure along with a discussion of the relevant theoretical points several days before the exercise takes place. This material includes the names and structures of ten aromatic carbonyl compounds to be analyzed (Figure 2). It is pointed out that amides display two bands in
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MATERIALS AND EQUIPMENT The ten carbonyl compounds were obtained from VWR and are commercially available from various suppliers. An ATR− FTIR spectrometer was used to obtain spectra, signal-averaged from 4 scans, over a range of 5000−600 cm−1 at 4 cm−1 resolution.
■ Figure 2. The set of aromatic carbonyl compounds used in this experiment: benzaldehyde (1), acetophenone (2), 2,2,2-trifluoroacetophenone (3), benzophenone (4), benzoic acid (5), salicylic acid (6), ethyl benzoate (7), benzamide (8), N-methylbenzamide (9), and benzoic anhydride (10).
HAZARDS Most of the compounds used in this experiment are classified as hazardous, and students should make sure they do not come into contact with their skin and eyes. To this end, they should wear gloves and safety glasses and ensure that they do not inhale fumes or dust. The liquid samples should be kept in a fume hood when not in use. 2,2,2-Trifluoroacetophenone is flammable, with a flash point of 41 °C.
the carbonyl region of their IR spectra and that it is the higherfrequency one (the “amide I” peak) that represents the carbonyl stretching frequency. Students are given an online, prelab quiz to test their understanding of the experiment before they carry it out. One question asks students to predict the relative carbonyl stretching frequencies of the ten compounds, using the three
RESULTS AND DISCUSSION The experiment has been run four times, each time by a group of 50−60 students over a 6 h lab period in parallel with another experiment. Alternatively, the exercise could be run on its own over a 3 h lab period. Representative, student-obtained carbonyl stretching frequencies of the ten compounds and a typical finger epidermis are given in Table 1 in order of descending
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Reactivities of Carbonyl Compounds
Table 1. Measured Carbonyl Stretching Frequencies of the Substances Used in This Experiment Substance
a
benzoic anhydride (10) 2,2,2 trifluoroacetophenone (3) ethyl benzoate (7) benzaldehyde (1) acetophenone (2) benzoic acid (5) benzamide (8) salicylic acid (6) benzophenone (4) finger epidermis N-methylbenzamide (9)
Carbonyl Frequency(ies)/cm
In their experimental report, students considered the relationship between the measured carbonyl stretching frequencies in the acid derivatives examined and these compounds’ reactivities toward nucleophilic attack. The carbonyl stretching frequencies, depending as they do on both inductive and mesomeric effects, reflect the magnitude of the partial positive charge on the carbonyl carbon and, thus, the compounds’ reactivities.17 The acid derivatives in this experiment become broadly more reactive toward nucleophilic attack as their stretching frequencies increase (in the order amide < acid < ester < anhydride). By linking these reactivity data with their own conclusions from this experiment, students gained an appreciation of resonance effects that would be hard to gain solely from lectures or textbooks. Good answers to the postlab questions in the student report showed that students realized that they had often underestimated resonance and hydrogen bonding effects, and many students were able to reflect on the connection between carbonyl electronic structure and reactivity as hoped. By recognizing such correlations, students were invited to consider the chemical logic by which evolution has come to use certain families of carbonyl compounds in biology. It would be hard to imagine a biochemistry that relied on acid anhydrides, for example, because they are too quickly hydrolyzed. Amides, by contrast, are kinetically stable to hydrolysis in the absence of catalysts and they, therefore, make robust backbone linkages in proteins. This was emphasized to students by asking them to identify the compound responsible for the carbonyl peak in their “IR fingerprint”. The compound is keratin, a major and usefully inert component of the epidermis that students recently encountered in the lecture course. Indeed, evolution in this instance has taken advantage of the same stability of amides toward nucleophilic attack that are valued in synthetic polyamide fibers. Given the importance of thioesters in metabolism one would like to include in the experiment a compound of this type, for instance S-methyl benzothioate or benzoyl sulfide. However, thioesters’ high costs and unpleasant odors make them easier to incorporate as a database exercise (see question 4 in the student report form, Supporting Information.) Students were directed to the Spectral Database for Organic Compounds16 (a free, online resource) and asked to compare the carbonyl stretching frequency of phenyl acetate with that of S-phenyl acetate, and to comment on their findings.18 Thioesters have lower carbonyl stretching frequencies than esters because sulfur is less electronegative than oxygen and so exerts a smaller inductive effect on the carbonyl carbon.19 However, thioesters are more reactive than estersthey are easily reduced by sodium borohydride, for instancebecause sulfur is unable to donate π electron density into the carbonyl group.19 Thioesters, therefore, provide another example of the practical importance of resonance effects in biochemistry.
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1772, 1713 1717 1714 1697 1681 1678 1654b 1653 1649 1645b 1635b
a
Numbers assigned to compounds in Figure 2 are given in parentheses. bAmide I stretch.
frequency. Example FTIR spectra are included in the Supporting Information. These data satisfactorily match literature values,16 although some discrepancies arise in the case of solid samples because the literature results were obtained from a KBr disk rather than by ATR−FTIR. Most students obtained the same frequency order, with benzamide and salicylic acid occasionally swapping places. The main challenge for them, and the focus of this exercise, came in matching their experimental results to theory and in evaluating their earlier predictions. Electronic Effects in Carbonyl Compounds
When asked to predict the frequency ranking of the ten compounds in the prelab quiz, there was found to be a strong correlation between the real, experimental frequency ranking and the student-predicted ranking (Pearson’s correlation coefficient = 0.84), but students tended to overestimate the frequency of those compounds whose frequencies are reduced by resonance and intermolecular hydrogen bonding effects (e.g., benzamide and benzoic acid) and correspondingly underestimated the frequency of other compounds (e.g., benzaldehyde and acetophenone). Students reflected on their unexpected experimental results in the expectation that they would recognize the importance of electron delocalization and hydrogen bonding effects. The former effect was particularly noticeable in the amides, the latter in the acids. The carbonyl stretching frequency of benzoic acid, for instance, was lower than that of ethyl benzoate, an observation that was put down to intermolecular hydrogen bonding in the acid. A strong, intramolecular hydrogen bond was responsible for markedly lowering salicylic acid’s carbonyl stretching frequency. Students were often surprised that the acetophenone frequency was lower than that of benzaldehyde, and Nmethylbenzamide lower than benzamide, because they knew that hydrogen has a lower Pauling electronegativity than carbon. The methyl group’s electron-donating character was explained to them by pointing out that hydrogen and carbon differ only slightly in their electronegativities and that the methyl group has a much greater electron density than a single hydrogen atom, making its electrons more available to bonding partners. Inquisitive students were directed toward the concept of hyperconjugation.
Partner Experiments
Because of the theoretical focus of this exercise, it may be convenient to have students perform a related, more practically based experiment alongside it, either simultaneously or consecutively. For this purpose, an extraction of piperine from peppercorns20 has been used with a modified procedure in which students use ATR−FTIR spectroscopy to confirm the identity of their product. Another attractive possibility would be 728
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(10) Del Bene, J. E. Molecular Orbital Theory of the Hydrogen Bond. X. Monosubstituted Carbonyls as Proton Acceptors. J. Chem. Phys. 1975, 62, 1314−1322. (11) Lewell, X. Q.; Hillier, I. H.; Field, M. J.; Morris, J. J.; Taylor, P. J. Theoretical Studies of Vibrational Frequency Shifts upon Hydrogen Bonding. J. Chem. Soc., Faraday Trans. 2 1988, 84, 893−898. (12) Gentner, D.; Medina, J. Similarity and the Development of Rules. Cognition 1998, 65, 263−297. (13) Schunn, C. D.; O’Malley, C. J. Now They See the Point: Improving Science Reasoning Through Making Predictions. In Proceedings of the Twenty-Second Annual Conference of the Cognitive Science Society; Gleitman, L. R., Joshi, A. K., Eds.; Psychology Press: New York, 2000; pp 889−894. (14) Bendit, E. G. Infrared Absorption Spectrum of Keratin. I. Spectra of Alpha-, Beta-, and Supercontracted Keratin. Biopolymers 1966, 4, 539−559. (15) Rogers, K. Skin and Connective Tissue; Britannica Educational Publishing: New York, 2012; pp 7−12. (16) Spectral Database for Organic Compounds, SDBS. http://sdbs. db.aist.go.jp/ (accessed Mar 2014). (17) Flett, M. St. C. Characteristic Infrared Frequencies and Chemical Properties of Molecules. Trans. Faraday Soc. 1948, 44, 767−774. (18) In the absence of an infrared spectrometer, the whole exercise may be carried out by referring to such online databases. Alternatively, students may use a suitable software package to compute the molecules’ carbonyl stretching frequencies. (19) El-Aasar, A. M. M.; Nash, C. P.; Ingraham, L. L. Infrared and Raman Spectra of S-Methyl Thioacetate: Toward an Understanding of the Biochemical Reactivity of Esters of Coenzyme A. Biochemistry 1982, 21, 1972−1976. (20) Epstein, W. W.; Netz, D. F.; Seidel, J. L. Isolation of Piperine from Black Pepper. J. Chem. Educ. 1993, 70, 598−599. (21) Baru, A. R.; Mohan, R. S. The Discovery-Oriented Approach to Organic Chemistry. 6. Selective Reduction in Organic Chemistry: Reduction of Aldehydes in the Presence of Esters Using Sodium Borohydride. J. Chem. Educ. 2005, 82, 1674−1675. (22) Rosenberg, R. E. A Guided-Inquiry Approach to the Sodium Borohydride Reduction and Grignard Reaction of Carbonyl Compounds. J. Chem. Educ. 2007, 84, 1474−1476.
for students to measure the different rates at which their carbonyl compounds are reduced by sodium borohydride.21,22
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CONCLUSIONS Molecular bioscience students are expected to learn a lot of carbonyl chemistry in their study of biology, and they may only make sense of it in the light of fundamental chemical principles. In this experiment they used three relationships, based on familiar electronic effects, to predict the relative carbonyl stretching frequencies of various aromatic compounds. In the prelab quiz, most students expected inductive effects to predominate but found, using ATR−FTIR spectroscopy, that all three effects are important in determining their frequencies. Students were led to consider the implications of electronic structure (particularly resonance effects) on biological carbonyl reactivity, particularly with regard to their own epidermis. Generally good answers to the postlab questions in the student report indicated that the pedagogical goals had been achieved.
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ASSOCIATED CONTENT
S Supporting Information *
Instructor’s notes, example FT-IR spectra, student handout and report sheet, prelab quiz and prelab quiz analysis. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS I gratefully acknowledge the technical support of Steve Richards and Ray Smith, the advice of Paul Finch, the financial support of Royal Holloway, University of London, and the helpful comments of anonymous reviewers.
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REFERENCES
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