In the Laboratory
W
Discovering Electronic Effects of Substituents in Nitrations of Benzene Derivatives Using GC–MS Analysis Malgorzata M. Clennan* and Edward L. Clennan Department of Chemistry, University of Wyoming, Laramie, WY 82071; *
[email protected] tures of those six substrates are then analyzed during the laboratory period with GC–MS.2 At the beginning of the next laboratory period students are given their chromatographs and the mass spectrum of each of the observed peaks. Students then identify the unreacted starting material and products and assign their structures based upon retention times and analysis of fragmentation patterns. The results of each section are then pooled and tabulated. Pooling results enables students to draw conclusions about the directing effects of substituents (ranging from strongly activating to strongly deactivating). In addition, writing down the resonance structures of possible intermediates allows them to draw conclusions about the mechanism of the nitration reaction. It is up to the instructor to decide how much additional information should be given to the students. The reactions are conducted on a millimolar scale to reduce the quantity of waste while providing sufficient material for GC–MS analysis. In addition, the MSDS categorized toxic nitration products are never isolated minimizing student contact.
The nitration of substituted benzenes is a classical example of the electrophilic aromatic substitution reaction and it is still, despite its age, commonly taught in the second semester of undergraduate second-year organic courses (1). This reaction is often carried out in the laboratory as an illustration of lecture material, but the majority of the existing procedures in laboratory textbooks focus on isolation of a major product and validation of its identity (2). Detection of other isomers formed in these nitrations has been virtually neglected thus giving students the misleading impression that other isomers are not formed. In fact, only a few experiments for the undergraduate laboratories described in the literature deal with the determination of isomer distribution in nitration reactions but their scope is limited to nitration of alkyl substituted benzenes (3a), aniline (3b), and benzoic acid (3c). We describe a discovery-based (4, 5) experiment in which students, by applying GC–MS analyses to the reaction mixtures, discover the identity of all isomers formed in the nitration of a series of substituted benzenes. The instructional value of introducing GC–MS analyses in the organic secondyear undergraduate laboratory is well established (3a, 6) and in this experiment students exercise their knowledge of principles of chromatographic separation (7a) as well as fragmentation patterns (7, 8) to identify the substrates and isomer products. The effectiveness of a discovery-based approached to learning has been discussed previously (4) but only Mohan et al. (5) have described a discovery-oriented nitration experiment. However, even in this experiment the isolation and purification protocol used allows NMR identification of the major product but does not allow detection of other isomers.
Experiment
Overview of the Experiment
Synthesis One of the substrates (4 mmol) (1 through 6 in Table 1) was added to 2 mL of 98% H2SO4 and the solution was chilled in an ice bath. A chilled nitrating mixture (0.5 mL of 70% HNO3 and 98% H2SO4, 1.25兾0.25, v兾v) was then added. The reaction mixture was kept at room temperature for 15 min followed by dilution with 20 mL water and extraction with two 5 mL portions of ethyl acetate. The combined extracts were dried with sodium sulfate and gravity filtered. This solution was then used for GC–MS analysis.
During a three-hour laboratory period each student performs the nitration of one of six1 substrates using identical reaction conditions (procedures). Representative product mix-
GC–MS Conditions An Agilent 6890 GC兾5973 MSD system with an automatic liquid sampler (split injection 100兾1) and a HP-5, 30 m
Table 1. Relative Peak Areas and Retention Times [min] of Nitration Products Isomers
Unreacted Substrates
Ortho
Meta
Para
o,p-Disub
N-Methylaniline, 1
trace [2.50]
09.8 [4.72]
27.3 [5.23]
62.9 [6.14]
none
Anisole, 2
01.1 [1.99]
04.0 [3.97]
none
06.3 [4.26]
84.1a [6.43]
Fluorobenzene, 3
36.1 [1.96]
02.2 [4.52]
none
52.3 [4.24]
09.4b [6.79]
Ethyl Benzoate, 4
42.2 [2.97]
10.8 [4.86]
45.5 [5.17]
01.5 [5.05]
none
Acetophenone, 5
62.1 [2.52]
07.0 [4.25]
30.9 [4.57]
not detected
c
none
α,α,α-Trifluorotoluene, 6
72.7 [2.05]
Trace [4.02]
25.3 [4.09]
01.9b [4.48]
none
Products
a
b
c
A second di-nitro isomer at 5.11 min was not identified (rel area 4.5). Original samples were not analyzed. The mass spectra of the original samples of 3’- and 4’-nitroacetophenone show only minute differences. The meta isomer may be contaminated with para, but attempts to separate them on our GC–MS failed.
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In the Laboratory A
B
Figure 1. (A) Chromatogram (TIC) of a crude reaction mixture obtained from nitration of ethyl benzoate. (B) Mass spectrum of the product with retention time 5.17 min identified as ethyl 3-nitrobenzoate.
× 0.25 mm column was used. Temperatures were inlet250 ⬚C, detector-280 ⬚C, and oven-110 ⬚C (or 60 ⬚C for fluorobenzene and α,α,α-trifluorotoluene) ramped to 250 ⬚C, 20 ⬚C兾min. Hazards Ethyl acetate and substrates 2–6 are irritants. Nmethylaniline and all products are toxic. Fluorobenzene, α,α,α-trifluorotoluene, and ethyl acetate are flammable. Sulfuric and nitric acids are corrosive. Gloves should be worn throughout the experiment. The reactions and the work up of the reaction mixtures should be carried out in hoods. Results An example of a total ion chromatogram from the nitration of ethyl benzoate and the mass spectrum of the major isomer formed are presented in Figure 1. The uncorrected (7a) peak areas for isomers expressed as a percent of total peak area in the chromatograms and the corresponding retention times are compiled in Table 1. All substrates exhibit molecular ions and their identifications by fragmentation patterns are accomplished by the students without major problems.3 However, the identification of the products create a real-life dilemma. On one hand, GC–MS analysis, using the “nitrogen rule” and the mass of the molecular ions as well as the diagnostic peaks (8) deriving from the loss of NO2 and NO from the molecular ions, allow students to identify those peaks that are from monoand those that are from di-substituted products. On the other hand, the distinction between the ortho, meta, and para mono-substituted isomers based on fragmentation patterns cannot be achieved. However, armed with retention times of authentic samples and a basic tenet of GC separation theory (7a) 4 the identifications can be accomplished. The unexpected formation of meta isomer in nitration of Nmethylaniline can be explained by existence of the protonated substrate in the acidic medium that directs nitration to the meta position (9).
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Conclusions In this experiment students discover that the reactivity and the substitution pattern in the nitration of aromatic rings depend strongly on the electronic properties of the substituent. Writing and examining the resonance structures for possible intermediates allow them to elucidate the directing mechanism. Especially appreciated by the students is the connection between the material presented in lecture and in this laboratory experiment that provide them with insight on how the data presented in the textbook are obtained. This simple discovery-based experiment keeps students involved and their enthusiasm intact throughout the laboratory session. Surprisingly, students involvement in data analysis increases when they are told that the distinction between isomers cannot be achieved based solely on fragmentation patterns and that NIST98 library program will not help. Acknowledgment The authors would like to thank the National Science Foundation for the funds necessary for the purchase of the GC–MS (DUE-0125911). W
Supplemental Material
Instructions for the students, notes for the instructor, chromatograms of reaction mixtures, and mass spectra of the major isomers are available in this issue of JCE Online. Notes 1. Numerous substrates were used in the preliminary testing (among them acetanilide, bromobenzene, and toluene) but the ones selected gave the most reproducible and illustrative results and were most amenable to chromatographic separations. 2. The rest of the samples are analyzed during the ensuing week. 3. Provided some GC–MS experiment(s) are introduced in the first semester of the course as the gradualism approach to teaching dictates, see ref 6a.
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In the Laboratory 4. Elution of compounds is in the order of their increasing boiling points (vapor pressure) with the exception of substrate 4. 5.
Literature Cited
6.
1. (a) Vollhardt, K. P. C.; Schore, N. E. Organic Chemistry: Structure and Function, 5th ed.; W. H. Freeman & Co: New York, 2007; pp 726–735. (b) Yurkanis Bruice, P. Organic Chemistry, 4th ed.; Prentice Hall: Upper Saddle River, NJ, 2004; pp 609– 610. (c) Solomon, G.; Fryhle, C. Organic Chemistry, 7th ed.; John Wiley & Sons, Inc.: New York, 2000; pp 667–680. 2. (a) Wilcox, C. F. W.; Wilcox, M. F. Experimental Organic Chemistry: A Small Scale Approach, 2nd ed.; Prentice-Hall: Englewood Cliffs, NJ, 1995; pp 392–396. (b) Campbell, B. N.; McCarthy Ali, M. Organic Chemistry Experiments. Microscale and Semi-Microscale; Brooks/Cole Publishing Company: Belmont, CA, 1994; pp 292–305. 3. (a) Asleson, G. L.; Doig, M. T.; Heldrich, F. J. J. Chem. Educ. 1993, 70, A290–A294. (b) Hurlbut, J. A. J. Chem. Educ. 1971, 48, 411–412. (c) Feldman, M.; Wheeler, J. W. J. Chem. Educ. 1967, 44, 464–465. 4. For example see: (a) Moroz, J. S.; Pelino, J. L.; Field, K. W. J.
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7.
8.
9.
Chem. Educ. 2003, 80, 1319–1321. (b) Burns, D. S.; Berka, L. H.; Kildahi, N. J. Chem. Educ. 1993, 70, A100–A102. McElveen, S. R.; Gavardinas, K.; Stamberger, J. A.; Mohan, R. S. J. Chem. Educ. 1999, 76, 535–536. For example see: (a) Clennan, M. M.; Clennan, E. L. J. Chem. Educ. 2005, 82, 1676–1678. (b) Fleisher, J. M. J. Chem. Educ. 2002, 79, 1247–1248. (c) Zahedkargaran, H.; Smith, L. R. J. Chem. Educ. 2001, 78, 1379–1380. (c) Pelter, M. W.; Macudzinski, R. M. J. Chem. Educ. 1999, 76, 826–828. (d) Kjonaas, R. A.; Soller, J. L.; McCoy, L. A. J. Chem. Educ. 1997, 74, 1104–1105. (a) Pavia, D. L.; Lampman, G. M.; Kriz, G. S.; Engel, R. G. Microscale and Macroscale Techniques in the Organic Laboratory; Harcourt College Publishers: Philadelphia, 2002; pp 333–354, 470–491. (b) Palleros, D. R. Experimental Organic Chemistry; John Wiley & Sons, Inc.: New York, 2000; pp 783–800. Silverstein, R. M.; Webster, F. X. Spectrometric Identification of Organic Compounds, 6th ed.; John Wiley & Sons: New York, 1998; pp 31–32. March, J. Odvanced Organic Chemistry. Reactions, Mechanisms and Structure, 3rd ed.; John Wiley & Sons: New York, 1985, pp 453–457.
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