Experimental Determination of Activation Energy of Nucleophilic

Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX-XXX

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Experimental Determination of Activation Energy of Nucleophilic Aromatic Substitution on Porphyrins Waqar Rizvi,*,†,‡ Emaad Khwaja,† Saim Siddiqui,† N. V. S. Dinesh K. Bhupathiraju,† and Charles Michael Drain*,†,§ †

Department of Chemistry and Biochemistry, Hunter College of the City University of New York, New York, New York 10065, United States ‡ Ph.D. Program in Chemistry, The Graduate Center of the City University of New York, New York, New York 10016, United States § The Rockefeller University, New York, New York 10065, United States S Supporting Information *

ABSTRACT: A physical organic chemistry experiment is described for second-year college students. Students performed nucleophilic aromatic substitution (NAS) reactions on 5,10,15,20tetrakis(2,3,4,5,6-pentafluorophenyl)porphyrin (TPPF20) using three different nucleophiles. Substitution occurs preferentially at the 4-position (para) because it is thermodynamically favored, and the 2- and 6- (ortho) positions are kinetically disfavored because of steric interactions with the porphyrin ring. The activation energy depends heavily on the nucleophile. Opensource software (ImageJ from NIH) was used to quantify relative intensities of spots on a TLC plate obtained from different times and varying temperatures. These data were used to generate Arrhenius plots allowing students to determine relative activation energies for three different primary nucleophiles. The experiment was developed by 5 undergraduates and evaluated by 40 organic chemistry II students and 8 students in a physical chemistry laboratory. Students gained a deeper understanding of the relationships between the NAS mechanism, Arrhenius plots, and activation energy. KEYWORDS: Second-Year Undergraduate, Organic Chemistry, Physical Chemistry, Collaborative/Cooperative Learning, Computer-Based Learning, Hands-On Learning/Manipulatives, Dyes/Pigments, Nucleophilic Substitution, Reactions, Chromatography

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INTRODUCTION Synthetic chemistry underpins modern society since this discipline supplies many valuable resources that enable production of the fertilizers, pesticides, insecticides, and medicines needed to feed and protect the growing world population, as well as produce the numerous customized materials for the advanced technologies.1,2 One simple way to functionalize an organic molecule is via nucleophilic aromatic substitution (NAS) where a nucleophile displaces a good leaving group, such as Br or NO2, on an aromatic ring without yielding many toxic byproducts. This facile and green method is employed in the pharmaceutical and medicinal industry. NAS reactions3 are key steps is the synthesis of therapeutics such as the antidepressant fluoxetine (trade name Prozac), the antibiotic ofloxacin,4 and arenes with 18F for use in positronemission tomography (PET) imaging.5 NAS is used in nature for the enzymatic degradation of nitroarenes.6 Cyanuric chloride undergoes NAS reactions to make triazine herbicides, such as atrazines, reactive dyes for clothing, and melaminebased polymers, which are used in whiteboards.7 Thus, chemicals derived from NAS reactions have global impacts on health, agriculture, materials, and the environment. © XXXX American Chemical Society and Division of Chemical Education, Inc.

One industrial concern is mass producing a cost-effective product. Understanding the energy required to run a chemical reaction (activation energy) is key to developing the optimum reaction conditions. While higher temperatures result in faster reactions, it is not always cost-effective to heat beyond the required temperature. The Arrhenius equation can be used to calculate activation energy (Ea) of a reaction, which allows optimization of commercial chemical transformations. Many students in organic chemistry exhibit a gap in understanding the relationships between rates, the Arrhenius equation, and Ea. In this experiment, we determine the Ea values of different NAS reactions using three different nucleophiles on a porphyrin dye. Other undergraduate NAS reactions are described that focus on the number, position, and nature of the electron-withdrawing groups, using varying experimental conditions and assays.8−18 There are no experiments that both compare the Ea of different nucleophiles, which gives insights into the mechanism, and use thin layer chromatography (TLC) as the analytical method. Received: December 7, 2016 Revised: October 24, 2017

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DOI: 10.1021/acs.jchemed.6b00940 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

Scheme 1. Nucleophilic Aromatic Substitution Reactions on a Tetra(pentafluorophenyl)porphyrin

NAS Substitution Chemistry

This lab experiment provides a basis to teach other concepts, such as nucleophile hardness and multistep reactions. The use of a porphyrin dye allows visualization of the products under ambient light, and cell-phone pictures of the TLC plates allow quantification of the products using free software (ImageJ) from the National Institutes of Health (NIH). Porphyrins are deeply colored tetrapyrrolic macrocycles that can chelate almost any metal ion in the periodic table. In addition to the naturally occurring porphyrins such as chlorophylls in photosynthesis and heme in blood, synthetic derivatives have applications in medicine, dyes, and solar cells among others. NAS on synthetic porphyrins is a simple and efficient means to synthesize a plethora of easily identifiable and stable products for these diverse applications (Scheme 1).19 In this laboratory experiment, three stock solutions of 5,10,15,20-tetrakis(2,3,4,5,6-pentafluorophenyl)porphyrin (TPPF20), a primary straight-chain alkane nucleophile, and K2CO3 are prepared. After placing appropriate amounts of each solution into three reaction vessels, students heat these solutions for 1−2 h at three different temperatures, and collect small aliquots over set time intervals. After 2 h, the aliquots are spotted on a silica-TLC plate and eluted until distinct spots are separated. Students use cell phones to take photos of the TLC plates, and import the images into ImageJ. Using the software, relative concentrations of each product are determined and plotted against time to determine the reaction rate at each temperature. These data were used to determine the activation energy for the substitution of each nucleophile. An alternative analysis of TLC plate images based on color vectors of pixels was reported.20 Different nucleophiles and temperatures were split evenly between the 10 students in each lab class. The experiment was conducted in four separate organic chemistry II lab classes, and one upper-division physical chemistry lab class. Results and TLC images were shared for individual analysis of data. Each student was responsible for analyzing class data using ImageJ and developing Arrhenius plots to calculate activation energies of NAS.

In this experiment, NAS reaction occurs preferentially at the 4′position (para) on the pentafluorophenyl group of TPPF20 because it is thermodynamically and sterically favored, and the 2′- and 6′-positions (ortho) are kinetically disfavored due to steric interactions with the porphyrin ring.19 In addition to the electron-withdrawing properties of the fluorine on the aryl group, the carbanion transition state can be delocalized into the porphyrin ring.23 Because there are four pentafluorophenyl groups, 4 equiv of the nucleophile will result in sequential substitution of the four para positions to give the tetrasubstituted product. Large excesses of the nucleophile and higher temperatures can result in further substitutions of either the ortho- or meta-fluoro groups. Because the pentafluorophenyl groups are orthogonal to the chromophore and very weakly coupled to each other, they react independently of one another; thus, the activation energies are the same. This is demonstrated by the statistical distribution of five possible products.24 The activation energy for substitution depends heavily on the nature of the nucleophile. Most undergraduate experiments currently used in laboratories concentrate on the number, position, and nature of the electron-withdrawing groups as factors in the mechanism of NAS reactions.16−18 Herein, through a combination of varying experimental conditions and visual techniques, students estimate relative activation energies for three different primary nucleophiles: butanethiol, butylamine, and butanol. A sample reaction mechanism is shown in Scheme 2.

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EXPERIMENTAL PROCEDURE A stock solution of TPPF20 (2 mM) and K2CO3 (20 mM) in Nmethyl-2-pyrrolidone (NMP) was prepared. Students withdrew 5 mL of stock solution using a syringe or a graduated cylinder and transferred this to a round-bottom flask before adding 6 molar equiv of one of the nucleophiles: butanethiol, butylamine, or butanol. Three reactions were prepared for each nucleophile. Students worked in groups of three. Each group handled a single nucleophile, and a student within each group ran the reaction at a given separate temperature. Heated stirring plates were used in the reaction. Butanethiol samples were allowed to react at room temperature (25 °C), 40, and 60 °C under nitrogen. Butylamine samples were allowed to react at 40, 60, and 85 °C. Butanol samples were allowed to react at 60, 85, and 120 °C. These samples were left to stir at the designated temperatures for 2 h.

Introductory Lecture

The introduction of NAS serves as the foundation of the lecture. Concepts of substitution chemistry, nucleophile hardness, activation energy, and the Arrhenius equation serve as the foundation for chemical knowledge.9,11,13,21,22 An overview of ImageJ and linearizing concentration versus time plots of second-order reactions creates a path by which students fully understand the rate of reaction and how to analyze it. B




Using the Arrhenius Equation To Calculate Activation Energy

Scheme 2. NAS Mechanism on TPPF20

Within each column, the relative proportion of each spot determined by ImageJ is multiplied by the initial concentration to generate a plot of concentration versus time (Supporting Information, Figure S7) at each temperature. From these plots, rate constants are determined that are used in the Arrhenius equation to determine the activation energies for substitution by the nucleophiles. The reaction is assumed to be second-order. Spots representing a unique product were selected because the four perfluorophenyl groups react independently of each other, and the rates of accumulation of all the products are assumed to be equal. To linearize the plot in Figure S7, these data are plotted as a graph of inverse concentration versus time (M−1 vs time in seconds) (Figure S8). The slopes of these graphs are negative rate constants, −k. The rate constants, k, from each temperature within the group are plotted as ln(k) versus 1/ temperature (K) (Figure S9). The slope of this graph is taken as −Ea/R. From here, multiplication by −8.314 J/mol K yields the activation energy for that particular nucleophile.

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HAZARDS All chemicals should be treated as potentially hazardous. Exposure through skin or nasal inhalation should be avoided. Thiols and other sulfur compounds are extremely smelly and should always be opened in a fume hood. Products of the reaction with substituted sulfur are not as smelly and can be used outside the hood. Dichloromethane and petroleum ether are hazardous to human health. Petroleum ether is flammable and is hazardous to the environment. TPPF20 and its derivatives may cause skin and eye irritation, as well as acute toxicity if inhaled or ingested. Eye protection, gloves, and protective clothing should be worn at all times in a lab environment. Methanol is toxic and may lead to damaging the kidneys, liver, or eyes. Exercise caution if using a UV lamp. All flammables and solvents should be handled with extra care.

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Figure 1. Representative student TLC plate of a reaction with butylamine. Each column represents a 15 min interval. Spots for six products can be seen at each time, with the relative intensity of each spot changing over time.

RESULTS AND DISCUSSION Six possible products are observable on the TLC plate including the starting material (Figure 1). The color, intensity, and area of the spots are directly proportional to the relative concentration of that product in the reaction mixture. From top to bottom, these spots are (1) TPPF20 starting material (2) monosubstituted product (3) trans-disubstituted product (4) cis-disubstituted product (5) trisubstituted product (6) tetrasubstituted product The starting material is run in the first (left) column of the TLC plate. This is regarded as zero minutes. Samples of the reaction mixture collected every 15 min for 120 min are then spotted with a capillary tube on the TLC plate as shown in Figure 1. At higher temperatures, it is possible for more spots to appear on the TLC plate, which are a result of substitution of the ortho or meta F and because of rotational isomers. These products can produce streaking seen at the bottom of the TLC plates (Figure S6). These spots may be visualized by eye, but use of a UV lamp enhances visibility and produces images with more distinct peaks in ImageJ. After quantification of the products using ImageJ, activation energies are calculated for

Product Distribution and Yield

Aliquots of the reaction mixture were collected in Eppendorf tubes every 15 min. Using a capillary tube, students spotted a small amount of each of these aliquots on a silica-TLC plate (6 cm × 10 cm) (Figure 1). The TLC plates were run in a freshly prepared solvent system of dichloromethane/petroleum ether (75:25 v/v), with one drop of methanol. The molecule becomes increasingly more polar with each substitution (Figure 1), which allows for easy separation on the TLC plate. After running the TLC plates, students took photos of the TLC plates with their cell phones. Since the porphyrin macrocycle is highly colored and fluorescent, photos can be taken under ambient light or under 365 nm UV lamp for enhanced visibility. ImageJ allows determination of the relative yield of the products in the reaction mixture based on the area, intensity, and position of each spot. Students imported these photos into ImageJ and obtained an intensity profile of each lane. The areas under the peaks were quantified for the products, and were imported into Microsoft Excel. C



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each nucleophile. Given the potential errors in using ImageJ analysis of the TLC plates to measure concentrations, there is a large standard deviation in the activation energies calculated. From the results of 48 student experiments, the activation energy of butanethiol was found to be 8 ± 5 kJ/mol, butylamine was 57 ± 14 kJ/mol, and butanol was 149 ± 16 kJ/mol. The relative activation energies are expected to be in the order of polarizability of the nucleophile: butanethiol < butylamine < butanol. All of the graphs and calculations can be found in the Supporting Information. All students were able to calculate activation energies from their data. Depending both on the nucleophile and TLC skills, 30% of the students were not able to observe all six spots of the reaction. To determine the rate versus temperature, only one spot on the TLC plate needs to be clearly resolved. Since the exact proportions of the other products are not necessary, the rest of the spots can simply be represented by the sum of intensities. TPPF20 is a commercially available molecule costing around $50/g, and only about 50 mg is needed for a laboratory experiment of 10 students. This experiment allowed discussion of topological isomers of products. For simpler experiments, where only one substitution is observed on the pentafluorophenyl ring, 5-(pentafluorophenyl)-10,15,20-tris(phenyl)porphyrin25,26 can be used. Although a dye such as 4-fluoro3-nitroaniline can be used and is extremely cheap, it cannot be readily visualized because of weak absorption and fluorescence. Students rated the experiment highly in terms of interest and knowledge gained in NAS chemistry and use of the Arrhenius equation to correlate temperature with rate constant (Supporting Information). Before the experiment there was a low level of understanding of the principles underlying the Arrhenius equation. Only 20% of students answered Arrhenius equation questions correctly in prelab assignments while 90% answered NAS and mechanism questions correctly. However, >90% of students demonstrated a better understanding of the relationship between activation energy, rate, and temperature in postlab assignments and a laboratory recitation exam. Results showed that combining NAS and Ea in one lab experiment allowed students to make a clear connection between the two concepts and allowed a more efficient method of teaching. More than 70% of the students received scores over 90% in their lab assignments, while the remaining 30% of the students showed scores between 70% and 90%. According to student surveys, the experiment was interesting and a useful hands-on experience, and students felt more independent during this experiment compared to other organic chemistry experiments. Significant independent exploration by students is possible, e.g., substitutions of the meta positions and rotational isomers. Depending on the size of the class and the time allotted to the experiment, students may divide into several groups and produce more data. The students can readily synthesize TPPF20 from pentafluorobenzaldehyde and pyrrole in refluxing propionic acid in a prior laboratory.27,28

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Waqar Rizvi: 0000-0002-2415-2485 Charles Michael Drain: 0000-0003-2541-2813 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

W.R. thanks David. R. Mootoo and Jacopo Samson for helping to organize the laboratory experiment and Riona Park for TLC elution. This work was supported by the National Science Foundation CHE-1610755; Hunter College science infrastructure is supported by the NSF, the National Institute on Minority Health and Health Disparities 8G12 MD007599, and the City University of New York.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.6b00940. Instructor notes and student handout (PDF, DOCX) Spreadsheet for data analysis (XLSX) D




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Experimental Determination of Activation Energy of Nucleophilic

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