Noncovalent Derivatization: A Laboratory Experiment for

Laboratory Experiment pubs.acs.org/jchemeduc

Noncovalent Derivatization: A Laboratory Experiment for Understanding the Principles of Molecular Recognition and SelfAssembly through Phase Behavior Amy S. Cannon,† John C. Warner,‡ Smaa A. Koraym,§ and Anne E. Marteel-Parrish*,§ †

Beyond Benign, 100 Research Drive, Wilmington, Massachusetts 01887, United States Warner Babcock Institute for Green Chemistry, 100 Research Drive, Wilmington, Massachusetts 01887, United States § Washington College, 300 Washington Avenue, Chestertown, Maryland 21620, United States ‡

S Supporting Information *

ABSTRACT: An experiment focusing on the creation of phase diagrams involving nonconvalent derivatives of hydroquinone and bis[N,N-diethyl]terephthalamide (HQ-DETPA) is presented. A phase diagram was assembled by taking samples of different compositions (i.e., 40% hydroquinone and 60% bis[N,N-diethyl]terephthalamide, 70%/30%, etc.) and determining the melting points of each sample. This experiment is suitable for students enrolled in a physical chemistry class or materials science course and was effectively accomplished by three pairs of students. The experiment requires two 3-h lab sessions. Background information, experimental procedure and hazards, and results of the research are detailed. Results indicate that the noncovalent derivatization successfully provides a co-crystal that assembles into a 50:50 molar ratio. The eutectic points are shown to take place at the 25:75 and 75:25 molar ratios, respectively. Because entropy was the driving force behind the assembly of the co-crystals, the presence of a maximum point on the phase diagram, which represents the highest value of enthalpy and lowest point of entropy, was also witnessed and occurred at the 50:50 molar ratio of HQ to DETPA. KEYWORDS: Upper-Division Undergraduate, Laboratory Instruction, Physical Chemistry, Hands-On Learning/Manipulatives, Green Chemistry, Materials Science, Molecular Recognition, Noncovalent Interactions, Phases/Phase Transitions/Diagrams

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BACKGROUND Chemists manipulate matter in order to make molecules perform in a predictable manner. Traditional methods involve identifying a molecule of interest, then synthesizing hundreds or even thousands of derivatives in order to “fine-tune” its properties. For example, in order to make a molecule less water-soluble, traditionally one would functionalize it by adding an alkyl group. The alkyl group increases the hydrophobicity of the molecule, intensifying the lipophilic−lipophilic interactions between the molecules and decreases the interactions with the surrounding water molecules, therefore making the molecule less water-soluble. This type of derivatization is done extensively in industry in order to manipulate the physical properties of a molecule to obtain the desired performance. Traditional derivatization of molecules is governed by the enthalpy of the system. Enthalpy (ΔH) is a measurement of energy changes that take place when two bonded atoms are dissociated, while in the gas phase and at constant pressure.1 High temperatures and reactive materials are typically used in order to make and break bonds, which increases the chance for unwanted byproducts and waste. However, by exploiting the entropy of a molecular system and understanding how to use and manipulate noncovalent interactions, the process becomes © XXXX American Chemical Society and Division of Chemical Education, Inc.

inherently more benign. Entropy is a calculation of disorder and when it is initiating the reaction, an increase in the enthalpy of the surrounding and a decrease in the entropy of a system take place.2 The reason behind why the entropy or disorder of the system decreases has to do with the noncovalent interactions taking place between complementary molecules. Understanding the noncovalent “tendencies” is crucial in the manipulation of the functions and properties of a target molecule that can still be altered without having to change its structure. For example, when making a molecule less water-soluble, rather than changing the structure of the molecule itself, one can change the environment in which the molecule resides. If the molecule has the appropriate functional groups, the molecule can be trapped within a noncovalent matrix with a second (benign) auxiliary molecule. The auxiliary molecule changes how the first molecule interacts with itself and its surroundings and, if designed correctly, can make the molecule less water-soluble. Therefore, the same end point, decreasing the water solubility of a molecule, can be achieved by not changing the molecular structure of the molecule itself. This type of manipulation of physical properties is termed noncovalent derivatization.3,4

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dx.doi.org/10.1021/ed400622r | J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

Figure 1. Types of noncovalent interactions.

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NONCOVALENT DERIVATIZATION SYSTEM To determine what type of auxiliary molecules is needed for noncovalent derivatization, the types of noncovalent interactions the molecule can undertake must first be identified. There are four main types of noncovalent interactions, including hydrogen bonding, electrostatic interactions, charge transfer (π-stacking), and lipophilic−lipophilic interactions (Figure 1). There have been several studies utilizing hydrogen bonding and looking into the formation of cocrystals with the properties of the interest molecule greatly altered. This technique is used extensively in the pharmaceutical sector in order to alter the physical properties of active pharmaceutical ingredients.5−8 In fact, the U.S. FDA is taking an active role in guiding researchers using this new method for creating pharmaceutical active ingredients.9 A previous study conducted by Warner et al. portrays the outcomes of two different co-crystals of hydroquinone with bis(alkyl)terephthalamides.10 Warner et al. used hydroquinone as their molecule of interest and combined it with the auxiliary molecules of bis(N,N-diethyl)terephthalamide and bis(N,N-dimethyl)terephthalamide to form 1:1 molecular complexes. The co-crystals were verified by solid-state NMR, differential scanning calorimetry (DSC) and through X-ray crystal structures.10,11 Further studies have shown the formation of co-crystals with other auxiliary molecules, demonstrating the application and versatility of noncovalent derivatization and revealing the extent to which noncovalent derivatization is an environmentally benign technique.3,4,10−14 Hydroquinone is a water-soluble molecule and a bis hydrogen bond donor. Therefore, a bis hydrogen bond acceptor molecule, such as bis[N,N-diethyl]terephthalamide, can be introduced in order to trap hydroquinone in a noncovalent hydrogen bonded matrix (Figure 2). This new complex is called a noncovalent derivative of hydroquinone.3,4 Figure 2 indicates that hydroquinone (HQ) and bis[N,Ndiethyl]terephthalamide (DETPA) combine in a 1:1 molar ratio; therefore, this is denoted as a 1:1 HQ:DETPA noncovalent derivative.

Figure 2. Hydroquinone and bis[N,N-diethyl]terephthalamide 1:1 noncovalent derivative.

involves grinding the two materials together with a mortar and pestle or with a ball mill grinder. Co-crystallization is achieved by dissolving the two solids in a solvent and allowing them to crystallize as a co-crystal complex out of solution. An aqueous grind is typically used for large-scale applications, where the materials are ground together in water. Solution deposition, which is the least ideal method, consists in dissolving the materials in an organic solvent and quickly evaporating the solvents in order to leave behind the co-crystal complex. Once the complexes are made, they must be analyzed to confirm the ratio of the derivative complexes, and to understand the phase behavior and the physical properties of the complex. The complexes are generally analyzed by differential scanning calorimetry (DSC), solid-state NMR spectroscopy, X-ray crystallography and FT-IR spectroscopy. DSC is the most important analytical method for understanding the phase behavior of the complexes. This instrument is used to

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SYNTHESIS OF NONCOVALENT DERIVATIVES Noncovalent derivative complexes are made by various methods, including solid grinding,10 co-crystallization,10 aqueous grinding14 or solution deposition.14 Solid grinding B




determine the melting point of the complexes. However, the experimental procedure presented here shows that this could also be done using a simple melting point apparatus. The melting points are then used to create a phase diagram of the co-crystal complex and to identify the ratio of the co-crystal complex.

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ANALYSIS OF NONCOVALENT DERIVATIVES Most compounds, when mixed together, do not form a cocrystal complex, but will form a eutectic mixture of the compounds. For example, when introducing molecule A into molecule B, a melting point depression and a widening of the melting point range will be experienced. This range represents impurities in the pure sample. As more and more of molecule A and molecule B are mixed, the melting point range gets smaller and smaller until a minimum point (the eutectic point) is reached, where the mixture is in its most disordered state and entropy is at a maximum. If the melting point versus the composition is plotted, a diagram such as the one shown in Figure 3 would be expected, where the area between the

Figure 4. Expected phase diagram representing the temperature vs composition for a noncovalent derivative containing a mixture of A and B.

where the purity of the samples can be assessed. To determine if a unique complex is being formed, versus just a mixing of two compounds, DSC curves can be observed to find either one melting point curve, or more. The observation of one melting point for a co-crystal complex represents the formation of a unique complex, whereas multiple melting points represent a mixture of compounds. This manuscript describes the construction of a phase diagram using a melting point apparatus, which does not allow one to view multiple melting points, but instead observe a widening of the melting point range representing a mixture (or impurity in the sample).

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EXPERIMENTAL OVERVIEW The two components of this experiment are hydroquinone (HQ) and bis(N,N-diethyl)terephthalamide (DETPA). Whereas the hydroquinone can be purchased, the DETPA has to be synthesized in the lab. If the students synthesize the DETPA, two 3-h laboratory periods are needed. During the first 3 h period, the students synthesize the DETPA and during the second 3 h period, the students collect and recrystallize the DETPA. Alternatively, the DETPA can be prepared by the instructor and given to the students. Instructions for both procedures are available in the Supporting Information. One or two laboratory periods are required to prepare the noncovalent derivative complexes and take melting point measurements. Student groups can either be tasked with performing the 11 solid grinds of HQ and DETPA required to construct the phase diagram (in Table 1) or the student groups can be assigned different ratios of the materials and the class can construct the phase diagram together by sharing their data. The students measure the appropriate masses of HQ and DETPA and grind them together for 15−20 min using a mortar and pestle. Each sample is stored in a small vial and labeled accordingly. After each sample is produced, the melting point is measured using a melting point apparatus (Mel-Temp) and recorded. The students then construct the phase diagram based on their collected melting points.

Figure 3. Expected diagram for melting point (on-set) vs composition of compound A (temperature vs composition).

temperature line at 140 °C and the two curves is the melting range and the data points at 140 °C represent the eutectic mixture of compounds A and B. With the use of a melting point apparatus, the melting point of the mixed compounds will seem like a range. To construct a phase diagram of a noncovalent derivative of molecules A and B, a matrix of samples with compositions varying from 0% A, 100% B to 100% A, 0% B would be produced. The melting points of the various samples would be collected and the melting point would be plotted versus composition. A phase diagram as the one shown in Figure 4 would be expected. This phase diagram has two eutectic points and also a maximum point at 50% A and 50% B indicating the presence of a 1:1 molar ratio of a complex of molecule A and molecule B. This maximum point is where entropy is at a minimum and enthalpy is at a maximum and the two minima is where entropy is at a maximum and enthalpy is at a minimum. Not every noncovalent derivative will give a 1:1 co-crystal; however, a phase diagram will indicate what molar ratio the two compounds will form (if any). It should be noted that phase diagrams are ideally constructed using a differential scanning calorimeter (DSC),

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HAZARDS Hydroquinone is a suspected carcinogen, mutagen, and an irritant. Terephthaloyl chloride is corrosive, a lachrymator, and an irritant. Diethyl amine is an irritant and a permeator. Methylene chloride is a carcinogen and an irritant. Anhydrous magnesium sulfate is an irritant. Hydrochloric acid is corrosive, C




compositions of 25:75 and 75:25 molar ratios of hydroquinone to DETPA and with respective eutectic temperatures of approximately 104 and 105 °C. The maximum point is also another important feature, for it displays at what point entropy is at its lowest value and enthalpy is at its highest. This point is important for two reasons: (1) it pin-points the molar ratio at which this cocrystal forms and (2) it validates that entropy is the driving force behind the derivatization. In the phase diagram, the maximum point occurs at a 50:50 molar composition, indicating that this noncovalent derivative provides a 1:1 molar composition of HQ and DETPA in the cocrystal at a temperature of about 140 °C. This point also provides an opportunity to discuss the interplay of entropy and enthalpy with respect to Gibbs free energy. One can see that entropy was the driving force because this is the point at which the two molecules are the most ordered. Knowing that entropy increases as the molecules become more disordered, the entropy is at its lowest value when molecules are in their most ordered state. Enthalpy, on the other hand, is at its highest value because if the structure of the co-crystal is thought of in terms of the multitude of hydrogen bonds present, then the amount of stored energy within the molecule increases tremendously.

an irritant, and a permeator. Sodium hydroxide is corrosive and an irritant. Ethanol is flammable and an irritant.

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RESULTS AND DISCUSSION Thirteen samples of co-crystal complexes were made by solidstate grinding different weights of HQ and DETPA reflecting different % molar compositions. Table 1 lists the characteristics of each co-crystal. Table 1. Student Data for Assembled Co-Crystal Masses and Melting Point Values Melting Point (°C)a Sample No.

Molar % HQ

Molar % DETPA

Mass HQ/g

Mass DETPA/g

Set 1

Set 2

1 2 3 4 5 6 7 8 9 10 11 12 13

100 90 80 75 66.7 60 50 40 33.3 25 20 10 0

0 10 20 25 33.3 40 50 60 66.7 75 80 90 100

0.4996 0.4271 0.3585 0.3254 0.2755 0.2405 0.1901 0.1455 0.1175 0.0854 0.0669 0.0318 0

0 0.0791 0.1499 0.1777 0.2254 0.2623 0.3175 0.3503 0.3836 0.4176 0.4398 0.4658 0.5002

168 150 122 105 111 116 140 126 112 104 116 146 148

168 150 122 105 110 116 138 126 112 103 118 146 148

a

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CONCLUSION After successfully developing phase diagrams that reflect the presence of eutectic points and a maximum point, the molar ratio formed by the co-crystal hydroquinone-bis(N,N-diethyl)terephthalamide is proved to be 1:1. After acquiring the molar ratio of this co-crystal, the next goal should be to take it a step further and isolate a co-crystal so that the structure can be properly characterized using X-ray crystallography, differential scanning colorimetry, and solid-state NMR spectroscopy.

Melting points were first onset measurements.

This experiment was performed by three groups of two students and each time two sets of melting points were recorded for each sample. Table 1 summarizes typical values of melting points for each co-crystal. The data in the table are the combined data obtained by three pairs of students in the same lab session. From the obtained melting point values, a phase diagram was assembled from an average of each set of melting points (Figure 5) and several characteristics were assessed. First, there were two minima. These points represent the eutectic points, with

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ASSOCIATED CONTENT

S Supporting Information *

A list of the detailed experimental procedure and hazards, as well as instructor notes. 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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REFERENCES

(1) Moore, J. W.; Stanitski, C. L.; Jurs, P. C. In Chemistry: The Molecular Science, 3rd ed.; Books/Cole Cengage Learning: Belmont, CA, 2008; p 1105. (2) Levine, I. N. In Physical Chemistry; McGraw Hill: Boston, MA, 2002; p 986. (3) Warner, J. C. Pollution Prevention via Molecular Recognition and Self Assembly: Non-Covalent Derivatization. In Green Chemistry: Frontiers in Benign Chemical Synthesis and Processes; Anastas, P., Williamson, T., Eds.; Oxford University Press: London, 1998; pp 336− 346. (4) Warner, J. C. Entropic Control in Green Chemistry and Materials Design. Pure Appl. Chem. 2006, 78 (11), 2035−2041. (5) Rager, T.; Hilfiker, R. Application of Phase Diagrams in Cocrystal Search and Preparation. In Pharmaceutical Salts and Co-Crystals,

Figure 5. Phase diagram representing the melting point temperature vs composition recorded for the co-crystal hydroquinone-bis(N,Ndiethyl)terephthalamide. The percent composition is based on the composition of hydroquinone (HQ). The melting points were first onset measurements. D




Wouters, J.; Quéré, L., Eds.; RSC Drug Discovery Series, Royal Society of Chemistry: Vol. 16, 2012, pp. 280−299. (6) McNamara, D. P. Use of a glutaric acid cocrystal to improve oral bioavailability of a low solubility API. Pharm. Res. 2006, 23 (8), 1888− 1897. (7) Asija, R.; Mangukia, D.; Asija, S. Pharmaceutical Cocrystals: An Overview. J. Drug Discovery Ther. 2013, 1 (3), 10−14. (8) Desiraju, G. R. Pharmacuetical Salts and Co-crystals: Retrospect and Prospects. In Pharmaceutical Salts and Co-Crystals, Wouters, J.; Quéré, L., Eds.; RSC Drug Discovery Series, Royal Society of Chemistry: Vol. 16, 2012, pp. 1−8. (9) Guidance for Industry: Regulatory Classification of Pharmaceutical Co-Crystals, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), April 2013. http://www.fda.gov/downloads/ Drugs/Guidances/UCM281764.pdf (accessed May 2014). (10) Guarrera, D.; Taylor, L. D.; Warner, J. C. Molecular SelfAssembly in the Solid State. The Combined Use of Solid State NMR and Differential Scanning Calorimetry for the Determination of Phase Constitution. Chem. Mater. 1994, 6, 1293. (11) Foxman, B. M.; Guarrera, D. J.; Taylor, L. D.; Warner, J. C. Environmentally Benign Synthesis Using Crystal Engineering: Steric Accommodation in Non-Covalent Derivatives of Hydroquinones. Cryst. Eng. 1998, 1, 109. (12) Foxman, B. M.; Guarrera, D. J.; Pai, R.; Tassa, C.; Warner, J. C. Non-Covalent Derivatives of Hydroquinone: Bis-(N,N-Dialkyl)Bicyclo[2.2.2]octane-1,4-dicarboxamide Complexes. Cryst. Eng. 1999, 2 (1), 55. (13) Cannon, A. S.; Foxman, B. M.; Guarrera, D. J.; Warner, J. C. Noncovalent Derivatives of Hydroquinone: Complexes with Trigonal Planar Tris(N,N-dialkyl)trimesamides. Cryst. Growth Des. 2005, 5, 407−411. (14) Guarrera, D. J.; Taylor, L. D.; Warner, J. C. Proceedings of the 22nd NATAS Conference; NATAS: Bowling Green, KY, 1993, Vol. 496.

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Noncovalent Derivatization: A Laboratory Experiment for

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