Preparation and Characterization of a Flavor Compound Inclusion

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

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Preparation and Characterization of a Flavor Compound Inclusion Complex in a Simple Experiment Guangyong Zhu*,† and Genfa Yu† Shanghai Institute of Technology, No. 100 Haiquan Road, Shanghai 201418, China

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S Supporting Information *

ABSTRACT: Flavor plays a crucial role and is the most important aspect of foods and beverages. β-Ionone is a common flavor compound with a characteristic violet-like odor. An undergraduate flavor compounds chemistry laboratory that provides preparation and characterization of the flavor compound inclusion complex between hydroxypropyl-β-cyclodextrin (HP-β-CD) as a host and β-ionone as a guest is reported. This simple laboratory experiment allows students to become familiar with the concepts of microencapsulation technology and flavor compound inclusion complexes by teaching students how to prepare and characterize the β-ionone−HP-β-CD inclusion complex. A glass flask with a long, graduated neck is used for fabrication of the inclusion complex, which facilitates observation the volume change of βionone with and without the addition of HP-β-CD. From the volume change, the β-ionone loading capacity can be calculated. The volume difference of β-ionone also provides an intuitive way for students to understand that β-ionone is encapsulated in HP-β-CD and that the water solubility of β-ionone is improved by encapsulation. This laboratory experiment also allows students to become familiar with the freeze-drying process, thermogravimetric analysis (TGA), and the application of TGA in characterization of the inclusion complex. Formation of the inclusion complex and analysis of the TGA results allow students to understand the characteristics of β-ionone release from the inclusion complex and the improved thermal stability of β-ionone. KEYWORDS: Second-Year Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Thermal Analysis

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INTRODUCTION Flavor plays a crucial role and is the most important aspect of foods and beverages.1,2 Flavor Compounds Chemistry is a basic course open to second-year undergraduate students who major in perfume and aroma technology at the Shanghai Institute of Technology. The course provides comprehensive information about the odor characteristics, properties, methods of manufacture, and applications of various natural and synthetic raw materials used in the flavor industry, as well as information about flavor technology. The prerequisites include a background in inorganic, organic, and analytical chemistry. Flavor microencapsulation technology is one important part of the curriculum. Microencapsulation is a technique in which tiny droplets or particles are surrounded by a coating to yield small capsules with many useful properties. Coacervation, cocrystallization, molecular inclusion, and interfacial polymerization are the main chemical techniques for microencapsulation. Spray drying, spray chilling, extrusion, and use of a fluidized bed are the main mechanical techniques for microencapsulation. In general, the interior contents of the capsules can be released through four different mechanisms: diffusion, fracturation, biodegradation, and melting or dissolution. The making of a flavor compound inclusion complex and characterization of the complex is a simple © XXXX American Chemical Society and Division of Chemical Education, Inc.

laboratory experiment designed to help students better understand flavor microencapsulation. For colleges that do not teach flavor chemistry as a separate course, this experiment can be used as an organic synthesis or analytical chemistry experiment. Flavor compounds are typically volatile chemicals, and many of them are insoluble in water. Microencapsulation technology can be used to prevent the loss of volatile aromatic compounds and improve their shelf life.3,4 Therefore, flavor microcapsule formation can provide protection and enhance the stability of aromatic compounds. The formation of flavor compound inclusion complexes is one method used to prepare flavor microcapsules. Cyclodextrins (CDs), which have a hydrophobic inner cavity and a hydrophilic outer side, can accommodate compounds to form inclusion complexes.5−7 Hydroxypropyl-β-cyclodextrin (HP-β-CD) has high water solubility and low toxicity.8 One function of HP-β-CD is to act as wall material to encapsulate flavor compounds. Another function is that it solubilizes insoluble flavor compounds through the formation of HP-β-CD inclusion complexes. Received: December 12, 2018 Revised: June 10, 2019

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DOI: 10.1021/acs.jchemed.8b00996 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

β-Ionone is a common flavor ingredient with a characteristic violet-like odor and fruity and woody notes that has been widely used in many products, such as beverages, candy, puddings, baked goods, frozen dairy foods, and chewing gum. β-Ionone can also be used in floral fragrances such as violet, osmanthus, rose, jasmine, lily, tuberose, and so on.9,10 Therefore, β-ionone was selected as the core material and HP-β-CD was selected as the wall material for students to prepare a flavor compound inclusion complex in the experiment. Furthermore, this laboratory experiment also allows students to perform analysis of the inclusion complex by thermal gravimetric analysis (TGA) and become familiar with its application in the characterization of inclusion complexes. Before performing TGA, HP-β-CD and the βionone−HP-β-CD inclusion complex should be dried. The freeze-drying process is a low temperature dehydration process also known as lyophilization or cryodesiccation. Because of the low temperature used in processing, freeze-drying results in a high-quality product. This laboratory experiment uses the freeze-drying process to dry HP-β-CD and the β-ionone−HPβ-CD inclusion complex. It is a multidisciplinary laboratory experiment for undergraduate students that integrates flavor compound chemistry, organic chemistry, and analytical chemistry. It is a straightforward experiment that provides an introduction to the concept of inclusion complexes and their application in the encapsulation of flavor compounds.

Figure 2. Glass flask with a long neck for complexation of β-ionone and HP-β-CD.

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inclusion complex. TGA is a method of thermal analysis in which the mass of a sample is measured over time as the temperature changes. It can provide information about chemical phenomena, such as chemisorption and thermal decomposition, as well as physical phenomena, such as absorption, desorption, and phase transitions.11,12 The thermal stability of a material can be evaluated by TGA. If a material is thermally stable in a desired temperature range, there will be minimal mass change. By comparison of weight loss curves of β-ionone, HP-β-CD, and the β-ionone−HP-β-CD inclusion complex, students studied the release characteristics of βionone from the inclusion complex, and the thermal stability of β-ionone before and after formation of the inclusion complex. This experiment was accomplished in a flavor laboratory course with 40 students. The lab period for the first part of the experiment was 5 h with a subsequent freeze-drying process for 48 h. The laboratory was equipped with one thermogravimetric analyzer. In the second part of the experiment, 40 students worked in five groups of four using the TGA. The duration of the second part of the experiment was 30 h. The whole experiment took a total of 83 h of lab time for all five groups to complete. The notes for instructors and the student handout are provided in the Supporting Information.

EXPERIMENTAL OVERVIEW The experiment consisted of two parts. In the first part of the experiment, students produced the β-ionone−HP-β-CD inclusion complex using instruments such as those shown in Figure 1, including a glass flask with long, graduated neck (Figure 2).

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EXPERIMENTAL PROCEDURES

Materials

β-Ionone (light yellow liquid, content: ≥95%, molecular weight: 192) and HP-β-CD (white powder, content: ≥98%, molecular weight: 1806) were received from Shanghai Shenbao Flavor and Fragrance Company, Ltd., and Shandong Binzhou Zhiyuan Bio-Technology Company, Ltd., respectively, and were used as received. β-Ionone is a common flavor raw material that can also be obtained from Givaudan, International Flavor and Fragrance, Firmenich, or Thermo Fisher Scientific. HP-β-CD can also be obtained from Thermo Fisher Scientific.

Figure 1. Instruments for the preparation of the β-ionone−HP-β-CD inclusion complex.

β-Ionone (log Kow = 4.42, specific gravity: 0.940−0.947 at 25 °C) is insoluble in water and will float on the surface of the water. By addition of a fixed amount of β-ionone, students compared the amount of β-ionone solubilized in the presence of HP-β-CD versus that in a control with no HP-β-CD. In the second part of the experiment, students performed TGA on β-ionone, HP-β-CD, and the β-ionone−HP-β-CD B



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Fabrication of the β-Ionone−HP-β-CD Inclusion Complex

HAZARDS Although the experiment does not present any particular hazards, approved safety gloves and proper laboratory clothing must be used in a laboratory. β-Ionone is volatile and flammable. HP-β-CD does not present any hazards as long as it is not swallowed or inhaled. In the case of skin or eye contact, flush hands or eyes with plenty of water for at least 15 min. The experiment should be done in a hood.

First, students began this experiment by preparing a 50% (w/ w) HP-β-CD aqueous solution in a beaker. Students then added 50 g of HP-β-CD aqueous solution (50%) to a 110 mL glass flask with a long, graduated neck, as shown in Figure 1. A scale (100−110 mL) was marked on the neck with minimum scale value of 0.1 mL. Students kept the flask in a thermostatic water bath at a constant temperature of 30 °C. Second, students added 2.8 g of β-ionone to the HP-β-CD aqueous solution. The mixtures were stirred using a magnetic stirrer for 2 h at 30 °C to encapsulate a portion of the β-ionone in the HP-β-CD cavities and solubilize it in the water. Finally, students cooled the mixture for about 15 min to room temperature and added 51 mL of deionized water to the scale mark (100−110 mL) of the flask. β-Ionone is difficult to dissolve in water, and its density is lighter than that of water. Unencapsulated β-ionone will float on the top, as shown in Figure 2, and can be quantified by the scale. The volume of unencapsulated β-ionone can be obtained by subtracting the low scale value from the high scale value. As shown in Figure 2, the yellow liquid is β-ionone. From the graduation marks, the high scale value (103.59 mL) and low scale value (102.18 mL) can be observed. In this way, the volume of unencapsulated β-ionone can be obtained. At the same time, students perform the experiment on a control sample of deionized water without the addition of HP-β-CD. Students calculate the loading capacity (LC), defined as the mass ratio of encapsulated β-ionone to HP-βCD, as in eq 1.

(

M1 − V1 × LC =

M3

M2 V2


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RESULTS AND DISCUSSION

Encapsulation of β-Ionone in HP-β-CD and Loading Capacity

The initial masses of β-ionone and HP-β-CD added by a typical student and the volume of β-ionone obtained from the long neck glass flask are shown in Table 1. Table 1. Initial Mass of β-Ionone and HP-β-CD and Volume of β-Ionone

HP-β-CD (g)

Initial β-Ionone for Encapsulation (g)

Initial β-Ionone for the Control (g)

β-Ionone after Encapsulation (mL)

β-Ionone Volume of the Control (mL)

25.010

2.804

2.807

0.63

2.98

β-Ionone is insoluble in water. Addition of HP-β-CD causes a volume decrease of β-ionone, as shown in Table 1. This is because of the formation of a water-soluble inclusion complex between β-ionone and HP-β-CD. When 25.010 g of HP-β-CD was added, 2.211 g of β-ionone was absorbed, which was calculated by [2.804 − 0.63 × (2.807/2.98)]. By observation of volume changes in the long neck of the glass flask, students could easily determine that most of the β-ionone molecules were encapsulated in HP-β-CD and that the solubility of βionone in water can be improved through the formation of an inclusion complex with HP-β-CD. According to eq 1 and data obtained as shown in Table 1, the LC can be calculated, and the value of the LC was 8.84%. This meant that 1 g of HP-β-CD accommodated 0.0884 g of βionone under the experimental conditions. The molecular weight of β-ionone is 192, and the molecular weight of HP-βCD is 1806. The moles of β-ionone solubilized and moles of HP-β-CD were 0.0115 and 0.0138 mol respectively. Therefore, the ratio of HP-β-CD to β-ionone for the inclusion complex is approximately 1:1.

) × 100% (1)

where M1 is the initial mass of β-ionone added for encapsulation, V1 is the unencapsulated volume of β-ionone after encapsulation, V2 is the volume of β-ionone in the control sample, M2 is the initial mass of β-ionone added for the control, and M3 is the mass of HP-β-CD added. In eq 1, V1(M2/V2) is the mass of unencapsulated β-ionone, whereas M1 − V1(M2/V2) is the mass of encapsulated βionone. In order to reduce the effect of error caused by βionone dissolving slightly in water, M2/V2 was adopted in eq 1 instead of the density of β-ionone. After removal of the unencapsulated β-ionone, the βionone−HP-β-CD inclusion complex aqueous solution was dried in am FD-1C-50 freezer-drier at a temperature lower than −50 °C and pressure of around 0.02 kPa for 48 h. The dried samples were collected and stored in a desiccator at room temperature for further characterization by TGA.

TGA Results

TGA was used to investigate the release of β-ionone from the β-ionone−HP-β-CD inclusion complex as the temperature increased in the experiment. During the process of heating a sample, the mass was continuously measured with time as the temperature increased. From the comparison of the weight loss curves of β-ionone and the β-ionone−HP-β-CD inclusion complex, the students learned that the thermal stability of βionone was enhanced by the formation of the inclusion complex. The weight losses of HP-β-CD and the β-ionone− HP-β-CD inclusion complex obtained when students optimized the experiment are shown in Figure 3. During the heating process, β-ionone could not withstand high temperatures; it vaporized quickly and was almost completely gone at 175 °C, although its boiling temperature range is 237−239 °C. Because of heating, gas flow, sample geometry, and the initial sample size, having the β-ionone

TGA Analysis

A TGA-Q500IR thermogravimetric analyzer (TA Instruments) was used for determining the weight loss of β-ionone, HP-βCD, and the β-ionone−HP-β-CD inclusion complex over time as a function of increasing temperature. In the experiment, students weighed approximately 7 mg of sample and spread it uniformly on the ceramic crucible of the thermal analyzer. The temperature of the furnace was programmed to rise from room temperature to 600 °C at a heating rate of 10 °C/min in a dynamic, high purity nitrogen flow of 20 mL/min. C




and analytical chemistry. In the experiment, the β-ionone−HPβ-CD inclusion complex is successfully synthesized by students using a glass flask with a long, graduated neck. By observation of volume changes in the long neck of the glass flask, students can easily learn that most of the β-ionone molecules are encapsulated in HP-β-CD and that the solubility of β-ionone in water can be improved by formation of an inclusion complex with HP-β-CD. The experimental protocol also has students analyze β-ionone, HP-β-CD, and the β-ionone−HP-β-CD inclusion complex using TGA. TGA reveals that β-ionone release from the β-ionone−HP-β-CD inclusion complex mainly occurs in the temperature range of 210 to 320 °C, whereas unencapsulated β-ionone is volatile almost completely at 175 °C. The TGA analysis can help students understand the improved thermal stability of β-ionone from the formation of the inclusion complex. The β-ionone−HP-β-CD inclusion complex has a characteristic violet-like odor that can be detected by smelling. Feedback from students on the experiment was positive. The experiment has proven to be an effective method for providing students with hands-on experience performing fabrication and TGA analysis. By completing this experiment, students will gain knowledge of preparation of flavor compound inclusion complexes and analytical skills.

Figure 3. Weight loss curves of HP-β-CD and the β-ionone−HP-βCD inclusion complex with increasing temperature.

evaporate completely before the boiling point is obvious. As shown in Figure 3, the weight loss curves of HP-β-CD and the β-ionone−HP-β-CD inclusion complex show three clear stages. The first stage goes from room temperature to 292 °C; the second stage goes from 292 to 405 °C; and the third stage goes from 405 to the final temperature of 600 °C. The majority of weight loss occurs in the second stage, when sharp declines are observed in the curves of the HP-β-CD and βionone−HP-β-CD inclusion complexes because of the degradation of HP-β-CD. Continued slight weight loss was observed in the third stage because the solid residuals of HP-βCD continuously decomposed at a very slow rate. In the first stage, the weight loss curve of HP-β-CD was almost parallel to the temperature axis, and the weight loss was very slight, whereas the weight loss curve of the β-ionone−HP-β-CD inclusion complex had a downward sloping trend; especially in the temperature range of 210 to 292 °C, relative larger weight loss was observed. From room temperature to 210 °C, the weight losses of HP-β-CD and the β-ionone−HP-β-CD inclusion complex were 1.12 and 1.33% respectively. From 210 to 292 °C, the weight losses of HP-β-CD and the βionone−HP-β-CD inclusion complex were 0.25 and 3.15% respectively. This difference can be attributed to the release of β-ionone. In the beginning, with decomposition of HP-β-CD from 292 to 320 °C, β-ionone molecules encapsulated in the cavities of HP-β-CD molecules could be released rapidly. The weight losses for HP-β-CD and the β-ionone−HP-β-CD inclusion complex were 2.34 and 5.22%, respectively, in the temperature range of 292 to 320 °C. This difference could also be attributed to the release of β-ionone. The differences in weight loss revealed that β-ionone release mainly occurred in the temperature range of 210 to 320 °C for the β-ionone−HPβ-CD inclusion complex. However, unencapsulated β-ionone was volatile almost completely at 175 °C. From this phenomenon, students could easily understand that the thermostability of β-ionone was improved by encapsulation technology.

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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.8b00996. Notes for Instructors (PDF, DOCX) Student Handout (PDF, DOCX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Guangyong Zhu: 0000-0002-0990-1118 Author Contributions †

G.Z. and G.Y. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Shanghai Alliance Program (LM201844) is gratefully acknowledged. REFERENCES

(1) Zhu, G.; Xiao, Z. Creation and Imitation of a Milk Flavour. Food Funct. 2017, 8, 1080−1084. (2) Epstein, J. L.; Castaldi, M.; Patel, G.; Telidecki, P.; Karakkatt, K. Using Flavor Chemistry To Design and Synthesize Artificial Scents and Flavors. J. Chem. Educ. 2015, 92, 954−957. (3) Zhu, G.; Feng, N.; Xiao, Z.; Zhou, R.; Niu, Y. Production and Pyrolysis Characteristics of Citral-monochlorotriazinyl-β-cyclodextrin Inclusion Complex. J. Therm. Anal. Calorim. 2015, 120, 1811−1817. (4) Zhu, G.; Xiao, Z.; Zhou, R.; Yi, F. Fragrance and Flavor Microencapsulation Technology. Adv. Mater. Res. 2012, 535−537, 440−445. (5) Mendicuti, F.; González-Alvarez, M. J. Supramolecular Chemistry: Induced Circular Dichroism to Study Host-Guest Geometry. J. Chem. Educ. 2010, 87, 965−968.

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CONCLUSIONS A simple experiment in a basic curriculum, Flavor Compounds Chemistry, was developed. It is intended to help undergraduate students become familiar with the concepts of microencapsulation technology and flavor compound inclusion complexes. This is a multidisciplinary laboratory experiment that includes flavor compound chemistry, organic chemistry, D




(6) Khalafi, L.; Kashani, S.; Karimi, J. Molecular Recognition: Detection of Colorless Compounds Based On Color Change. J. Chem. Educ. 2016, 93, 376−379. (7) Jones, D. R.; DiScenza, D. J.; Mako, T. L.; Levine, M. Environmental Application of Cyclodextrin Metal-Organic Frameworks in an Undergraduate Teaching Laboratory. J. Chem. Educ. 2018, 95, 1636−1641. (8) Zhu, G.; Xiao, Z.; Zhu, G.; Zhou, R.; Niu, Y. Encapsulation of Lmenthol in Hydroxpropyl-β-cyclodextrin and Release Characteristics of the Inclusion Complex. Pol. J. Chem. Technol. 2016, 18, 110−116. (9) Ansari, M.; Emami, S. β-Ionone and Its Analogs as Promising Anticancer Agents. Eur. J. Med. Chem. 2016, 123, 141−154. (10) Burdock, G. A. Fenaroli’s Handbook of Flavor Ingredients, 6th ed.; CRC: Boca Raton, 2006; pp 955−956. (11) Iwanek, E.; Gliński, M. Application of Thermal Analysis in Determining Properties of Herbaceous Materials. J. Chem. Educ. 2018, 95, 1359−1364. (12) Zhu, G.; Xiao, Z.; Zhou, R.; Zhu, Y. Study of Production and Pyrolysis Characteristics of Sweet Orange Flavor-β-cyclodextrin Inclusion Complex. Carbohydr. Polym. 2014, 105, 75−80.

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Preparation and Characterization of a Flavor Compound Inclusion

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