Determining the Deacetylation Degree of Chitosan - ACS Publications

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

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Determining the Deacetylation Degree of Chitosan: Opportunities To Learn Instrumental Techniques Leyre Pérez-Á lvarez,* Leire Ruiz-Rubio, and Jose Luis Vilas-Vilela Department of Physical Chemistry, Faculty of Science and Technology, University of the Basque Country, UPV/EHU, Barrio Sarriena s/n, 48940 Leioa, Spain S Supporting Information *

ABSTRACT: To enhance critical thinking and problem-solving skills, a project-based learning (PBL) approach for “Instrumental Techniques” courses in undergraduate physical chemistry was specifically developed for a pharmacy bachelor degree program. The starting point of this PBL was an open-ended question that is close to the student scientist’s interest and related to this lab: “How can we determine the deacetylation degree of chitosan?” Chitosan is a polysaccharide used in a broad range of pharmacy applications because of its unique properties. Many of these properties derive from the presence of primary amino groups (−NH2) in its structure. These −NH2 groups are usually formed by the deacetylation of chitin. For this reason, a proper quantification of the deacetylation degree (DD) of chitosan is important to know whether it can be used in a particular application. This driving question requires an experimental procedure that can be carried out by a wide range of instrumental techniques. In this project, techniques such as conductometry, potentiometry, and 1H NMR were addressed to present an easily reproducible experience for first-year undergraduate students. Students evaluated the applicability of the available instrumental techniques, designed their own laboratory procedures, obtained experimental results, and analyzed data in a challenging experience in which high order thinking and knowledge transfer skills were endorsed. The project described here promotes students’ knowledge of instrumental techniques, and offers them an appropriate scenario to develop transferable skills, such as team-working, problem-solving, and written communication, among others. KEYWORDS: Laboratory Instruction, Physical Chemistry, Carbohydrates, First-Year Undergraduate/General, Hands-On Learning/Manipulatives, Inquiry-Based/Discovery Learning

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INTRODUCTION

In the light of this, the project presented herein aims to solve an experimental question that requires the use of different instrumental techniques and leads to a practical learning of analytical and physical chemistry. The present PBL experience is centered on the open-ended question “how can we determine the deacetylation degree of chitosan?” which is a specific problem that presents several correct solutions and can be solved by different instrumental techniques. Certainly, learning chemistry using instrumental techniques applied to a kind of molecule within the framework of a PBL approach is not a novelty. However, the most extended choice along the years has been focused on developing experiences based on a unique instrumental technique.6−11 Those cases were limited to the better specific understanding of the principles and practical operation of a technique. However, taking into account the huge importance of the capability of students to discriminate between different techniques in a pharmacy curriculum, examples including a collection of techniques seem to be more adequate in promoting critical

Project-Based Learning

Active learning, this is, a learning process in which students are actively involved and engaged with a higher-order task,1 has been increasingly applied around the world over the past decades. Project-based learning (PBL) is noteworthy among current active learning teaching methods. PBL tries to gain knowledge and skills by investigating a challenging question for an extended period of time2 and is widely known for promoting critical thinking and problem-solving skills.3 These skills are of great interest for undergraduate students, especially for pharmacists, because of the current need in the area for professionals able to meet changing demands of the society.4,5 Although some PBL examples have been applied to clinical pharmacy over the past years, only a few isolated miniprojects, being related to general courses of general chemistry or analytical chemistry, have been made on instrumental techniques. As a consequence, there is an actual necessity to improve the practical support and increase the number of examples available for instructors in this field that justifies present work. © XXXX American Chemical Society and Division of Chemical Education, Inc.

Received: November 23, 2017 Revised: April 11, 2018

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DOI: 10.1021/acs.jchemed.7b00902 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

thinking. This was the proposal of Grannas et al.,12 who exposed students to several instrumental techniques within the same problem scenario. Nonetheless, with concerns regarding methodology, Grannas’ approach required application of typical protocols described in a standards text, restricting high order thinking and knowledge transferring. To avoid this, there are also a few examples about the integration of PBL in the instrumental techniques laboratory that try to escape from “cooking recipe” experiments and in which students are requested to design, perform, and correct their own experiments in whole.13 Considering all of the above, in order to promote critical thinking and knowledge transfer, the present project combines the above cited approaches in a real-life problem. Four main learning outcomes of students are specifically worked in this PBL experiment. The first outcome is the capability to connect and discriminate among instrumental techniques. The second learning objective consists of being able to design entire experimental procedures that do not result from standard protocols, but require extrapolation of ideas, and knowledge about instrumental techniques principles and specifications. Third, students will be skilled on learning from their own mistakes, and, finally they will be able to analyze, interpret, and report the results of such experiments. This project is also applicable to areas outside of pharmacy, such as food chemistry or general chemistry. In addition, this project combines the understanding of the general principles of characterization techniques with the development of transferable skills, such as teamwork, decision making, or writing skills.

one of the most promising biomaterials in the past decades. Chitosan is degraded by lysozymes in the body and excreted as nontoxic, nonimmunogenic, and noncarcinogenic degradation products.15,16 In addition, chitosan exhibits many exceptional properties, such as low toxicity, biocompatibility, immune stimulation, suppression of tumor growth, and antifungal and antibacterial properties; it is an enhancer of humoral and cellmediated immune responses, and it presents a cholesterol lowering effect, wound-healing, and mucoadhesive properties.17−19 Due to this wide range of favorable features, as well as its low cost, chitosan has become a relevant compound in various fields closely related to pharmacy, such as cosmetics, dietetics, and biomedicine.20,21 In addition, chitosan is a cationic polysaccharide in an acidic medium. This is a unique characteristic among polysaccharides that enhances drug penetration across the mucosa. As a consequence, chitosan has been extensively studied as a carrier for mucosal drug delivery22,23 and as a material for ophthalmology.24 This interesting polymer has even been employed in the development of a laboratory experiment for undergraduate students in order to investigate the use of hydrogels in targeted drug delivery.25 The degree of deacetylation, DD, of chitosan is the parameter that indicates the molar percentage of glucosamine monomeric units and varies from 0 (chitin) to 100 (fully deacetylated chitin). DD of chitin/chitosan is the most important parameter that influences their biological, physicochemical, and mechanical properties and, subsequently, the effectiveness of chitosan and its derivatives.26 In general, chitosan with a higher DD exhibits better biological properties than poorly deacetylated chitosan, due to the higher concentration of the −NH2 groups versus the N-acetyl glucosamine moiety. Therefore, the determination of the DD of chitosan is essential to predict its properties and validate it for specific applications. Several techniques have already been employed to determine the DD of chitin/chitosan, such as proton nuclear magnetic resonance (1H NMR,27 solid 13C NMR,28 and 15N NMR29), UV spectrophotometry,26 conductometric and potentiometric titration,30 differential scanning calorimetry,31 or CHN elemental analysis,26,30 among others. Some of these techniques are expensive and dependent on a technician (solid NMR, elemental analyses), while others, are simple, inexpensive, and suitable for first-year students’ laboratory (conductometry, potentiometry, and so on). The election of the most appropriate method continues to be a difficult task, and due to this, some review studies have reported on this issue in past years.26,27 Thus, the novelty of this work lies in the compilation of some of these works resulting in a simple PBL experiment specifically adapted for a instrumental techniques course. In this context, students designed and performed experiments to determine the DD of chitosan by 1H NMR, conductometry, and potentiometry, as well as reported the obtained experimental data in a publication style report.

Background of the Open-Ended Driving Question: Deacetylation Degree of Chitosan

Chitosan, poly[β-(1−4)-linked-2-amino-2-deoxy-D-glucose], is a biodegradable biopolymer usually obtained by alkaline partial deacetylation of chitin. Chitin is the second most plentiful organic resource in the world and is the main constituent of the exoskeleton of crustaceans and insects present in plants, cell walls of some fungi, and microorganisms.14 Figure 1 shows the chemical structures of fully N-acetylated chitin and completely N-deacetylated chitosan. Thus, chitosan is a copolymer of 2acetamido-2-deoxy-D-glucose (N-acetyl glucosamine, GlcNAc) and 2-amino-2-deoxy-D-glucose (glucosamine, GlcN) bonded by with β-D-(1−4) glycoside linkages. Chitosan poses unique biological and physicochemical properties that have made it

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EXPERIMENTAL METHODS

Reagents

Acetic acid (for analysis, 99.8%, Panreac) or hydrochloric acid (Panreac, 37%) and sodium hydroxide (≥98%, anhydrous, pellets, Panreac) were used as received. Chitosan (Aldrich, low molecular weight, 66,000 g mol−1 viscosity average molecular weight measured by an Ubbelohde capillary viscometer in HAc

Figure 1. Chemical structure of (A) chitin and (B) chitosan. B




0.3 M/NaAc 0.2 M, 25 °C) was used without further purification. Distilled water, deionized water, CD3COOD (Aldrich), and D2O (Aldrich) were also needed.

Table 1. Project Assignments and Assessments

1

H NMR Spectroscopy

Sessions

Assignments

Class: 1

Problem analysis and experimental design

Lab: 1, 2

Experimental trial

Lab: 3, 4 Class: 2

Experiments performing Data treatment and elaboration of the written report

1

H NMR spectra were recorded on a Bruker Avance 500 MHz instrument. The samples were dissolved in 2% w/w CD3COOD/D2O. Spectra were recorded from 0 to 10 ppm.

Potentiometric Titration

Chitosan (∼0.05 g) was dissolved in an excess aqueous acidic solution (0.1 mol L−1 HCl solution, or acetic acid 1%). This solution was then titrated with 0.1 mol L−1 NaOH measuring pH with a pH-meter Crison (BASIC 20). Potentriometric tritations were made in duplicate.

Assessments Definition of the objective of the project General scheme of experimental procedures Experimental procedure draft Corrections to experimental procedure draft Chemicals, materials and instrumentation sheet Data sheet Data treatment Written report

Conductometric Titration

A chitosan solution was prepared by the dissolution of a known mass of chitosan (∼0.2 g) in 40 mL of a 0.05 mol L−1 HCl solution (or acetic acid 1%) at room temperature (20 °C). Deionized water (DI) was employed in the preparation of all solutions for conductometric determinations in order to avoid external ions’ presence. After the addition of 100 mL of water, conductometric titration was carried out with NaOH (0.15 mol L−1) using a conductivity meter Crison (EC-Metro BASIC 30). All the titrations were done in duplicate.

applications (see student’s guide in Supporting Information). On the basis of this description, during the first class session (before starting lab), students conducted an analysis of the raised problem and a revision of the principles and applications of studied instrumental techniques (see Supporting Information student’s guide: problem analysis sheet). This gave them the opportunity to compare and gain better insight into the possibilities and accuracies of the different instrumental techniques that were available for this experience: potentiometry, conductometry, and 1H NMR spectroscopy (learning outcome 1). In addition, students defined their own preliminary laboratory procedure (learning outcome 2) and completed a risk assessment. Once the design was approved by the lecturer, the students were allowed to perform their lab experiments on a trial session (the first 2 lab sessions) (see Supporting Information student’s guide: work designing sheet and modifications to experimental procedure draft). In this test session, students checked their design and modified their laboratory project according to their findings, observations, and mistakes (learning outcome 3). After designing their own practical work for conductometry, potentiometry and 1H NMR spectroscopy, students performed it in practice (2 last lab sessions), except in the case of 1H NMR spectroscopy. Due to the high technical complexity and responsibility that 1H NMR entails for first/second years, measurements were carried out by the instructor or a technical specialist using samples that were previously prepared by students. Students were provided with acid and base solutions (see Supporting Information Instructor’s Guide) before starting the experiments in order to ease the design of the experimental procedure. Finally, students accurately collected experimental data for each technique.

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HAZARDS Chitosan is not classified as hazardous. However, it can be hazardous in the case of eye contact, skin contact (irritant), ingestion, or inhalation (lung irritant). Sodium hydroxide is highly corrosive and causes severe burns which are particularly dangerous to the eyes. It gives out heat when added to water. Acetic acid is flammable as liquid and vapor, is highly corrosive to the skin and eyes, and, because of this, must be handled with extreme care. Acetic acid can also be damaging to the internal organs if ingested or in the case of vapor inhalation. Hydrochloric acid is highly corrosive and causes severe skin burns and eye damage and as well as respiratory irritation. For this reason, students are not required to handle acetic acid or hydrochloric acid, and diluted solutions are prepared in advance by the instructor. Students should wear safety latex gloves, goggles, and a lab coat throughout the laboratory sessions.

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RESULTS AND DISCUSSION This work describes a PBL approach applied in an Instrumental Techniques compulsory course of the department of Physical Chemistry for undergraduate students in the Bachelor Degree on Pharmacy at the University of the Basque Country. Enrolled students had background knowledge on mathematics, physics, and general, inorganic, analytical, physical, and organic chemistry. The present experience was offered to a unique learning group of 50−60 students and was developed, as summarized in Table 1, during 2 class sessions (2 h/session) and 4 laboratory sessions (4 h/session) simultaneously with master classes and tutorial lectures in which fundamentals of spectroscopic, optic, and electrochemical methods were introduced. Thus, techniques addressed in this PBL were thoroughly introduced. Laboratory sessions were designed for 20 students, and they worked in small groups of 2−3 people. Students received a short description of a driving problem in which the open-ended question “How can we determine the deacetylation degree of chitosan?” was presented in a real application framework close to pharmacy interests and

1

H NMR Spectroscopy

Students dissolved chitosan in a previously prepared solution of deuterated acetic acid in deuterated water, as has been described in Experimental Section, and the technician provided them with the 1H NMR spectrum of the sample. Students were allowed to find the assignments and chemical shifts of the 1H NMR signals for predicting spectra using ChemDraw software. A typical 1H NMR spectrum of chitosan is shown in Figure 2, and obtained assignments and chemical shifts of the 1H NMR signals are given as follows according to Figure 2. Chitosan 1H NMR (D2O/CD3COOD, 500 MHz, 20 °C): δ = 4.85 (H1 of GlcN), 4.75 (H1 of GlcNAc), 3.45−4 (H3, H4, H5, H6, H2 of GlcNAc), 3.21 (H2 of GlcN), and 2.08 (H7 of −HN−COCH3). C




Figure 2. 1H NMR spectrum, signal assignments, and integrations of the most relevant signals for chitosan.

are the averages and the standard deviations of three measurements).

The signal at 2.0−2.1 ppm corresponds to the three protons of N-acetyl glucosamine (GlcNAc) residues, and it possess the highest resolution and, so, is typically used as reference for quantitative determinations. The H2 proton of glucosamine (GlcN) units presents a characteristic peak at 3.1−3.2 ppm with high resolution, while the nonanomeric protons (H1) resonate around 4.6−4.8 ppm with low resolution and overlapped with the signal of water. The rest of the signals (H3, 4, 5, 6) show a broad envelope of peaks between 3.5 and 4 ppm. Thus, several possibilities could be considered in order to determine the deacetylation degree of chitosan from its 1H NMR spectrum. The simplest and clearest ones are represented by (Figure 2): (a) the integration of the signal corresponding to H7 methyl protons of GlcNAc units and the sum of the integrals of H2, H3, H4, H5, and H6 (b) the integration of the signal corresponding to H7 methyl protons of GlcNAc units and the integration of the peak of resonance of H2 proton in GlcN units DD can be defined according to eq 1: nD DD = × 100 nD + nA (1)

Potentiometric Titration

DD of chitosan was also determined by acid−base titration with a standardized solution of NaOH 0.1 mol L−1. Around pH = 5.5, chitosan begins to precipitate, and this suggests that one should carry out the titrations in a heterogeneous medium. The variation of pH for chitosan solution as a function of NaOH added volume was plotted. The titration curve (Figure 3) was

Here, nD is the number of moles of deacetylated units and nA is the number of moles of acetylated units. With the integration of the above signals, DD can be calculated as follows by eqs 2 and 3: ⎡ 1/3 × IN−COCH3 ⎤ ⎥ × 100 DD = ⎢1 − ⎢⎣ 1/6 × I(H2−H6) ⎥⎦

Figure 3. Chitosan potentiometric titration versus NaOH 0.1 mol L−1 solution.

(2)

where IN−COCH3 is the integral of the signal corresponding to H7 methyl protons of GlcNAc units and I(H2−H6) is the sum of the integrals of H2, H3, H4, H5, and H6. DD also can be calculated when signal of H2 is adequately separated by eq 3 ⎡ 1/3 × IN−COCH3 ⎤ ⎥ × 100 DD = ⎢1 − ⎢⎣ IH2(GlcNAc) ⎥⎦

obtained, where two equivalent points could be distinguished. The first point corresponds to the neutralization of the excess of HCl (H+ + OH− → H2O). The second point is ascribed to the protonation of amine groups of GlcN residues of chitosan. In acidic pH’s primary amino groups are protonated, and as NaOH is added during titration, they are neutralized (−NH3+ + OH− → −NH2 + H2O) and their concentration can, thus, be quantified. The DD is calculated from the NaOH volume added during titration, by calculating the number of moles of −NH2 groups titrated with respect to the total number of moles of acetylated and deacetylated monomeric units. Thus, DD could be determined by the following expression assuming that the

(3)

where IH2(GlcNAc) corresponds to the integration of the peak of resonance of H2 proton in GlcN units. The obtained DD values calculated with the above equations were 68.7 ± 4.3% and 73.5 ± 2.1%, respectively (reported data D




consequence of the increase on the concentration of Na+ and OH− ions in solution. The intercept between the first region line and the second region line gives the volume in which protonated amine groups of GlcN units of chitosan start to be neutralized, and the interception between the second region and the third region lines, that in which neutralization of primary amine groups is over. From conductometric titration curves students calculated the number of moles of −NH 2 groups titrated and, consequently, the DD degree of chitosan sample. The number of moles of deacetylated or aminated (nD) units can be easily calculated as follows

molecular weight of chitosan unit corresponds to that of the deacetylated unit32 (for high DD values) ⎛M⎞ DD = 161 × 10−3 × (y − x)⎜ ⎟ × 100 ⎝w⎠

(4)

where 161 is the molar mass of the monomeric unit of fully deacetylated chitosan (g mol−1), y and x are the second and first equivalent points (mL), respectively, and M is the concentration of NaOH solution (mol L−1) and w is the chitosan weight (g). A more general expression can be obtained for DD when acetylated and deacetylated different molecular weights are considered in above expression for any value of DD. Thus, being 203.19 g mol−1, the molecular weight of acetylated monomeric units, DD, can be calculated as follows: nD DD = × 100 nD + nA =

nD = (y − x) × 10−3 × M

where (y − x) is the volume (mL) of added NaOH solution for neutralization of −NH3+ groups of GlcN units, and M is the concentration (mol L−1) of NaOH solution. Thus, the number of moles of acetylated (nA) units can be expressed as

(y − x) × 10−3 × M (y − x) × 10−3 × M + × 100

(6)

[w − ((y − x) × 10−3 × M × 161.16)] 203.19

nA =

(5)

The obtained DD values by potentiometric titration were 82.9 ± 2.1% and 87.2 ± 1.2% for eqs 4 and 5, respectively (reported data are the averages and the standard deviations of three measurements).

=

w − nDMD mA = MA MA w − (y − x) × 10−3 × M × MD MA

(7)

where mA is the mass of acetylated units (g), w is the mass of chitosan (g), and MD and MA are the molar masses of the acetylated (203.19 g mol−1) and deacetylated (161.16 g mol−1) units, respectively. According to DD definition (eq 1), substituting eq 6 and eq 7 in eq 1 leads to the final expression for DD

Conductometric Titration

Chitosan acidic solution was titrated with diluted NaOH solution, and its conductivity was measured. When conductivity was represented against NaOH added volume, three different regions could be appreciated in the curve (Figure 4), and they

DD = MA ×

(y − x) × 10−3 × M (y − x) × 10−3 × M × ΔM + w

× 100 (8)

where ΔM is the difference between the molar mass of acetylated and deacetylated units (42.04 g mol−1). A simplified expression can be also defined for high DD values, assuming that the molecular weight of the chitosan unit corresponds to that of the deacetylated unit (eq 3). The DD values determined by conductometric tritation were 80.1 ± 1.9% and 75.2 ± 2.2% for eqs 8 and 3, respectively (reported data are the averages and the standard deviations of three measurements). Data treatment of experimental results was presented as a written laboratory report with journal article style. Thus, students provided Materials, Methods, Experimental Procedure, and Results and Discussion sections, including calibration procedures and data analysis (learning outcome 4). Finally, students were asked to draw conclusions, to assess the accuracy of the manufacturer’s label (see Instructor’s Guide), and to discuss the limitations of employed methods. Optionally, students can be required to complete their written report with a full literature review. This teaching approach implies a student-centered active lab learning that tries to avoid text- and manual-based knowledge (nevertheless, students were provided with health and safety information and experimental lab skills were taught in previous years) and allows students to acquire some basic professional standards, such as team-working, problem solving, or task planning, among others.

Figure 4. Conductometric tritation curve for chitosan. The first region line (green) corresponds to the excess of H+. The second region line (purple) corresponds to the titration of −NH2 groups of chitosan, and the third region line (blue) corresponds to the excess of added OH−.

could be fitted to straight lines. In the first region of the curve (green, Figure 4) conductivity decreases as NaOH is added due to the neutralization of H+ of HCl in chitosan solution (H+ mobility is higher than that of Na+). In the second region (pink, Figure 4), conductivity increases as a consequence of the neutralization of the −NH3+ groups present in chitosan acidic solution, because added Na+ ions have higher mobility than protonated chitosan. Finally, in the third region (blue, Figure 4) conductivity increases as the addition of alkali is carried out as a E



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CONCLUSIONS A PBL-based pedagogical approach is described for being used in an Instrumental Techniques laboratory course for undergraduate students of Pharmacy within the Physical Chemistry curriculum. The experimental determination of the deacetylation degree of chitosan allowed implementation of PBL in a new environment and developing a thorough analysis of the fundamentals and applications of conductometry, potentiometry and 1H NMR in a real-world scenario. These methods are proposed as simple and available techniques that enrich the experimental performance in laboratories of first-year students of bachelor degrees. Presenting this challenging learning experience promoted self-reflective learning and provided evidence of being an interesting tool to acquire higher capacity for knowledge transfer to novel situations, which is a basic skill for a future pharmacist.

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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.7b00902. Instructor materials (PDF, DOCX) Student materials (PDF, DOCX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Leyre Pérez-Á lvarez: 0000-0003-0543-4134 Notes

The authors declare no competing financial interest.

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REFERENCES

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Determining the Deacetylation Degree of Chitosan - ACS Publications

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