Phương pháp tạo màng calcium alginate and calcium pectinate

UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ 10/24/2007 Robert B. Wieland Master of Science Chemistry Preparation of Calcium Alginate and Calcium Pectinate Films and Determinations of Their Permeabilities Dr. James E. Mark Dr. Estel Sprague Dr. Carl Seliskar Preparation of Calcium Alginate and Calcium Pectinate Films and Determinations of Their Permeabilities A dissertation to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in the Department of Chemistry by Robert B. Wieland Bachelors of Science in Chemical Technology University of Cincinnati, June 2001 Committee Chair: Dr. James E. Mark ABSTRACT Small amounts of polymers are typically used in flavor and food applications. Polymers are typically applied in thin coatings which allow for a cost-effective encapsulation with desirable barrier properties. Understanding the properties of thin barrier coatings is essential to obtaining optimal encapsulation performance. Many of the polymers used in the flavor and food industry are cross-linked hydrogels, which are water insoluble but water swellable. Hydrogel barriers allow water soluble components to be extracted from the encapsulation system. Flavor components having a large affinity for water will be extracted from the encapsulation system while more hydrophobic flavor components will remain encapsulated. Preferential flavor extraction is a large problem for the flavor industry because flavors are complicated mixtures of both hydrophilic and hydrophobic components. Understanding diffusion and permeability coefficients is desirable for creating optimized encapsulation systems. However, creating thin uniform films reproducibly can be challenging and expensive. In the past, thick polymer films were cast onto a metal sheet and cross-linked with the appropriate chemicals. The method produced wrinkled and inconsistent film thicknesses. The inconsistent films produced irreproducible diffusion and permeability coefficient data. New testing methods were developed to understand flavor partitioning across thin hydrogel membranes. One focus of the present work was to create 10μm to 50μm polymer films reproducibly with uniform thicknesses. The second focus of this project was to determine thin film diffusion and permeability coefficients of the created polymer films. The first portion of this thesis discusses the creation of thin polymer films. Calcium  ii  alginate and calcium pectinate films were created using a lightly scuffed metal sheet. The sheet was then used in a leveling apparatus which provided a level surface for film casting. The polymer films were characterized by micrometer measurement, environmental scanning electron microscopy (ESEM) and swelling ratio experiments. Micrometer measurements demonstrated the successful preparation of 21 to 23 (+/- 1) μm calcium alginate films and 19 to 20 (+/- 1) μm calcium pectinate films. The 4 to 6% relative standard deviation was considered acceptable for the present work. The calcium alginate and calcium pectinate films were also analyzed by ESEM. Both sides of the films were analyzed at 200X and 1500X magnifications. The polymer film surface exposed to the scuffed metal sheet produced a rough and irregular surface. The polymer film surface not exposed to the scuffed metal sheet had a smooth and uniform surface. Film thickness measurements were also performed using the ESEM computer software to further verify the film thickness measurements obtained from the micrometer. The ESEM film thickness measurements demonstrated a 20.9 (+/- 1.1) μm calcium alginate film and a 20.2 (+/- 0.7) μm calcium pectinate film had been produced. Both films demonstrated an average relative standard deviation of 4 to 6% which was considered acceptable for the present work. The ESEM measurements of film thickness demonstrate the methodology for creating thin polymer films is reproducible and within the desired thickness range. However, the scuffed metal sheet creates films that are rough on one side and smooth on the other side. Preliminary polymer swelling ratio experiments in distilled water showed calcium alginate films swell to 2.4 times their original dry weight and calcium pectinate films swell by a factor of 3.8. The large  iii  swelling ratios for the films indicated that distilled water was an appropriate solvent for determining film permeability and diffusion coefficients. The second portion of this thesis focused on determining film diffusion and permeability coefficients. A new thin-film diffusion cell (TFD) was built and coupled to a UV/VIS spectrophotometer fitted with a fiber optic probe which allowed for in-situ measurement of analytes which absorb ultraviolet radiation such as benzaldehyde. Permeability measurements using benzaldehyde demonstrated a permeability coefficient of 3X 10 -5 cm/sec. (+/- 5%) for the 22 (+/- 1) μm calcium alginate film and 2 X 10 -4 cm/sec. (+/-6%) for the 20 (+/- 1) μm calcium pectinate film. Diffusion coefficients were then calculated for the two films. The diffusion coefficient for a 22 (+/-1) μm calcium alginate film was 6.5 X 10 -8 (+/- 11%) cm 2 /sec while the diffusion coefficient for a 20 (+/-1) μm calcium pectinate film was 3.9 X 10 -7 (+/- 12%) cm 2 /sec. The relative standard deviations for the permeability and diffusion coefficients were considered acceptable for this study. The permeability and diffusion coefficients indicated a calcium pectinate film is more permeable than a calcium alginate film of equal thickness.  iv  ACKNOWLEDGEMENTS I would like to express my gratitude to Dr. Dave Siegel for the guidance and support shown to me while obtaining a graduate degree. I acknowledge my graduate achievement would not have been possible without the patience, flexibility and understanding shown by this man. I would also like to thank Dr. Robert Eilerman for his flexibility allowing me to achieve an academic milestone while maintaining full time duties at the Givaudan Flavor Corporation. I would like to acknowledge the financial support of the Givaudan Flavor Corporation. The financial support allowed for critical glassware to be purchased and allowed me to obtain a graduate degree. I would like to give a heartfelt thank you to Dr. Jing Zhang for helping me understand diffusion and permeability. In this way, Dr. Zhang helped me become a better chemist with an increased knowledge of polymer systems. I appreciate the guidance Dr. James Mark has given while writing my graduate thesis and during my academic career. His insight has been valuable and informative. I would like to thank my mother Brenda Wilson for her endless encouragement. Your determination, work ethic and loving support has enabled me to be successful in the workplace and in academia. I am grateful for the support and encouragement shown by my wife Jessica Wieland. Without her support this achievement would not be possible. Thank you for understanding the extra hours at school and the extra hours of homework which kept us apart. I could not ask for a more supportive and inspiring wife.  v  TABLE OF CONTENTS ABSTRACT……………………………………………………………………………….… ii ACKNOWLEDGEMENTS……………………………………………………………….… v TABLE OF CONTENTS………………………………………………………………….…vi LIST OF FIGURES……………….…………………………… …………………… … viii LIST OF TABLES……………… …………………………… ……………….………… xi 1. INTRODUCTION…………………………………………………………………………1 1.1: Pectin: Sourcing, Manufacture and Functionality……………………………… 5 1.2: Pectin: Structure…………………………………………………………………….7 1.3: Alginate: Sourcing, Manufacture and Functionality……………………………. 8 1.4: Alginate: Structure…………………………………….………… ………………10 2. EXPERIMENTAL SECTION………………………………………………………… 12 2.1: Experimental Objectives………………………………………………………….12 2.2: Raw Materials…………………………………………………………… ……….13 2.3: Procedures………………………………………… …………………………….14 2.3.1: Film Sheet Preparation………………………………………………… …….14 2.3.2: Leveling Film Sheet Apparatus……………………………………………… 15 2.4: Procedures-Sample Preparation……………………………………………… 16 2.4.1: Creating 1.0% Sodium Alginate Solutions……………………………………16 2.4.2: Creating Calcium Alginate Films………………………………………………17 2.4.3: Creating 1.0% Pectin Solutions 18 2.4.4: Creating Calcium Pectinate Films…………………………………………… 18 2.4.5: Creating 1000PPM Benzaldehyde Standards in Miglyol 812………………20  vi  2.4.6: Creating Benzaldehyde Standards in Distilled Water………………….……20 2.5: Characterization Techniques…………………………………………………….21 2.5.1: Micrometer Measurements……………… 21 2.5.2: Environmental Scanning Electron Microscopy (ESEM)….….….…….…….22 2.5.3: Swelling Ratio Measurement (SR) 22 2.5.4: Polymer Film Diffusion Analysis……………………………………….………23 2.5.4.1: UV/VIS Absorbance Linear Regression Calibration….….…………….….23 2.5.4.2: UV/VIS Thin Film Diffusion Analysis (TFD)……………………………… 24 3. RESULTS and DISCUSSION…………………………………………………………27 3.1: Micrometer Measurements…………………… 27 3.2: Environmental Scanning Electron Microscopy (ESEM)……….….….……….28 3.3: Swelling Ratio Measurement (SR) 31 3.4: Polymer Film Diffusion Analysis………….…………………………… ………33 3.4.1: UV/VIS Absorbance Linear Regression Calibration….….………………….33 3.4.2: UV/VIS Thin Film Diffusion Analysis (TFD)…………….….………… …….35 4. CONCLUSIONS……………………………………………………… ……………….38  vii  LIST OF FIGURES Figure 1.2-1: Structure of the pectin molecule……………………………………………7 Figure 1.2-2: Structure of the calcium pectinate molecule……………….…………… 8 Figure 1.4-1: Alginate monomers…………… …………………………………………. 10 Figure 1.4-2: Alginate block types…………………………………………………… 11 Figure 1.4-3: Calcium binding site in polyguluronate dimer… …………………… 12 Figure 1.4-4: Calcium alginate cross-linked “Egg box” model………………………… 12 Figure 2.2-1: Structure of benzaldehyde… …………………….…………………… 14 Figure 2.3.1-1: Scuffed cookie sheet…………………………………………………… 15 Figure 2.3.2-1: Leveling film apparatus with key measurements………….………… 16 Figure 2.3.2-2: Leveling film apparatus containing film sheets……………………… 16 Figure 2.4.2-1: 20μm calcium alginate film……………………………………… ….…18 Figure 2.4.4-1: 20μm calcium pectinate film………………………………………….…19 Figure 2.5.4.1-1: UV/VIS spectrophotometer……………… ……………………… 23 Figure 2.5.4.1-2: Fiber optic probe fitted with UV/VIS spectrophotometer…… … 24 Figure 2.5.4.2-1: Schematic of thin film diffusion (TFD) apparatus…….…………… 25 Figure 3.2-1: ESEM analysis of calcium alginate and pectinate rough film side…… 29 Figure 3.2-2: ESEM analysis of calcium alginate and pectinate smooth film side …. 30 Figure 3.4.1-1: Linear regression chart for benzaldehyde standards……….………. 35 Figure 3.4.2-1: Permeability coefficient for calcium alginate film…….……….………. 37 Figure 3.4.2-2: Permeability coefficient for calcium alginate film………………… …37  viii  [...]... Benzaldehyde standard dilutions……………………………………………….21 Table 2: Micrometer measurements for thin calcium alginate films……… .28 Table 3: Micrometer measurements for thin calcium pectinate films……… ……… 28 Table 4: Calcium alginate film thickness measurements by ESEM analysis……… 31 Table 5: Calcium pectinate film thickness measurements by ESEM analysis … .31 Table 6: Swelling ratio measurements of calcium alginate. .. 1000.0  Table 1: Benzaldehyde standard dilutions 2.5 Characterization Techniques 2.5.1 Micrometer Measurements A micrometer was used to measure the thickness of the calcium alginate and calcium pectinate films The films were cut into 5cm X 5cm squares and ten micrometer measurements were performed and recorded An average micron thickness,   21  standard deviation and relative standard deviation was then... Microscopy (ESEM) Calcium alginate and calcium pectinate films were sampled and sent to the University of Cincinnati for ESEM analysis The polymer films were cut into small pieces and adhered to a metal plate using carbon tape The smooth surface and scuffed surface of the films were analyzed at 200X and 1500X magnifications Photographs of the film surface structure were obtained to better understand the film... Creating Calcium Alginate Films Approximately 250.0g of the 1.0% sodium alginate solution was poured into a lightly scuffed cookie sheet and allowed to stand in a fume hood for 24 hours on a leveling apparatus The 1% sodium alginate solution delivered 2.5g of sodium alginate across 1205 cm2 of surface area on the cookie sheet As the water evaporated from the sodium alginate solution a thin film of sodium alginate. .. and allowed to stand for 30 minutes The 2% calcium chloride solution delivered 5g of calcium chloride to 2.5g of sodium alginate The calcium displaced the sodium in the alginate film forming a cross-linked water insoluble calcium alginate film The calcium chloride solution was then decanted from the cookie sheet The cookie sheet containing the cross-linked alginate film was rinsed three times with 250.0g... hood and allowed to stand for 24 hours The film was slowly peeled off the cookie sheet Thin uniform   17  calcium alginate films were created containing no wrinkles or high spots The film was cut into 5cm X 5cm squares and stored in a sealed container The dried calcium alginate film demonstrated a moisture content of 1.8 to 2.3% 2.4.3 Creating 1.0% pectin solutions Figure 2.4.2-1: 20μm calcium alginate. .. seaweed to swell and form a viscous solution The highly viscous solution is diluted and the insoluble residues are removed Chlorine is added to the liquid fraction containing the alginate and treated with calcium chloride to form a water insoluble precipitate The calcium alginate is then recovered, pressed to remove excess water and treated with a hydrochloric acid solution The acid-washed alginate cake... drugs and cellular materials 25 Alginate has also been used to dehydrate products such as paper and textiles, as flame retardants for fabrics, and as blood detoxifiers 1.4 Alginate: Structure Alginate is a complex carbohydrate comprised of glucuronic and mannuronic acid monomers Based on the seaweed source and processing, alginate can be produced with varying glucuronic and mannuronic acid contents 26... site of D-glucuronate and Figure 1.4-4 illustrates the egg box structure of calcium crosslinked alginate Guluronate Guluronate Figure 1.4-3: Calcium binding site in polyguluronate dimer Figure 1.4-4: Calcium alginate cross- linked “Egg box” model 2 Experimental Section 2.1 Experimental Objectives The objectives of the present work are as follows: a) Prepare uniform films of calcium alginate reproducibly... films become very brittle, brittle and cracked films lead to ineffective barriers, and thin films are hard to cast uniformly The thin films created were uniform and reproducible which ensured a robust method and reliable data For the present work, two calcium cross-linkable polymers were chosen for study Alginate and pectin were chosen for their acceptance in the food and flavor industry The thin film