OS SEGREDOS DAS
PANELINHAS
POR CARLOS BRAGA
Panelinhas de todos os tipos importadas e distribuídas com exclusividade pela SR Grupo. Agora a pronta entrega no Brasil
Hermética Al TA Instruments
Compatíveis com todas as marcas: *TA Instruments
Standard Al TA Instruments
Tzero TA Instruments
TGA Pt TA Instruments
Keep Discovering
TGA Pt
TGA Alumina ou Safira
*PerkinElmer *Mettler *Netzsch
Netzsch Std - Al
PerkinElmer Std Al
*Shimadzu *Seiko Disponível também em safira, aluminio, platina e alumina.
Mettler com Pin Al
Mettler Std Al
DSC clean brush TA Instruments
PARA MAIS INFORMAÇÃO
Braga
Carlos Braga
Como participar da nossa próxima importação, por favor entre em contato conosco atráves do (11) 3259-5978 ou vendas@srgrupo.com.br
Representante exclusivo no Brasil: SR&GR Instrumentos Científicos Computadorizados Ltda CNPJ 52960697/0001-46
A Borealis é líder no fornecimento de soluções inovadoras nas áreas de poliolefinas, químicos básicos e fertilizantes. Com sede em Viena, Áustria, a empresa conta com cerca de 6.600 colaboradores e opera em mais de 120 países. Possui presença em toda a América do Sul e fábrica na cidade de Itatiba-SP, onde desenvolve, produz e comercializa compostos de polipropileno para diversos segmentos de mercado, como automotivo, linha branca, eletroeletrônicos, entre outros. Com desenvolvimento local e alta capacidade técnica, somos o parceiro ideal da indústria na busca por soluções inovadoras em compostos de polipropileno.
www.borealisgroup.com
ISSN 0104-1428 (printed) ISSN 1678-5169 (online)
P o l í m e r o s - I ss u e I I I - V o l u m e X X V I I I - 2 0 1 8 I n d e x e d i n : “ C h e m ic a l A b s t r a c t s ” — “ RA P RA A b s t r a c t s ” — “A l l - R u s s i a n I n s t i t u t e o f S ci e n c e T e c h n ic a l I n f o r m a t i o n ” — “ R e d d e R e v i s t a s C i e n t i f ic a s d e A m e r ic a L a t i n a y e l C a r i b e ” — “ L a t i n d e x ” — “ W e b o f S ci e n c e ”
and
Polímeros E d i t o r i a l C o u nci l Antonio Aprigio S. Curvelo (USP/IQSC) - President
Editorial Committee Sebastião V. Canevarolo Jr. – Editor-in-Chief
Members Adhemar C. Ruvolo Filho (UFSCar/DQ) Ailton S. Gomes (UFRJ/IMA) Alain Dufresne (Grenoble INP/Pagora) Antonio Aprigio S. Curvelo (USP/IQSC) Bluma G. Soares (UFRJ/IMA) César Liberato Petzhold (UFRGS/IQ) Cristina T. Andrade (UFRJ/IMA) Edson R. Simielli (Simielli - Soluções em Polímeros) Edvani Curti Muniz (UEM/DQI) Elias Hage Jr. (UFSCar/DEMa) Eloisa B. Mano (UFRJ/IMA) João B. P. Soares (UAlberta/DCME) José Alexandrino de Sousa (UFSCar/DEMa) José António C. Gomes Covas (UMinho/IPC) José Carlos C. S. Pinto (UFRJ/COPPE) Júlio Harada (Harada Hajime Machado Consutoria Ltda) Laura H. de Carvalho (UFCG/DEMa) Luiz Antonio Pessan (UFSCar/DEMa) Luiz Henrique C. Mattoso (EMBRAPA) Marco-Aurelio De Paoli (UNICAMP/IQ) Osvaldo N. Oliveira Jr. (USP/IFSC) Paula Moldenaers (KU Leuven/CIT) Raquel S. Mauler (UFRGS/IQ) Regina Célia R. Nunes (UFRJ/IMA) Richard G. Weiss (GU/DeptChemistry) Rodrigo Lambert Oréfice (UFMG/DEMET) Sadhan C. Jana (UAKRON/DPE) Sebastião V. Canevarolo Jr. (UFSCar/DEMa) Silvio Manrich (UFSCar/DEMa)
A ss o ci at e E d i t o r s Adhemar C. Ruvolo Filho Alain Dufresne Bluma G. Soares César Liberato Petzhold José António C. Gomes Covas José Carlos C. S. Pinto Paula Moldenaers Richard G. Weiss Rodrigo Lambert Oréfice
Sadhan C. Jana
D e s k t o p P u b l is h in g
www.editoracubo.com.br
“Polímeros” is a publication of the Associação Brasileira de Polímeros São Paulo 994 St. São Carlos, SP, Brazil, 13560-340 Phone: +55 16 3374-3949 emails: abpol@abpol.org.br / revista@abpol.org.br http://www.abpol.org.br Date of publication: July 2018
Financial support:
Polímeros / Associação Brasileira de Polímeros. vol. 1, nº 1 (1991) -.- São Carlos: ABPol, 1991Available online at: www.scielo.br
Quarterly v. 28, nº 3 (June/July 2018) ISSN 0104-1428 ISSN 1678-5169 (electronic version)
Website of the “Polímeros”: www.revistapolimeros.org.br
1. Polímeros. l. Associação Brasileira de Polímeros. Polímeros, 28(3), 2018
E1
E E E E E E E E E E E E E E E E E E E E E E E E E E E E
I I I I I I I I I I I I I I I I I
Editorial Section News....................................................................................................................................................................................................E3 Funding Institutions.............................................................................................................................................................................E4 Agenda.................................................................................................................................................................................................E6
O r i g in a l A r t ic l e Surface treated bagasse fiber ash on rheological, mechanical properties of PLA/BFA biocomposites Watcharin Sitticharoen, Chet Uthiyoung, Nateechai Passadee and Chanokpol Wongprom............................................................................ 187
Water-uptake properties of a fish protein-based superabsorbent hydrogel chemically modified with ethanol Vilásia Guimarães Martins, Jorge Alberto Vieira Costa and Carlos Prentice................................................................................................ 196
Characterization of additives in NR formulations by TLC-IR (UATR) Lidia Mattos Silva Murakami, Joyce Baracho Azevedo, Milton Faria Diniz, Leandro Mattos Silva and Rita de Cássia Lazzarini Dutra........................................................................................................................................................................ 205
Effect of disinfection techniques on physical-mechanical properties of a microwave-activated acrylic resin Carmen Beatriz Borges Fortes, Fabrício Mezzomo Collares, Vicente Castelo Branco Leitune, Priscila Raquel Schiroky, Stéfani Becker Rodrigues, Susana Maria Werner Samuel, Cesar Liberato Petzhold and Valter Stefani......................................................... 215
Synthesis of poly(ethyl methacrylate-co-methyl methacrylate) obtained via ATRP using ruthenium benzylidene complexes Maria Beatriz Alves Afonso, Lucas Gomes Gonçalves, Talita Teixeira Silva, José Luiz Silva Sá, Nouga Cardoso Batista, Beatriz Eleutério Goi and Valdemiro Pereira Carvalho Júnior....................................................................................................................... 220
Polystyrene and cornstarch anti-corrosive coatings on steel Cinthia de Souza, Ricardo Luiz Perez Teixeira, José Carlos de Lacerda, Carla Regina Ferreira, Cynthia Helena Bouças Soares Teixeira and Valdir Tesch Signoretti.............................................................................................................. 226
Chitosan and gum arabic nanoparticles for heavy metal adsorption Flavia Oliveira Monteiro da Silva Abreu, Nilvan Alves da Silva, Mateus de Sousa Sipauba, Tamara Fernandes Marques Pires, Tatiana Araújo Bomfim, Oyrton Azevedo de Castro Monteiro Junior and Maria Madalena de Camargo Forte............................................ 231
UATR and NIRA evaluation in the quantification of ATBC in NC blends Talita de Souza Dias Mello, Milton Faria Diniz and Rita de Cássia Lazzarini Dutra.................................................................................... 239
Polyurethane derived from Ricinus Communis as graft for bone defect treatments Tatiana Peixoto Telles de Sousa, Maria Silvana Totti da Costa, Renata Guilherme, Wilson Orcini, Leandro de Andrade Holgado, Elcia Maria Varize Silveira, Orivaldo Tavano, Aroldo Geraldo Magdalena, Sérgio Augusto Catanzaro-Guimarães and Angela Kinoshita.............................................................................................................................................................................................. 246
Polyvinyl alcohol (PVA) molecular weight and extrusion temperature in starch/PVA biodegradable sheets Juliano Zanela, Ana Paula Bilck, Maira Casagrande, Maria Victória Eiras Grossmann and Fabio Yamashita............................................ 256
Molecular dynamics studies of amylose plasticized with Brazilian Cerrado oils: part I Felipe Azevedo Rios Silva, Maria José Araújo Sales, Leonardo Giordano Paterno, Mohamed Ghoul, Latifa Chebil and Elaine Rose Maia............................................................................................................................................................................................. 266
R e vi e w A r t ic l e Review of fungal chitosan: past, present and perspectives in Brazil Anabelle Camarotti de Lima Batista, Francisco Ernesto de Souza Neto and Weslley de Souza Paiva........................................................... 275
Cover: Figure 5 (pg 272). Arts by Editora Cubo.
E2
Polímeros, 28(3), 2018
Borealis and Henkel cooperation produces plastic bottle and nozzle composed 100% of post‑consumer recycled material Packaging solution made of 100% plastic recyclate delivers circular economy proof point. Borealis, a leading provider of innovative solutions in the fields of polyolefins, base chemicals and fertilizers, announces the successful launch of a new packaging solution produced entirely with post-consumer recycled (PCR) material. Developed in close collaboration with the German consumer and industrial goods company Henkel and two additional value chain partners, this truly sustainable packaging solution is further evidence of how mtm plastics GmbH, a member of the Borealis Group, is helping increase the circularity of plastics. The launch has significance for the consumer goods industry: the robustness of this new packaging solution provides further evidence that plastic recyclate is indeed suitable for a variety of demanding packaging applications, in this case a popular adhesive brand marketed by Henkel. Value chain collaboration yields plastic bottle and nozzle composed of 100% PCR material. In 2016, Borealis acquired leading German recycler mtm plastics GmbH, which is now a member of the Borealis Group. By leveraging their respective areas of expertise and decades of experience as a virgin polyolefins producer and “upcycler”, respectively, Borealis and mtm plastics are exploring new growth opportunities with joint forces. A success story originating from this exploration is a recently completed pilot project with Henkel, the global leader for adhesives, sealants and functional coatings. The companies have worked to develop a new packaging solution based on recycled material for the Made-at‑Home all-purpose glue bottle and cap, which Henkel is marketing under its well-known Pattex brand. The aim was to replace the virgin plastic material traditionally used for this packaging with a recyclate-based resin. The resin, however, had to fulfil the diverse material demands for packaging of an adhesive product. After extensive and joint application development, a new bottle was developed with Borealis’ proprietary product brand Purpolen PE, a high-quality polyethylene regranulate produced by mtm at its facilities in Niedergebra, Germany. Value chain partner KKT Kaller Kunststoff Technik GmbH, a plastics processor also based in Germany, manufactured the bottles. For the three separate components of the adjustable applicator nozzle, which is used for both filigree and wide‑area gluing, high‑quality Purpolen polypropylene regranulate produced by mtm was identified as the ideal solution. German plastic components manufacturer bomo trendline Technik GmbH produced the applicator nozzles. The new Pattex Made‑at‑Home packaging solution successfully passed extensive application tests, including a three-month storage test and other tests of mechanical properties. It was launched on the European market in 2018. “As a virgin polyolefins producer, Borealis is thrilled to be among the pioneers in using plastic recyclate in new applications,” says Günter Stephan, Head of Borealis Circular Economy Solutions. “Even though momentum is gaining in the drive to increase the circularity of plastics, we still need to prove without a doubt within the industry that using recyclates – and even 100% PCR – is a suitable and effective option, even for demanding applications. Thanks to this successful value chain cooperation with our partners Henkel, KKT and bomo, we are giving plastics a second life and are thus one step closer to the goal of a more circular economy of plastics.” “Our commitment to leadership in sustainability is deeply embedded in our companies´ values,” explained Matthias Schaefer, Project Manager for Global Packaging Engineering at Henkel Adhesive Technologies. “We are at the forefront of the industry when it comes to new sustainability strategies in packaging. Thus, we identified Pattex Made-at-Home as a candidate for exploring the use of recyclate instead of virgin plastics. This constructive collaboration with our partners proves the viability of 100% PCR material for an adhesive product like Made-at Home. It also underscores our efforts at Henkel to drive leadership in sustainability in the consumer goods sector.” Source: Borealis Circular Economy Solutions www.borealisgroup.com
Shape-Memory Polymer Market: 2017 – 2025 Investments, Strategies, Key Companies and Future Trends Shape-memory polymers are polymeric smooth materials that have the skill to yield from a temporary shape (deformed state) to their permanent (original) shape through an external provocation (trigger), such as change in temperature. The major driver of the shape-memory polymer market is growing application of shape-memory polymer for smart drug delivery in healthcare, orthopedic braces, orthodontic implants and splints, catheters, cardiovascular stents, etc. The second main application of shape-memory polymer is in the building of self-repairing concrete, foams for building insulation, window sealants, etc. The comparatively low values of shape-memory polymer, when compared to other resources, is a disadvantage for the polymer, which can be changed by strengthening shapememory polymer with additional materials, like Kevlar and fiber glass. The major drivers of the shape-memory polymers market are growing application in the healthcare sector and rising construction industry in Asia Pacific countries such as India and China. However, as the market of shape-memory polymers is growing, there are certain setbacks, restraining the market such as relatively low stiffness values. The growing demand for other shape-memory materials of SMP, like shapememory alloys (SMAs) and ceramics, is a huge opportunity for the market. Invention of better manufacturing techniques, like mnemosynation, enable mass production of cross-linked SMP devices, which would otherwise be cost-prohibitive if made using traditional thermoset polymerization techniques. The shape-memory polymers market is segmented by stimulus or type, by application, and by region. The shape‑memory polymers market is segmented by type into temperature-induced, light-induced, electricity-induced and others (PH, Magnetic, etc.). Furthermore, by application the market is segmented into aerospace, automotive, construction, healthcare and others (robotics, textile, etc.). The global shape-memory polymers market has been studied for five geographic regions, namely Asia Pacific (China, Japan, India, and South Korea), Europe (Germany, France, Italy, and the U.K.), North America (Canada, Mexico, and the U.S.), South America (Brazil and Argentina), and Middle East & Africa (MEA). These innovative materials are being used in automotive industry for vehicle subsystems which during any damage can self-heal, or which is made to transform its appearance or color. This original material offers new high-tech technologies while improving performance of automobile which is inexpensive Utilizing materials which change shape in the motors replacement gives lot of advantages such as improved fuel budget, lessen vehicle mass and component size/complexity and increase improve/reliability performance of vehicle. Lately, these polymers are being used for deployable structures and mechanisms in aerospace. The key applications include hinges, trusses, optical reflectors, morphing skins, booms, and antennas. Furthermore, there are many copyrights filed by important polymer chameleon companies in relation to shape-memory polymers applications, such as intravascular distribution system, hood/seat assembly automobiles, tunable automotive supports, and gripper. In addition, shape-memory polymers also present additional potential in the areas of drug and pharmaceutical. Shape-memory polymers are chemically framed so that they electro-actively sense changes in environment in biological systems. These polymers are also a valuable tool for metabolic regulator mechanisms. Polymer chameleon market is typically used as smart medication implants and delivery scheme followed by textile engineering. Encouraging demand in main application industries is anticipated to drive the complete polymer chameleon market. Asia Pacific is expected to show the highest growth, due to the thriving construction industry and the healthcare applications of SMP in smart drug delivery. North America and Europe are expected to experience considerable growth, owing to their established aerospace, robotics, and healthcare industries. Source: True Industry Newsb - trueindustrynews.com
Polímeros, 28(3), 2018 E3
N N N N N N N N N N N N N
ABPol Associates Sponsoring Partners
Institutions UFSCar/ Departamento de Engenharia de Materiais, SP SENAI/ Serviço Nacional de Aprendizagem Industrial Mario Amato, SP UFRN/ Universidade Federal do Rio Grande do Norte, RN
E4
PolĂmeros, 28(3), 2018
ABPol Associates Collective Members A. Schulman Plásticos do Brasil Ltda. Aditive Plásticos Ltda. Avamplas – Polímeros da Amazônia Ltda. CBE – Grupo Unigel Colorfix Itamaster Indústria de Masterbatches Ltda. Cromex S/A Cytec Comércio de Materiais Compostos e Produtos Químicos do Brasil Ltda. Formax Quimiplan Componentes para Calçados Ltda. Imp. e Export. de Medidores Polimate Ltda. Innova S/A Instituto de Aeronáutica e Espaço/AQI Jaguar Ind. e Com. de Plásticos Ltda Master Polymers Ltda. Milliken do Brasil Comércio Ltda. MMS-SP Indústria e Comércio de Plásticos Ltda. Nexo International Ltda. Nitriflex S/A Ind. e Com. Politiplastic Politi-ME. Premix Brasil Resinas Ltda. QP - Químicos e Plásticos Ltda. Radici Plastics Ltda. Replas Comércio de Termoplásticos Ltda. Uniflon - Fluoromasters Polimeros Ind .Com. Imp. Export.Ltda
Polímeros, 28(3), 2018
E5
November
A A A A A A A A A A A A A A A A A A A A A
Polymers + 3D Date: November 1-2, 2018 Location: Houston, United States Website: www.poly3d.org 9th International Conference on Biopolymers and Polymer Sciences Date: November 1-2, 2018 Location: Bucharest, Romania Website: biopolymers.materialsconferences.com Regional Conference of the Polymer Processing Society (PPS-Americas) Date: November 5-9, 2018 Location: Boston, United States Website: www.pps2018boston.com. Feira e Congresso Internacionais de Composites, Poliuretano e Plásticos de Engenharia (FEIPLAR COMPOSITES & FEIPUR 2019) Date: November 6-8, 2018 Location: São Paulo, Brazil Website: www.feiplar.com.br XVI Latin-American Polymer Symposium (SLAP 2018) and XIV Iberoamerican Polymer Congress (CIP 2018) Date: November 6-8, 2018 Location: Mar del Plata, Argentina Website: www.slap2018.com European Thermoplastic Compounding Summit Date: November 21-22, 2018 Location: Dusseldorf, Germany Website: www.wplgroup.com/aci/event/european-thermoplasticcompounding-summit 47th National Symposium of the French Group of Polymers Studies and Applications Date: November 26-29, 2018 Location: Toulouse, France Website: gfp2018.sciencesconf.org
December 13th European Bioplastics Conference Date: December 4-5, 2018 Location: Berlin, Germany Website: www.european-bioplastics.org/events/eubp-conference 12th SPSJ International Polymer Conference (IPC 2018) Date: December 4-7, 2018 Location: Hiroshima, Japan Website: main.spsj.or.jp/ipc2018 Plastics Regulations – 2018 Date: December 11-12, 2018 Location: Pittsburgh, United States Website: www.ami.international/events/event?Code=C0946 Silicon-Containing Polymers and Composites Date: December 16-19, 2018 Location: San Diego, United States Website: polyacs.net/Workshops/18SiliconC/home.html International Conference on Advanced and Applied Petroleum, Petrochemicals, and Polymers (ICAPPP 2018) Date: December 18-20, 2018 Location: Bangkok, Thailand Website: www.icappp2018.com
January 7th Future of Polyolefins Summit Date: January 16-19, 2019 Location: Antwerp, Belgium Website: www.wplgroup.com/aci/event/polyolefins-conference 6th Maximizing Propylene Yields Date: January 23-24, 2019 Location: Barcelona, Spain Website: www.wplgroup.com/aci/event/maximising-propyleneyields
E6
22nd Thermoplastic Concentrates Date: January 29-31, 2019 Location: Coral Springs, United States Website: www.ami.international/events/event?Code=C0937
February 17th Polyethylene Films Date: February 5-7, 2019 Location: Coral Springs, United States Website: www.ami.international/events/event?Code=C947 International Conference on Polymers and Plastics, Artificial Intelligence (ICPPAI 2019) Date: February 23-24, 2019 Location: Shanghai, China Website: irnest.org/icppai-feb-2019 1st International Symposium on Polymer Chemistry and Applications (SICPA-2019) Date: February 23 -25, 2019 Location: Taghit, Algeria Website: www.sicpa-dz.com Polymers for Fuel Cells, Energy Storage, and Conversion Date: February 24-27, 2019 Location: Pacific Grove, United States Website: polyacs.net/Workshops/19Fuel/home.html
March Polymers in Footwear Date: March 5-6, 2019 Location: Woburn, United States Website: www.ami.international/events/event?Code=C971 4th Edition of International Conference and Exhibition on Polymer Chemistry Date: March 28-30, 2019 Location: Rome, Italy Website: polymerchemistry.euroscicon.com
May 6th International Symposium Frontiers in Polymer Science Date: May 5-8, 2019 Location: Budapest, Hungary Website: www.elsevier.com/events/conferences/frontiers-inpolymer-science International Conference of the Polymer Processing Society (PPS-35) Date: May 26-30, 2019 Location: Çeşme-Izmir, Turkey Website: www.pps-35.org
June Polymers: Gordon Research Seminar - Innovations in Design, Fabrication and Application of Polymeric Materials Date: June 8-9, 2019 Location: South Hadley, United States Website: www.grc.org/polymers-grs-conference/2019 Polymers: Gordon Research Conference Date: June 9-14, 2019 Location: South Hadley, United States Website: www.grc.org/polymers-conference/2019 13th International Workshop on Polymer Reaction Engineering Date: June 11-14, 2019 Location: Hamburg, Germany Website: dechema.de/en/PRE2019.html
July Polymer Composites and High Performance Materials Date: July 22-25, 2019 Location: Sonoma, United States Website: polyacs.net/Workshops/19Composites/home.html
Polímeros, 28(3), 2018
ISSN 1678-5169 (Online)
http://dx.doi.org/10.1590/0104-1428.0010
Surface treated bagasse fiber ash on rheological, mechanical properties of PLA/BFA biocomposites Watcharin Sitticharoen1*, Chet Uthiyoung1, Nateechai Passadee1 and Chanokpol Wongprom1 Department of Industrial Engineering, Faculty of Engineering, Rajamangala University of Technology Lanna – RMUTL, Muang, Chiang Mai, Thailand
1
*wsitticharoen@yahoo.com
Abstract The use of silica based bagasse fiber ash (BFA) as a reinforcing filler in polylactic acid (PLA) biocomposites was examined. The effects of surface-treated BFA on the rheological, swelling behavior, and mechanical properties and water absorption of biocomposites were studied. BFA was treated using a silane coupling agent (Bis-[3-(triethoxysilyl)propy]‑tetrasulfide). Composites with BFA were varied from 5 to 25wt.%. The PLA/BFA composite melts were pseudoplastic non -Newtonion fluid and exhibited a shear thinning behavior. The viscosity of the surface-treated BFA biocomposites was higher than that of the untreated. The viscosity of the composites decreased with increasing BFA content and shear rate. The extrudate swell of the composites increased with increasing shear rate, whereas it decreased with increasing BFA content and die temperature. The extrudate swell tended to be suppressed when surface-treated BFA was used. Silane treated BFA composites showed improvement in their mechanical properties, and exhibited significantly reduced water absorption. Keywords: bagasse fiber ash, polylectic acid, rheology, extrudate swell, mechanical properties.
1. Introduction Nowadays, plastics are used at very high rates all over the world. Plastics are strong, durable and long‑lasting, and have become a major environmental problem. The use of polymer composites from renewable resources has advantages over synthetic resources, particularly as a solution to the environmental problem caused by plastic waste. Thus, bio-based composite materials have become a hot issue for study and industrial research. Polylactic acid (PLA) is the first commodity plastic produced from annually renewable resources due to its great mechanical strength, thermal plasticity, easy processability and compostable polymer. Besides its advantages, PLA also has some practical shortcomings, such as its low elongation at break, melt strength, and expensive price. To address these disadvantages, PLA is modified by a variety of methods including reinforcement with nature fibers[1-3]. Natural fiber has gained much attention due to its low price, high toughness, easy availability and biodegradability[4]. Bagasse ash is a by-product from burning sugar cane, and is used as fuel for power generation within the sugar factory. The resulting bagasse ash represents approximately 0.62% of the sugarcane weight[5], or 656,000 tons per year in Thailand, and is now becoming an environmental burden[6]. Bagasse ash can be considered an important potential reinforcing filler for polymer composites. Agunsoye and Aigbodion[7] studied the effect of carbonized bagasse particles (CBp) on mechanical properties of recycled low-density polyethylene (RLDPE) composites, and found that the tensile, bending strength and hardness properties of the composites increased with increasing carbonized bagasse content. Aigbodoin et al.[8] found that recycled low-density polyethylene (RLDPE) filled
Polímeros, 28(3), 187-195, 2018
with carbonized bagasse ash particles increased the wear resistance of the composites greatly. Sitticharoen et al.[9] suggested that bagasse fiber ash (BFA) particles contained 78-80% of silica which could be used as reinforcing or extending filler in recycled high-density polyethylene composites, and found that the tensile and flexural modulus and hardness properties of the BFA treated composites with vinyltrimethoxysilane were higher than that of the untreated particles. Additionally, Sitticharoen et al.[9] found that BFA particle’s composites exhibited a pseudoplastic non-Newtonoin flow behavior. Moreover, bagasse fiber ash (BFA) can be utilized as a property enhancing filler in natural rubber (NR) compounds[10]. An understanding of the rheological behavior of fiber/polymer composites is beneficial to the design of polymer processing equipment, and can be used to predict final polymer products and to correlate with the mechanical properties of the composites. Awal et al.[11] studied the cellulose fibers on rheological and mechanical properties of polylactic acid (PLA) biocomposites, and found that the tensile modulus, tensile strength and impact strength properties of the biocomposites increased significantly with increasing bioadimide and its biocomposites exhibited a shear thinning behavior. Dangtungee et al.[12] investigated the rheology behavior and extrudate swell of polylactic acid (PLA) filled with nanoclay and found that its composites exhibited a pseudoplastic behavior as the shear stress and die swell increased with increasing shear rate. The objective of this study is to develop biocomposites using PLA as matrix filled with surface-treated BFA obtained from agricultural waste, give value-added applications to
187/195 187
O O O O O O O O O O O O O O O O
Sitticharoen, W., Uthiyoung, C., Passadee, N., & Wongprom, C. an undervalued material and investigate its melt rheology, extrudate swell, mechanical properties and water absorption of the produced biocomposites.
2. Materials and Methods 2.1 Raw materials 1. Polylactic acid (PLA 2003D) with a melt flow rate of 6 g/10 min was supplied in granular form by NatureWorks LLC (Minnetonka, MN, USA); 2. Bagasse fiber ash (BFA) was supplied by the biomass power plant station of Kaset Thai International Sugar Corp Co., Ltd. (Nakhonsawan, Thailand). The dimensions and shape of the BFA particles were examined using a JEOL JSM-5910LV SEM machine (Tokyo, Japan), and found that BFA had round-shaped particles with relatively smooth surfaces. The average particle size of the BFA used was 53μm. An X-ray fluorescence spectrometer (XRF, MESA-500W, Horiba, Japan) was used for analyzing the chemical compositions of the BFA. It was found that the major component of BFA was SiO2, which constituted about 80% of BFA.
2.2 Surface treatment of BFA Bis-[3-(triethoxysilyl)-propy]-tetrasulfide (Couplink 89), supplied by Behn Meyer Chemical (T) Co., Ltd. (Bangkok, Thailand), was used as a chemical coupling agent for BFA surface treatment. A solution of 2.0 wt% silane coupling agent was slowly dropped into 100 ml of ethanol and then stirred for 30 minutes. 100 g of the BFA particles were then added into the solution and stirred for 15 min to ensure a uniform distribution of the coupling agent on the BFA surfaces. After treatment, the BFA particles were dried in an oven at 100 °C for 24 h.
2.3 Preparation and fabrication of PLA/BFA composites PLA granules were dry-blended with desired amounts of BFA particles and silane coupling agent using a high speed mixer for 2 min before being melt-blended in a single‑screw extruder (Model SMC500, Thailand) to obtain PLA/BFA composites. The BFA content was varied from 0 to 25 wt%. The blending temperature profiles on the extruder were 150, 160, 170 and 180 °C from hopper to die zones and the screw rotation speed was 40 rpm. A two-strand die having a diameter of 3 mm for each strand and coupled with a pelletizing unit was used to produce the PLA/BFA composites pellets, which were held in an oven for 24 h at 80 °C. The PLA/BFA composites were injection-molded at 185 °C using a HYF-1000 injection moulding machine (Charoen Tut, Samut Prakan, Thailand), to produce test specimens for determinations of their mechanical properties.
2.4 Rheological and extrudate swell measurements and apparatus Melt rheology and extrudate swell of neat PLA and PLA/BFA composites samples were studied by using an extrusion rheometer and connected at the end of a single screw extruder as shown in Figure 1. A single screw extruder 188 188/195
Figure 1. Experimental arrangement for measuring the flow properties and extrudate swell of PLA/BFA composite melts in a single-screw extruder.
(RMUTL-SE001 MUSHIKNG PolyLab) supplied by RMUTL (Chiang Mai, Thailand) was varied to investigate the melt rheological and extrudate swell behavior of the composites. The barrel rig was assembled at the end of the single screw extruder. The exact length-to-diameter (L/D) ratio of the barrel was 600/25 mm/mm, and the temperature profiles on the extruder from hopper to die zones were 150, 170, 180 and 160 °C, respectively. The die used in this work was 40 mm in length and 6 mm in diameter. The die temperature was varied from 160 to 170 °C. The rotational speed of the screw was varied from 3 to 20 rpm in order to generate shear rates from 8.0 to 49.4 s-1. The flow characteristics of the neat PLA and PLA/BFA composite melts were evaluated during extrusion from the barrel through a capillary die in the single screw extruder. The pressure drop at the entrance was recorded in real time using a high speed data-logging and recording system and a personal computer through a visual basic program. The pressure drop at the die entrance was used to calculate the shear stress. The volumetric flow rate from the extruder was varied by changing the rotational speed of the screw and was used to calculate the shear rate. It should be noted that Bagley corrections were not applied to the flow data generated in this work due to two reasons: First, the wall shear stress and wall shear rate data were used solely for comparative reasons to illustrate the magnitude of the changes observed in the flow characteristics of the materials as a function of the rotational speed of the screw and the BFA content. Second, the die dimensions used were constant throughout this work which implies that Bagley’s corrections could be neglected in this case. The apparent wall shear stress ( τapp ) of the capillary can be expressed by Equation 1[9,11-12], ∆PR τapp = 2L
(1)
where ΔP is the pressure drop along the die, L is the length of the die. The apparent wall shear rate γ °app is defined as:
(
3n + 1 4Q γ °app = 4n 3 πR
)
(2)
where Q is the volumetric flow rate along the capillary die, and n is the power law index. The apparent viscosity ( ηapp ) of the PLA/BFA composites is practically expressed by the Polímeros, 28(3), 187-195, 2018
Surface treated bagasse fiber ash on rheological, mechanical properties of PLA/BFA biocomposites ratio of the apparent wall shear stress ( τapp ) to the apparent wall shear rate γ °app .
( )
Extrudate swell of neat PLA and PLA/BFA composites was measured using a color video-camera (WATASHI CCD and 700TVL lines), and a high-resolution macro-zoom lens (WLA008) was used to record the flows for further calculations of extrudate swell. All results were recorded and displayed in real time using a personal computer and used a motic images plus 2.0 ML program for extrudate swell measurement. The extrudate swell (S) is reported as the percentage of extrudate swell (%S), as follows[13]: %S =
( Dext − Dd ) ×100 Dd
(3)
Where Dext and Dd are the diameter of the extrudate and the die, respectively.
2.5 Mechanical properties The mechanical properties of neat PLA and PLA/BFA composites were evaluated via tensile, flexural, impact and hardness properties. All the mechanical property results reported in this work were averaged from at least five independent determinations. The tensile test was performed on a Instron universal testing machine (Model Instron 5569, UK) using a crosshead speed of 50 mm/min and the test procedure followed the ASTM D638 (2010) specimen Type I. The flexural test was performed according to ASTM D790 (2007) at the test speed of 1 mm/min. The Izod impact test was conducted in accordance with ASTM D256 (1993) using a Gotech impact tester (Model GT-7045, Taiwan) with the notched side facing the pendulum. The hardness test was assessed using a Durometer Shore D (Model HH‑337-11, Japan). The test procedure was specified by ASTM D2240 (2005).
2.6 Water absorption tests Water absorption tests of the composites were conducted following the modified ISO-62 standard. Samples measuring 19 x 19 x 3.2 mm were cut from the head of the tensile test specimens and the cut cross sections were polished. Five specimens were tested in each sample. The samples were immersed in water and the samples were weighed after 2 weeks of water immersion.
2.7 Scanning Electron Microscopy (SEM) investigations Fracture surface analysis is usually required for mechanical testing of the PLA/BFA composites. In this work, fracture surfaces of the PLA/BFA composites were obtained after a 2-min immersions in liquid nitrogen. All the samples were sputter coated with a thin layer of gold before analysis and investigated with JEOL (JSM-5910LV, Tokyo, Japan) SEM machine at 15 kV of accelerating voltage.
3. Results and Discussion 3.1 Rheological properties The flow behavior of the composite in the molten state is important to characterize the processing properties of the polymer melt and is usually reported by the flow curve relationship between shear stress and shear rate. Figure 2 shows results of the apparent wall shear stress as a function of the apparent wall shear rate for the neat PLA and PLA/BFA composites at a test temperature of 160 °C with various content of untreated (a) and surface-treated (b) BFA particles. It can be seen that the neat PLA and its biocomposites exhibited a pseudoplastic non-Newtonion flow behavior as the wall shear stress increased with increasing wall shear rate in a nonlinear manner. At a given wall shear rate, the wall shear stress decreased with increasing BFA content. With increasing BFA filler content, the difference
Figure 2. Flow curves of neat PLA and PLA/BFA composites at 160 °C with various content of untreated (a) and surface-treated (b) BFA particles. Polímeros, 28(3), 187-195, 2018
189/195 189
Sitticharoen, W., Uthiyoung, C., Passadee, N., & Wongprom, C. between the wall shear stress values of PLA/BFA composites with untreated and neat PLA at a given apparent shear rate gradually increased, with the value of PLA/BFA composites with untreated BFA particles being lower than that of neat PLA and the maximum difference between the shear stress values being observed at the filler content of 25 wt.%. Comparison between the results shown for PLA/BFA composites with untreated and surface-treated BFA revealed that, at a given wall shear rate, values of the shear stress for PLA/BFA composites were higher in the case of surfacetreated BFA particles. It was obvious that the apparent shear stress of the PLA/BFA composite melts with a high filler content (25 wt.%) of untreated BFA particles was 67.5 x103 Pa, versus 72.4 x103 Pa for surface-treated BFA. This fact can be explained by the surface treatment effect, leading to an increased apparent shear stress. This result was in agreement with Dangtungee et al.[12] who stated that the shear stress values for the PLA composite melts that were surface- treated were higher than those that were untreated. Figure 3 shows the apparent shear viscosity against the wall shear rate for the neat PLA and PLA/BFA composites at a test temperature of 160 °C for various concentrations of untreated (a) and surface-treated (b) BFA particles. Generally, the shear viscosity of the neat PLA and its biocomposites gradually decreased with increasing apparent shear rate. These results confirm that all materials exhibited a shear thinning (pseudoplastic) non-Newtonion behavior due to molecular orientation and relaxation of PLA chains under shear deformation, leading to the values of viscosity decreasing. This view could be supported by the work of Sitticharoen et al., Dangtungee et al. and Muksing et al.[9, 12-13] who stated that under shear, molecular chains become oriented, relaxation of the chains, and the numbers of entanglement were reduced; as a result of which the viscosity decreases. It is noteworthy that the apparent shear viscosity of the composite melts, both untreated and surface-treated BFA, were found to decrease with increasing BFA content. The viscosity of PLA/BFA
composite melts with surface-treated BFA particles became higher than that of untreated BFA particles when the shear rate was increased, and obviously the maximum difference between the viscosity values of PLA/BFA composites observed at the filler concentration were 25 wt.%. This was because the surface-treated BFA particles, resulted in a uniform dispersion of the BFA particles in the polymer matrix and also can be intercalated between PLA molecular chains, which would cause the increase in the melt viscosity of the biocomposites.
3.2 Extrudate swell behavior Figure 4 shows the dependence of the extrudate swell as a function of the apparent shear rate at 160 °C for the neat PLA and PLA/BFA composites with various content of untreated (a) and surface-treated (b) BFA particles. Extrudate swell of sample materials increased with increasing apparent shear rate in a nonlinear manner. It was observed that at the lower apparent shear rate range (8.0-25.9 s-1), the extrudate swell of the PLA/BFA composite melts sharply increased with increasing apparent shear rate and then gradually increased. This can be attributed to the increase of apparent shear rate, which then increased the force on the composite melts while flowing into the capillary die. This force occurs in the form of an apparent shear stress, which was found to increase with increasing shear rate or extrusion rate. At the higher apparent shear rates (25.9-49.4 s-1), the effect of shear rate was associated with higher extrusion rates generating greater elastic energies stored in the molten polymer flow increases, which caused the enhanced extrudate swell for both composite systems[13-15]. The neat PLA showed a greater swelling than that of the PLA/BFA composites with untreated and surface-treated BFA particles. Apparently, the extrudate swell of the PLA/BFA composites with surface-treated BFA particles was lower than in the case of untreated ones. This could be explained by the fact that the inorganic silica particles present in the
Figure 3. Apparent shear viscosity as a function of wall shear rate for neat PLA and PLA/BFA composites at 160 °C with various content of untreated (a) and surface-treated (b) BFA particles. 190 190/195
Polímeros, 28(3), 187-195, 2018
Surface treated bagasse fiber ash on rheological, mechanical properties of PLA/BFA biocomposites BFA showed a good dispersion and distribution in the PLA matrix and can be intercalated between PLA chains, resulting in an inhibition of the polymer chain motion by the filler particles, leading to decreased extrudate swell. It was also found that the composites both untreated and surface-treated BFA particles reduced the severity of the extrudate swell with increasing BFA content. This view could be supported by the work of Musking et al. [13], Dangtungee et al.[16] and Liang[17] who stated that the movement of the matrix molecular chains would be made more difficult due to the
presence of filler particles and that the elastic recovery would be blocked when the composite melt emerges from the die, leading to a decrease in extrudate swell. Figure 5 shows photographs of extrudates of PLA/BFA composites with 10 wt.% of untreated (a) and surface-treated (b) BFA particles for different wall shear rates which were taken during extrusion. Figure 6 illustrates the dependence of the extrudate swell as a function of the apparent shear rate of the neat PLA and PLA/BFA composites with various content of
Figure 4. Percentage of extrudate swell as a function of wall shear rate at 160 °C for neat PLA and PLA/BFA composites with various content of untreated (a) and surface-treated (b) BFA particles.
Figure 5. Shows photographs of extrudates of PLA/BFA composites with 10 wt.% of untreated (a) and surface-treated (b) BFA particles. Polímeros, 28(3), 187-195, 2018
191/195 191
Sitticharoen, W., Uthiyoung, C., Passadee, N., & Wongprom, C. surface-treated BFA particles for different temperatures. The extrudate swell of the neat PLA and biocomposite melts at various shear rates decreased with a rise of test temperature, and it rose with increasing apparent shear rate. With a rise of temperature, the molecular mobility of the PLA chains will be intensified, and the relaxation process of the composite melt is correspondingly shortened, leading to a reduction of the elastic recovery of the composite melt when it leaves the die. Therefore, the extrudate swell of the biocomposite is extremely reduced[13,18].
Figure 6. Percentage of extrudate swell as a function of wall shear rate of neat PLA and PLA/BFA composites with various content of surface-treated BFA particles for different temperatures.
3.3 Mechanical properties Figure 7 shows the tensile modulus and flexural modulus of the PLA/BFA composites with untreated and surface‑treated BFA particles. It was found that the amount of added reinforcement filler contributes to variation of the tensile and flexural modulus of the composites, both with untreated and surface-treated BFA particles. Obviously, the tensile and flexural modulus of the composites increased progressively when increasing the BFA filler content. Silica‑based BFA particles insertion can contribute to an increase of the composite’s modulus due to Young’s modulus of the BFA particles being higher than the thermoplastic modulus[7,9]. The tensile and flexural modulus of both untreated and surface-treated BFA gradually increased when the filler concentration increased up to 25 wt.%. The maximum tensile and flexural modulus of the composites with untreated particles were 4.23 x109 and 3.95 x109 Pa, versus 4.61 x109 and 4.24 x 109 Pa, respectively, for surface‑treated BFA particles. Tensile strength and flexural strength of the composites with untreated and surface-treated BFA particles are shown in Figure 8. It was found that the tensile and flexural strength generally tended to decrease as the BFA filler loading increased. The values of the tensile and flexural strength of the composites with 25 wt.% untreated BFA particles were 44.03 x106 and 76.14 x106 Pa, versus 47.93 x106 and 81.26 x 106 Pa, respectively, for surface‑treated particles. The reduction in the tensile and flexural strength of the composites with treated BFA were likely caused by the decreasing interfacial area in PLA matrix as the filler concentration increased[7,9]. Figure 9 shows the fracture surfaces of the PLA/BFA composites with untreated and surface-treated BFA particles. It can be seen that in the case of untreated BFA, voids and pores within the PLA matrix are present, as shown in Figure 9a. This presence may be due to the agglomeration of silica-based BFA filler,
Figure 7. Effect of bagasse fiber ash content on tensile modulus (a) and flexural modulus (b) for PLA/BFA composites with untreated and surface-treated BFA particles. 192 192/195
Polímeros, 28(3), 187-195, 2018
Surface treated bagasse fiber ash on rheological, mechanical properties of PLA/BFA biocomposites
Figure 8. Effect of bagasse fiber ash content on tensile strength (a) and flexural strength (b) for PLA/BFA composites with untreated and surface-treated BFA particles.
Figure 9. SEM micrographs of PLA/BFA composites with 25 wt% of untreated (a) and surface-treated (b) BFA particles.
resulting in the incompatibilities between the hydrophobic PLA and hydrophilic BFA particles[9,19]. The surface-treated BFA particles significantly improved their compatibility between the PLA matrix and BFA particles, and the matrix was well bonded to the BFA particles, leading to a smoother fracture surface than that of untreated particles, as shown in Figure 9b. Figure 10 illustrates the impact strength of the PLA/BFA composites with untreated and surface-treated BFA particles. As can be seen, the impact strength of both composite systems tended to rise with increasing BFA filler content. This was because as the BFA content increased, more interfaces existed on the cracked path, which then led to absorbtion of more energy. The value of the impact strength Polímeros, 28(3), 187-195, 2018
of the composite with 25 wt.% untreated BFA particles was 4.42 kJ/m2, versus 4.86 kJ/m2 for surface-treated particles. The results of hardness values are shown in Figure 11, the hardness values of the biocomposites increase because of greater rigidities of the filler. The value of Shore D hardness of the composite with 25 wt.% for untreated and treated BFA particles was 86.1 and 88.3, respectively.
3.4 Water absorption Figure 12 shows the results of water absorption of the PLA/BFA composites after 2 weeks with untreated and surface-treated BFA particles. Neat PLA has more hydrophobic characteristics than biocomposites and therefore 193/195 193
Sitticharoen, W., Uthiyoung, C., Passadee, N., & Wongprom, C.
Figure 10. Effect of bagasse fiber ash content on impact strength for PLA/BFA composites with untreated and surface-treated BFA particles.
Figure 11. Effect of bagasse fiber ash content on hardness for PLA/BFA composites with untreated and surface-treated BFA particles.
absorbs less water. The addition of BFA particles into the polymeric matrix obviously contributes to more efficient water absorption because of its hydrophilicity, allowing the water to diffuse within the samples. The surface-treatment of BFA particles significantly reduced water absorption of the composites compared to the untreated ones. This view could be supported by Saenghirunwattana et al.[20] who stated that the chemical surface-treated with silane coupling agent increased the hydrophobicity of the fibers due to improving a strong interfacial adhesion between the fiber and polymeric matrix. 194 194/195
Figure 12. Water absorption of PLA/BFA composites after 2 weeks with untreated and surface-treated BFA particles.
4. Conclusions This research presents the study of the effects of surface-treated silica based bagasse fiber ash on the melt rheological and swell behavior, and mechanical properties and water absorption of biocomposites. The flow behavior studies demonstrated the neat molten PLA and PLA/BFA composites with untreated and surface-treated BFA particles exhibited a shear thinning behavior. The melt viscosity of the biocomposites with surface-treated BFA particles was higher than that of the untreated ones. The viscosity of the composite melts decreased with increasing BFA content and shear rate. The extrudate swell of sample materials increased with increasing wall shear rate in a non-linear manner, whereas it reduced with increasing BFA content and die temperature. The extrudate swell tended to be suppressed when surface-treated BFA particles were used. Silane treated BFA composites showed an improvement in their mechanical properties (tensile and flexural modulus, impact strength and hardness), and exhibited significantly reduced water absorption, as compared to the untreated composites. The tensile and flexural strength for both composite systems tended to decrease with increasing BFA content. The BFA particles added to the neat PLA showed an increase in tensile and flexural modulus, impact strength and hardness values of the composites.
5. Acknowledgements The authors thank the Rajamangala University of Technology Lanna for financial support throughout this work through RMUTL’s grant number 2/2558. Special thanks also go to Kaset Thai International Sugar Corp Co., Ltd. (Nakhonsawan, Thailand) and Behn Meyer Chemical (T) Co., Ltd. (Bankok, Thailand) for kindly providing the raw materials. Polímeros, 28(3), 187-195, 2018
Surface treated bagasse fiber ash on rheological, mechanical properties of PLA/BFA biocomposites
6. References 1. Arao, Y., Fujiura, T., Itani, S., & Tanaka, T. (2015). Strength improvement in injection-molded jute-fiber-reinforced polylactide green-composites. Composites. Part B, Engineering, 68, 200206. http://dx.doi.org/10.1016/j.compositesb.2014.08.032. 2. Yuqiong, X., Min, Y., & Jinping, Q. (2009). Melt rheology of poly (lactic acid) plasticized by epoxidized soybean oil. Wuhan University Journal of Natural Sciences, 14(4), 349-354. http:// dx.doi.org/10.1007/s11859-009-0413-4. 3. Sawpan, M. A., Pickering, K. L., & Fernyhough, A. (2011). Improvement of mechanical performance of industrial hemp fibre reinforced polylactide biocomposites. Composites. Part A, Applied Science and Manufacturing, 42(3), 310-319. http:// dx.doi.org/10.1016/j.compositesa.2010.12.004. 4. Peltola, H., Pääkkönen, E., Jetsu, P., & Heinemann, S. (2014). Wood based PLA and PP composites: Effect of fibre type and matrix polymer on fibre morphology, dispersion and composite properties. Composites. Part A, Applied Science and Manufacturing, 61, 13-22. http://dx.doi.org/10.1016/j. compositesa.2014.02.002. 5. Cordeiro, G. C., Toledo, R. D., Fo., Fairbairn, E. M. R., Luis, M. M. T., & Oliveira, C. H. (2004). Influence of mechanical grind on the pozzolanic activity of residual sugarcane bagasse ash. In International RILEM Conference on Use of Recycled Materials in Building and Structure (p. 731-740). Bagneux, France: RILEM Publications Sarl. 6. Office of Cane and Sugar Board. (2015). Report on total cane crushing and sugar production 2014/2015. Bangkok, Thailand: Office of Cane and Sugar Board. 7. Agunsoye, J. O., & Aigbodion, V. S. (2013). Bagasse filled recycled polyethylene bio-composites: Morphologicaland mechanical properties study. Results in Physics, 3, 187-194. http://dx.doi.org/10.1016/j.rinp.2013.09.003. 8. Aigbodion, V. S., Hassan, S. B., & Agunsoye, J. O. (2012). Effect of bagasse ash reinforcement on dry sliding wear behavior of polymer. Materials & Design, 33(1), 322-327. http://dx.doi.org/10.1016/j.matdes.2011.07.002. 9. Sitticharoen, W., Chainawakul, A., Sangkas, T., & Kuntham, Y. (2016). Rheological and mechanical properties of silica-based bagasse-fiber-ash-reinforced recycled HDPE composites. Mechanics of Composite Materials, 52(3), 421-432. http:// dx.doi.org/10.1007/s11029-016-9594-z. 10. Kanking, S., Niltui, P., Wimolmala, E., & Sombatsompop, N. (2012). Use of bagasse fiber ash as secondary filler in silica or carbon black filled natural rubber. Materials & Design, 41, 74-82. http://dx.doi.org/10.1016/j.matdes.2012.04.042.
Polímeros, 28(3), 187-195, 2018
11. Awal, A., Rana, M., & Sain, M. (2015). Thermorheological and mechanical properties of cellulose reinforced PLA biocomposites. Mechanics of Materials, 80, 87-95. http://dx.doi. org/10.1016/j.mechmat.2014.09.009. 12. Dangtungee, R., Petcharoen, K., Pinijsattawong, K., & Siengchin, S. (2012). Investigation of the rheological properties and die swell of polylactic acid/nanoclay composites in a capillary rheometer. Mechanics of Composite Materials, 47(6), 663-670. http://dx.doi.org/10.1007/s11029-011-9246-2. 13. Muksing, N., Nithitanakul, M., Grady, B. P., & Magaraphan, R. (2008). Melt rheology and extrusion swell of organobentonitefilled polypropylene nanocomposites. Polymer Testing, 27(4), 470-479. http://dx.doi.org/10.1016/j.polymertesting.2008.01.008. 14. Liang, J. Z. (2008). Effects of extrusion conditions on dieswell behavior of polypropylene/diatomite composite melts. Polymer Testing, 27(8), 936-940. http://dx.doi.org/10.1016/j. polymertesting.2008.08.001. 15. Intawong, N., Udomsom, S., Sugtakchan, K., & Sitticharoen, W. (2015). Influence of flow pattern development at die entrance and inside annular die on extrudate swell behavior of NR compound. Polímeros: Ciência e Tecnologia, 25(5), 508-513. http://dx.doi.org/10.1590/0104-1428.2021. 16. Dangtungee, R., Yun, J., & Supaphol, P. (2005). Melt rheology and extrudate swell of calcium carbonate nanoparticle-filled isotactic polypropylene. Polymer Testing, 24(1), 2-11. http:// dx.doi.org/10.1016/j.polymertesting.2004.08.006. 17. Liang, J. Z. (2002). The melt elastic behavior of polypropylene/ glass bead composites in capillary flow. Polymer Testing, 21(8), 927-931. http://dx.doi.org/10.1016/S0142-9418(02)00036-3. 18. Liang, J. Z., Yang, J., & Tang, C. Y. (2010). Die-swell behavior of PP/Al(OH)3/Mg(OH)2 flame retardant composite melts. Polymer Testing, 29(5), 624-628. http://dx.doi.org/10.1016/j. polymertesting.2010.03.014. 19. Dharmalingam, U., Dhanasekaran, M., Balasubramanian, K., & Kandasamy, R. (2015). Surface treated fly ash filled modified epoxy composites. Polímeros: Ciência e Tecnologia, 25(6), 540-546. http://dx.doi.org/10.1590/0104-1428.2152. 20. Saenghirunwattana, P., Noomhorm, A., & Rungsardthong, V. (2014). Mechanical properties of soy protein based “green” composites reinforced with surface modified cornhusk fiber. Industrial Crops and Products, 60, 144-150. http://dx.doi. org/10.1016/j.indcrop.2014.06.010. Received: Jan. 20, 2017 Revised: Apr. 12, 2017 Accepted: May 12, 2017
195/195 195
ISSN 1678-5169 (Online)
http://dx.doi.org/10.1590/0104-1428.0117
O O O O O O O O O O O O O O O O
Water-uptake properties of a fish protein-based superabsorbent hydrogel chemically modified with ethanol Vilásia Guimarães Martins1, Jorge Alberto Vieira Costa2 and Carlos Prentice1* Laboratório de Tecnologia de Alimentos, Escola de Química e Alimentos – EQA, Universidade Federal do Rio Grande – FURG, Rio Grande, RS, Brasil 2 Laboratório de Engenharia Bioquímica, Escola de Química e Alimentos – EQA, Universidade Federal do Rio Grande – FURG, Rio Grande, RS, Brasil
1
*dqmprent@furg.br
Abstract Hydrophilic polymers can form hydrogels, which are able to absorb and retain as much water as one hundred times their weight. Polymers based on natural products have been drawing attention since they are biocompatible, biodegradable and nontoxic. The aims of this study were to produce and to characterize a biopolymer with superabsorbent properties from fish protein isolates. Hydrogels were produced from protein isolates from Whitemouth croaker processing wastes chemically modified. The extension of change in lysine residues, kinetics in water-uptake capacity, pH effect, ionic strength over the absorption of water by hydrogels and the behavior of the biopolymer when subject to successive hydration and dehydrations were investigated. Results showed that acid modified protein without ethanol treatment reached a maximum absorption of 103.25 gwater/gdry gel, while the same sample modified with ethanol reached 216.05 gwater/gdry gel. Keywords: biopolymer, ethanol, fish wastes, protein isolates, superabsorbent.
1. Introduction Protein-based materials have the potential to solve a vast array of technical challenges[1]. The recovery and modification of fish proteins present in by-products and their application, such as, in industrial products is a promising and exciting alternative[2]. For industry, developing processes to recover and to use the wastes from fish processing becomes economically feasible instead of discarding them[3,4]. Approximately 50% of the whole fish is considered waste after processing and it is not applied as food[5,6]. The world population has grown and the fish quantity caught nowadays values of approximately 100 million tons/year suggest that the estimate of sustainability is exceeded and obviously this increase leads to the necessity of more intelligent and cautious use of water resources[5]. Alternatively, chemical hydrolysis focuses on biomass recovery from fish results in a soluble product, known as fish protein isolate[7]. The soluble isolate is submitted to dehydration, which increases the stability and protein content[8]. Fish protein tends to have higher application, with inherent capacity of water uptake since the fish proteins are richer in lysine than vegetables[9]. Some of lysine residues (-NH2) of proteins could be replaced by carboxylic group using a reaction with ethylenediamine tetraacetic dianhydride and such modification in the protein may lead to a hydrogel with higher water retention capacity and transparency[5]. Chemical changes in protein became common in the 1950s, when the techniques were originally developed to help the structural analysis of protein molecules. The purpose of such changes was to find out the number of residual
196 196/204
amino acids in a protein molecule in the physical-chemical state, as well as, to identify amino acid residues that have a particular role in the protein[10]. Protein chemical changes allow the introduction and exposure of functional groups, which are unattainable through conventional techniques of mutagenicity[11].This technique is also applied to change the specificity of enzymes and in the pharmaceutical industry it helps to understand how drugs works[12,13]. Such modifications do not change the characteristics of protein biodegradability and biocompatibility. However, suitable properties may be enhanced and then, some highly toxic synthetic polymers, unwanted in many applications, can be replaced by these natural polymers. In the last years, there has been an increasing interest in biodegradable polymers from renewable sources. Hydrophilicity and water absorption are the most required characteristics when absorbent materials are developed. Polymers designed from natural products, such as, polysaccharides and proteins, consist in materials of interest and are available at low cost[14]. Hydrogels are solid polymer structures, which contain a significant fraction of water, regularly above 90%. The tridimensional polymeric structure of hydrogel is usually made by cross-link, not only by physical interactions, such as, Van der Waals or hydrogen links, but by covalent links created by crosslinking agents or gamma-irradiation[5,15]. Due to their high water content, tissue-mimic physical and mechanical properties and biocompatibility, hydrogels have potential to be used in numerous biomedical applications
Polímeros, 28(3), 196-204, 2018
Water-uptake properties of a fish protein-based superabsorbent hydrogel chemically modified with ethanol such as tissue repair, reconstructive surgery, drug delivery and cell encapsulation[16,17]. The development of new hydrogels with high capacity of absorption from natural polymers has been of invaluable importance due to a great potential of its application as material to immobilize enzymes and the large capacity of water retention[18]. Hydrogels are highly biocompatible because they have a close structural resemblance to the natural extracellular matrix (ECM)[17,19]. Biodegradable polymers may be consumed by microorganisms and are reduced to simple compounds, such as, carbon dioxide, water and ammonia[20,21]. Hydrogels based on proteins are able to absorb between 80 and 300 g of water per gram of dry gel, depending on the extension of modification, crosslinking density and protein concentration used during the process[22]. It has been reported that after adding the crosslinking agent, a significant amount of folded structures remain, such as, α-helix, and β-sheet, in protein polymers, and these structures enable the hydrogel to absorb a smaller amount of liquid, as these structures may oppose to relaxing the protein chain, affecting water diffusion inside the tridimensional chain[17,22]. By converting these twisted protein structures in a more linear chain, it is possible to increase the capacity of water uptake, in order to proceed with the denaturation of β-sheet structure and this is accomplished by adding organic solvents[5]. This study aimed to produce and characterize a superabsorbent protein hydrogel, through the treatment of fish protein isolates, obtained from Whitemouth croaker, modified chemically with ethanol, an organic solvent.
was separated in three phases, the superior phase (lipids) and the lower phase (insoluble proteins) were discarded, and the intermediate phase (soluble proteins) was submitted to acid and alkaline precipitation until it reached proteins isoelectric point (pH = 5), where 1 N HCl was applied as acidifying and 1 M NaOH as the alkalizing one, with exposure time of 20 min at 30 °C under agitation. Afterwards, a new centrifugation was done at 7500 x g for 15 min, where the supernatant was discarded and the precipitate, which is the protein isolate, was stored at -18 °C until it was lyophilized.
2. Materials and Methods
2.4 Determination of protein content
2.1 Material
Determination of protein content from the modified isolate was carried out with the method described by Scopes[25], which applies ultraviolet absorption at two wave lengths (205 and 280 nm) and two protein concentrations, 5-25 μg mL-1 at 205 nm and 50-1000 μg mL-1 at 280 nm, for such analysis the samples must be purified, generally dialyzed and lyophilized. The error in this method is estimated at less than 2%. The protein content was determined according to Equation 1. A250 (1) P(mg / mL) = 27 + 120 A280 A205
Whitemouth croaker (Micropogonias furnieri) were obtained from fish processing industries of Rio Grande, Southern Brazil. The fishes were transported in ice-filled containers to the Laboratory of Food Technology, where fish protein isolation was carried out. Firstly, the fish was filleted, after having separated the residue, it was washed up with chlorine water and grounded in equipment with an endless screw, and it was afterwards frozen at -18 °C until its use. Ethylenediamine tetraacetic dianhydride (EDTAD) and glutaraldehyde were purchased from Sigma-Aldrich, United States. All the other reagents used were analytical grade.
2.2 Isolation of Whitemouth croaker protein Two types of chemical extraction processes to isolate protein were performed, alkaline and acid solubilization, applying the pH shifting process according to Martins et al. [5] and Martins et al.[23]. The samples were homogenized with distillated water at a ratio of 1:5. The chemical processes were carried out in a jacketed reactor associated with a thermostatic bath and coupled to an agitator. 1 M NaOH was applied as the alkaline agent and 1 N HCl as the acidifying acid, the alkaline solubilization was done during 20 min at 20 °C at pH 11 and the acid solubilization during 20 min at 30 °C at pH 2.5. After solubilization, the hydrolysate was centrifuged at 7500 x g for 15 min. In this centrifugation, the sample Polímeros, 28(3), 196-204, 2018
2.3 Modification of Whitemouth croaker protein isolates The fish protein isolates were modified with ethylenediamine tetraacetic dianhydride (EDTAD) according to the method described by Hwang and Damodaran[24]. A protein solution of 1% was adjusted to pH 12 with a solution of 1 N NaOH and then heated at 65 °C for 30 min and cooled in an ice bath in order to return to room temperature. The pre-treated protein was reacted with solid EDTAD. During the period of reaction of 2-3 h, the protein solution was mixed constantly; incremented amounts of dianhydride were added during the first 30-90 min, the reaction occurred at room temperature, and the pH of the protein solution, throughout the reaction, was kept constant by adding 1 N NaOH. The reaction ended when pH remained constant for 30 min. Then, the pH of protein solution was adjusted to 7 and dialyzed exhaustively with deionized water for 24 h at 4 °C in membranes of molecular weight 6000-8000 kDa. The modified protein was then lyophilized at a pressure of 121 x 10-3 Mbar and collector at -48 °C was applied in a Liotop model L108 (Liobrás, Brazil)[5].
(
)
2.5 Extension of modification The extension of acetylation was expressed as a percentage of the total modified residual lysine. The unmodified lysine content and acetylated proteins were determined by the acid method 2,4,6-trinitrobenzenosulfonic (TNBS) as described by Hall et al.[26]. For 1 mL of 4% of NaHCO3, 0.8 mL of a solution containing less than 5 mg of protein was added, followed by the addition of 0.2 mL solution of TNBS (12.5 mg mL-1). The mixture was incubated at 40 °C for 2 h, and 3.5 mL of HCl concentrate was added. The tube was covered and kept at 110 °C for 3 h and then, after cooling, the volume was filled up to 10 mL with deionized water. The solution was extracted twice with ethylic ether. The tube was uncovered and kept at 40 °C to allow the residue of ether 197/204 197
Martins, V. G., Costa, J. A. V., & Prentice, C. to evaporate. Absorbance of yellow solution (ε-TNP lysine) was measured at 415 nm. The amount of residue reactive to the lysine from acetylated and non-acetylated proteins was determined from standard curve using lysine.
2.6 Crosslinking by glutaraldehyde A solution of 10% of fish modified isolates with EDTAD was adjusted to pH 9 by adding a solution of 1 N NaOH and homogenized, its volume was 10 mL. A solution of glutaraldehyde 25% at a ratio of 0.02:1 (glutaraldehyde/protein, p/p) was then added to it. After adding the crosslinking agent, the solution was homogenized, and left overnight at room temperature[5].
2.7 Treatment with ethanol After crosslinking, the sample was divided into two parts, one of them placed in an oven at 40 °C for 48 h, in order to be dried and grounded, and the other part was suspended in ethanol for 3 h, during this period ethanol was changed at least twice. The treatment with ethanol causes denaturation of protein and dehydration of gel. At the end of the treatment with ethanol, the gel was in the shape of dry particles, although the gel was put in an oven at 40 °C for 2 h to remove the ethanol residue and moisture[27].
2.8 Kinetics of gel water-uptake A sample of gel of 20-30 mg was placed in an envelope (4 × 6 cm) from nylon and a paper complex (Bolmet Inc.), which was later hot sealed. This envelope was submerged in Milli-Q water for 2, 4, 6, 8, 10 and 24 h, then it was centrifuged at 214 x g for 5 min, and weighted immediately. All the studies of water absorption were carried out at room temperature (25 ± 2 °C). The amount of water absorbed by gel was determined by the weight of water absorbed divided by the weight of dry gel. Drying of gel took place in an oven at 105 °C until constant weight[5]. All the swelling experiments were performed in triplicate.
2.9 Effect of ionic strength over water uptake capacity of gel The influence of ionic strength over swelling property of gels was tested by immersion of the samples in solutions with different concentrations of NaCl, from 0.01 to 0.15 M for a period of 24 h at room temperature (25 ± 2 °C). Later, the samples were centrifuged at 214 x g for 5 min and weighted immediately. The amount of water absorbed was calculated by the weight of water obtained divided by the weight of dried gel. The gel was dried in an oven at 105 °C until constant weight. A control sample was also made, without adding salt.
2.10 Effect of pH on the water-uptake capacity The influence of pH over the behavior of water absorption of hydrogel was determined by placing dried gel samples in buffers of different pH, ranging from 3 to 10 at room temperature for a period of 24 h. The buffer applied in this study were pH 3 (formic acid); pH 4 (benzoic acid); pH 5 (acetic acid); pH 6 (MES); pH 7 (phosphate buffer); 198 198/204
pH 8 (tris base); pH 9 (tris base) and pH 10 (CAPS). All the buffers were prepared with the same ionic strength of 0.01[5].
2.11 Gel absorption and reversibility For the study of swelling kinetics, the samples were immersed in deionized water. At regular intervals of time, the samples were removed, centrifuged at 214 x g for 5 min and weighted. Reversibility of absorption and dissolution of the gel was determined by the use of the same samples for sequential absorption in deionized water and dissolution in solution of 0.15 M NaCl.
3. Results and Discussion The lyophilized fish waste presented 64.3% of protein content. The acid and alkaline isolate reached 86.9 and 72.3% of protein, respectively. The yield of each isolate was 55.87% (acid isolate) and 46.48% (alkaline isolate).
3.1 Extent of chemical modification Chemical modification of protein isolates was carried out using ethylenediamine tetraacetic dianhydride (EDTAD) in proportions which range from 0.05:1 to 0.5:1 (EDTAD:protein, w/w)[5], the best results were achieved in the isolates modified with 0.2:1 and 0.5:1 (EDTAD:protein, w/w). Therefore, only these ones were tested in the remaining part of the study. Table 1 shows the results obtained for each isolate. The extent of modification is related to the amount of residue of lysine present in the isolates, as well as, the amount of chemical agent added during the process of hydrogel production. In the alkaline isolate, the initial amount of lysine found was 11.73% and in the acid isolate it was 11.86%, which is in agreement with the literature, as fish protein presents between 10 and 12% of lysine[28]. The chemical modification of lysyl residues with a tetracarboxylic dianhydride introduces a large number of carboxyl groups into a protein molecule. These added carboxyl groups, in addition to causing extensive unfolding of the protein molecule via intramolecular electrostatic repulsion, would impart a polyanionic character to the protein with numerous sites for water binding[5]. It is possible to verify the modification of the isolate through the increase in the number of lysine modified residues with the increase of EDTAD (Table 1). During the chemical modification of proteins, it is ideal to stop the process when it reaches lysine modification residues between 50 and 80%, since an extension in the process would lead to the production of a hydrogel with reduced water-uptake capacity. An extensively modified protein would have many linking sites for water, although it would have less space for its absorption, the opposite would be also undesirable, since a slightly modified protein would, Table 1. Extent of modification of Whitemouth croaker protein isolates in different proportion of EDTAD:protein (w/w). Acid (%) Alkaline (%)
0.05:1 16.63 13.42
0.08:1 24.61 15.86
0.1:1 46.2 33.69
0.2:1 61.35 45.76
0.5:1 72.41 63.5
Polímeros, 28(3), 196-204, 2018
Water-uptake properties of a fish protein-based superabsorbent hydrogel chemically modified with ethanol probably, has fewer water binding sites, and consequently a hydrogel with low capacity of water uptake. Rathna et al.[22] applied modified albumin with EDTAD to produce hydrogel, however, heating was applied to form the gels. The researchers observed that the larger extension of modification resulted in higher water-uptake capacity, although proteins with high degree of modification had less density of crosslinking and they eventually disintegrated, for instance, modified hydrogel 100% disintegrated after 3 h, while hydrogel prepared with 83% took 5 h to disintegrate. According to Hwang and Damodaran[27], through chemical modification of lysine residues with an ethylenediamine tetracarboxylic it is possible to introduce a large amount of carboxylic groups inside the protein molecule. For each lysine residue modified, up to three carboxylic groups can be introduced in the protein. The modified protein with EDTAD is nontoxic; there are no reactive groups, besides the carboxylic groups which are introduced in the molecule of the protein. During the reaction, EDTAD reacts with water and it is converted in sodium salt EDTA, the Na EDTA is considered a safe food additive, thus, it does not pose risks to human health[29].
3.2 Kinetics of water-uptake For practical applications, as diapers, plants water supply and any other product that need absorb water or keep water to release, both high capacity of water-uptake and high rate of retention are required. Buchholz[30] suggests that the kinetics of water-uptake of superabsorbent materials is influenced by many factors, such as, capacity of water retention, size of particles and polymer composition. During the hydrogel production, glutaraldehyde was added at proportion of 0.02:1 (glutaraldehyde:protein, p/p). It has been proved by several authors that an increase in the amount of glutaraldehyde interferes in water uptake. Some studies show that the higher amount of crosslinking agent during hydrogel production leads to a drop in the amount of water absorbed by the gel[31,32]. According to Hwang and
Damodaran[27], this is due to the capacity of water-uptake, since by raising the crosslinking density between the protein molecules, reduces the space for water absorption inside the tridimensional network. It is reported that after the stage of adding the cross linking agent during the process of hydrogel production, a significant amount of folded structures remain, such as, α-helix and β-sheet, these structures may lead the hydrogel to absorb less amounts of liquid, these structures may oppose the protein chain relaxation, affecting water diffusion inside the tridimensional network[27]. One way to increase water absorption is to turn these twisted structures into more linear ones, using organic solvents. This way it is possible to denature such structures and make them more linear, thus more sites are exposed for water binding and there are more spaces for swelling inside the protein molecule. By denaturing the polypeptide chains in situ in the protein matrix, it is apparently possible to avoid refolding these structures, even after removing the reagent that provides denaturation. This procedure increases the flexibility of protein chains and relaxes the tridimensional gel network, such as, water diffusion inside the polymeric chain[9]. Figure 1 presents the kinetics of water-uptake of protein modified isolates during a period of 24 h. It is possible to verify that the samples treated with ethanol had higher water absorption than the samples treated only with glutaraldehyde. In 24 h, the control samples obtained maximum water uptake of 7.03 gwater/gdrygel and 9.22 gwater/gdrygel, for hydrolyzed, alkaline and acid, respectively. Samples modified chemically reached much higher values. The highest rate of water absorption occurred within the first hours of immersion, in the alkaline modified isolates it is observed until 2 h of immersion that the water absorption became virtually constant until the last period of 24 h, after that only a slight increase in water retention is noticed. In the acid modified isolates the same trend was observed, although both modified isolates treated with ethanol had
Figure 1. Kinetics of water absorption of hydrogels. (a) Alkaline modified isolates (b) acid modified isolates. ♦ alkaline and acid isolates without chemical modification; ■ modified 0.2:1 (EDTAD:protein, p/p); ▲ modified 0.5:1 (EDTAD:protein, p/p); × modified 0.2:1 treated com ethanol; * modified 0.5:1 treated com ethanol. Polímeros, 28(3), 196-204, 2018
199/204 199
Martins, V. G., Costa, J. A. V., & Prentice, C. sharper increase between 2 and 10 h, when compared with other samples, after this period, they reached stability until the period of 24 h. The alkaline modified isolates showed lower water retention capacity than the acid modified isolates, as expected, the extent of modification was larger in acid modified isolates. A higher water uptake is reached when it has more carboxylic groups inside of the protein molecule and it is possible because of the chemical modification of the lysine residues, the chemical reaction with the EDTAD make the substitution of NH2 for carboxyl groups. The alkaline and acid modified isolates 0.5:1 exhibited water retention capacity 11 times higher than the non-modified hydrolysates and the modified 0.5:1 treated with ethanol showed water retention capacity 19 times higher for alkaline and 23 times higher for acid. In a similar study carried out by Rathna and Damodaran[9] with fish modified hydrolysates, it was shown that the modified isolates treated with ethanol had increased capacity of water-uptake of approximately 50% when compared with those without treatment with ethanol. According to García Sánchez and Cortés Ortega[16], the aqueous solution with ethanol stabilizes growing particles, running as the continuous phase, a system similar to emulsion or microemulsion, depending on the amount of ethanol in the reaction mixture. It is interesting to point out that the protein isolate used did not have any fat residual. In this study, the increase in water absorption rate of modified treated with ethanol was much higher than 50%, it might be related with the amount of fat present in the hydrolysates (ranging from 7 to 9%). In this case, ethanol improves the denaturation of β-sheets structures and also removes fat residuals. Such fat, present in modified protein may have prevented the modified isolates without the presence of ethanol from absorbing more water.
3.3 Effect of ionic strength over the swelling capacity of gels The rate of water-uptake is mainly related to the characteristics of the external solution, for example, ionic power, nature of the polymer, elasticity of tridimensional
network, presence of hydrolytic functional groups and extent of modification. The ability of swelling of “anionic” hydrogels in several saline solutions is lower than in distillated water[33]. Thus, loss of capacity in water-uptake is regularly justified by the “screening effect” of cations added, which cause flaws in electrostatic anion-anion repulsions[34]. Figure 2 shows the behavior of modified isolates, treated or not, with ethanol, when exposed to different salt concentrations for 24 h. Hydrogels produced from fish isolates are very sensitive to saline solutions; a drop in water absorption between 60 and 90% was detected when a lower concentration of 0.01 M NaCl was applied. All of the immersed gels in saline solution 0.15 M had reductions higher than 90% in the water uptake capacity while compared to the same immersed in distillated water. Similar studies of hydrogel production with soy and fish protein modified by ethylenediamine tetraacetic dianhydride and added with glutaraldehyde demonstrated the same behavior in terms of ionic strength[5]. Sadeghi and Hosseinzadeh[35] investigated a hydrogel formed from starch and evaluated at different concentrations of NaCl. It demonstrated to be more resistant to saline conditions than the protein hydrogel produced in this study, as it absorbed 87 gwater/gdry gel in a concentration of 0.01 M and in 0.15 M it was still able to absorb 58 gwater/ gdry gel. Pourjavadi et al.[36] produced hydrogel from hydrolysis of collagen, reached a water absorption of 58 gwater/ggel in a concentration of 0.15 M NaCl. Mahdavinia et al.[37] studied the ionic strength of κ-carrageenan/sodium alginate hydrogel in salt solutions, suc as CaCl2, NaCl and KCl. The authors observed a different swelling behavior for each salt solution, the hydrogel disintegrated after 160 min in the NaCl solution, for the CaCl2 and KCl solution the hydrogel swell without any disintegration. Mahdavinia et al.[38] investigated a swelling behavior of a hydrogel made from modified chitosan in two salt solutions (NaCl and CaCl2), and observed that the hydrogel absorbed much more in the NaCl solution than CaCl2 solution, then the swelling behavior of hydrogels in
Figure 2. Effect of ionic strength over hydrogels. (a) Alkaline modified isolates (b) acid modified isolates. ♦ alkaline and acid isolate without chemical modification; ■ modified 0.2:1 (EDTAD:protein, p/p); ▲ modified 0.5:1 (EDTAD:protein, p/p); × modified 0.2:1 treated with ethanol; * modified 0.5:1 treated with ethanol. 200 200/204
Polímeros, 28(3), 196-204, 2018
Water-uptake properties of a fish protein-based superabsorbent hydrogel chemically modified with ethanol general depends of their structure and the saline solution which it is immersed.
3.4 Effect of pH over water-uptake capacity The pH affects the water-uptake capacity of protein-based hydrogels produced from fish isolates, as shown in Figure 3. Also, pH of solutions affects ionization of carboxylic and other ionizable groups[18]. All the samples evaluated reached the maximum of water absorption at pH 8. The same behavior was found by Sadeghi and Hosseinzadeh[35], who studied the influence of pH on water absorption by a hydrogel produced from starch. According to Hwang and Damodaran[24], the values of pK1, pK2 and pK3 of the carboxylic groups of EDTA are 2, 2.6 and 6.2, respectively. Hence, theoretically, all carboxylic groups must be fully ionized around pH 8, providing maximum water absorption of water around this pH value. However, in the study made by these authors, which the hydrogel was produced from fish muscle, water absorption of the hydrogel increased from pH 3 to pH 10, with the maximum reached in the latter. It is interesting to observe that the non-modified isolates presented a higher value of water absorption at pH 3 when compared with the modified isolate, this may be associated to a higher amount of lysine present in non-modified isolate, since they are protonated in pH 3. The increase in the positive charge of protein, due to protonation of carboxylic groups below pH 4, may cause electrostatic repulsions within the protein matrix allowing water retention at pH 3. At pH 3, most of the carboxylic groups are in the form of COOH and the hydrogel has low values of water retention, it can be justified by the presence of hydrolytic non-ionic groups COOH and -OH in the polymeric network of hydrogel[35], as well as, many carboxyl anions are protonated, the main anion-anion repulsion forces are eliminated and, consequently, the capacity of water absorption decreases[36]. As pH increases from 4 to 8, the rate of water absorption increases significantly. Reis et al.[39] noticed that the higher the pH, the higher the water uptake, which was associated to
more COOH groups dissociate to COO-, raising the number of ionized groups in the hydrogel structure. It generates electrostatic repulsion in the ionized groups in the polymer network, which increase water absorption of hydrogels. Similar behaviors on water absorption in different pH have been reported for other types of hydrogels[29,40,41]. The ionic strength of all the buffers applied in the study of water absorption by hydrogels was 0.01 M, it was probably the explanation of low water retention. For example, the acid modification 0.5:1 (EDTAD:protein, p/p) when treated with ethanol absorbed 216.05 gwater/gdrygel, while the maximum absorption of the same sample at pH 8 was 66.69 gwater/gdrygel. Hydrogel produced from alkaline isolates, also treated with ethanol reached 137.19 gwater/gdrygeland at pH 8, the maximum of water retention was 28.76 gwater/gdrygel.
For pH higher than 8, where all the samples had maximum water holding capacity, there was a slight reduction in pH 9, probably because of the lower ionization of amino acids residues that could keep the water absorption, followed by a new increase in water absorption in pH 10. This increase in swelling capacity of gels at pH 10, according to Hwang and Damodaran[27], is associated with electrostatic repulsion force which is directly proportional to the square charge of molecular network, thus, even though there is a small increase in the charge of the network due to ionization of tyrosine residues (pK3 = 9.6) there will be an increase in electrostatic repulsion inside the network of the gel, resulting in an increase of gel expansion and water retention. Other authors[16] state that growing particles are surrounded by layers of aqueous ethanol solution, which allow the stability of the particles growing in establishing a colloidal system.
3.5 Reversibility of hydrogels According to Kong and Li[42], protein-based hydrogels have attracted considerable interest due to their potential biomedical applications. Although various methods have been developed to engineer self-assembling, physically‑crosslinked protein hydrogels, exploring novel driving forces to engineer such hydrogels remains challenging. Since the hydrogels
Figure 3. Effect of pH over hydrogels. (a) Alkaline modified isolates (b) acid modified isolates. ♦ alkaline and acid isolates without chemical modification; ■ modified 0.2:1 (EDTAD:protein, p/p); ▲ modified 0.5:1 (EDTAD:protein, p/p); × modified 0.2:1 treated with ethanol; * modified 0.5:1 treated with ethanol. Polímeros, 28(3), 196-204, 2018
201/204 201
Martins, V. G., Costa, J. A. V., & Prentice, C. produced in this study proved to be sensitive to exposition to saline solutions, the capacity of reversibility, absorption and dehydration of gels was investigated. Therefore, a solution of 0.15 M NaCl was used, as dehydration agent and deionized water for hydration. Figures 4 and 5 show the behavior of the gels during a 36 h period, in which hydrogels were submitted to subsequent hydrations and dehydrations. When the gel hydrated for 24 h was exposed to a saline solution of 0.15 M NaCl, for a period of 2 h, there was a drop of more than 90% in water content, it occurred for hydrogels produced with both alkaline and acid modified protein. The control samples also had a loss in water content, although it was between 55 and 64%. Figure 4 clearly shows that the hydrogels produced had good water retention capacity, even after dehydration. Nonetheless, it is noticed that their capacity of water absorption was slightly reduced, after the first dehydration. Once dehydrated the second time, it is observed that there was an increase in the capacity of hydration, when compared to the first dehydration, which can be explained, partly, due to an increase in flexibility of the gel matrix, after repeated dehydrations and hydrations. Hwang and Damodaran[24] studied reversibility of the hydrogel produced from alkaline isolates of fish muscle and obtained a higher water retention capacity when the gel was dehydrated and hydrated again for the first time. However, when the gel is submitted to dehydration for the second time and returned to hydration, it took long to absorb water, 7 times lower than the first time. Figure 5 shows the behavior of modified protein derived from acid isolates, they had similar behaviors to
Figure 4. Kinetics of absorption and dehydration of hydrogels produced from alkaline isolates. ♦ alkaline isolates without chemical modification; ■ modified 0.2:1 (EDTAD:protein, p/p); ▲ modified 0.5:1 (EDTAD:protein, p/p); × modified 0.2:1 treated with ethanol; * modified 0.5:1 treated with ethanol.
protein alkaline modified. These hydrogels also had good hydration capacity, after successive dehydrations. Another fact noticed was that dehydration in 0.15 M NaCl is faster than next hydration. While gels lose approximately 90% of water content in 2 h immersion, these take approximately 4 h to hydrate again. This slower water absorption may be justified by a low dissociation rate of ions linked to the polymer network.
4. Conclusion The protein isolates produced from fish wastes and modified chemically were able to form biopolymers as hydrogels with superabsorbent properties. The results of this study indicate that protein hydrogels produced with crosslinking when treated with ethanol enhance their water swelling properties. For the acid solubilized protein, it was obtained an increase of 209% and 172% for the alkaline solubilized protein. Besides raising the water retention capacity of hydrogels, the modification with ethanol offers other advantages, such as, dehydration of the gel that does not require dryness for a long period, extraction of odorous compounds of low molecular weight, which improves its acceptance by the consumer, mainly when it comes from a hydrogel formulated from fish, it also eliminates any non-reactive residue of glutaraldehyde which may be present in the gel. Ethanol applied in the process can also be easily recovered and recycled. The reversibility of swelling capacity of hydrogels shown proves that hydrogels produced from fish protein isolates can be repeatedly applied, without losing their hydration capacity, hence; they can be used in the dehydration processes in many industries, such as, pharmaceutical, food, chemical, among others. The potential applications of such hydrogels could be in diapers and plants water supply, the main advantage of those hydrogels over the conventional ones is the degradability, they are made from proteins, and can degrade much faster than the conventional polymers. These hydrogels have an eco-friendly nature, the use of fish wastes to produce them is a good destiny for those sub-products, because in most of the cases the fish wastes are disposed directly in the environment causing a great environmental problem.
5. Acknowledgements This work was supported by grants from the National Council for Scientific and Technological Development of Brazil (CNPq), and the Coordination for Improvement of Higher Education Personnel of Brazil (CAPES).
6. References
Figure 5. Kinetics of absorption and dehydration of hydrogels produced from alkaline isolates. ♦ alkaline isolates without chemical modification; ■ modified 0.2:1 (EDTAD:protein, p/p); ▲ modified 0.5:1 (EDTAD:protein, p/p); × modified 0.2:1 treated with ethanol; * modified 0.5:1 treated with ethanol. 202 202/204
1. Huang, P.-S., Boyken, S. E., & Baker, D. (2016). The coming of age of de novo protein design. Nature, 537(7620), 320-327. http://dx.doi.org/10.1038/nature19946. PMid:27629638. 2. Martins, V. G., Palezi, S. C., Costa, J. A. V., & Prentice, C. (2014). Hydrolysis of insoluble fish protein residue from Whitemouth croaker (Micropogonias furnieri) by fungi. Brazilian Archives of Biology and Technology, 57(1), 96-102. http://dx.doi.org/10.1590/S1516-89132014000100014. Polímeros, 28(3), 196-204, 2018
Water-uptake properties of a fish protein-based superabsorbent hydrogel chemically modified with ethanol 3. Kristinsson, H. G., & Rasco, B. A. (2000). Fish protein hydrolysates: production, biochemical, and functional properties. Critical Reviews in Food Science and Nutrition, 40(1), 43-81. http://dx.doi.org/10.1080/10408690091189266. PMid:10674201. 4. Freitas, I. R., Cortez-Vega, W. R., & Prentice, C. (2015). Recovery of anchovy (Engraulis anchoita) and whitemouth croaker (Micropogonias furnieri) proteins by alkaline solubilisation process. Acta Alimentaria, 44(2), 221-228. http://dx.doi. org/10.1556/AAlim.2014.0005. 5. Martins, V. G., Costa, J. A. V., Damodaran, S., & Prentice, C. (2011). Chemical modification and structural analysis of protein isolates to produce hydrogel using Whitemouth croaker (Micropogonias furnieri) wastes. Applied Biochemistry and Biotechnology, 165(1), 279-289. http://dx.doi.org/10.1007/ s12010-011-9250-y. PMid:21505805. 6. Halim, N. R. A., Yusof, H. M., & Sarbon, N. M. (2016). Functional and bioactive properties of fish protein hydrolysates and peptides: a comprehensive review. Trends in Food Science & Technology, 51, 24-33. http://dx.doi.org/10.1016/j. tifs.2016.02.007. 7. Cortez-Vega, W. R., Pizato, S., Souza, J. T. A., & Prentice, C. (2014). Using edible coatings from Whitemouth croaker (Micropogonias furnieri) protein isolate and organo-clay nanocomposite for improve the conservation properties of fresh-cut Formosa papaya. Innovative Food Science & Emerging Technologies, 22, 197-202. http://dx.doi.org/10.1016/j. ifset.2013.12.007. 8. Guerard, F., Guimas, L., & Binet, A. (2002). Production of tuna waste hydrolysates by a commercial neutral protease preparation. Journal of Molecular Catalysis. B, Enzymatic, 19-20(2), 489498. http://dx.doi.org/10.1016/S1381-1177(02)00203-5. 9. Rathna, G. V. N., & Damodaran, S. (2002). Effect of nonprotein polymers on water-uptake properties of a fish protein-based hydrogel. Journal of Applied Polymer Science, 85(1), 45-51. http://dx.doi.org/10.1002/app.10566. 10. Matsushima, A., Kodera, Y., Hiroto, M., Nishimura, H., & Inada, Y. (1996). Bioconjugates of proteins and polyethylene glycol: potent tools in biotechnological processes. Journal of Molecular Catalysis. B, Enzymatic, 2(1), 1-17. http://dx.doi. org/10.1016/1381-1177(96)00003-3. 11. DeSantis, G., & Jones, B. (1999). Chemical modification of enzymes for enhanced functionality. Current Opinion in Biotechnology, 10(4), 324-330. http://dx.doi.org/10.1016/ S0958-1669(99)80059-7. PMid:10449313. 12. Kurniawan, L., Qiao, G. G., & Zhang, X. (2007). Chemical modification of wheat protein-based natural polymers: grafting and cross-linking reactions with poly(ethylene oxide) diglycidyl ether and ethyl diamine. Biomacromolecules, 8(9), 2909-2915. http://dx.doi.org/10.1021/bm0703719. PMid:17663528. 13. Solanki, K., Shah, S., & Nath Gupta, M. (2008). Chemical modification of alpha-chymotrypsin for obtaining high transesterification activity in low water organic media. Biocatalysis and Biotransformation, 26(4), 258-265. http:// dx.doi.org/10.1080/10242420801897361. 14. Iannace, S., Nicolais, L., & Huang, S. J. (1997). Water sorption of glycol-modified cross-linked gelatin-based hydrogels. Journal of Materials Science, 32(6), 1405-1408. http://dx.doi. org/10.1023/A:1018533429283. 15. Kunioka, M., & Choi, H. J. (1998). Hydrolytic degradation and mechanical properties of hydrogels prepared from microbial poly(amino acid)s. Polymer Degradation & Stability, 59(1-3), 33-37. http://dx.doi.org/10.1016/S0141-3910(97)00181-X. 16. García Sánchez, L. G. J., & Cortés Ortega, J. A. (2014). Síntesis de hidrogeles de acrilamida en soluciones acuosas de etanol. Polímeros, 28(3), 196-204, 2018
Polímeros: Ciência e Tecnologia, 24(6), 752-756. http://dx.doi. org/10.1590/0104-1428.1663. 17. Chen, J., Ma, X., Dong, Q., Song, D., Hargrove, D., Vora, S. R., Ma, A. W. K., Lu, X., & Lei, Y. (2016). Self-healing of thermally-induced, biocompatible and biodegradable protein hydrogel. RSC Advances, 6(61), 56183-56192. http://dx.doi. org/10.1039/C6RA11239K. 18. Park, T. G., & Hoffman, A. S. (1992). Synthesis and characterization of pH-and/or temperature sensitive hydrogels. Journal of Applied Polymer Science, 46(4), 659-671. http:// dx.doi.org/10.1002/app.1992.070460413. 19. Bae, K. H., & Kurisawa, M. (2016). Emerging hydrogel designs for controlled protein delivery. Biomaterials Science, 4(8), 1184-1192. http://dx.doi.org/10.1039/C6BM00330C. PMid:27374633. 20. Hideki, O. (1992). Handbook of polymer degradation. New York: Marcel Dekker. 21. Hellriegel, J., Günther, S., Kampen, I., Bolea Albero, A., Kwade, A., Böl, M., & Krull, R. (2014). Biomimetic gellanbased hydrogel as a physicochemical biofilm model. Journal of Biomaterials and Nanobiotechnology, 5(2), 83-97. http:// dx.doi.org/10.4236/jbnb.2014.52011. 22. Rathna, G. V. N., Li, J., & Gunasekaran, S. (2004). Functionallymodified egg white albumen hydrogels. Polymer International, 53(12), 1994-2000. http://dx.doi.org/10.1002/pi.1611. 23. Martins, V. G., Costa, J. A. V., & Prentice-Hernández, C. (2009). Hidrolisado proteico de pescado obtido por vias química e enzimática a partir de corvina (Micropogonias furnieri). Química Nova, 32(1), 61-66. http://dx.doi.org/10.1590/S010040422009000100012. 24. Hwang, D. C., & Damodaran, S. (1997). Synthesis and properties of fish protein-based hydrogel. Journal of the American Oil Chemists’ Society, 74(9), 1165-1171. http://dx.doi.org/10.1007/ s11746-997-0041-0. 25. Scopes, R. K. (1974). Measurement of protein by spectrophotometry at 205 nm. Analytical Biochemistry, 59(1), 277-282. http:// dx.doi.org/10.1016/0003-2697(74)90034-7. PMid:4407487. 26. Hall, R. J., Trinder, N., & Givens, D. I. (1973). Observations on the use of 2,4,6-trinitrobenzene sulphonic acid for the determination of available lysine in animal protein concentrates. Analyst, 98(1170), 673-686. http://dx.doi.org/10.1039/ an9739800673. PMid:4753164. 27. Hwang, D. C., & Damodaran, S. (1996). Chemical modification strategies for synthesis of protein-based hydrogel. Journal of Agricultural and Food Chemistry, 44(3), 751-758. http:// dx.doi.org/10.1021/jf9503826. 28. Thiansilakul, Y., Benjakul, S., & Shahidi, F. (2007). Compositions, functional properties and antioxidative activity of protein hydrolysates prepared from round scad (Decapterus maruadsi). Food Chemistry, 103(4), 1385-1394. http://dx.doi.org/10.1016/j. foodchem.2006.10.055. 29. Hwang, D., & Damodaran, S. (1998). Carboxyl modified superabsorbent protein hydrogel. US Patent No 5,847,089. Alexandria. 30. Buchholz, F. L. (1994). Preparation methods of superabsorbent polyacrylates. In American Chemical Society. Superabsorbent polymers: science and technology (ACS Symposium Series, Vol. 573). Washington: ACS. 31. George, M., & Abraham, T. E. (2007). pH sensitive alginateguar gum hydrogel for the controlled delivery of protein drugs. International Journal of Pharmaceutics, 335(1-2), 123-129. http://dx.doi.org/10.1016/j.ijpharm.2006.11.009. PMid:17147980. 32. Tsai, H.-S., & Wang, Y.-Z. (2008). Properties of hydrophilic chitosan network membranes by introducing binary crosslink 203/204 203
Martins, V. G., Costa, J. A. V., & Prentice, C. agents. Polymer Bulletin, 60(1), 103-113. http://dx.doi. org/10.1007/s00289-007-0846-x. 33. Pourjavadi, A., Amini-Fazl, M. S., & Hosseinzadeh, H. (2005). Partially hydrolyzed crosslinked alginate-graft-polymethacrylamide as a novel biopolymer-based superabsorbent hydrogel having pH-responsive properties. Macromolecular Research, 13(1), 45-53. http://dx.doi.org/10.1007/BF03219014. 34. Peppas, N. A., & Mikes, A. G. (1986). Hydrogels in Medicine and Pharmacy. Vol.1, CRC Press: Boca Raton, FL. 35. Sadeghi, M., & Hosseinzadeh, H. (2008). Synthesis of starch– poly(sodium acrylate-co-acrylamide) superabsorbent hydrogel with salt and pH-responsiveness properties as a drug delivery system. Journal of Bioactive and Compatible Polymers, 23(4), 381-404. http://dx.doi.org/10.1177/0883911508093504. 36. Pourjavadi, A., Kurdtabar, M., Mahdavinia, G. R., & Hosseinzadeh, H. (2006). Synthesis and super-swelling behavior of a novel protein-based superabsorbent hydrogel. Polymer Bulletin, 57(6), 813-824. http://dx.doi.org/10.1007/s00289-006-0649-5. 37. Mahdavinia, G. R., Rahmani, Z., Karami, S., & Pourjavadi, A. (2014). Magnetic/pH-sensitive κ-carrageenan/sodium alginate hydrogel nanocomposite beads: preparation, swelling behavior, and drug delivery. Journal of Biomaterials Science, 25(17), 1891-1906. http://dx.doi.org/10.1080/09205063.2014.95616 6. PMid:25197770. 38. Mahdavinia, G. R., Pourjavadi, A., Hosseinzadeh, H., & Zohuriaan, M. J. (2004). Modified chitosan 4. Superabsorbent hydrogels from poly(acrylic acid-co-acrylamide) grafted
204 204/204
chitosan with salt and pH-responsiveness properties. European Polymer Journal, 40(7), 1399-1407. http://dx.doi.org/10.1016/j. eurpolymj.2004.01.039. 39. Reis, A. V., Guilherme, M. R., Cavalcanti, O. A., Rubira, A. F., & Muniz, E. C. (2006). Synthesis and characterization of pH-responsive hydrogels based on chemically modified Arabic gum polysaccharide. Polymers, 47(6), 2023-2029. http://dx.doi. org/10.1016/j.polymer.2006.01.058. 40. Toit, L. C. D., Pillay, V., & Danckwerts, M. P. (2006). Application of synergism and variation in ionic compatibilities within a hydrophilic polymeric sodium starch glycolate-kappacarrageenan combination: textural profiling of the suspension behavior. Journal of Bioactive and Compatible Polymers, 21(2), 107-122. http://dx.doi.org/10.1177/0883911506062975. 41. Zhang, J., Yuan, K., Wang, Y. P., Zhang, S. T., & Zhang, J. (2007). Preparation and pH responsive behavior of poly(vinyl alcohol) - chitosan- poly(acrylic acid) full-IPN hydrogels. Journal of Bioactive and Compatible Polymers, 22(2), 207218. http://dx.doi.org/10.1177/0883911506076046. 42. Kong, N., & Li, H. (2015). Protein fragment reconstitution as a driving force for self-assembling reversible protein hydrogels. Advanced Functional Materials, 25(35), 5593-5601. http:// dx.doi.org/10.1002/adfm.201502277. Received: Oct. 06, 2016 Revised: Jan. 24, 2017 Accepted: June 05, 2017
Polímeros, 28(3), 196-204, 2018
ISSN 1678-5169 (Online)
http://dx.doi.org/10.1590/0104-1428.06317
Characterization of additives in NR formulations by TLC-IR (UATR) Lidia Mattos Silva Murakami1,2, Joyce Baracho Azevedo1,2, Milton Faria Diniz3, Leandro Mattos Silva4 and Rita de Cássia Lazzarini Dutra1* Instituto Tecnológico de Aeronáutica, São José dos Campos, SP, Brasil 2 Tenneco Automotive Brasil, Cotia, SP, Brasil 3 Divisão de Química – AQI, Instituto de Aeronáutica e Espaço – IAE, São José dos Campos, SP, Brasil 4 Petroquímica Brasken S.A., Capuava, Santo André, SP, Brasil 1
*ritacld@ita.br
Abstract It is a well-established fact that rubber accelerator is essential to provide solution in different sectors. However, there is a reversal process which can reduce the material performance. Sulfur accelerators donors and organic peroxides have been presented as a solution to the problem. The methodology development that can separate or characterize those components is a challenge and still allows gaps, explained by the application of conventional technique to reach this goal. This study aimed at contributing to the use of off-line coupling of thin layer chromatography (TLC)/infrared spectroscopy (IR) by Universal Attenuated Total Reflection (UATR) for analysis of N-cyclohexyl-2-benzotiazolsulfenamide (CBS), tetraethylthiuram disulfide (TMTD) and dicumyl peroxide (DCP), in natural poly-cis-isoprene (NR) formulations, containing naphthenic oil. The best results were obtained for the plasticizer and DCP, in formulations that had a greater proportion of these compounds. The separation of CBS and TMTD was made with less effectiveness, due to bands overlapping. Keywords: additives, characterization, NR, TLC, UATR.
1. Introduction The competitive industrial environment has demanded of companies more agility in terms of response to the market. Companies have embraced some strategies to accomplish that specific objective and knowing better their own product substantially is one of them. Not only does this route lead to better process variable evaluation, but also it improves the employment of technology. Products based on polymeric materials play a major role in Aerospace and Automotive Industry considering they can withstand harsh environments, reduces production costs and offers flexibility in processing. As a light solution, the polymers successfully replace metals and traditional compounds due to its mechanical resistance, corrosion resistance and ease in the manufacture of parts with narrower tolerance. A vulcanized rubber is a widely applied polymer throughout several industrial areas. Its counterpart Natural rubber (NR) for instance, is applied in the automotive industry, like tires and hoses[1], and in aerospace industry, as flexible joints[2]. It contains elastomer, vulcanization agent and accelerator, reinforcing filler, stabilizers, among other additives. This mixture is vulcanized after passing through thermal processes or after been exposed to high energy radiation[3]. Among these additives, it is possible to highlight the accelerators, which reduce the cure time and consequently the process costs. They are classified according to their chemical composition and / or their speed of action
Polímeros, 28(3), 205-214, 2018
in vulcanization. In Table 1, a list of the most common accelerators[4] is included. Acknowledge the accelerators and vulcanizing agent of a formulation is paramount for new polymers development. Not only does it aid in reducing process costs, but also it increases the solution spectrum for industry. Taking this into account, the reversion problem is brought to our attention, when the polysulfide bond breaks causing reduction of crosslink density, changing the distribution of the bonds types and modification in main chain structure. These facts lead to reduced article performance. Reversion resistance of rubber compounds has been obtained by controlling the sulfur content in the crosslink bonds, by applying sulfur donor accelerators and organic peroxides. In terms of thermal stability, their higher bond strength gives more stability than the carbon/sulfur/carbon bond and gives good properties for aging resistance[5]. Study of sulfur vulcanization has been mentioned in the literature[6]. TMTD, among others accelerator polysulfides such as N,N-pentamethylenethiuram disulfide (CPTD) were employed and it was concluded that CPTD and its polysulfides are thermally less stable than is TMTD. As reported by Joseph et al.[7], among the various organic sulphur containing compounds, TMTD has been the most studied. Vulcanizates obtained using this compounds in combination with ZnO have superior thermal and oxidative
205/214 205
O O O O O O O O O O O O O O O O
Murakami, L. M. S., Azevedo, J. B., Diniz, M. F., Silva, L. M., & Dutra, R. C. L. Table 1. Most usual accelerators and vulcanizing agent for elastomeric compositions. Acronym / nomenclature MBT (2-mercaptobenzothiazole) MBTS (benzothiazole disulfide) CBS (N-cyclohexyl-2-benzothiazolesulfenamide) TBBS (N-tert-butyl-di (2-benzothiazolesulfenamide) MBS (2- (4-Morpholinothio) benzothiazole) TMTD (Tetramethylthiuram Disulfide) TMTM (Tetramethylthiuram Monosulfide) TETD (tetraethylthiuram disulphide)
stability and negligible modulus reversion. According to radical mechanism, at vulcanization temperatures, interactive recombination will lead to the formation of accelerator polysulphides (TMTPs). Influence of sulfenamide accelerators, such as CBS, on cure kinetics and properties of NR has been studied as well[8]. CBS accelerator shows the fastest sulfur vulcanization rate and the lowest activation energy (Ea) because CBS accelerator produces higher level of basicity of amine species than other sulfenamide accelerators. The effect of temperature and peroxide concentration has been studied[9] . DCP was evaluated among other peroxides. It was observed that DCP and di-tert-butyl peroxide (DTBP) showed lower amounts of decomposition products compared to other peroxides studied. DCP and DTBP were suggested as better curing agents for NR based rubber compounds at higher peroxide concentrations. TMTD, CBS and DCP were the additives used in this current paper to be separated and characterized by reason of their chemical characteristics, good properties, including thermal stability and lower content decomposition products. Fourier Transform Infrared Spectroscopy (FT-IR) is one of the techniques that has been largely applied for materials identification and characterization. However, due to the wide variety of products in the extracts of rubbers and the small concentration of additives contained, it is necessary to perform a separation step by thin layer chromatography (TLC). The association of Infrared Spectroscopy Technique (TLC/IR)[10,11] is required for chemical structure characterization, although it is hardly used in the literature. In the TLC/ IR technique, using the KBr pyramids, the extract is dissolved in the solvent used for the extraction and applied on a suitable silica plate, which is developed in a closed chromatographic vat using a mobile phase (eluent). The plate is dried and the spots revealed, with specific product. The spots are grated and placed in a tube containing the KBr pyramids. A small amount of solvent used in the extraction is added to conduct the sample to the top of the pyramid. After solvent evaporation, this top is removed, scraped and pressed as a KBr disc for IR analysis. This technique was started in our laboratories, in the Brazilian Institute, Instituto de Aeronáutica e Espaço (IAE), in 1996[10] and it was used for additives analysis, in rubbers [12] as in paints, with positive results[11]. Chauveau et al.[13] separated and identified several vulcanization agents (CBS, MBT, TMTM, DPG, MBT and TMTM) and antioxidants (IPPD and 6PPD) present in 206 206/214
Acronym / nomenclature ZDBC (Dibutyldithiocarbamate Zinc) ZDEC (zinc diethyldithiocarbamate) ZDMC (zinc dimethyldithiocarbamate) DPG (N, N’-diphenyl guanidine) DOTG (Diortotolylguanidine) DTDM (4,4’-dithiomorpholine) ETU (2-mercaptoimidazoline) / (ethylene thiourea) DCP (Dicumyl peroxide)
hospital rubbers (synthetic polyisoprene-isoprene - IR and copolymer of butadiene and styrene - SBR), by means of the TLC technique. In parallel, the identification of additives by GLC coupled to a mass spectrometer (MS) method was made to confirm the presence or absence of additives. The samples were extracted in acetone and two types of eluents were used: toluene/ethyl acetate/ammonia (100/5/1) to extract CBS, MBT, IPPD, 6PPD, TMTM and toluene/acetone/ammonia (45/65/1) to extract DPG, MBT, TMTM. UV fluorescent light was applied to reveal the eluted deposits. It was demonstrated that the TLC method was more efficient in the identification of additives, since it presented greater thermal stability, without degradation of it, during the extraction with acetone at 66°C. Despite of it, most additives were separated by TLC, even in small amounts of formulations analyzed. and by MS, through fragmented products, there were difficulties in separating some components, such as TMTM and CBS, under the conditions used. Other recent studies, in IAE laboratories, were carried out to identify additives in polymers using the TLC/IR technique (off-line) and selective extraction. Among them is the Rodrigues et al.[11] publishing, where a painting formulation containing polyurethane (PU) and nitrocellulose (NC) was evaluated. In this study, three eluent systems were used: ethyl acetate/ethyl alcohol (70/35/30), pure toluene and toluene/ethyl acetate (70/30), applying Gibbs developer. Among the four additives of the formulation, it was possible to characterize two of them (ATBC plasticizer and oleamide slider) by UATR. Damazio et al. [12] applied the same technique, TLC/IR technique (off-line), for the analysis of MBT, TMQ, TMTM and TMTD additives, in ethylene propylene diene monomer terpolymer based rubber (EPDM). Two types of EPDM were evaluated with two different kinds of eluent system: an eluent system with hexane, diethyl ether and acetic acid - 70: 30: 5, and Gibbs developer. The formulation studied contained more than one additive, and thus generated band overlap, but the characteristic absorptions of sulfide additives were revealed by UATR, even though they were in a lower proportion. Other researches have been done to study rubber additives. For example, the potential of FT-IR analysis of gaseous pyrolyzates (PY-G/FT-IR) for characterization of EPDM additives has also been evaluated[14] TMTM, TMTD, and MBT were employed in this study. Results demonstrated that the PY-G/FT-IR technique can identify additives containing sulfur in concentrations as low as 1.4 phr (1.26%) in EPDM. Polímeros, 28(3), 205-214, 2018
Characterization of additives in NR formulations by TLC-IR (UATR) However, the method showed some limitation to detect TMTM and TMTD due to overlapping and to similarities of their PY-G/FT-IR spectra, which could not be distinguished from each other. Although the quoted papers presented favorable results for rubber additives, formulations of NR containing peroxides were not analyzed in comparison with others containing sulfur accelerator or vulcanizing agent systems. There are also limitations for detecting of some sulfur additives. Then, in this paper, the applicability of the TLC/UATR technique was evaluated to characterization of TMTD, CBS and DCP, which are frequently found in NR and EPDM rubbers formulations, used in the industries mentioned earlier. In short, the interest of Automotive and Aerospace Companies in methodology development for problem solving and polymer article improvement is the same.
2.2 Rubbers preparation The raw material was weighed on a precision scale and all components, except for accelerators (TMTD and CBS) and vulcanizing agent (DCP and Sulfur), were mixed in a laboratory banbury 2 liters for 240 sec., with a pylon pressure of 4 kgf/cm2, rotation of 70 rpm and initial temperature of 40°C, to produce the masterbatch. Then it was processed in a roller form six times in an open mill (laboratory cylinder), with rotation of 40 rpm. Later, the masterbatch was accelerated in banbury, with the rest of the weighted components (TMTD, CBS or DCP and sulfur) for 120 seconds, with 4 kgf/cm2 pylon pressure, rotation of 70 rpm and initial temperature of 40°C. Eventually, the rubber was homogenized in roller form six times in an open mill (laboratory cylinder) with a rotation of 40 rpm and removed in blanked.
2.1 Samples
The rheometer curve interferes in the vulcanization temperature setting of a rubber part. High temperatures can promote a reversal process, where bonds breakage occurs, and consequently the degradation of the polymer[1].
The sample of NR was kindly provided by Tenneco Automotive. In Table 2, are presented the formulations developed for NR containing CBS or TMTD or DCP, named, respectively, NR (CBS), NR (TMTD) and NR (DCP).
However, the vulcanization times obtained in rheometric tests should be performed at the same temperature as the part will be processed. The value of T90 should be the time to be used when the material thickness is between 1.5 and 2.5 mm[1].
2. Materials and Methods
Table 2. NR System (phr). NR
NR
NR
(CBS)
(TMTD)
(PEROXIDE)
Natural Rubber (NR)
100
100
100
Naphthenic oil
7.7
5.4
5.4
4.8
4.8
4.8
1.6
1.6
1.6
Components
Functional Group / Structural Formula
Zinc oxide
ZnO
Stearic acid Carbon Black N550 Sulfur
C
24
53
53
S
2.1
3.2
---
TMTD
---
1.1
---
CBS
1.1
---
---
DCP
---
---
6.4
Polímeros, 28(3), 205-214, 2018
207/214 207
Murakami, L. M. S., Azevedo, J. B., Diniz, M. F., Silva, L. M., & Dutra, R. C. L. In this current paper, the compounds vulcanization times were determined from the rheometric curves using a rheometer, from Alpha Technologies, model MDR 2000, at the same vulcanization temperature (T90@160ºC), and the specimens thickness were 2.5 mm[1]. For the preparation of vulcanized rubbers slabs, a hydraulic press with vulcanization time of 6 min., temperature in the plateaus of 160°C and 150 kgf/cm2 of closing pressure was used.
2.3 Characterization equipment/conditions For the TLC analysis, Merck glass chromatography plates, measuring 20 × 20 cm and covered with Silica Gel D60 and glass vat were applied. In the FT-IR analysis, a spectrometer FT-IR Spectrum One PerkinElmer (resolution 4 cm-1, gain 1, 4000 to 400 cm-1, 20 scans) was used. FT-IR spectra were obtained, by reflection technique, using the UATR accessory. It was used 20 scans for UATR analysis, based on other papers that they have successfully published for paint [11], rubber [12] and oil analysis[15]. This scan number is a suitable to smooth noises in the FT-IR reflection techniques, such as UATR, diffuse reflectance (DRIFT) and attenuated total reflection (ATR).
2.4 Methodology 2.4.1 Extraction and analysis by Infrared spectroscopy (IR) of rubbers and additives Vulcanized rubber slabs were cut into small pieces, in sizes of approximately 3.0 x 3.0 x 3.0 mm and placed for extraction in acetone in Soxhlet extractor. The rubber
samples were prepared by pyrolysis (thermal degradation) and analyzed as liquids by transmission, by IR. The extracts containing the additives were analyzed by reflection, UATR. 2.4.2 TLC analysis The TLC plates were labeled and identified with pencils and placed in an oven for 15 minutes at 105°C for activation, to remove moisture from the silica. With the aid of a micro syringe, approximately 15 μL of extract and pure additives were deposited, side by side, for use as a color reference and retention factor (Rf) on the TLC plate. The additives (TMTD, CBS and DCP), which were powders, were previously solubilized in acetone. The TLC plate was placed in the glass vat and the eluent was added until it reached the 1 cm mark. The run time of the eluent on the plate was timed until it reached the upper marking of 2 cm. Rf is the ratio of the distance traveled by the sample to the distance traveled by the eluent in the system (Figure 1). After running the eluent, excess solvent from the plate was evaporated at room temperature in the exhaust hood, then eliminated in an oven for 15 minutes at 105°C. Four eluent systems were used, based on literature data: Toluene[16]; Toluene/Acetone (45/65) and Toluene/Ethyl Acetate (100/5)[13], but in the last two cases ammonia was removed, due it is a product considered to be hazardous to health and the fluorescent developer replaced by the Gibbs reagent. The Toluene/Acetone system (65/45) was also used. After oven dried and cooled to room temperature, the plates were revealed with a solution of 0.3g of the Gibbs reagent (2,6-dichloro-p-benzoquinone-4-chloroimine) in 30ml of ethyl alcohol. The Rf values and developed colors were noted for each eluted spot which was separated on the TLC plate from extract and pure additives. 2.4.3 TLC/UATR analysis The silica that contained the eluted deposits was scraped washed with 10 ml of acetone and filtered through filter paper on a watch glass. After the eluent evaporation, at room temperature to avoid degrade the material, the samples were analyzed by UATR (Figure 2).
3. Results and Discussion 3.1 UATR analysis of NR extracts, containing different additives
Figure 1. Preparation of the TLC plate for the deposition of the extracts and the RF marking.
For the initial evaluation of the additives, extraction was carried out with the appropriate solvent, acetone[10]. The extracts were analyzed by UATR as casting films, to avoid the appearance of KBr moisture bands around 3300 and 1640 cm-1, as a result of solid sample pellets
Figure 2. Scraping process of the separated spots by the TLC plate and prepared for UATR analysis. 208 208/214
Polímeros, 28(3), 205-214, 2018
Characterization of additives in NR formulations by TLC-IR (UATR) preparation, for transmission analysis, that could interfere on the observation of NH bands of CBS accelerator. In Figure 3, it is included the UATR spectra of extracts, in acetone, from the vulcanized NR rubbers samples, compared to additives spectra, taken as references. The objective is verifying if by analyzing of the spectra of NR extracts, it is possible to indicate the different additives presence. Although the extract spectrum of a rubber shows absorptions of all soluble additives in the used solvent, meaning there are overlapping bands of other formulation additives, it is possible to make some considerations: • the spectra of Figures 3a, 3c and 3e are different, confirming that the formulations are not the same in terms of organic additives; • a small number of the major CBS bands, approximately (cm-1), assigned to the following functional groups[17,18]: 3200 (NH) (very low-intensity band, better viewed when the extract spectrum was analyzed separately), 750 (ortho-substituted aromatic ring) and 730 (CH2), is present in the extract spectrum of NR (CBS). However, bands that should appear around 1500, related to benzothiazole[18], were not visualized. There is also overlapping, in other regions, of naphthenic plasticizer bands, that have aliphatic CH groups, which absorb at 3000-2900, 1460-1400 and 700-750, and which is in greater proportion, relative to the formulation additives (see Table 2); • a small number of bands (cm-1) around 1240 (C=S, C-S), 1140 (C-N), 560 (S-S) characterizes the presence of TMTD[12,17] in the NR (TMTD) extract spectrum. Despite of a greater similarity between NR extract (TMTD) and TMTD spectra that was observed in the fingerprint region, there were overlaps relative to CH3 groups bands between 3000-2900, 1400-1350, present in other additives; • a small number of bands (cm-1) around 1250-1100, 980-870, of variable intensity, weak to strong, assigned to the C-O (stronger) and O-O (weaker) groups[18], may serve as the basis for characterization of DCP presence. However, this typical intensity variation[12] makes their characterization difficult compared to other
additives. Due to other additives band interference in the formulation, it was decided to try to separate the additives, by TLC and TLC/UATR, from the others. It does not mean that the analysis of rubber extracts was already the first step in the methodology to evaluate the indication of different additives presence.
3.2 TLC analysis of NR extracts and the different additives Four eluent systems were used: A) toluene, already used in research on the antioxidant N-phenyl-N’-isopropyl-p-phe nylenediamine (IPPD) in NR[10], B) toluene/acetone (65/45), C) toluene/acetone (45/65) and D) toluene/ethyl acetate (100/5); B, C and D systems being used in our laboratories and in research of different accelerators and antioxidants of NR or poly (cis-isoprene) rubber (IR)[13]. Gibbs reagent, already used in other NR research[10], was the developer.
3.3 NR (CBS) In Table 3, TLC data for NR (CBS) rubber are included, using toluene, toluene / acetone (65/45), toluene / acetone (45/65), toluene/ethyl acetate (100/5), as eluents, and Gibbs reagent as developer, which will respectively be referred to as: NR(CBS) – Toluene/Gibbs, NR(CBS) – Toluene/acetone (65/45)/Gibbs, NR(CBS) – Toluene/acetone (45/65) / Gibbs and NR(CBS) – Toluene / ethyl acetate (100/5)/Gibbs. In relation to TLC analysis, only the Toluene/Acetone (45/65) / Gibbs system indicated a possible CBS separation. The other systems did not show efficiency for this procedure, since the colors of deposits and/or RF were different from those observed for respective accelerator. It is apparently caused by the greater plasticizer content and the presence of different products concentrations in the formulation[10] (Table 2, already shown).
3.4 NR (TMTD) In Table 4, is included TLC data for NR(TMTD) rubber, using toluene, toluene/acetone (65/45), toluene/acetone (45/65), and toluene / ethyl acetate (100/5) as eluents and Gibbs reagent as developer, that will be, respectively, referred to as systems: NR (TMTD) -Toluene / Gibbs, NR (TMTD) -
Figure 3. UATR spectra of the acetone extracts of the vulcanized NR, containing additives and references additives: (A) NR (CBS); (B) CBS; (C) NR (TMTD); (D) TMTD; (E) NR (DCP); (F) DCP. Polímeros, 28(3), 205-214, 2018
209/214 209
Murakami, L. M. S., Azevedo, J. B., Diniz, M. F., Silva, L. M., & Dutra, R. C. L. Table 3. TLC data for NR(CBS) Systems. Sample Eluent/Developer Running time (h: min: sec) NR (CBS) Toluene/Gibbs 1:04:13 NR (CBS) Toluene/Acetone (65/45)/Gibbs 0:53:25 NR (CBS) Toluene/Acetone (45/65)/Gibbs 0:48:06 NR (CBS) Toluene/Ethyl Acetate (100/5)/Gibbs 1:02:00
Eluted deposit - Color of eluted deposit
Distance traveled by the eluent (cm)
Rf
CBS extract – Purple NR (CBS) extract 1°Rf – Grey NR (CBS) extract 2°Rf - Light yellow NR (CBS) extract 3°Rf - Yellow CBS extract – Purple NR (CBS) extract 1°Rf - Yellow
8.6 2.4 3.0 15.0 14.6 14.0
0.54 0.15 0.19 0.94 0.91 0.88
CBS extract - Yellow NR (CBS) extract 1°Rf - Light yellow
15.6 15.5
0.98 0.97
CBS extract - Purple NR (CBS) extract 1°Rf - Light yellow NR (CBS) extract 2°Rf - Light brown NR (CBS) extract 3°Rf - Strong yellow NR (CBS) extract 4°Rf - Light grey
10.1 2.4 7.0 7.6 14.5
0.67 0.15 0.44 0.48 0.91
Eluted deposit (Color of eluted deposit)
Distance traveled by the eluent (cm)
Rf
TMTD extract - Dark yellow NR (TMTD) extract 1°Rf - Light yellow NR (TMTD) extract 2°Rf - Light purple
3.6 6.6 8.7
0.23 0.41 0.54
TMTD extract - Dark purple NR (TMTD) extract 1°Rf - Light purple
13.0 14.0
0.81 0.87
TMTD extract - Yellow NR (TMTD) extract 1°Rf - Light yellow NR (TMTD) extract 2°Rf - Light yellow
14.3 15.0 15.9
0.89 0.94 0.99
TMTD extract - Dark purple NR (TMTD) extract 1°Rf - Light purple NR (TMTD) extract 2°Rf - Light purple
8.9 8.9 12.0
0.56 0.56 0.75
Table 4. TLC data for the NR (TMTD) system. Sample Eluent/Developer Running time (h: min: sec) NR (TMTD) Toluene / Gibbs 1:12:44 NR (TMTD) Toluene/Acetone (65/45)/Gibbs 0:46:45 NR (TMTD) Toluene/Acetone (45/65)/Gibbs 0:48:06 NR (TMTD) Toluene/Ethyl Acetate (100/5)/Gibbs 1:00:00
Toluene/acetone (65/45)/Gibbs, NR (TMTD) - Toluene/acetone (45/65)/Gibbs and NR (TMTD) - Toluene/ethyl acetate (100/5)/Gibbs. TLC analysis suggested that is possible to separate this accelerator, especially, by the toluene/ethyl acetate (100/5)/Gibbs (1°Rf) system.
3.5 NR (DCP) In Table 5, are included TLC data for NR(DCP) rubber, using toluene, toluene/acetone (65/45), toluene/acetone (45/65), and toluene/ethyl acetate (100/5), as eluents, and Gibbs reagent as developer, that will be referred to, respectively, as systems: NR (DCP) Toluene/Gibbs, NR (DCP) - Toluene/acetone (65/45)/Gibbs, NR (DCP) - Toluene/acetone (45/65)/Gibbs By the TLC analysis, all the eluent systems showed RF closer to those observed for DCP, although some colors presented differences, possibly because of plasticizer presence, which suggests that the separation is feasible to the accelerator by these eluent systems, with higher accuracy for the Toluene/Acetone (45/65)/Gibbs and NR (DCP) - Toluene/ethyl acetate/Gibbs. 210 210/214
4. TLC/UATR analysis 4.1 NR(CBS) By the IR spectra analysis, the separation of CBS presumably did not occur, due to the greater presence of plasticizer (see Table 2). There are only indications of separation beside the evaluation of the spectrum referring to the extract in toluene/acetone (45/65), through bands in (cm-1), most likely assigned[17] to the groups: 3329 (NH), 2922, 2853 and 722 (CH2) and 755 (C-H ortho substitution) (Figure 4). An interesting fact is under conditions (toluene/Gibbs) for similar formulations of NR [6], the rubber extract usually produces an eluted deposit, in higher RF and yellow color, which is essentially the plasticizer. Thus, the analysis of the 3°RF deposition in toluene was made to characterize the naphthenic plasticizer in the formulation, succeeding in the applied methodology (Figure 5), since that the separated product spectrum (3°Rf) showed the same absorptions of referred additive. Polímeros, 28(3), 205-214, 2018
Characterization of additives in NR formulations by TLC-IR (UATR) Table 5. TLC data for the NR (DCP) system. Sample - Eluent/Developer Running time (h: min: sec) TLC Plate NR (DCP) Toluene / Gibbs 1:04:13 NR (DCP) Toluene/Acetone (65/45)/Gibbs 0:53:45 NR (DCP) Toluene/Acetone (45/65)/Gibbs 0:48:06 NR (DCP) Toluene/Ethyl Acetate (100/5)/Gibbs 0:54:51
Eluted deposit - Color of eluted deposit
Distance traveled by the eluent (cm)
Rf
DCP extract - Purple NR(DCP) extract 1°Rf - Yellow
14.6 14.7
0.91 0.92
DCP extract - White NR(DCP) extract 1°Rf - Yellow
15.0 14.4
0.94 0.90
DCP extract NR(DCP) extract 1° Rf - Light yellow
16.0
1.00 1.00
DCP extract - White NR(DCP) extract 1°Rf – White NR(DCP) extract 2°Rf - Light yellow NR(DCP) extract 3°Rf - Yellow
12.9 2.4 4.7 12.4
0.81 0.15 0.29 0.78
Figure 4. UATR spectra (after TLC, in toluene/acetone (45/65): (a) Eluted deposit of CBS (reference); (b) eluted deposit of vulcanized NR (CBS) (1º. Rf).
4.2 NR (TMTD) Despite the fact there would be loss of material and spectral resolution in the TLC/IR technique, perhaps caused by the conditions applied, mainly due to transmission/pellet technique[8] application, Figure 6 shows that in the UATR spectrum of extract, in toluene, of TMTD after TLC, there are very similar absorptions to the reference TMTD UATR spectrum, also to the UATR spectrum of the extract, in toluene/acetone (45/65), which does not happen with the TMTD spectra, obtained in other eluent/developer systems. The Figure suggests that in the analysis of NR spectra, in TLC/UATR technique, the reference spectrum of analyzed accelerator should be used, under the same conditions as the TLC analysis. By TLC analysis, toluene/ethyl acetate (100:5)/Gibbs that would be used to separate the TMTD, and this can be better visualized by comparing NR(TMTD) (1°Rf) and TMTD spectra, after TLC, in this system (Figure 7), and with discussion of the spectra being made according to data found in the literature[12]. Polímeros, 28(3), 205-214, 2018
In recent paper[12], it was evaluated the additives separation, including TMTD, in ethylene propylene diene monomer terpolymer (EPDM), with other eluent systems. It was mentioned that despite the fact there was some overlap of bands, it was possible to indicate in spectra obtained after TLC, in conditions (70% of hexane / 30% of ethyl ether and 5% of acetic acid), two absorptions in 1240 cm-1 (C=S, C-S) and 560 cm-1 (S-S) of TMTD, also associated to the bands shape of sulfur compounds, even in a small proportion in the formulation (also around 1phr, as in current paper). In Figure 7, albeit there is also overlap, notably in the region of 3000-2800 and 1400-1460 cm-1, of CH2 and CH3 groups, the C-N band, around 1140 cm-1, common bands to other additives, the absorptions of geminated methyl group, between 1380-1350 cm-1 and around 560 cm-1 (S-S), of TMTD[12,17], as well as their shape, are better visualized in the rubber extract spectra, in comparison with the TMTD spectrum, after TLC, suggesting that the used conditions, toluene / ethyl acetate (100:5) / Gibbs, are suitable for the NR rubber, for this purpose. In addition, the TLC analysis 211/214 211
Murakami, L. M. S., Azevedo, J. B., Diniz, M. F., Silva, L. M., & Dutra, R. C. L.
Figure 5. UATR spectra (after TLC): (a) Eluted deposit, in toluene, of vulcanized NR (CBS) (3º Rf); (b) naphthenic oil.
Figure 6. UATR spectra: (a) Eluted deposit of TMTD, after TLC, in toluene/acetone (45/65); (b) Eluted deposit of TMTD, after TLC, in toluene; (c) TMTD, as received; (d) Eluted deposit of TMTD, after TLC, in toluene/acetone (65/45); (e) Eluted deposit of TMTD, after TLC, in toluene/ethyl acetate (100/5).
Figure 7. UATR (after TLC) spectra: (a) eluted deposit of TMTD, in toluene/ethyl acetate (100/5); (b) eluted deposit of vulcanized NR (TMTD) (1º. Rf), in toluene/ethyl acetate.
showed Rf of the NR(TMTD) rubber extract equal to that observed for the TMTD (reference), indicating the TLC/UATR coupling was effective, as an indication of this accelerator in the formulation. 212 212/214
4.3 NR (DCP) By the TLC analysis, all eluent systems used were efficient for the separation of DCP, with better result for Toluene / Ethyl Acetate (100/5) / Gibbs, 3°Rf. This is also Polímeros, 28(3), 205-214, 2018
Characterization of additives in NR formulations by TLC-IR (UATR)
Figure 8. UATR spectra: (a) eluted deposit of DCP, in toluene; (b) eluted deposit of vulcanized NR (DCP), in toluene; (c) eluted deposit of DCP, in toluene/acetone (65/45); (d) eluted deposit of vulcanized NR (DCP), in toluene/acetone (65/45); (e) eluted deposit of DCP, in toluene/ethyl acetate (100/5); (f) eluted deposit of vulcanized NR (DCP) (3°Rf), in toluene/ethyl acetate (100/5).
demonstrated by the UATR analysis, Figure 8. In this figure, the similarity of the spectra can be noted as well and, with the bands (cm-1) being around 1250-1100, 980-870, of variable intensity, weak to strong, assigned to the C-O (stronger) and O-O (weaker) groups[12], that may fit as reference in the characterization of the DCP presence, which can be better observed, meeting the TLC data. After these results, the TLC-IR(UATR) analysis applied to separate and characterize NR additives such as CBS, TMTD and DCP, was considered available to rubber quality control in laboratories because is faster and simpler than methodologies involving other coupling types such as TG/IR[19]. In this study[19], although good results were reached for sulfur additives such as MBT and TMTM, they were characterized by a complex methodology of its degradation products. Plasticizer was not detected, whereas in the TLC/UATR showed in this current paper, naphthenic oil was separated and characterized. Degradation studies are not necessary too. However, such methods can be considered complementary, if it necessary wide characterization of rubber additives.
5. Conclusion UATR analysis of acetone NR extracts showed some differences in the formulations, the TMTD presence were better demonstrated. However, there are overlaps of bands of other additives. Therefore, it is necessary a separation/identification step, such as TLC/IR. By TLC and TLC/UATR analysis, it was only evidenced the CBS separation/identification using the toluene/acetone system (45/65). By TLC/UATR analysis, it was possible to characterize the naphthenic oil presence, with the system (toluene/Gibbs). Perhaps, this separation/identification has been facilitated by this additive, paraffinic oil, being present in a higher proportion in the formulation (7.7 phr) than the other accelerators like CBS and TMTD (5.4 phr) in their specific formulations. Polímeros, 28(3), 205-214, 2018
TLC analysis suggested that is achievable to separate TMTD, specifically by the toluene/ethyl acetate (100/5) / Gibbs (1°Rf) system. TLC/UATR analysis confirmed this indication, despite of some bands overlaps, which was once registered in EPDM rubber formulations, apparently because of the small TMTD amount in referred formulations. By the TLC/UATR analysis, all eluent systems used were efficient for the separation of DCP, predominantly the Toluene/Ethyl Acetate (100/5)/Gibbs, 3°Rf, constituting the best methodology result in terms of characterization (TLC) and identification (UATR). It may be explained by the higher quantity of the additive (6.4 phr) in relation to the others (1.1 phr), in the specific formulations. The decision to use the same curing time for all formulations did not affect the analysis because the results showed the additives separation by TLC and their identification by FT-IR (UATR), by characteristics bands of DCP (Figure 8) and TMTD (Figure 7). The unambiguous separation and identification of CBS may have been impaired due to the higher content of plasticizer in its formulation, which may have been the factor that facilitated the separation and identification of naphthenic oil (Figure 5). In short, it was concluded that the developed TLC/UATR methodology for studying the additives not only has it contributed to the potential of formulations characterization in the automotive industry, but also could be applied in the aerospace area. The reason is related to these compounds that can be present in NR and EPDM formulations. It is a multiplier effect of acquired technical knowledge, being extremely useful to predict specific properties for different applications.
6. Acknowledgements This study was supported in part by the National Senior Professor Program (PVNS) from the Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES). 213/214 213
Murakami, L. M. S., Azevedo, J. B., Diniz, M. F., Silva, L. M., & Dutra, R. C. L.
7. References 1. Ciência e Tecnologia da Borracha. Retrieved from in 2017, March 28, from http://ctborracha.com 2. Mohan, C. H. V. R., Ramanathan, J., Kumar, S., & Gupta, A. V. S. S. K. S. (2011). Characterization of Materials Used in Flex Bearings of Large Solid Rocket Motors. Defence Science Journal, 61(3), 264-269. http://dx.doi.org/10.14429/dsj.61.52. 3. Chemikeys. Retrieved from in 2017, April 18, from http://www. chemkeys.com/blog/wp-content/uploads/2008/09/polimeros. pdf 4. Grinson, E. C. (2010). Borrachas e seus aditivos - Componentes, Influências e Segredos. Porto Alegre: Ed. Letra & Vida. 5. D’Angelo, A. (2008). Peróxidos resistentes ao oxigênio para vulcanização contínua em túnel de ar quente. Revista Borracha Atual, 1-35. Retrieved in 2017, May 25, from Retrieved from http://www.retilox.com.br/astecnicos/download/peroxidos1. pdf 6. Reyneke-Barnard, C. P., Gradwell, M. H. S., & Mcgill, W. J. (2000). N,N’Pentamethylenethiuram disulfide- and N,N´´Pentamethylenethiuram Hexasulfide-accelerated Sulfur Vulcanization. I. Interaction of Curatives in the Absence of Rubber. Journal of Applied Polymer Science, 77(12), 2718-2731. http:// dx.doi.org/10.1002/1097-4628(20000919)77:12<2718::AIDAPP200>3.0.CO;2-E. 7. Joseph, A. M., George, B., Madhusoodanan, K. N., & Alex, R. (2015). Current status of sulphur vulcanization and devulcanization chemistry: Process of vulcanization. Rubber Science, 28(1), 82-121. 8. Charoeythornkhajhornchai, P., Samthong, C., & Somwangthanaroj, A. (2017). Influence of sulfenamide accelerators on cure kinetics and properties of natural rubber foam. Journal of Applied Polymer Science, 134(19), 44822. http://dx.doi.org/10.1002/ app.44822. 9. Kruželák, J., Sýkora, R., & Hudec, I. (2014). Peroxide vulcanization of natural rubber. Part I: effect of temperature and peroxide concentration. Journal of Polymer Engineering, 34(7), 617-624.
214 214/214
10. Dutra, R. C. L. (1996). Aplicação de técnica de TLC-IR em estudos de separação, identificação e quantificação de aditivos em borrachas. Polímeros: Ciência e Tecnologia, 6(2), 26-31. 11. Rodrigues, V. C., Diniz, M. F., Mattos, E. C., & Dutra, R. C. L. (2016). Separação e identificação de aditivos em tinta por TLC-IR/ UATR e extração seletiva. Polímeros: Ciência e Tecnologia, 26(Special Number), 68-74. 12. Damazio, D., Campos, E. A., Diniz, M. F., Mattos, E. C., & Dutra, R. C. L. (2016). TLC/IR (UATR) off-line coupling for the characterization of additives in EPDM rubber compositions. Polímeros: Ciência e Tecnologia, 26(1), 74-80. 13. Chauveau, S., Hamon, M., & Leleu, E. (1991). Separation and identification of various vulcanization agents and antioxidants in two types of rubber by chromatographic and spectrometric methods. Talanta, 38(11), 1279-1283. http:// dx.doi.org/10.1016/0039-9140(91)80106-A. PMid:18965298. 14. Sanches, N. B., Cassu, S. N., Diniz, M. F., & Dutra, R. C. L. (2014). Characterization of additives typically employed in EPDM formulations by using FT-IR of gaseous pyrolyzates. Polímeros: Ciência e Tecnologia, 24(3), 269-275. 15. Mueller, D., Ferrão, M. F., Marder, L., Costa, A. B., & Schneider, R. C. S. (2013). Fourier Transform Infrared Spectroscopy (FTIR) and multivariate analysis for identification of different vegetable oils used in biodiesel production. Sensors, 13(4), 4258-4271. http://dx.doi.org/10.3390/s130404258. PMid:23539030. 16. Wake, W. C., Tidd, B. K., & Loadman, M. J. R. (1983). Analysis of rubber and rubber-like polymer (3rd ed.). New York: Applied Science. 17. Smith, A. L. (1979). Applied infrared spectroscopy. New York: John Wiley & Sons. 18. Wolfang, W. (1987). Tópicos de espectroscopia no infravermelho. São José dos Campos: ITA. 19. Sanches, N. B., Cassu, S. N., Diniz, M. F., & Dutra, R. C. L. (2015). TG/FT-IR characterization of additives typically employed in EPDM formulations. Polímeros: Ciência e Tecnologia, 25(3), 247-255. Received: June 20, 2017 Revised: Sept. 28, 2017 Accepted: Dec. 07, 2017
Polímeros, 28(3), 205-214, 2018
ISSN 1678-5169 (Online)
http://dx.doi.org/10.1590/0104-1428.004616
Effect of disinfection techniques on physical-mechanical properties of a microwave-activated acrylic resin Carmen Beatriz Borges Fortes1, Fabrício Mezzomo Collares1*, Vicente Castelo Branco Leitune1, Priscila Raquel Schiroky1, Stéfani Becker Rodrigues1, Susana Maria Werner Samuel1, Cesar Liberato Petzhold2 and Valter Stefani2 Laboratório de Materiais Dentários – LAMAD, Departamento de Odontologia Conservadora – DOC, Faculdade de Odontologia, Universidade Federal do Rio Grande do Sul – UFRGS, Porto Alegre, RS, Brasil 2 Departamento de Química Orgânica – DQO, Instituto de Química, Universidade Federal do Rio Grande do Sul – UFRGS, Porto Alegre, RS, Brasil 1
*fabricio.collares@ufrgs.br
Abstract The effects of disinfection by microwave irradiation and immersion in peracetic acid on the physical-mechanical properties of a microwave-activated acrylic resin were evaluated. Specimens of acrylic resin were divided into a control group (specimens not disinfected) and 2 test groups subjected to one disinfection method: microwave irradiation at 850 W for 1 minute or immersion in 50 mL of 0.2% peracetic acid for 5 minutes. Specimens were submitted to Knoop hardness, flexural strength, flexural modulus, Izod impact, water sorption and solubility, glass transition temperature, and degree of conversion tests. Microwave disinfection significantly increased the mean Knoop hardness, Izod impact strength, water sorption, water solubility and glass transition temperature, whereas the flexural properties remained unaffected. Microwave disinfection increased the degree of conversion. Peracetic acid disinfection showed no changes in any properties. Both disinfection techniques did not adversely affect the evaluated properties. Keywords: acrylic resins, microwave disinfection, peracetic acid.
1. Introduction Complete dentures are colonized and infected by microorganisms, forming a denture biofilm and leading to oral fungal infection. Candida albicans is commonly found in the oral cavity at denture base, changing the oral environment by increasing part of the oral microflora[1]. The presence of Candida albicans in denture biofilm is a major factor in the multifactorial etiology of denture-related stomatitis, a common opportunistic infection found in denture wearers[2,3]. Poor denture hygiene is associated with denture-related stomatitis, as it could lead to fast establishment of a biofilm[4,5]. This biofilm could be removed by mechanical methods (brushing or ultrasound), chemical methods (soaking in chemicals), using a microwave oven or an association of different methods[6]. However, a mechanical biofilm removal requires a degree of manual dexterity that is often lacking among older people[5]. In addition, complete dentures contaminated by Candida albicans are a potential source of infection and re-infection of the oral soft tissues, and its disinfection has been recommended as an adjuvant in the treatment of stomatitis and as an essential procedure for maintaining a healthy oral mucosa[7,8]. Many protocols for denture disinfection have been proposed, including immersion in chemical solutions such as glutaraldehyde[9], sodium perborate[10], sodium hypochlorite[11,12], chlorhexidine digluconate[7], peracetic acid[11,12], and microwave irradiation[7,13]. Ideally, the choice of the disinfectant method should be made with regard to
Polímeros, 28(3), 215-219, 2018
its effectiveness in inactivating microorganisms without detrimental effects on the acrylic resins. Originally used to polymerize microwave-activated acrylic resins[14,15], microwave irradiation has been used for post-polymerization treatment[16] and has been shown to disinfect effectively[7,17,18]. Different time (3, 5, and 6 minutes) and power (650 and 720 W) settings of microwave irradiation have been proposed in the literature for denture disinfection[10,19-22]. Despite its effectiveness in disinfection, these protocols have reached contradictory findings, including deleterious effects on some physical and mechanical properties due to the material heating during the irradiation, which could affect the polymer structure[19-21]. Previous studies suggested that the time the acrylic resins are subjected to heat during disinfection could be reduced in order to produce adequate disinfection without any adverse effects[7,23,24]. Microwave exposure time of 1 minute has been found to be effective for disinfection against Candida albicans[17,18]. However, only the effects on the hardness and flexural strength were verified with 1 minute of microwave irradiation exposure at high power[22]. Peracetic acid (C2H4O3) has been suggested to be used for acrylic resins disinfection[11,12,25], acting even at low concentrations with a broad antimicrobial spectrum and producing no harmful by products[25,26]. The disinfection efficacy of immersion in 0.2% peracetic acid-based disinfectant has been shown in the literature, including against
215/219 215
O O O O O O O O O O O O O O O O
Fortes, C. B. B., Collares, F. M., Leitune, V. C. B., Schiroky, P. R., Rodrigues, S. B., Samuel, S. M. W., Petzhold, C. L., & Stefani, V. Candida albicans[17,25]. Likewise, immersion in a chemical solution may cause water sorption and polymer solubility, altering the polymer structure[9]. Despite color stability and surface roughness, its effect on the physical-mechanical properties of microwave-activated denture base acrylic resins have not been evaluated[11,12]. The aim of this study was to investigate the effects of two methods of disinfection, microwave irradiation at 840 W for 1 minute and immersion in 0.2% peracetic acid for 5 minutes, on the Knoop hardness, flexural strength, flexural modulus, Izod impact, water sorption and solubility, glass transition temperature, and degree of conversion of one type of microwave-activated denture base acrylic resin. The null hypothesis was that microwave irradiation and peracetic acid disinfection protocols would not detrimentally affect the properties of the material.
2. Materials and Methods The material selected for this study was a microwave-activated denture base acrylic resin (powder: PMMA; liquid: MMA/EDMA) (VIPI WAVE; Dental VIPI Ltda., São Paulo, Brazil). Rectangular (64.00 × 10.00 × 3.00 ± 0.5 mm) and disc-shaped (50.00 × 0.5 ± 0.5 mm) test specimens were produced in casts prepared by the investment of stainless steel casts in addition silicone, further supported by a type II dental die within the flask. After dental die setting (2 hours), the acrylic resin was prepared with a powder-liquid ratio of 2:1 by weight. The monomer and then the powder were placed in a glass mixing vessel and hand mixed with a spatula until homogenization. When the plastic phase was reached, the acrylic resin was inserted into the casts. The flask was packed and pressed with a 500 kg load and then instantly opened to remove the excess resin. Afterwards, it was kept under 1000-kg pressure for 30 minutes. The specimens were submitted to a microwave polymerization cycle at 140 W for 20 minutes plus 5 minutes at 420 W. After polymerization and flask cooling for 4 hours, the specimens were removed and any excess was trimmed with a tungsten bur and an aluminum oxide stone bur, as well as polished with progressively finer grades (280, 400, 600, 1000) of silicon carbide sandpapers. The accuracy of the specimens’ dimensions was verified with a digital caliper. The final polishing was reached by using a polishing machine, pumice, and calcium carbonate, resulting in a smooth and bright surface. The rectangular and disc-shaped specimens were randomly divided into one control group and two experimental groups. In the control group (CG), the specimens were not disinfected. The specimens from the microwave group (MW) were immersed in 50 mL of deionized water and irradiated at 840 W for 1 minute in a microwave oven (MB-315 Ml Intellowave; LG Electronics, Manaus, Brazil) to simulate the disinfection. The specimens from the peracetic acid group (PA) were immersed in 50 mL of 0.2% peracetic acid (Sterilife; Lifemed Produtos Médicos Comércio Ltda., São Paulo, Brazil) for 5 minutes to simulate the disinfection. 216 216/219
2.1 Knoop hardness Knoop hardness was measured by using a microhardness tester (MICROMET; Bueller, Dusseldorf, Germany). Indentations were made under a 25-g load for 10 seconds. Five indentations were made on each disc-shaped specimen (n=5), one in the center and four 100 µm from the center. The average of the five indentations was considered to be the specimen’s hardness.
2.2 Flexural strength and flexural modulus The flexural strength and flexural modulus were tested according to ISO 1567[27]. The rectangular specimens (n=10) were stored in distilled water at 37 °C for 50 ± 2 hours. The three-point bending test was performed with an increasing load from 0 N, at a crosshead speed of 5 mm/min (± 1 mm/min).
2.3 Izod impact strength Thirty rectangular specimens (n=10) were submitted to an impact strength test in the Izod configuration, according to the specifications of ASTM D256 – Izod impact modified, because the specimens were not notched[28]. An impact tester (EMIC AIC-1; Equipamentos e Sistemas de Ensaio Ltda., São José dos Pinhais, Brazil) was used to measure the impact energy required to fracture the specimens.
2.4 Water sorption and solubility Fifteen disc-shaped specimens (n=5) were used to determine the water sorption and solubility according to the method described in ISO 1567[27]. Disc-shaped specimens were conditioned to a constant mass in a desiccator placed in an oven at 37 °C for 24 hours. The specimens were weighed and reweighed until the weight loss was not greater than 0.0002 g in any 24-hour period. They were immersed in 50 mL of deionized water at 37 °C for 7 days. Afterwards, the specimens were removed from the water, dried with a paper towel, weighed, and reconditioned to a constant mass in the desiccator.
2.5 Glass transition temperature (Tg) The glass transition temperature was assessed by using the differential scanning calorimetry (DSC) method with a calorimeter (DSC-4; Perkin Elmer, Beaconsfield, England). The specimens (n=3), weighing approximately 10 mg, were contained in aluminum pans and subjected to a temperature range of 50 °C to 90 °C at a heating rate of 10 °C or 20 °C per minute. The glass transition temperature was determined in the second heating cycle.
2.6 Degree of Conversion (DC) The degree of conversion was measured by micro-Raman spectroscopy, one specimen of each group was used (n=1). The spectrum peaks of the double bonds between the unreacted carbon atoms (C=C) around 1650 cm−1 and the C=O bonds around 1750 cm−1 were analyzed. The percentage of double bonds was determined based on the peak areas in the monomer and polymer spectra. Polímeros, 28(3), 215-219, 2018
Effect of disinfection techniques on physical-mechanical properties of a microwave-activated acrylic resin Table 1. Mean and standard deviation values of evaluated properties in control, microwave (MW) and peracetic acid (PA) groups. Properties Knoop hardness (KHN) Flexural strength (MPa) Flexural modulus (MPa) Izod impact strength (J/m) Water sorption (µg/mm3) Water solubility (µg/mm3) Glass transition temperature (°C)
Control 21 ± 1 b 93 ± 10 a 2551 ± 63 a 98 ± 15 b 25 ± 2 b 0.8 ± 0.1 b 103 ± 2 b
MW 24 ± 1 a 93 ± 10 a 2555 ± 68 a 129 ± 15 a 8±1a 0.2 ± 0.1 a 109 ± 2 a
PA 21 ± 1 b 96 ± 11 a 2546 ± 64 a 98 ± 11 b 25 ± 2 b 0.8 ± 0.1 b 103 ± 2 b
F 15.000 0.28 0.0481 16.830 160.556 60.000 9.000
P <.001 .758 .953 <.001 <.001 <.001 .016
Different letters in the same line denotes statistically significant difference (one-way ANOVA and Tukey test, p<.05). MW, microwave group; PA, peracetic acid group; P, p-value; F, Fisher-Snedecor distribution.
2.7 Statistical analysis The normality of the data was evaluated using the Kolmogorov-Smirnov test. The data were statistically analyzed with one-way ANOVA and the Tukey post hoc test with α=0.05 level of significance. Analyzes were performed on SigmaPlot 12.0 Software (Systat Software Inc., San Jose, USA).
3. Results All data are reported on Table 1. The microwave group (MW) showed the highest values, differing significantly (p<.05) from the control and peracetic acid (PA) groups for the following properties: Knoop hardness (24 ± 1 KHN), Izod impact strength (129 ± 15 J/m), water sorption (8 ± 1 µg/mm3), water solubility (0.2 ± 0.1 µg/mm3), and glass transition temperature (109 ± 2 °C). There was no significant difference between the control and peracetic acid (PA) groups. For flexural strength and flexural modulus, there were no significant differences among the 3 groups. The microwave group (MW) showed a degree of conversion of 92%, versus 88% for the control group and 87% for the peracetic acid group (PA).
4. Discussion This study examined the effects of microwave irradiation and peracetic acid disinfection protocols on a denture base acrylic resin activated by microwave energy. It is desirable for microwave and chemical disinfection to not affect any physical–mechanical properties of denture base resins. The null hypothesis that microwave irradiation and peracetic acid would not detrimentally affect the properties of the material was accepted. Knoop hardness, impact strength, water sorption, water solubility, glass transition temperature, and degree of conversion were improved by microwave irradiation, while the peracetic acid disinfection did not affect any of the properties. Also, all of the groups fulfilled the requirements regarding water sorption (<32 mg/mm3), water solubility (<1.6 mg/mm3), flexural strength (>65 MPa), and flexural modulus (>2500 MPa), according to ISO 1567[27]. The use of microwave irradiation exposure at high power during 1 minute was shown to be effective as a disinfection method for dentures with Candida albicans biofilm[17,18], and three microwave irradiation cycles (850 W/1 minute) were shown to completely sterilize the denture surface[7]. Previous studies have shown contradictory results about the effects Polímeros, 28(3), 215-219, 2018
of disinfection by microwave irradiation on properties of the acrylic resins. Microwave irradiation (650 W/3 minutes) was observed to decrease the Knoop hardness of acrylic resin[19]. In the present study, the Knoop hardness significantly increased after microwave polymerization, as did the Izod impact strength and glass transition temperature. Others suggested that the protocol (720 W/5 minutes) reduced[20] or did not affect (650 W/3 and 5 minutes) the impact strength[19,23]. A protocol of 650 W/3 minutes decreased the glass transition temperature of acrylic resin[21]. In this study, the flexural strength and flexural modulus did not differ significantly in the microwave-disinfected specimens, compared with the control specimens. Previous studies (650 W/3 minutes, 650 W/1 minute, and 650 W/5 minutes) corroborate the flexural strength results[19,22,23]. With a protocol of 720 W/5 minutes, another study showed a decrease in the flexural modulus[20]. The water sorption and solubility of complete denture bases in a previous study (650 W/6 minutes) showed no significant changes[13]. In the present study, both properties significantly decreased after microwave disinfection. Conflicts in the literature may be attributed to the differences in the disinfection time, power of the microwave oven, water volume and types of materials used, leading to contradictory conclusions concerning the safety of microwave disinfection[20,24]. While the microwave irradiation lasted only 1 minute in the present investigation, most of the studies were conducted using at least 3 minutes of exposure and showed deleterious effects on physical-mechanical properties. This could explain the main differences in the results. Reduced microwave irradiation time did not cause adverse effects on acrylic resins, corroborating with preliminary studies[22,24]. During microwave irradiation, the amount of unreacted monomer reduces due to further polymerization[10,18], and the residual monomer released into water increases[21,22]. The heating generated could increase the water sorption, inducing a plasticizing effect that may affect the material[13], besides the plasticizing effect of residual monomers[14]. All of these mechanisms must be considered as factors that can change the physical-mechanical properties of the acrylic resins[21]. It could be assumed that, in this study, further polymerization and release of residual monomer into water may have overcome the detrimental plasticizing effect of water uptake and residual monomers on polymeric chains. The increased degree of conversion for the microwave group (MW) reinforces this assumption, as the supposed lower residual monomer content after the further polymerization 217/219 217
Fortes, C. B. B., Collares, F. M., Leitune, V. C. B., Schiroky, P. R., Rodrigues, S. B., Samuel, S. M. W., Petzhold, C. L., & Stefani, V. is the result of a higher degree of conversion[15]. In addition, a high degree of conversion results in increased glass transition temperature[21]. The effect of 2% peracetic acid disinfection with 30 and 60 minutes of immersion on the color stability and surface roughness of acrylic resins was previously investigated and both properties were altered, but without clinical significance[11]. When a lower peracetic acid concentration (1%) with 30 minutes of immersion was evaluated, it did not affect the surface roughness[12]. The acrylic resin surfaces roughness is an important property, as an increased roughness can promote attachment of microorganisms and the colonization of the surface[4]. In the present study, disinfection with 0.2% peracetic acid for 5 minutes did not affect the evaluated properties. Immersion in disinfectant solutions could affect the physical-mechanical properties due the sorption of the solution and its plasticizing effect into the polymer, and the detrimental effect appears to be time and concentration dependent[29]. The low concentration and the immersion time of only 5 minutes used in this study could explain the findings. Hence, the use of higher concentration or immersion time seems to be not justified, as the immersion in 0.2% peracetic acid-based disinfectant for 5 minutes has been found to be effective against Candida albicans[17]. The results of this study suggest that the evaluated disinfectant protocols of microwave irradiation and immersion in peracetic acid are shown to be promising in disinfecting complete dentures. It is important to remember that the results may only partially predict the clinical performance of complete dentures. Other properties of acrylic resins should be investigated, such as dimensional stability. Additionally, further research evaluating the effects of cumulative disinfections should be performed.
5. Conclusions Disinfection of acrylic resin by microwave irradiation exposure at high power (850 W) for 1 minute and immersion in 0.2% peracetic acid for 5 minutes cause no damage to the physical-mechanical properties of the microwave-activated denture base acrylic resin. These findings indicate that both protocols shown to be safe for disinfection of complete dentures maintaining the physical-mechanical properties of the acrylic resin.
6. References 1. Radford, D. R., Challacombe, S. J., & Walter, J. D. (1999). Denture plaque and adherence of Candida albicans to denturebase materials in vivo and in vitro. Critical Reviews in Oral Biology and Medicine, 10(1), 99-116. http://dx.doi.org/10.11 77/10454411990100010501. PMid:10759429. 2. Barbeau, J., Seguin, J., Goulet, J. P., Koninck, L., Avon, S. L., Lalonde, B., Rompre, P., & Deslauriers, N. (2003). Reassessing the presence of Candida albicans in denture-related stomatitis. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, 95(1), 51-59. http://dx.doi.org/10.1067/ moe.2003.44. PMid:12539027. 3. Ramage, G., Tomsett, K., Wickes, B. L., Lopez-Ribot, J. L., & Redding, S. W. (2004). Denture stomatitis: a role for Candida biofilms. Oral Surgery, Oral Medicine, Oral Pathology, Oral 218 218/219
Radiology, and Endodontology, 98(1), 53-59. http://dx.doi. org/10.1016/j.tripleo.2003.04.002. PMid:15243471. 4. Gendreau, L., & Loewy, Z. G. (2011). Epidemiology and etiology of denture stomatitis. Journal of Prosthodontics, 20(4), 251-260. http://dx.doi.org/10.1111/j.1532-849X.2011.00698.x. PMid:21463383. 5. Kulak-Ozkan, Y., Kazazoglu, E., & Arikan, A. (2002). Oral hygiene habits, denture cleanliness, presence of yeasts and stomatitis in elderly people. Journal of Oral Rehabilitation, 29(3), 300-304. http://dx.doi.org/10.1046/j.1365-2842.2002.00816.x. PMid:11896849. 6. de Souza, R. F., de Freitas Oliveira Paranhos, H., Lovato da Silva, C. H., Abu-Naba’a, L., Fedorowicz, Z., & Gurgan, C. A. (2009). Interventions for cleaning dentures in adults. Cochrane Database of Systematic Reviews, 7(4), CD007395. http://dx.doi. org/10.1002/14651858.CD007395.pub2. PMid:19821412. 7. Banting, D. W., & Hill, S. A. (2001). Microwave disinfection of dentures for the treatment of oral candidiasis. Special Care in Dentistry, 21(1), 4-8. http://dx.doi.org/10.1111/j.1754-4505.2001. tb00216.x. PMid:11795452. 8. Garcia-Cuesta, C., Sarrion-Perez, M. G., & Bagan, J. V. (2014). Current treatment of oral candidiasis: a literature review. Journal of Clinical and Experimental Dentistry, 6(5), e576-e582. http:// dx.doi.org/10.4317/jced.51798. PMid:25674329. 9. Schwindling, F. S., Rammelsberg, P., & Stober, T. (2014). Effect of chemical disinfection on the surface roughness of hard denture base materials: a systematic literature review. The International Journal of Prosthodontics, 27(3), 215-225. http://dx.doi.org/10.11607/ijp.3759. PMid:24905261. 10. Machado, A. L., Breeding, L. C., Vergani, C. E., & da Cruz Perez, L. E. (2009). Hardness and surface roughness of reline and denture base acrylic resins after repeated disinfection procedures. The Journal of Prosthetic Dentistry, 102(2), 115-122. http://dx.doi.org/10.1016/S0022-3913(09)60120-7. PMid:19643225. 11. Fernandes, F. H., Orsi, I. A., & Villabona, C. A. (2013). Effects of the peracetic acid and sodium hypochlorite on the colour stability and surface roughness of the denture base acrylic resins polymerised by microwave and water bath methods. Gerodontology, 30(1), 18-25. http://dx.doi.org/10.1111/j.17412358.2012.00640.x. PMid:22486758. 12. Guiraldo, R. D., Sczepanski, F., Sczepanski, C. R. B., Berger, S. B., Consani, R. L. X., & Gonini-Junior, A. (2014). Effect of sodium hypochlorite and peracetic acid on the surface roughness of acrylic resin polymerized by heated water for short and long cycles. European Journal of Dentistry, 8(4), 533-537. http:// dx.doi.org/10.4103/1305-7456.143638. PMid:25512737. 13. Seó, R. S., Vergani, C. E., Giampaolo, E. T., Pavarina, A. C., Reis, J. M. S. N., & Machado, A. L. (2008). Effect of disinfection by microwave irradiation on the strength of intact and relined denture bases and the water sorption and solubility of denture base and reline materials. Journal of Applied Polymer Science, 107(1), 300-308. http://dx.doi.org/10.1002/app.27120. 14. Azzarri, M. J., Cortizo, M. S., & Alessandrini, J. L. (2003). Effect of the curing conditions on the properties of an acrylic denture base resin microwave-polymerised. Journal of Dentistry, 31(7), 463-468. http://dx.doi.org/10.1016/S0300-5712(03)00090-3. PMid:12927457. 15. Blagojevic, V., & Murphy, V. M. (1999). Microwave polymerization of denture base materials: a comparative study. Journal of Oral Rehabilitation, 26(10), 804-808. http://dx.doi. org/10.1046/j.1365-2842.1999.00456.x. PMid:10564437. 16. Urban, V. M., Machado, A. L., Oliveira, R. V., Vergani, C. E., Pavarina, A. C., & Cass, Q. B. (2007). Residual monomer of reline acrylic resins: effect of water-bath and microwave postPolímeros, 28(3), 215-219, 2018
Effect of disinfection techniques on physical-mechanical properties of a microwave-activated acrylic resin polymerization treatments. Dental Materials, 23(3), 363-368. http://dx.doi.org/10.1016/j.dental.2006.01.021. PMid:16620950. 17. Fortes, C. B. B., Leitune, V. C. B., Collares, F. M., Dornelles, N. B., Jr., Rodrigues, S. B., Samuel, S. W., Petzhold, C. L., & Stefani, V. (2015). Acrylic resin disinfection by peracetic acid and microwave energy. Revista Gaucha de Odontologia, 63(3), 315318. http://dx.doi.org/10.1590/1981-863720150003000093013. 18. Senna, P. M., Silva, W. J., & Del Bel Cury, A. A. (2012). Denture disinfection by microwave energy: influence of Candida albicans biofilm. Gerodontology, 29(2), e186-e191. http:// dx.doi.org/10.1111/j.1741-2358.2010.00439.x. PMid:21083738. 19. Consant, R. L. X., Vieira, E. B., Mesquita, M. F., Mendes, W. B., & Arioli-Filho, J. N. (2008). Effect of microwave disinfection on physical and mechanical properties of acrylic resins. Brazilian Dental Journal, 19(4), 348-353. http://dx.doi. org/10.1590/S0103-64402008000400011. PMid:19180326. 20. Hamouda, I. M., & Ahmed, S. A. (2010). Effect of microwave disinfection on mechanical properties of denture base acrylic resin. Journal of the Mechanical Behavior of Biomedical Materials, 3(7), 480-487. http://dx.doi.org/10.1016/j.jmbbm.2010.05.002. PMid:20696412. 21. Lombardo, C. E., Canevarolo, S. V., Reis, J. M., Machado, A. L., Pavarina, A. C., Giampaolo, E. T., & Vergani, C. E. (2012). Effect of microwave irradiation and water storage on the viscoelastic properties of denture base and reline acrylic resins. Journal of the Mechanical Behavior of Biomedical Materials, 5(1), 53-61. http://dx.doi.org/10.1016/j.jmbbm.2011.09.011. PMid:22100079. 22. Ribeiro, D. G., Pavarina, A. C., Machado, A. L., Giampaolo, E. T., & Vergani, C. E. (2008). Flexural strength and hardness of reline and denture base acrylic resins after different exposure times of microwave disinfection. Quintessence International, 39(10), 833-840. PMid:19093060. 23. Konchada, J., Karthigeyan, S., Ali, S. A., R, V., Amirisetty, R., & Dani, A. (2013). Effect of simulated microwave disinfection on the mechanical properties of three different types of denture
Polímeros, 28(3), 215-219, 2018
base resins. Journal of Clinical and Diagnostic Research, 7(12), 3051-3053. http://dx.doi.org/10.7860/JCDR/2013/7376.3850. PMid:24551725. 24. Senna, P. M., Da Silva, W. J., Faot, F., & Del Bel Cury, A. A. (2011). Microwave disinfection: cumulative effect of different power levels on physical properties of denture base resins. Journal of Prosthodontics, 20(8), 606-612. http://dx.doi. org/10.1111/j.1532-849X.2011.00770.x. PMid:21980952. 25. Chassot, A. L., Poisl, M. I., & Samuel, S. M. (2006). In vivo and in vitro evaluation of the efficacy of a peracetic acid-based disinfectant for decontamination of acrylic resins. Brazilian Dental Journal, 17(2), 117-121. http://dx.doi.org/10.1590/ S0103-64402006000200006. PMid:16924337. 26. Subha, N., Prabhakar, V., Koshy, M., Abinaya, K., Prabu, M., & Thangavelu, L. (2013). Efficacy of peracetic acid in rapid disinfection of Resilon and gutta-percha cones compared with sodium hypochlorite, chlorhexidine, and povidone-iodine. Journal of Endodontics, 39(10), 1261-1264. http://dx.doi. org/10.1016/j.joen.2013.06.022. PMid:24041388. 27. International. Organization for Standardization – ISO. (1999). ISO 1567: 1999: dentistry-denture base polymers. Geneva: ISO. 28. American Society for Testing and Materials – ASTM. (2010). ASTM D256: standard test methods for determining the izod pendulum impact resistance of plastics. West Conshohocken: ASTM. 29. Shen, C., Javid, N. S., & Colaizzi, F. A. (1989). The effect of glutaraldehyde base disinfectants on denture base resins. The Journal of Prosthetic Dentistry, 61(5), 583-589. http://dx.doi. org/10.1016/0022-3913(89)90281-3. PMid:2501480. Received: Apr. 11, 2016 Accepted: Aug. 06, 2016
219/219 219
ISSN 1678-5169 (Online)
http://dx.doi.org/10.1590/0104-1428.06917
O O O O O O O O O O O O O O O O
Synthesis of poly(ethyl methacrylate-co-methyl methacrylate) obtained via ATRP using ruthenium benzylidene complexes Maria Beatriz Alves Afonso1, Lucas Gomes Gonçalves1, Talita Teixeira Silva2, José Luiz Silva Sá2, Nouga Cardoso Batista2, Beatriz Eleutério Goi1 and Valdemiro Pereira Carvalho Júnior1* Faculdade de Ciências e Tecnologia – FCT, Universidade Estadual Paulista – UNESP, Presidente Prudente, SP, Brasil 2 Núcleo Interinstitucional de Estudo e Geração de Novas Tecnologias – GERATEC, Departamento de Química – DQ, Universidade Estadual do Piauí – UESPI, Teresina, PI, Brasil 1
*valdemiro@fct.unesp.br
Abstract Atom-Transfer Radical Copolymerization (ATRP) of methyl methacrylate (MMA) and ethyl methacrylate (EMA) under different reaction conditions was conducted using Grubbs 1st (1) and 2nd (2) generation catalysts. Initially, the study focused on the reactivity of the catalysts in ATRP of EMA individually, then the syntheses of poly(MMA-co-EMA) were also conducted in different mixtures of monomers ([MMA]/[EMA] = 100/200 and [MMA]/[EMA] = 200/100). Conversion and semilogarithmic kinetic plots as a function of time were related to the different catalysts and reaction conditions. The values of Mn and PDI also changed when different catalysts were used in the presence of Al(OiPr)3, and more controlled polymerizations were achieved using 1. In the syntheses of poly(MMA)-co-(EMA), conversion of 60% was reached for both catalysts at different [MMA]/[EMA] ratios for 16 h; however, for shorter time, 4 h, better conversion values were obtained using 1 as catalyst for both [MMA]/[EMA] = 100/200 or 200/100. Keywords: methyl methacrylate, ethyl methacrylate, copolymer, ATRP, Grubbs catalysts.
1. Introduction Atom-transfer radical polymerization (ATRP) was pioneered by the research groups of Kato et al.[1] and Wang and Matyjaszewski[2,3]. ATRP is based on atom transfer radical addition (ATRA) - a modified Kharasch addition in which a transition metal complex catalyzes the addition of an alkyl halide across a carbon-carbon double bond. A radical species is generated by the transfer of a halogen atom from the alkyl halide to the transition metal complex. Radicals are generated in ATRP through a reversible redox process, catalyzed by a metal-ligand complex which undergoes a one-electron oxidation and abstracts a halogen atom from a dormant species (Scheme 1). ATRP has proven versatile for the synthesis of copolymers with desired molecular weight in various forms and compositions because of the wide range of vinyl monomers susceptible to this polymerization[1-3]. Moreover, ATRP is attractive due to its simple experimental setup and commercial availability of initiators and catalyst components, while maintaining exquisite control and versatility[1-12]. ATRP is a catalyst-based process in which the growing radicals can be reversibly activated or deactivated via dynamic equilibrium using a transition metal complex with exchange of halide species between the chain end and the metal complex[13]. The metal should present two readily accessible oxidation states separated by one electron, reasonable affinity towards the halogen,
220 220/225
expandable coordination sphere upon oxidation to accept the halogen atom, and be relatively strongly complexed by the ligand[13]. Although a variety of metal complexes has been used as ATRP catalysts[14-17], ruthenium has probably received the most attention because of its wide versatility in the coupling of different reactions via tandem process. In particular, Grubbs 1st (1) and 2nd (2) generation metathesis catalysts have shown excellent application in promoting two reactions with such markedly different mechanisms via various tandem reactions in which olefin metathesis and atom transfer radical reactions occur in one pot[18]. As complexes 1 and 2 were known to be active for ROMP and ATRP reactions[19-21], we expanded our investigations towards exploration of the activity of both catalysts in the copolymerization of methacrylate monomers. Methacrylate copolymers are a class of functional polymer materials and have been widely used in many fields because of their resistance to enzymatic attack, biocompatibility, and high optical qualities. Particularly, copolymerization of MMA and EMA is rare, with few cases reported in the literature, in which the researchers used both monomers to produce copolymers via conventional polymerization. Kitayama et al.[22,23] and Zune et al.[24] investigated the synthesis of MMA/EMA copolymers by anionic polymerization. Dzulkurnain et al.[25] reported the synthesis of random copolymers with three different ratios of
Polímeros, 28(3), 220-225, 2018
Synthesis of poly(ethyl methacrylate-co-methyl methacrylate) obtained via ATRP using ruthenium benzylidene complexes
Scheme 1. Illustration of ATRP reaction; the termination step is not shown for clarity.
EMA and MMA using the free-radical bulk polymerization method. Copolymerization of MMA with EMA conducted in a continuous stirred-tank reactor was reported by Shin and Seul[26]. Liu et al.[27] performed bulk copolymerization of MMA with EMA via free radical polymerization using azo initiator (AIBN) and studied the copolymer thermal properties. Free radical dispersion copolymerization of methyl and ethyl methacrylate in supercritical carbon dioxide was reported by Giles et al.[28]. The present study aimed to optimize the reaction conditions for the controlled copolymerization of MMA and EMA by ATRP using ruthenium-based metathesis catalysts. The homopolymerization of EMA and its copolymerization with MMA via ATRP using Grubbs catalysts in different reaction conditions were investigated. At first, the aim of this work was to evaluate the reactivity of 1 and 2 in ATRP of EMA reaction individually, then poly(MMA-co-EMA) were also obtained in different mixtures of monomers. The complexes 1 and 2 were able to mediate these polymerizations with acceptable rate and level of control. Random copolymerization of the previously mentioned monomers was conducted to establish the most favorable conditions to obtain polymers with desired molecular weights and compositions.
Toluene was dried overnight over calcium chloride, filtered and distilled from sodium benzophenone ketyl and degassed by three vacuum–nitrogen cycles under nitrogen before use. The monomers methyl methacrylate (MMA) and ethyl methacrylate (EMA) were washed with 5% NaOH solution, dried over anhydrous MgSO4, vacuum distilled from CaH2 and stored at −18 °C before use. Grubbs 1st and 2nd generation catalysts, anisole, 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), aluminium isopropoxide (Al(OiPr)3) and ethyl 2-bromoisobutyrate (EBiB) were used as acquired.
2.2 Homopolymers synthesis A ruthenium complex (23.5 µmol) was placed in a Schlenk tube containing a magnet bar and capped with a rubber septum. Air was expelled by three vacuum–nitrogen cycles before the monomer (EMA; 4.71 mmol) and the initiator solution (EBiB; 48.2 µmol) were added. All liquids were handled with dried syringes under nitrogen. The tube was capped under N2 atmosphere using Schlenk techniques, then the reaction mixture was magnetically stirred and heated in a thermostated oil bath at 85 °C. Aliquots (20 µL) were removed at appropriate intervals.
2.3 Copolymer synthesis
2. Materials and Methods 2.1 General details All reagents were purchased from Aldrich Chemical Co. All reactions and manipulations were conducted under a nitrogen atmosphere using standard Schlenk techniques. Polímeros, 28(3), 220-225, 2018
The same procedure was used for the copolymerizations, except for the fact that two monomers were added into a Schlenk tube. In a typical ATRP experiment for a [MMA]200[EMA]100 solution, a mass of MMA corresponding to 4.71 mmol was added into a reaction tube, resulting in a [Monomer]/[Ru] = 200 (means, [MMA]200). After that, 221/225 221
Afonso, M. B. A., Gonçalves, L. G., Silva, T. T., Sá, J. L. S., Batista, N. C., Goi, B. E., & Carvalho Júnior, V. P. a mass of EMA for a [Monomer]/[Ru] = 100 was added ([EMA]100= 2.35 mmol). The tube was capped under N2 atmosphere using Schlenk techniques, the reaction mixture was then magnetically stirred and heated in a thermostated oil bath at 85 °C. Aliquots (20 µL) were removed at appropriate intervals.
2.4 Analyses Conversion was determined from the concentration of residual monomer measured by gas chromatography (GC) using a Shimadzu GC-2010 gas chromatograph equipped with flame ionization detector and a 30 m (0.53 mm I.D., 0.5 μm film thickness) SPB-1 Supelco fused silica capillary column. Anisole was added to polymerization and used as an internal standard. Analysis conditions: injector and detector temperature, 250 °C; temperature program, 40 °C (4 min), 20 °C min−1 until 200 °C, 200 °C (2 min). The molecular weights and the molecular weight distribution of the polymers were determined by gel permeation chromatography using a Shimadzu Prominence LC system equipped with a LC-20AD pump, a DGU-20A5 degasser, a CBM-20A communication module, a CTO-20A oven at 40 °C, and a RID-10A detector equipped with two Shimadzu columns (GPC-805: 30 cm, Ø = 8.0 mm). The retention time was calibrated with standard monodispersed polystyrene using HPLC-grade THF as an eluent at 40 °C with a flow rate of 1.0 mL min−1. Theoretical molecular weights were calculated without considering the end groups according to the following equation: Mn,th = ([Monomer]0/[Initiator]0) × Conversion ×MWmonomer. The 1H NMR spectra were obtained in CDCl3 at 298 K on a Bruker DRX-400 spectrometer operating at 400.13 MHz. The obtained chemical shifts were reported in ppm relative to TMS. DSC experiments were performed using a SDT Q600 (V20.9 Build 20). The samples (10.0 ± 0.1 mg) in an aluminum open sample holder were heated at 10 °C min-1 from 20 to 180 °C in N2 atmosphere (20 cm3/min).
3. Results and Discussion ATRP of EMA was conducted in the presence of Al(OiPr)3 with initial molar ratio of [Monomer]/[EBiB]/[Ru] = 200/2/1 using 1 or 2 as catalysts. With 1, poly(EMA) was obtained with 8% of conversion in 4 h, increasing to 65% when reaction time is increased to 16 h; whereas with 2 as catalyst under similar conditions, 6% of conversion was observed in 4 h, increasing to 44% after 16 h of reaction (Figure 1). Semilogarithmic plots of the reaction time for ATRP of EMA showed a linear profile, with kobs = 2.14 × 10−5s−1 for 1 and kobs = 1.21 × 10−5 s−1 for 2, indicating that the radical concentration was constant throughout the polymerization (Figure 2). Mn values increased linearly with increased conversion and were followed by a decrease in the PDI values (Figure 3). The results are consistent with a certain degree of control for the polymerizations (low dispersity; Mn increasing with conversion for both catalyts). However, when comparing the polymerization control of EMA mediated by 1 and 2, the molecular weights of polyEMA obtained with 1 are in better agreement with the theoretical values for a controlled process. 222 222/225
Figure 1. Dependence of conversion on the reaction time for ATRP of EMA in the presence of Al(OiPr) 3 using 1 or 2 in toluene at 85 °C; (●) [EMA]/[EBiB]/[Al]/[1] = 200/2/4/1; (□) [EMA]/[EBiB]/[Al]/[2] = 200/2/4/1.
Figure 2. Semilogarithmic plots on the reaction time for ATRP of EMA in the presence of Al(OiPr)3 using 1 or 2 in toluene at 85 °C; (●) [EMA]/[EBiB]/[Al]/[1] = 200/2/4/1; (□) [EMA]/[EBiB]/[Al]/[2] = 200/2/4/1.
Figure 3. Dependence of Mn (solid) and PDI (hollow) values on conversion for ATRP of EMA in the presence of Al(OiPr)3 using 1 or 2 in toluene at 85 °C; (●) [EMA]/[EBiB]/[Al]/[1] = 200/2/4/1; (■) [EMA]/[EBiB]/[Al]/[2]/ = 200/2/4/1. Polímeros, 28(3), 220-225, 2018
Synthesis of poly(ethyl methacrylate-co-methyl methacrylate) obtained via ATRP using ruthenium benzylidene complexes Copolymerization conversion of MMA and EMA was sensitive to [MMA]/[EMA] ratio and catalyst type (Figure 4). With 1, using [MMA]/[EMA]/[EBiB]/[Al]/[Ru] = 200/100/2/4/1, the conversion increased from 45% in 4 h to 60% after 16 h; when the [MMA]/[EMA] ratio was reversed to 100/200, lower conversion was obtained in 4 h, but it also reached 60% after 16 h. With 2, using [MMA]/[EMA]/[EBiB]/[Al]/[Ru] = 200/100/2/4/1, 35% of conversion was obtained in the beginning of the reaction, when reversing the [MMA]/[EMA] ratio to 100/200, 25% of conversion was obtained in 4 h. Initially, lower conversions were achieved at [MMA]/[EMA] = 100/200 ratio for both catalysts. This can be explained by the lower reactivity of the EMA radical, considering that the pendant ethyl group provides greater stabilization of this monomeric radical. Similar conversion was obtained after 16 h regardless of [MMA]/[EMA] ratio and catalyst type.
Figure 4. Dependence of conversion on the reaction time for copolymerization of MMA/EMA via ATRP using 1 or 2 from different starting [MMA]/[EMA] compositions in toluene at 85 °C; [MMA]/[EMA]/[EBiB]/[Al]/[1] = 200/100/2/4/1 (●), [MMA]/[EMA]/[EBiB]/[Al]/[1] = 100/200/2/4/1 (■), [MMA]/[EMA]/[EBiB]/[Al]/[2] = 200/100/2/4/1 (▼), [MMA]/ [EMA]/[EBiB]/[Al]/[2] = 100/200/2/4/1 (▲).
Detailed information on the molecular structure of poly(MMA-co-EMA) using 1 or 2 at both [MMA]/[EMA] ratios was obtained from the 1H NMR spectra (Figure 5). In the 1H NMR spectra of copolymers, signals at 3.59 and 4.04 ppm are assigned to protons of the pendant methyl and ethyl group units, respectively. Signals between 0.87 and 1.03 ppm are assigned to methyl protons of the MMA and EMA units. Therefore, the presence of MMA and EMA in the polymer backbones is evident, as copolymers are present and characterized by MMA/EMA dyad signals. Table 1 shows the FMMA/FEMA ratio, which corresponds to the relative amounts of MMA and EMA units in the isolated copolymer chains determined from the pendant methyl and ethyl group NMR signals. The amount of EMA in the copolymer increased using both 1 or 2 as the starting amount of EMA increased. Similar behavior was observed when the concurrent tandem catalysis for MMA/EMA copolymers was conducted by varying EtOH concentration (EMA source by in situ transesterification of MMA into EMA with EtOH in the presence of Al(OiPr)3)[29]. The dependences of glass transition temperatures (Tg) of poly(MMA)-co-(EMA) in the mixture composition are shown in Table 1. As expected, Tg decreases as the amount of EMA increases in the mixture using both catalysts. When comparing the Tg values obtained with G1 and G2, higher Tg values are observed for the poly(MMA)-co-(EMA) produced with G1 in both mixtures, corroborating the higher insertion of MMA units (FMMA/FEMA) in the copolymer.
Copolymerization of MMA and EMA mediated by 1 showed living characteristics, as evidenced by the linear increase in molecular weight and decrease in polydispersity with conversion at both [MMA]/[EMA] ratios (Figure 6). Mn values were at least one order of magnitude higher with [MMA]/[EMA] = 100/200 compared with 200/100. When evaluating the relationship between theoretical and experimental molecular weights (Mn,exp/Mn,theor), the molecular weights closely agree with those predicted theoretically with PDI < 1.5 at [MMA]/[EMA] = 200/100 using 1 as catalyst; in contrast, when the [MMA]/[EMA] ratio is inverted to 100/200, the molecular weights are higher than the theoretical values for both catalysts. This can be attributed to the number of growing
Figure 5. 1H NMR spectra of poly(MMA)-co-(EMA) obtained with 1 (above) and 2 (below) from different starting [MMA]/[EMA] compositions in toluene at 85 °C. Polímeros, 28(3), 220-225, 2018
223/225 223
Afonso, M. B. A., Gonçalves, L. G., Silva, T. T., Sá, J. L. S., Batista, N. C., Goi, B. E., & Carvalho Júnior, V. P. Table 1. Copolymerization of MMA and EMA in the presence of Al(OiPr)3[a] using 1 or 2 in toluene in 16 h at 85 °C. Catalyst
[MMA]/[EMA]
1
200/100 100/200 200/100 100/200
2
Conv.[b] (%) 58 58 56 58
Mn[c]
(×103) 3.9 12.6 26.2 24.2
Mn,theor (×103) 8.7 8.7 8.4 8.7
PDI 1.33 1.64 1.89 1.60
FMMA/FEMA (%)[d] 72/28 43/57 69/31 40/60
Tg (K) 343 338 340 332
[Al(OiPr]/[Ru] = 4 molar ratio; [b]Determined from the concentration of residual monomer measured by gas chromatography (GC); [c]Determined with size exclusion chromatography (SEC) with polystyrene calibration; [d]Calculated from 1H NMR. [a]
with 1 (Figure 7). The bulkier properties of the N-heterocyclic ligand in 2, compared with the PCy3 ligand in 1, seem to hinder the reaction between catalyst and initiator or polymer‑halide, adversely affecting the polymerization control. Therefore, when comparing the catalytic efficiency of 1 and 2 in the copolymerization of MMA and EMA, 1 provides greater control over polymerization in both [MMA]/[EMA] ratios evaluated. This greater catalytic efficiency for 1 may be associated with higher steric accessibility to the Ru center, considering that that the bulkier properties of the N-heterocyclic in 2 seem to hinder the reaction between catalyst and initiator or polymer-halide.
4. Conclusions Figure 6. Dependence of Mn (solid) and PDI (hollow) values on conversion for poly(MMA)-co-(EMA) obtained with 1 from different starting [MMA]/[EMA] compositions in toluene at 85 °C; [MMA]/[EMA]/[EBiB]/[Al]/[1] = 200/100/2/4/1 (●,○) and [MMA]/[EMA]/[EBiB]/[Al]/[1] = 100/200/2/4/1 (■,□).
Figure 7. Dependence of Mn and PDI values on conversion for poly(MMA)-co-(EMA) obtained with 2 from different starting [MMA]/[EMA] compositions in toluene at 85 °C; [MMA]/[EMA]/[EBiB]/[Al]/[2] = 200/100/2/4/1 (●,○) and [MMA]/[EMA]/[EBiB]/[Al]/[2] = 100/200/2/4/1 (■, □).
radical chains being lower than expected, resulting in an effective increase in the monomer concentration. With 2, the molecular weight also increased linearly with conversion from 1.96 × 104 to 2.42 × 104 g mol−1 for [MMA]/[EMA] = 100/200 and from 1.98× 104 to 2.62 × 104 g mol−1 for 200/100, but the molecular weights were systematically higher than expected with PDI values slightly higher (2.1-1.6) than those obtained 224 224/225
Catalysts 1 or 2 were successfully applied for ATRP of EMA. Kinetics for the EMA polymerization demonstrated linear dependence on semilogarithmic coordinates and good agreement between of the experimental and theoretical molecular weights, and PDIs below 1.5. Most polymerizations occurred in a living fashion and were reasonably controlled with both catalysts. Poly(MMA)-co-(EMA) were synthesized in a reasonably controlled manner at different [MMA]/[EMA] ratios with both catalysts. The results show that control over polymerization and the polymerization yields are highly dependent on the catalyst. 1 was more efficient in controlling the polymerizations than 2, where the molecular weights increased in linear proportion with conversion and were closer to the calculated values, with narrower PDIs. Apparently, this greater catalytic efficiency for 1 may be associated with higher steric accessibility to the Ru center, considering that that the bulkier properties of the N-heterocyclic in 2 seem to hinder the reaction between catalyst and initiator or polymer-halide.
5. Acknowledgements The authors are indebted to the financial support from FAPESP (Proc. 2013/10002-0) and Prope (Proc. 2180/002/14-PROPe/CDC). The 400 MHz-NMR analyses were performed at the Instituto de Química de São Carlos - IQSC/USP, São Carlos, SP, Brazil.
6. References 1. Kato, M., Kamigaito, M., Sawamoto, M., & Higashimura, T. (1995). Polymerization of methyl methacrylate with the carbon tetrachloride/dichlorotris- (triphenylphosphine)ruthenium(II)/ methylaluminum Bis(2,6-di-tert-butylphenoxide) initiating system: possibility of living radical polymerization. Macromolecules, 28(5), 1721-1723. http://dx.doi.org/10.1021/ma00109a056. Polímeros, 28(3), 220-225, 2018
Synthesis of poly(ethyl methacrylate-co-methyl methacrylate) obtained via ATRP using ruthenium benzylidene complexes 2. Wang, J. S., & Matyjaszewski, K. (1995). Controlled/”living” radical polymerization: atom transfer radical polymerization in the presence of transition-metal complexes. Journal of the American Chemical Society, 117(20), 5614-5615. http://dx.doi. org/10.1021/ja00125a035. 3. Wang, J. S., & Matyjaszewski, K. (1995). Controlled/”living” radical polymerization: halogen atom transfer radical polymerization promoted by a Cu(I)/Cu(II) redox process. Macromolecules, 28(23), 7901-7910. http://dx.doi.org/10.1021/ma00127a042. 4. Matyjaszewski, K., & Tsarevsky, N. V. (2009). Nanostructured functional materials prepared by atom transfer radical polymerization. Nature Chemistry, 1(4), 276-288. http://dx.doi. org/10.1038/nchem.257. PMid:21378870. 5. Di Lena, F., & Matyjaszewski, K. (2010). Transition metal catalysts for controlled radical polymerization. Progress in Polymer Science, 35(8), 959-1021. http://dx.doi.org/10.1016/j. progpolymsci.2010.05.001. 6. Lee, H., Pietrasik, J., Sheiko, S. S., & Matyjaszewski, K. (2010). Stimuli-responsive molecular brushes. Progress in Polymer Science, 35(1-2), 24-44. http://dx.doi.org/10.1016/j. progpolymsci.2009.11.002. 7. Siegwart, D. J., Oh, J. K., & Matyjaszewski, K. (2012). ATRP in the design of functional materials for biomedical applications. Progress in Polymer Science, 37(1), 18-37. http://dx.doi. org/10.1016/j.progpolymsci.2011.08.001. PMid:23525884. 8. Matyjaszewski, K. (2012). Atom Transfer Radical Polymerization (ATRP): current status and future perspectives. Macromolecules, 45(10), 4015-4039. http://dx.doi.org/10.1021/ma3001719. 9. Wever, D. A. Z., Raffa, P., Picchioni, F., & Broekhuis, A. A. (2012). Acrylamide homopolymers and Acrylamide-Nisopropylacrylamide block copolymers by atomic transfer radical polymerization in water. Macromolecules, 45(10), 4040-4045. http://dx.doi.org/10.1021/ma3006125. 10. Matyjaszewski, K., & Tsarevsky, N. V. (2014). Macromolecular engineering by atom transfer radical polymerization. Journal of the American Chemical Society, 136(18), 6513-6533. http:// dx.doi.org/10.1021/ja408069v. PMid:24758377. 11. Chmielarz, P., Park, S., Simakova, A., & Matyjaszewski, K. (2015). Electrochemically mediated ATRP of acrylamides in water. Polymer, 60(9), 302-307. http://dx.doi.org/10.1016/j. polymer.2015.01.051. 12. Wu, W., Wang, W., & Li, J. (2015). Star polymers: advances in biomedical applications. Progress in Polymer Science, 46, 55-85. http://dx.doi.org/10.1016/j.progpolymsci.2015.02.002. 13. Matyjaszewski, K., & Davis, T. (2002). Handbook of radical polymerization. Hoboken: Wiley-Interscience. http://dx.doi. org/10.1002/0471220450. 14. Kamigaito, M., Ando, T., & Sawamoto, M. (2004). Metal-catalyzed living radical polymerization: discovery and developments. Chemical Record, 4(3), 159-175. http://dx.doi.org/10.1002/ tcr.20011. PMid:15293337. 15. Matyjaszewski, K. (1999). Transition metal catalysis in controlled radical polymerization: atom transfer radical polymerization. Chemistry, 5(11), 3095-3102. http://dx.doi.org/10.1002/(SICI)15213765(19991105)5:11<3095::AID-CHEM3095>3.0.CO;2-#. 16. Matyjaszewski, K., & Xia, J. (2001). Atom transfer radical polymerization. Chemical Reviews, 101(9), 2921-2990. http:// dx.doi.org/10.1021/cr940534g. PMid:11749397. 17. Kamigaito, M., Ando, T., & Sawamoto, M. (2001). Metalcatalyzed living radical polymerization. Chemical Reviews, 101(12), 3689-3746. http://dx.doi.org/10.1021/cr9901182. PMid:11740919. 18. Dragutan, V., & Dragutan, I. (2006). A resourceful new strategy in organic synthesis: tandem and stepwise metathesis/nonmetathesis catalytic processes. Journal of Organometallic Chemistry, 691(24-25), 5129-5147. http://dx.doi.org/10.1016/j. jorganchem.2006.08.012. 19. Simal, F., Demonceau, A., & Noels, A. F. (1999). Highly efficient ruthenium-based catalytic systems for the controlled Polímeros, 28(3), 220-225, 2018
free-radical polymerization of vinyl monomers. Angewandte Chemie International Edition, 38(4), 538-540. http://dx.doi. org/10.1002/(SICI)1521-3773(19990215)38:4<538::AIDANIE538>3.0.CO;2-W. 20. Lee, J., Grandner, J. M., Engle, K. M., Houk, K. N., & Grubbs, R. H. (2016). In situ catalyst modification in atom transfer radical reactions with ruthenium benzylidene complexes. Journal of the American Chemical Society, 138(22), 7171-7177. http:// dx.doi.org/10.1021/jacs.6b03767. PMid:27186790. 21. Afonso, M. B. A., Gonçalves, L. G., Borim, P., Sá, J. L. S., Goi, B. E., & Carvalho-Júnior, V. P., (2007). Atom transfer radical polymerization of methyl methacrylate mediated by Grubbs 1st and 2nd generation catalysts: insight into the active species. Journal of the Brazilian Chemical Society, 28(8), 1407-1413. http://dx.doi.org/10.21577/0103-5053.20160314. 22. Kitayama, T., Ute, K., Yamamoto, M., Fujimoto, N., & Hatada, K. (1990). Highly isotactic and living polymerization of ethyl methacrylate with t-C4H9MgBr in toluene and the preparation of block and random copolymers with high stereoregularity. Polymer Journal, 22(5), 386-396. http://dx.doi.org/10.1295/ polymj.22.386. 23. Kitayama, T., Nakagawa, O., & Hatada, K. (1995). Stereospecific polymerization of stereoregular poly(methyl methacrylate) macromonomer and determination of main-chain tacticity of resulting polymacromonomer. Polymer Journal, 28(2), 150-154. http://dx.doi.org/10.1295/polymj.28.150. 24. Zune, C., Zundel, T., Dubois, P., Teyssié, P., & Jerômé, R. (1999). New initiator system for the anionic polymerization of (meth)acrylates in toluene. IV. Random copolymerization of (meth)acrylates in toluene initiated by s-BuLi ligated by lithium silanolates. Journal of Polymer Science. Part A, Polymer Chemistry, 37(14), 2525-2535. http://dx.doi.org/10.1002/(SICI)10990518(19990715)37:14<2525::AID-POLA26>3.0.CO;2-M. 25. Dzulkurnain, N. A., Hanifah, S. A., Ahmad, A., & Mohamed, N. S. (2015). Characterization of random methacrylate copolymers synthesized using free-radical bulk polymerization method. International Journal of Electrochemical Science, 10(1), 8492. Retrieved in 2017, July 10, from https://ukm.pure.elsevier. com/en/publications/characterization-of-random-methacrylatecopolymers-synthesized-us 26. Shin, B. S., & Seul, S. D. (1993). Solution copolymerization of methyl methacrylate and alkyl methacrylates in a continuous stirred tank reactor(CSTR). Korean Journal of Chemical Engineering, 10(1), 1-9. http://dx.doi.org/10.1007/BF02697371. 27. Liu, G., Zhang, L., Wang, Y., & Zhao, P. (2009). Studies on binary copolymerization and glass transition temperatures of methyl methacrylate with ethyl methacrylate and n-butyl methacrylate. Journal of Applied Polymer Science, 114(6), 3939-3944. http:// dx.doi.org/10.1002/app.30961. 28. Giles, M. R., Hay, J. N., & Howdle, S. M. (2000). The copolymerisation of methyl and ethyl methacrylate in supercritical carbon dioxide. Macromolecular Rapid Communications, 21(15), 1019-1023. http://dx.doi.org/10.1002/1521-3927(20001001)21:15<1019::AIDMARC1019>3.0.CO;2-M. 29. Nakatani, K., Ogura, Y., Koda, Y., Terashima, T., & Sawamoto, M. (2012). Sequence-regulated copolymers via tandem catalysis of living radical polymerization and in situ transesterification. Journal of the American Chemical Society, 134(9), 4373-4383. http://dx.doi.org/10.1021/ja211436n. PMid:22296320. Received: July 10, 2017 Revised: Aug. 16, 2017 Accepted: Aug. 17, 2017 225/225 225
ISSN 1678-5169 (Online)
http://dx.doi.org/10.1590/0104-1428.015816
O O O O O O O O O O O O O O O O
Polystyrene and cornstarch anti-corrosive coatings on steel Cinthia de Souza1, Ricardo Luiz Perez Teixeira2*, José Carlos de Lacerda2, Carla Regina Ferreira1, Cynthia Helena Bouças Soares Teixeira2 and Valdir Tesch Signoretti2 Curso de Engenharia Metalúrgica, Faculdade de Engenharia – FaEnge, Universidade do Estado de Minas Gerais – UEMG, João Monlevade, MG, Brasil 2 Grupo de Pesquisa em Sistemas de Exaustão – GPESE, Universidade Federal de Itajubá – UNIFEI, Itabira, MG, Brasil 1
*ricardo.luiz@unifei.edu.br
Abstract This work aims to evaluate the performance of anticorrosive thermoplastic coatings in cold rolled steel sheets. Two types of thermoplastic coatings were studied: polystyrene (PS) and cornstarch. These types of coatings are applied for protection against corrosion during transport and storage of steel plates after cold rolling until delivery to stamping or other processing. The good performance for these coatings is suitably standardized to ABNT NBR 5915-2: 2013 and ASTM A1008 / A1008M-2016. According to corrosion tests carried out in saline chamber, the coatings were satisfactory in different degrees of guarantee of corrosion protection on ASTM 1080 steel, in accordance with standards mentioned. Keywords: aging, anti-corrosive coatings, polystyrene, cornstarch films.
1. Introduction Coating metal surfaces with polymers is a protective method used as a physical barrier on metallic surfaces to protect against attack by corrosive species (e.g. O2 and H+). However, polymeric coatings are not permanently impenetrable; once there are defects formed in the coatings, pathways will be created for the corrosive species to damage the metallic substrate, and localized corrosion will occur[1]. According to ABNT NBR 5915-2:2013[2], loads lower than 2 kbar are expected for the use of sheet steel coatings for stamping. In this case, polystyrene coatings become perfectly applicable, considering that its resistance is 4 kbar[3]. According to the literature[4], starch coatings with modified structures are renewable alternatives when compared with coatings of fossil sources, such as polystyrene. The starch coating presents hydrophilicity which can be worked in terms of biodegradability of the coating. This type of coating may also be modified to a less hydrophilic condition[5]. Starch is chemically composed of two polysaccharides, amylose and amylopectin[6]. Starch may be converted into thermoplastic materials by conventional methods (extrusion, injection molding) in the presence of plasticizers, such as water and low molecular weight polyols. The casting technique can also be applied. In this case, starch is processed by heating in the presence of an excess of water, which causes irreversible swelling of the granules. Swelling is accompanied by disruption of the native crystalline structure and solubilization of amylose (gelatinization)[7]. The advantages of thermoplastic starch when it is compared to other similar polymers are its renewability, low cost, and wide availability. It is also biodegradable and can be processed on common equipment of conventional plastics[8].
226 226/230
The main aims of this work is to produce an anticorrosive coating for the Cold Rolled Steel According to the standard ABNT NBR 5915-2: 2013[2] (ASTM A1008 / A1008M-16[9]) using two types of thermoplastic coatings: starch and polystyrene.
2. Materials and Methods 2.1 Substrate cleaning of steel (ASTM 1080) For the treatment of steel substrate samples used in the deposition of anti-corrosion polyester film, 6 samples of steel wire rod (ASTM 1080 standard) 11 cm long and 0.5 cm in diameter were selected. The samples were sanded by hand with grit sizes of sandpaper 220 #, 400 # and 600 # inline (unidirectional sanding). After sanding, the samples were cleaned by etching in sulphuric acid solution 2% in volume to remove possible oxides and hydroxides. Later, they were cleaned with neutral detergent and deionized water to remove grease and oils. After they were scrubbed and the clean substrate samples were dried in an oven at 150 °C over a period of 30 minutes[10,11]. The applied time of 30 minutes also aimed at the removal of eventual organic residue on the surface.
2.2 Preparation of films 2.2.1 Cast cornstarch film The biodegradable anti-corrosive layer was produced by gelling of cornstarch composed of 26-30% amylose and 74‑70% amylopectin, and with less than 0.5% gluten, supplied by Corn Products Brazil (Sao Paulo, Brazil). Cornstarch
Polímeros, 28(3), 226-230, 2018
Polystyrene and cornstarch anti-corrosive coatings on steel (5 g) was dispersed in 50 mL distilled and deionized water at room temperature. The suspensions were diluted with 50 mL water previously heated to 95 °C and then held under stirring for 5 min. Glycerol (0.75 g) was added as a plasticizer[12]. ASTM 1080 steel substrates were immersed (dip coating) for 30 minutes in dispersion of starch-glycerol and held suspended for oven drying for 48 hours at 70 °C. 2.2.2 Cast polystyrene film The film of PS were prepared by dip coating from 30 wt% toluene solutions (analytical grade) onto steel substrates (ASTM1080) at 2 minutes and held suspended for drying at room temperature[12].
2.3 Test in saline solution After a week the samples were dry and were withdrawn under the atmosphere temperature. All samples (ASTM 1080 steel coated with starch and polystyrene and not covered) were placed in a saturated solution of NaCl using two petri plates for storage. These samples were kept for a period of 6 months in saline solution for corrosive attack and evaluation by microscopy[11,13,14].
2.4 Contact angle The measurement of the contact angle was performed using the goniometer Ramé-Hart 100-00 NRL (operated in air and atmosphere temperature). Measurements were obtained in three different regions of the samples and the medium values evaluated. A drop of 2.5 μl fluid was placed on the specimen surface, and a digital camera connected to the equipment captured the drop image. The contact angles were calculated automatically by the computer connected to the equipment, as shown schematically in Figure 1. The performance evolution of the drop was measured in the range of 15 seconds in a total time of 600 seconds[12].
2.5 Microscopic characterization of films Optical microscopy was used for preliminary characterization of the morphology and texture of the thermoplastic polystyrene films. Used in this procedure was a Leica optical microscope (model DMRM) coupled with a digital camera. High-resolution images of the surface of the films were obtained by atomic force microscope (AFM) for visualization of surface morphology and its texture (roughness was expressed in terms of root mean square roughness, RMS) with high lateral and depth resolution (nanometric ranges). The amplitude of deviation of RMS roughness of
Figure 1. Schematic contact angle dynamics of (A) polystyrene coating fluid on steel and (B) the starch coating fluid on steel. Polímeros, 28(3), 226-230, 2018
the oscillation varies per the morphological characteristics of the specimen surface and provides, in general, the most relevant information of the surface texture. The analyses were performed in air, in intermittent contact with a Micromesh NSC 16 rod (spring constant of 5 N/m), and on the atomic force microscope, model 1 M plus from JPK Instruments. Also used was a scanning electron microscope that allows obtaining images of the surface with a great depth of field, high resolution, and easy interpretation of the three‑dimensional looking images. Microscopy were obtained by scanning with energy-dispersive with an acceleration voltage of 20.0 kV spectrometer (Oxford Instruments), which provided the thickness and composition of the film on the steel wire (ASTM 1080). The samples were previously covered with 250 Angstroms of conductive gold-palladium alloy vacuum-evaporated (PVD) to provide a suitable microanalysis. Surface roughness and thickness of samples were measured with stylus roughness Sloan DekTak II surface profilometer (Dektak, Veeco Instruments Inc., Plainview, NY, USA).
3. Results and Discussion Figure 1 shows the schematic contact angle dynamics from photos of water droplets of (A) polystyrene coating fluid on steel (about 45°) and (B) the starch coating fluid on steel (about 50°). All contact angles were similar and less than 90° (low contact angle), which indicated that the wetting of the surface was favorable, and the fluid would spread over a large area of the steel surface. Figure 2a shows the visual outcome by optical microscopy of ASTM 1080 steel covered with polystyrene which was attacked by corrosion in a saline chamber (after six months). The polystyrene-coated steel presented no signs of corrosion in its structure. Thus, the steel was protected against corrosion by the polystyrene film. Figure 2b presents the visual outcome of the surface of the ASTM 1080 steel with the starch coating showed signs of corrosion after 2 weeks in the saline solution test, but it is important to note that the film dissolved differently on the surface. In the range of 2 weeks, there was no significant visual change in the starch film over the ASTM 1080 steel. Conclusively, it was observed by optical microscopy that there was collapse of the starch coating after 2 weeks of its application on the steel surface. There were no alterations in the PS coating until 6 months. By ABNT NBR 5915-2:2013 standard and visual corrosion results, as shown in Figure 2a and Figure 2b, polystyrene on steel can cover a thin cold-rolled steel sheet forming the same quality as EEP grade (protecting until 6 months) and starch on steel can cover a thin cold-rolled steel sheet forming the same quality as EM grade (protecting until 2 weeks)[2]. For the uniformity analysis of polystyrene film coating on the ASTM 1080 steel, an analysis of wettability was performed. Polystyrene wettability results indicated a contact angle of about 45.0° and a film thickness of about 40 μm (Figure 3a, by scanning electron microscopy analysis). In Figure 3a, the carbon sign in the steel was not revealed in the EDS spectrum due to its low intensity compared to the starch and PS coatings. A good covering 227/230 227
Souza, C., Teixeira, R. L. P., Lacerda, J. C., Ferreira, C. R., Teixeira, C. H. B. S., & Signoretti, V. T.
Figure 2. (a) Visual outcome by optical microscopy of the surface of the steel with polystyrene (PS) coating after a six-month saline test for corrosion; (b) visual outcome by optical microscopy of the surface of the steel with starch after 2 weeks of analysis of saline corrosion.
Figure 3. (a) Photomicrography for secondary electrons 600× substrate profile (SEM) of anticorrosive film of polystyrene (PS) on ASTM 1080 steel (Steel) and the energy-dispersive X-ray spectroscopy spectrum (EDS) for ASTM 1080 steel (Steel) and polystyrene (PS); (b) photomicrography for secondary electrons 600× substrate profile (SEM) of anticorrosive film of starch (Starch) on ASTM 1080 steel (Steel) and the energy-dispersive X-ray spectroscopy (EDS) only to the spectrum for starch. 228 228/230
Polímeros, 28(3), 226-230, 2018
Polystyrene and cornstarch anti-corrosive coatings on steel
Figure 5. (a) AFM height image for polystyrene surface on steel; (b) AFM height image for starch surface on steel. Figure 4. (a) Photomicrography for secondary electrons 10,000× anticorrosive film of polystyrene (PS) on ASTM 1080 steel; (b) Photomicrography for secondary electrons 10,000× of anticorrosive film of starch (Starch) on substrate of ASTM 1080 steel.
of polystyrene film-toluene was obtained on the surface of the ASTM 1080 steel (Figure 4a). The Dektak II results of film thickness indicated a thickness between 34 μm to 43 μm with a low average roughness value of (0.7 ± 0.4) μm and a diversion of RMS roughness by atomic force microscopy (AFM) of 35 nm (Figure 5a). For the uniformity analysis of the starch-coated film on the ASTM1080 steel, it was made sure that the wettability analysis indicated a contact angle of about 50° (contact angle analysis) and a thickness of about 4 μm (Figure 3b, analysis by electron microscopy by scanning). Then there was the possibility of a good spreading of starch film on the surface of the ASTM 1080 steel (Figure 4b). The results of Dektak II indicated a thickness between 3 to 4 μm with a low average roughness value of (0.7 ± 0.4) μm and a range of RMS roughness by atomic force microscopy (AFM) of 150 nm (Figure 5b). Polímeros, 28(3), 226-230, 2018
4. Conclusions As the optical and electronic microscopy results showed that the film was uniform and had a thickness of about 40 μm, polystyrene film offers effective protection against corrosion and has good coverage on the steel surface by saline chamber and results of contact angle (polystyrene presents good wettability on the ASTM 1080 steel). It also presented similar performance to that EEP grade of ABNT NBR 5915-2:2013 standard. The starch-produced coating is attributed to the precipitation of amylose-lipid complexes that are formed after starch gelatinization and presents itself by microscopy results as uniform and with a thickness about 4 μm. Starch coating revealed effective corrosion protection for up to 2 weeks, as tested in saline solution. It also presented similar performance to that EM grade of ABNT NBR 5915-2:2013 standard.
5. References 1. Deveci, H., Ahmetli, G., Ersoz, M., & Kurbanli, R. (2012). Modified polystyrenes: Corrosion, physicomechanical and thermal properties evaluation. Progress in Organic Coatings, 73(1), 1-7. http://dx.doi.org/10.1016/j.porgcoat.2011.08.011. 229/230 229
Souza, C., Teixeira, R. L. P., Lacerda, J. C., Ferreira, C. R., Teixeira, C. H. B. S., & Signoretti, V. T. 2. Associação Brasileira de Normas Técnicas – ABNT. (2013). ABNT NBR 5915-2: chapas e bobinas de aço laminadas a frio. Parte 2: aços para estampagem. Rio de Janeiro: ABNT. Retrieved in 2017, May 8, from http://www.abntcatalogo.com. br/norma.aspx?ID=195852 3. Matsushige, K., Radcliffe, S. V., & Baer, E. (1975). The mechanical behaviour of polystyrene under pressure. Journal of Materials Science, 10(5), 833-845. http://dx.doi.org/10.1007/ BF01163078. 4. Shogren, R. L. (2012). Starch polymer as advanced material for industrial and consumer products. In J. Ahmed, B. K. Tiwari, S. H. Imam & M. A. Rao (Eds.), Starch-based polymeric materials and nanocomposites: chemistry, processing, and applications. Boca Raton: CRC Press. 5. Thiré, R. M., Simão, R. A., Araújo, P. J., Achete, C. A., & Andrade, C. T. (2004). Reduction of hydrophilicity of biodegradable starch-based films by plasma polymerization. Polímeros: Ciência e Tecnologia, 14(1), 57-62. http://dx.doi. org/10.1590/S0104-14282004000100015. 6. García, M. T., Gracia, I., Duque, G., Lucas, Ad., & Rodríguez, J. F. (2009). Study of the solubility and stability of polystyrene wastes in a dissolution recycling process. Waste Management, 29(6), 1814-1818. http://dx.doi.org/10.1016/j.wasman.2009.01.001. PMid:19217275. 7. Mali, S., Grossmann, M. V. E., & Yamashita, F. (2010). Filmes de amido: produção, propriedades e potencial de utilização. Semina: Ciências Agrárias, 31(1), 137-156. http://dx.doi. org/10.5433/1679-0359.2010v31n1p137. 8. Bastos, D. C., Santos, A. E., Silva, M. L., & Simão, R. A. (2009). Hydrophobic corn starch thermoplastic films produced by plasma treatment. Ultramicroscopy, 109(8), 1089-1093. http:// dx.doi.org/10.1016/j.ultramic.2009.03.031. PMid:19345017.
230 230/230
9. American Society for Testing and Materials – ASTM. ASTM A1008/A1008M-16: standard specification for steel, sheet, coldrolled, carbon, structural, high-strength low-alloy, high-strength low-alloy with improved formability, solution hardened, and bake hardenable. West Conshohocken: ASTM. Retrieved in 2017, May 8, from https://www.astm.org/Standards/A1008. htm 10. Barbosa, L. C. A. (2004). Introdução à química orgânica. São Paulo: Pearson-Prentice Hall. 11. Winston, R. (2011). Society uhlig’s corrosion handbook. Ontario: The Electrochemical Society. Retrieved in 2017, May 8, from http://onlinelibrary.wiley.com/doi/10.1002/ 9780470872864. fmatter/pdf 12. Bastos, D. C., Santos, A. E. F., & Simão, R. A. (2014). Acetylene coating on cornstarch plastics produced by cold plasma technology. Starch, 66(2-3), 267-273. http://dx.doi. org/10.1002/star.201200219. 13. Associação Brasileira de Normas Técnicas – ABNT. (1983). NBR 8094: material metálico revestido e não revestido: corrosão por exposição à névoa salina: método de ensaio. Rio de Janeiro: ABNT. Retrieved in 2017, May 8, from http:// www.abntcatalogo.com.br/norma.aspx?ID=5373 14. American Society for Testing and Materials – ASTM. (2011). ASTM G85: standard practice for modified salt spray (fog) testing. West Conshohocken: ASTM. Retrieved in 2017, May 8, from https://www.astm.org/Standards/G85.htm Received: May 08, 2017 Revised: Aug. 02, 2017 Accepted: Sept. 04, 2017
Polímeros, 28(3), 226-230, 2018
ISSN 1678-5169 (Online)
http://dx.doi.org/10.1590/0104-1428.02317
Chitosan and gum arabic nanoparticles for heavy metal adsorption Flavia Oliveira Monteiro da Silva Abreu1*, Nilvan Alves da Silva1, Mateus de Sousa Sipauba1, Tamara Fernandes Marques Pires2, Tatiana Araújo Bomfim2, Oyrton Azevedo de Castro Monteiro Junior2 and Maria Madalena de Camargo Forte3 Laboratório de Química Analítica e Ambiental – LAQAM, Centro de Ciências Tecnólogicas – CCT, Universidade Estadual do Ceará – UECE, Fortaleza, CE, Brasil 2 Laboratório de Química Tecnológica, Centro de Ciências Tecnológicas – CCT, Universidade de Fortaleza – UNIFOR, Fortaleza, CE, Brasil 3 Laboratório de Materiais Poliméricos – LaPol, Departamento de Engenharia dos Materiais – DeMat, Escola de Engenharia – EE, Universidade Federal do Rio Grande do Sul – UFRGS, Porto Alegre, RS, Brasil 1
*flavia.monteiro@uece.br
Abstract Chitosan (CT) is a polysaccharide with the ability to adsorb metals on its surface. In this work, CT-based nanoparticles (NPs) are produced by complex formation with gum arabic (GA) to increase their adsorbent potential for removal of heavy metals in aqueous medium. Adsorption efficiency is evaluated as a function of NP composition and polysaccharide concentration. NPs are sized from 250 to 375 nm at a zeta potential up to -25 mV, suggesting stability to adsorb metals. In particular, CTGA56 and CTGA80 NPs adsorbed a substantially higher amount of copper ions than pure CT. Adsorption kinetics studies showed that the reaction process followed a pseudo second-order model and the adsorption isotherm results fit a Langmuir model, highlighting the monolayer adsorption process with prominent adsorption capacity. These findings indicate the adsorbent potential of CTGA NPs and suggest that these particles can be used for removal of metal ions from contaminated water sources. Keywords: adsorption, chitosan, nanoparticles, polyelectrolytes.
1. Introduction Water is a natural resource that is essential for life. As such, problems related to water quality, such as contamination by highly toxic and non-biodegradable heavy metals that are a by-product of industrial, domestic, or agricultural activities, are of significant concern[1,2]. Recent ecological studies have reported high levels of metal in urban environments such as rivers that exceed the safe limit for drinking water and create harmful conditions for surrounding biota and inhabitants[3]. There are several methods for removing heavy metals from wastewater, including chemical precipitation, filtration with activated carbon, reverse osmosis, and coagulation[4]. Biopolymers such as chitosan (CT) have been tested for their potential for metal adsorption, especially given their low cost and abundance in nature. Biosorption is a physico-chemical and metabolically independent process for the removal or recovery of organic and inorganic substances from solution using biological materials that involves a variety of mechanisms including absorption, ion exchange, surface complexation, and precipitation[5]. CT is derived from deacetylated chitin from the exoskeleton of crustaceans and exhibits hydrophilic characteristics, biocompatibility, and biodegradability. The adsorption capacity is attributed to the presence of a large number of amino and hydroxyl groups in the polymer chain that act as active sites of metal coordination[6-9].
Polímeros, 28(3), 231-238, 2018
Amino groups of CT composites and derivatives presents well stabillished binding properties with some heavy metal ions such as Cu(II), Zn(II), Ni(II), Cd(II), Pb(II), Hg(II), and Cr(VI)[10]; cations and anions of dyes; and protein molecules in aqueous medium via various types of interactions including electrostatic attraction, coordinated bonds and chelation[11]. Various studies have evaluated the absorbing capacity of CT combined with other materials, such as with cellulose, alginate, and silica[9,12]. Gum arabic (GA) is a natural anionic polysaccharide derived from tree exudates of Acacia senegal and Acacia seyal whose chemical composition can vary with the age of the tree, climate, and regional soil conditions[13]. Although GA has been used for protein encapsulation and delivery[14], its potential for metal absorption has not yet been reported. Nevertheless, GA presents carboxyl groups which may act as a binding agent to enhance the adsorption potential of chitosan. In the present study, CT and GA nanoparticles (NPs) were synthesized by polyelectrolytic complexation based on electrostatic interactions arising from the opposite charges of the CT amino group (cation) and the anionic carboxylic group of GA in aqueous solution. The stability of such complexes depends on the composition and structure of the polymers as well as environmental factors such as temperature, pH, and solvent characteristics[15,16]. Here, we examined the
231/238 231
O O O O O O O O O O O O O O O O
Abreu, F. O. M. S., Silva, N. A., Sipauba, M. S., Pires, T. F. M., Bomfim, T. A., Monteiro Junior, O. A. C., & Forte, M. M. C. potential of the NPs for adsorption of heavy metal (Cu) to evaluate their applicability to the treatment of contaminated bodies of water. The complexed polysaccharides in the form of NPs could present greater adsorption capacity than CT alone owing to a larger surface area and lack of internal diffusion resistance, where GA may offer additional binding sites thourgh their carboxylic groups[17,18].
2. Materials and Methods 2.1 Materials Gum arabic (GA) (Sigma-Aldrich Brasil, Ltda, São Paulo, SP, BR) with an average molecular weight of 400 kDa, low viscosity, and high solubility in aqueous medium and Chitosan (CT) Polymar, Fortaleza, CE, BR. with an average molar mass of 200 kDa and deacetylation degree (DD) of 72¨% were used for experiments. The DD was determined by dissolving the CT sample in 20 ml HCl 0.100 M followed by titration with NaOH 0.05M.
2.2 Preparation of CTGA NPs To obtain 2% w/w GA solution, 2 g of GA was added to 100 mL of distilled water with magnetic stirring for 2 h. The stock solution was used to prepare 0.1%-0.3% solutions in a volumetric flask. A 2% w/w CT solution was prepared by adding 2 g of CT to 10 mL of 2% acetic acid (v/v) with stirring at a temperature of 60°C for 2 h, and was diluted to obtain a 0.2% CT solution. NPs were prepared by the complex coacervation method using CT as the nucleus. The 0.2% w/v CT solution was added dropwise to 30 mM sodium pyrophosphate (Na4P2O7•10H2O) using a 26-G needle, followed by gelification under magnetic stirring for 30 min. A molar ratio of 1:2 CT:P2O7-4 was used for formation of the complexes. After gelification process, the pre-nucleus were coated with GA by controlled addition of a specific amount of GA solution, using 0.1% or 0.3%, forming NPs with diferent final volume. The samples were homogenized for 1h in order to form GA-CT NPs. The NPs were rinsed three times with distilled water by centrifugation at 3500 rpm for 20 min and stored in distilled water under refrigeration for 12 h. Afterwords they were spray-dried with inlet and outlet temperatures of 140°C and 60°C, respectively, at a rate of 0.62 L/h and air volume of 40 L/min.
2.3 NPs Characterization NPs yield was determined from the ratio of the dry NP mass obtained by spray-drying to the total mass added to the reaction system. Average particle size and zeta potential were determined using a Zetasizer NanoZen3500 instrument (Malvern Instruments, Westborough, MA, USA). NPs were characterized by Fourier transform infrared (FTIR) spectroscopy in a Spectrum 1000 instrument using KBr disks (Perkin Elmer, Waltham, MA, USA).
2.4 Adsorption studies The amount of adsorbed metal after 24 h was determined by the batch method in order to find the NPs with the best sorption capacity. The samples were compared with CT and GA, which were used as received from the 232 232/238
manufacter. It was weighted 1g of the samples CT, GA and the NPs, each one of them were immersed individually in a 1M CuSO4 solution and the remaining amount of copper after 24h was determined by complexometric titration using EDTA. A 2 mL aliquot was added to 50 mL of distilled water in a flask; the pH was adjusted to 10, and the indicator Murexide P.A 99% (Diadema, SP, BR) was added to the solution. The number of moles of adsorbed copper (Nad) was calculated using Equation 1: = N ad
( N f − Ni )
/ m
(1)
Where Ni and Nf are the initial and final number of moles, respectively, and m is the mass of the NPs immersed in CuSO4 solution of known concentration. Kinetics of adsorption were evaluated by placing 50 mg of NPs into 100 mL of copper sulfate solution at initial concentrations of 250, 1250, and 2500 mg/L at constant pH 6. Cu(II) adsorbed by NPs was measured after continuous stirring with a magnetic stirrer, where aliquots were taken at specified time points between 5 and 180 min and the Cu content was determined using an atomic spectrophotometer (Shimadzu, model AA-7000). The amount of adsorption at equilibrium qe (mg/g) and at a given time qt (mg/g) were calculated by the following Equation 2 and 3: qe =
( C0 –
Ce ) . V / m
(2)
qt =
( C0 –
Ct ) .V / m
(3)
where C0 is the initial Cu(II) concentration, Ct is the concentration at a given time t, and Ce is the concentration at equilibrium in mg/L, and V is the volume of the solution and m is the mass of the adsorbent.
3. Results and Discussions CTGA NPs were produced using a primary complex of sodium pyrophosphate Na4P2O7 as a NP core to which GA was added at pH 6. Various CTGA NPs samples were produced by varying the CT/GA ratio and the %GA solution. NP properties were dictated by the intrinsic characteristics of the constituent polymer and the extent of interaction between polymers in solution. CTGA NPs were characterized by analyzing the zeta potential, yield, particle size and metal adsorption capacity. NP yield was calculated based on the total mass added to the reaction. The overall yield was low because of loss of material during the spray-drying process, in comparison with freeze-drying process which tends to present higher yield[19]. Nonetheless, NPs with CTGA56 and CTGA48 (e.g., 56% wt and 48% wt CT content) had a higher yield than those in which there was an excess of one of the polysaccharides (e.g., 80% wt and 25% wt CT content), as shown in Table 1. The CT had a lower molecular weight than GA, although they were both in the same order of magnitude (200 and 400 kDa, respectively). Thus, to obtain a higher yield of CTGA complexes, an Polímeros, 28(3), 231-238, 2018
Chitosan and gum arabic nanoparticles for heavy metal adsorption Table 1. Nanoparticle Composition, Yield and Zeta Potential. NP Code CTGA80 CTGA56 CTGA48 CTGA25
CT/GA mass ratio CT content (%wt) 4/1 1.3/1 1/1.1 1/3
80 56 48 25
GA
Yield
Zeta Potential
solution (%wt) 0.1 0.3 0.1 0.3
(%) 13.9 25.8 24.8 13.5
(mV) -30.8 -24.9 -23.7 -31.6
Particle size (nm) 650 221 340 28 (70%) 350 (30%)
equivalent amount of GA with acidic groups is needed for electrostatic interactions with the amino groups of CT, which would favor electrostatic interactions. As discussed in the literature, during complexation, polyelectrolytes are formed when both polymers are ionised and bear opposite charges. Depending upon the strength of the interactions, the polyeletroctrolytes can either coacervate, or form a more or less compact hydrogel and if ionic interactions are too strong, precipitation can occur[15]. In this case %GA solution didn’t affected the Yield in a direct manner, where neither NPs produced showed PEC precipitation. In a previous related work regarding Chitosan-Alginate microparticles[19], chitosan with lower MW, resulted in a higher yield in comparison with a chitosan with higher MW. Smaller chains presented less available amino groups per molecular chain and were used ina greater quantity to bind to the acid groups present in the ALG chains.
3.1 FTIR spectroscopy CT, GA and CT/GA NPs were analyzed by FTIR spectroscopy. Vibration modes of the main groups of polysaccharides and their profiles are shown in Figure 1. CT pure presents glucosamine units with stretching vibrations at 1420 cm–1 and 3379 cm–1. Their principal vibration modes are asymmetrical and symmetrical bending of amine and amide II bending at 1651 and 1556 cm-1, respectively. Also their acetyl-glucosamine groups appears at 1351 cm–1. For the GA, characteristic absorption bands were present, with a broad asymmetrical band at 1600 cm-1 and a narrower symmetrical band at 1415 cm-1, which can be attributed to C–O–O bond stretching and carboxylic ion axial stretching, respectively. An even broader absorption was observed near 1030 cm-1, which can be attributed to COH stretching. Similar absorption bands are found in polyssacarides such as alginate[14,18,19], and cashew gum[20,21] in the literature. As expected, all NPs showed similar profiles. A hydrocarbon group was detected in the CTGA NPs near 2900 cm−1[20]. Stretching vibrations from charged amino (NH3+) groups were observed near 3380 cm−1. The main stretching of GA acid (COO−) groups overlapped with the asymmetric bending of NH3+ around 1600 and 1400 cm−1. It was notice that none of the CTGA NPs showed the usual CT amino band at 1566 cm-1. In another study, the disappearance of this particular amino band were a evidence of the complex formation of chitosan with a anionic polyssacaride[19]. With increasing GA content, a broadening of the band at 1400 cm−1 was observed, revealing a second smaller peak at 1344 cm−1. The absorption band at 1129 cm−1 was attributed Polímeros, 28(3), 231-238, 2018
Figure 1. FTIR spectra of CT, GA and CTGA NPs.
to the stretching of COH, whereas the amino group of CT in the 1414 cm−1 band presented an intensified peak[21].
3.2 Particle size and zeta potential Particle size and zeta potential were evaluated as a function of the total volume fraction of NPs. Particles with zeta potential values > 30 or ≤ 30 mV avoid undesirable fluctuations. In this study, all NPs had large negative zeta potential values that were ≤ 24 mV at pH 6 (Table 1). This result indicates that the CTGA NPs presents enough stability, where GA, an anionic polysaccharide, successfully coated the surface of the particle, formed by the CT in the inner core. Particle distribution pattern and average size are important factors that influence the ability of NPs to adsorb heavy metals, as showed in Figure 2. The CT/GA mass ratio were altered in order to investigate the effect of the excess of one of the components in the final average particle size. Also, the GA concentration was tested in two levels in order to investigate the effect on the NPs formation process. Theoretically, higher concentration of one component in a lower total volume could favor an excess of interactions and form inter-agregation complexes to the particles under formation and cause higher particle size[19,22,23]. Results showed a largely unimodal distribution pattern for NPs, indicating uniformity in the production process. 233/238 233
Abreu, F. O. M. S., Silva, N. A., Sipauba, M. S., Pires, T. F. M., Bomfim, T. A., Monteiro Junior, O. A. C., & Forte, M. M. C.
Figure 2. NP size distribution as a function of total particle volume. (a) CTGA56; (b) CTGA43; (c) CTGA33; (d) CTGA80.
Figure 3. Formation of CTGA NPs without aggregation (left) and with possible aggregation of CT:P2O7-4 inner core (right).
NPs generally presented an average size of 250 - 350 nm, and this size range comprised > 95% of the fraction volume. This size range would tend to enhance adsorption because of the higher surface area:volume ratio. However, NPs produced with a higher CT content presented a higher size (~650 nm), suggesting that the excess CT and their amino groups may have favored intra- and interionic interactions. These interactions would lead to aggregation of a CT:P2O7-4 inner core, followed by a GA coating that would result in the formation of larger particles. This type of aggregation has been reported with other polysaccharides[19,22,23] and is represented schematically in Figure 3.
Figure 4. Adsorption of copper ions by CTGA NPs, CT, and GA.
3.3 Adsorption properties Pure CT and GA as well the CTGA NPs were evaluated for their ability to adsorb copper ions (Figure 4). The adsorption properties of pure CT were superior to those of pure GA (300 vs. 122 mg of copper ions per gram of polymer). It is well known that chitosan is a powerful chelating agent that easily forms complexes with transition metals and heavy metals. Amino and hydroxyl groups are mainly involved in the binding of metal ions by chitosan[7-9]. Hence, the complex- forming properties and durability of a metal-chitosan complex depend on several parameters, such as the degree of 234 234/238
deacetylation, the length of the polymer chain, and finally, the physical form of the adsorbent[24,25]. Accordingly, NPs produced with a higher CT content had a greater capacity for adsorption than those with a higher GA content, which was independent of average particle size. CTGA80 NPs presented the highest CT content, which presented the greatest amount of amino groups available for binding with copper ions. However, they presented lower adsorption Polímeros, 28(3), 231-238, 2018
Chitosan and gum arabic nanoparticles for heavy metal adsorption properties at these conditions because of their higher particle for CTGA56 and to 109, 940 and 2170 mg/g for CTGA80 for Cu (II) concentrations of 250, 1250 and 2500 mg/L. size (>600 nm). CTGA56 NPs (with 56% wt CT) had This profile agrees with those reported in the literature[26-29]. markedly higher copper adsorption (549 mg/g) than pure Other studies showed different adsorption capacity; magnetic CT (300 mg/g). In this case, the combination of the high CT chitosan microspheres modified with thiourea adsorbed content and the nanoscaled particle size (250 nm) enhanced 80 mg/g with a copper ions concentration of 350 mg/L[28]; the adsorption capacity. The NPs in this study showed granular semi-IPN hydrogels based on chitosan and gelatin superior results compared to those in other studies of CTpresented an adsorption capacity of 300 mg/g with a copper based systems, such as CT-bound Fe3O4 NPs (21.5 mg/g)[24], ions concentration of 1750 mg/L[29]. These results show that chitosan-coated perlite beads (104.0 mg/g)[25], or chitosan/ the prepared adsorbent has a very high Cu(II) adsorption cellulose (26.5 mg/g) and chitosan/alginate (67.7 mg/g)[9]. capacity compared to reported in literature, particularly for CT is already being used as alternative to activated carbon higher concentration of Cu(II) in aqueous media. for the treatment of contaminated water bodies[1,6]. However, it is worth noting that our NPs had a different pattern from previously reported CT-core NPs[18]. The presence of GA 3.4 Kinetics of adsorption in the NP coating may provide advantages in terms of To investigate the mechanisms controlling adsorption stability because it does not swell under acidic conditions, processes such as mass transfer and chemical reaction, as in the case of CT. Kinetics studies were conducted for reaction and diffusion models were used to describe sorption CTGA56 and CTGA80 NPs because of their improved kinetics. The kinetics of the processes were described by adsorption properties. pseudo-first and pseudo-second order equations, represented The effect of contact time on the adsorption capacities respectively by Equation 4 and Equation 5: (mg/g) for NPs CTGA56 and CTGA80 as a function of the initial concentration of Cu(II) is shown in Figure 5. (4) log ( qe − qt ) = log qe – ( k1 / 2,303) t From these data, the equilibrium time for the adsorption was established as equal to 60 min for NPs CTGA56 (5) and CTGA80, independently of the initial concentration. = 1/ qt 1/ k2 qe2 + (1/ qe ) t It was also noted that the adsorption rate of the metal complexes was higher at the beginning and thereafter where qe and qt are the sorption capacities for Cu(II) on achieved saturation level at 180 min. This also shows CTGA NPs at equilibrium (mg/g) and at time t (mg/g), that the adsorption capacities increased according to the respectively; k1 is the pseudo-first order sorption rate constant increase of [Cu(II)] concentration: 89, 961 and 2217 mg/g (min-1); k2 is the pseudo-second order sorption rate constant (g/mg.min); and t is the time (min). In the diffusion model considering an adsorbent with a spherical shape and a relatively short time, the simplified Equation 7 is represented using Equation 6:
(
= qt
Figure 5. Effect of the phase contact time on the adsortion capacity of CTGA56 and CTGA80, depending on the concentration of Cu(II).
)
Kd . t 0.5 + C
(6)
where Kd is the diffusion intraparticle coefficient (mg/g.min-0.5), and C is a constant related to diffusion resistance (mg/g). Kd was determined as a result of linearization of qt as a function of time t. The results for the kinetic parameters are shown in Table 2. Taking into account the higher values of the correlation coefficients (R2), it is evident that these data best fit pseudo-second
Table 2. Kinetic parameters for sorption of Cu(II) on CTGA56 and CTGA80 NPs. CTGA56 Kinetics
Parameters
1ª Order
qe1 k1 R2 qe2 k2 R2 C Kd R2
2ª Order
Difusion
Polímeros, 28(3), 231-238, 2018
250 78.47 0.002 0.418 0.088 4.01 0.736 87.90 -3.77 0.173
1250 48.89 0.017 0.725 909.1 -0.0015 0.999 985.34 -2.99 0.578
CTGA80 Concentration (mg/L) 2500 250 253.75 61.86 0.039 0.016 0.780 0.558 2500 0.008 0.0003 -4057.9 0.9999 0.94 2048.1 117.93 18.35 0.625 0.939 0.006
1250 36.39 0.018 0.948 909.09 -0.0024 1 987.24 -3.529 0.863
2500 31.17 0.013 0.390 2000 -0.0025 0.9999 2160.00 -1.142 0.076
235/238 235
Abreu, F. O. M. S., Silva, N. A., Sipauba, M. S., Pires, T. F. M., Bomfim, T. A., Monteiro Junior, O. A. C., & Forte, M. M. C. order kinetics. This model takes into consideration the fact that the adsorption process is limited and controlled by chemical reaction[30], or chemisorption. The adsorption capacity calculated by the model (qe2) is also similar to those determined in the experiments, particularly with increased initial copper concentration (1250 and 2500 mg/L). Table 2 also shows that the second order model presented qe2 theoretical values of 909 and 2500 for CTGA56 and 910 and 2000 for CTGA80. These values are very similar with those provided by the experimental data (961 and 2217 for CTGA56 and 940 and 2170 for CTGA80, from Figure 5). k1 and k2 are related with time scale, where the higher the value, faster the system reaches equilibrium, which may or may not be dependent from the operational conditions[31]. In this study, k2, regarding second order kinectis, presented a relative high value only at lower concentration (250ppm). In this case, the low concentration of copper aided to a fast adsorption. The low R2 presented at this particular concentration gives a indications that at lower concentration could be a combined kinetics process.
3.5 Adsorption Isotherm In the next step the experimental data, were fitted by Langmuir and Freundlich isotherm models. This investigation is fundamental in describing the interaction between the solute and the adsorbent, and it represents the surface properties and affinity of the adsorbent at equilibrium. The widely known Langmuir equation, valid for monolayer sorption onto a surface with a finite number of identical sites, can be described in the linear form[32] as in Equation 7: = 1/ qe
(1/ qmáx )
+ (1/ K L qmáxCe )
(7)
where qe and qmáx are the equilibrium adsorption and maximum adsorption at monolayer coverage (mg/g); KL is a constant related to the affinity of binding sites (L/mg); Ce is the final concentration in the solution (mg/L). Alternatively, the Freundlich equation also can be used to fit experimental isothermal data to explain adsorption systems, usually in the log-linearized form according to Equation 8: = logqe
logK F + (1/ n ) logCe
(8)
where qe and qmáx are the equilibrium adsorption and maximum adsorption (mg/g); Ce is the final concentration in the solution (mg/L); KF is a Freundlich constant representing the adsorption capacity; and n is a dimensional constant depicting the adsorption intensity. The adsorption isotherm parameters were calculated from linear plots of Langmuir and Freundlich isotherm models as 1/qe versus 1/Ce and logqe versus logCe, respectively, and the results are shown in Table 3. The correlation coefficients demonstrate that both models are acceptable, with R2 > 0.9. However, the Langmuir model presented a higher correlation coefficient of R2 > 0.995; the parameters also described the experimental data with more accuracy. Further analysis of the Langmuir equation enables investigation of the dimensionless equilibrium parameter 236 236/238
RL given by Equation 9, where the given value provides an indication of whether or not the adsorption is favorable. = RL
1/ (1 + K LC0 )
(9)
where C0 is the highest initial solute concentration (mg/L) and KL is the Langmuir constant, which indicates the nature of adsorption. Briefly, the RL value indicates that the adsorption is favorable if it takes values of 0 < RL < 1, and when RL > 1, adsorption is unfavorable, which means that the metallic ions have more affinity in the liquid phase than in the adsorbent material[33]. As shown in Table 3, the RL factor for Cu(II) sorption on the CTGA80 and CTGA56 NPs was 0.24 and 0.31, respectively. In this case, it is beneficial to consider Cu(II) monolayer sorption onto surfaces with a finite number of identical sites such as those of CTGA80 and CTGA56 NPs, as the present data suggest that Cu(II) adsorption on CTGA NPs is favorable. The sorption capacities were equal to 344.83 and 303.03 mg/g for CTGA80 and CTGA56 NPs, respectively. These results show that the prepared adsorbent has a very high adsorption capacity compared to previously established values in the literature[24,25,28,29,34-39] as listed in Table 4. Therefore, the present study confirms that NPs CTGA may be an effective adsorbent for the removal of Cu(II) ions from aqueous solutions.
Table 3. Isotherms parameters for sorption of Cu(II) on CTGA56 and CTGA80 NPs. Isotherms Langmuir
Freundlich
Parameters KL
CTGA56 0.01275
CTGA80 0.00907
qmáx
303.03
344.83
RL
0.24
0.31
R2
0.9947
0.9999
KF
2.2x10-09
1.9x10-07
n
0.208
0.253
R2
0.9200
0.9610
Table 4. Maximum adsorption capacity of materials based on chitosan for Cu(II) ions. Adsorbent Materials
qmáx (mg/g) -
Ref.
Chitosan modified by chelating agents 113.6-55.6 [24] Chitosan chemically modified with the 109.9 [25] complexation agent Thiourea-modified magnetic chitosan 66.7 [28] microspheres Semi-IPN hydrogel based on chitosan 277.04-300.3 [29] and gelatin Chitosan with epichlorohydrin 35.5 [34] Chitosan-clay nanocomposites 181.5 [35] Chitosan–zeolite composites 14.8-51.3 [36] Xanthate-modified magnetic chitosan 34.5 [37] Chitosan loaded with Reactive Orange 16 107.3 [38] Recycled chitosan nanofibril 227.3 [39] CTGA56 303 This work CTGA80 344.8 This work
Polímeros, 28(3), 231-238, 2018
Chitosan and gum arabic nanoparticles for heavy metal adsorption
4. Conclusions In summary, core-shell chitosan-arabic gum NPs were produced, characterized, and employed for the adsorption of copper ions in aqueous solution. The results showed that stable particles were produced, with zeta potential values below −24 mV. The stability may be attributed to the carboxylic groups of GA, which successfully coated the CT inner core. Particle size was dependent on the CT content, where the smallest particles were obtained with CTGA56 (250 nm) and the largest were obtained with CTGA80 (660 nm). In the latter case, the excess CT and their amino groups may have favored intra- and inter-ionic interactions, leading to aggregation of a CT:P2O7-4 inner core followed by GA coating to form larger particles. The copper adsorption on CTGA56 and CTGA80 NPs was higher than that on pure chitosan. The adsorption of Cu(II) by CTGA NPs occurred through chemical adsorption that took place on the homogeneous surface of the NPs. In the resulting monolayer adsorption, the functional groups of nitrogen and oxygen atoms likely chelated the Cu(II) from aqueous solution. With increasing Cu(II) concentration, CTGA56 provided better performance than CTGA80 because of its smaller particle size, which provides an increased surface area: volume ratio, thereby maximizing the number of active binding sites. Thus, the present study confirms that CTGA NPs may be an effective adsorbent in the treatment of waste water for removal of Cu(II) ions.
5. Acknowledgements The authors thank Professor Dr. Ana Cristina de Oliveira Monteiro Moreira from Centro de Ciencias da Saúde of Universidade de Fortaleza for use of the spray-dryer, Professor Haroldo César Beserra de Paula from the Laboratório de Biopolímeros of Universidade Federal do Ceará for zeta potential and particle size analysis. This work was supported by the Conselho Nacional de Desenvolvimento Científico - CNPq [Projeto Universal 442965/2014-1]
6. References 1. Bhatnagar, A., & Sillanpää, M. (2009). Applications of chitin- and chitosan-derivatives for the detoxification of water and wastewater-a short review. Advances in Colloid and Interface Science, 152(1-2), 26-38. http://dx.doi.org/10.1016/j. cis.2009.09.003. PMid:19833317. 2. Wu, S. J., Liou, T. H., Yeh, C. H., Mi, F. L., & Lin, T. K. (2013). Preparation and characterization of porous chitosantripolyphosphate beads for copper(II) ion adsorption. Journal of Applied Polymer Science, 127(6), 4573-4580. http://dx.doi. org/10.1002/app.38073. 3. Islam, M. S., Ahmed, M. K., Raknuzzaman, M., HabibullahAl-Mamun, M., & Islam, M. K. (2015). Heavy metal pollution in surface water and sediment: a preliminary assessment of an urban river in a developing country. Ecological Indicators, 48, 282-291. http://dx.doi.org/10.1016/j.ecolind.2014.08.016. 4. Dean, J. G., Bosqui, F. L., & Lanouette, K. H. (1972). Removing heavy metals from waste water. Environmental Science & Technology, 6(6), 518-522. http://dx.doi.org/10.1021/ es60065a006. Polímeros, 28(3), 231-238, 2018
5. Fomina, M., & Gadd, G. M. (2014). Biosorption: current perspectives on concept, definition and application. Bioresource Technology, 160, 3-14. http://dx.doi.org/10.1016/j. biortech.2013.12.102. PMid:24468322. 6. Crini, G., & Badot, P.-M. (2008). Application of chitosan, a natural amino polysaccharide, for dye removal from aqueous solutions by adsorption processes using batch studies: a review of recent literature. Progress in Polymer Science, 33(4), 399477. http://dx.doi.org/10.1016/j.progpolymsci.2007.11.001. 7. Modrzejewska, Z., Rogacki, G., Sujka, W., & Zarzycki, R. (2016). Sorption of copper by chitosan hydrogel: Kinetics and equilibrium. Chemical Engineering and Processing, 109, 104-113. http://dx.doi.org/10.1016/j.cep.2016.08.014. 8. Miretzky, P. J., & Cirelli, A. F. (2009). Hg(II) removal from water by chitosan and chitosan derivatives: a review. Journal of Hazardous Materials, 167(1-3), 10-23. http://dx.doi. org/10.1016/j.jhazmat.2009.01.060. PMid:19232467. 9. Wan Ngah, W. S., Teong, L. C., & Hanafiah, M. A. K. M. (2011). Adsorption of dyes and heavy metal ions by chitosan composites: A review. Carbohydrate Polymers, 83(4), 14461456. http://dx.doi.org/10.1016/j.carbpol.2010.11.004. 10. Wu, F. C., Tseng, R. L., & Juang, R. S. (2010). A review and experimental verification of using chitosan and its derivatives as adsorbents for selected heavy metals. Journal of Environmental Management, 91(4), 798-806. http://dx.doi.org/10.1016/j. jenvman.2009.10.018. PMid:19917518. 11. Lui, B., Wang, D., Yu, G., & Meng, X. (2013). Adsorption of heavy metal ions, dyes and proteins by chitosan composites and derivatives — A review. Journal of Ocean University of China, 12(3), 500-508. http://dx.doi.org/10.1007/s11802-0132113-0. 12. Li, N., & Bai, R. (2005). Copper adsorption on chitosan–cellulose hydrogel beads: behaviors and mechanisms. Separation and Purification Technology, 42(3), 237-247. http://dx.doi. org/10.1016/j.seppur.2004.08.002. 13. Ali, B. H., Ziada, A., & Blunden, G. (2009). Biological effects of gum arabic: a review of some recent research. Food and Chemical Toxicology, 47(1), 1-8. http://dx.doi.org/10.1016/j. fct.2008.07.001. PMid:18672018. 14. Yang, J., Han, S., Zheng, H., Dong, H., & Liu, J. (2015). Preparation and application of micro/nanoparticles based on natural polysaccharides. Carbohydrate Polymers, 123, 53-66. http://dx.doi.org/10.1016/j.carbpol.2015.01.029. PMid:25843834. 15. Berger, J., Reist, M., Mayer, J. M., Felt, O., & Gurny, R. (2004). Structure and interactions in chitosan hydrogels formed by complexation or aggregation for biomedical applications. European Journal of Pharmaceutics and Biopharmaceutics, 57(1), 35-52. http://dx.doi.org/10.1016/S0939-6411(03)001607. PMid:14729079. 16. Lee, J. W., Kim, S. Y., Kim, S. S., Lee, Y. M., Lee, K. H., & Kim, S. J. (1999). Synthesis and characteristics of interpenetrating polymer network hydrogel composed of chitosan and poly (acrylic acid). Journal of Applied Polymer Science, 73(1), 113-120. http://dx.doi.org/10.1002/(SICI)10974628(19990705)73:1<113::AID-APP13>3.0.CO;2-D. 17. Thinh, N. N., Hanh, P. T. B., Ha, L. T. T., Anh, L. N., Hoang, T. V., Hoang, V. D., Dang, L. H., Khoi, N. V., & Lam, T. D. (2013). Magnetic chitosan nanoparticles for removal of Cr(VI) from aqueous solution. Materials Science and Engineering C, 33(3), 1214-1218. http://dx.doi.org/10.1016/j.msec.2012.12.013. PMid:23827563. 18. Yu, K., Ho, J., McCandlish, E., Buckley, B., Patel, R., Li, Z., & Shapley, N. C. (2013). Copper ion adsorption by chitosan nanoparticles and alginate microparticles for water purification applications. Colloids and Surfaces. A, Physicochemical and 237/238 237
Abreu, F. O. M. S., Silva, N. A., Sipauba, M. S., Pires, T. F. M., Bomfim, T. A., Monteiro Junior, O. A. C., & Forte, M. M. C. Engineering Aspects, 425, 31-41. http://dx.doi.org/10.1016/j. colsurfa.2012.12.043. 19. Abreu, F. O. M. S., Bianchini, C., Forte, M. M. C., & Kist, T. B. L. (2008). Influence of the composition and preparation method on the morphology and swelling behavior of alginatechitosan hydrogels. Carbohydrate Polymers, 74(2), 283-289. http://dx.doi.org/10.1016/j.carbpol.2008.02.017. 20. Paula, H. C. B., Sombra, F. M., Cavalcante, R. F., Abreu, F. O. M. S., & Paula, R. C. M. (2011). Preparation and characterization of chitosan/cashew gum beads loaded with Lippia sidoides essential oil. Materials Science and Engineering C, 31(2), 173-178. http://dx.doi.org/10.1016/j.msec.2010.08.013. 21. Abreu, F. O., Oliveira, E. F., Paula, H. C., & Paula, R. C. (2012). Chitosan/cashew gum nanogels for essential oil encapsulation. Carbohydrate Polymers, 89(4), 1277-1282. http://dx.doi. org/10.1016/j.carbpol.2012.04.048. PMid:24750942. 22. Zhu, A., Chan-Park, M. B., Dai, S., & Li, L. (2005). The aggregation behavior of O-carboxymethylchitosan in dilute aqueous solution. Colloids and Surfaces. B, Biointerfaces, 43(34), 143-149. http://dx.doi.org/10.1016/j.colsurfb.2005.04.009. PMid:15941653. 23. Lin, Y. H., Liang, H. F., Chung, C. K., Chen, M. C., & Sung, W. H. (2005). Physically crosslinked alginate/N,O-carboxymethyl chitosan hydrogels with calcium for oral delivery of protein drugs. Biomaterials, 26(14), 2105-2113. http://dx.doi.org/10.1016/j. biomaterials.2004.06.011. PMid:15576185. 24. Kołodyńska, D. (2012). Adsorption characteristics of chitosan modified by chelating agents of a new generation. Chemical Engineering Journal, 179, 33-43. http://dx.doi.org/10.1016/j. cej.2011.10.028. 25. Justi, K. C., Fávere, V. T., Laranjeira, M. C., Neves, A., & Peralta, R. A. (2005). Kinetics and equilibrium adsorption of Cu(II), Cd(II), and Ni(II) ions by chitosan functionalized with 2[-bis-(pyridylmethyl)aminomethyl]-4-methyl-6-formylphenol. Journal of Colloid and Interface Science, 291(2), 369-374. http://dx.doi.org/10.1016/j.jcis.2005.05.017. PMid:15992808. 26. Chang, Y. C., & Chen, D. H. (2005). Preparation and adsorption properties of monodisperse chitosan-bound Fe3O4 magnetic nanoparticles for removal of Cu(II) ions. Journal of Colloid and Interface Science, 283(2), 446-451. http://dx.doi.org/10.1016/j. jcis.2004.09.010. PMid:15721917. 27. Hasan, S., Ghosh, T. K., Viswanath, D. S., & Boddu, V. M. (2008). Dispersion of chitosan on perlite for enchancement of copper(II) adsorption capacity. Journal of Hazardous Materials, 152(2), 826-837. http://dx.doi.org/10.1016/j. jhazmat.2007.07.078. PMid:17850957. 28. Zhou, L., Wang, Y., Liu, Z., & Huang, Q. (2009). Characteristics of equilibrium, kinetics studies for adsorption of Hg(II), Cu(II), and Ni(II) ions by thiourea-modified magnetic chitosan microspheres. Journal of Hazardous Materials, 161(2-3), 995-1002. http://dx.doi.org/10.1016/j.jhazmat.2008.04.078. PMid:18538924. 29. Wang, W. B., Huang, D. J., Kang, Y. R., & Wang, A. Q. (2013). One-step in situ fabrication of a granular semi-IPN hydrogel based on chitosan and gelatin for fast and efficient
238 238/238
adsorption of Cu2+ ion. Colloids and Surfaces. B, Biointerfaces, 106, 51-59. http://dx.doi.org/10.1016/j.colsurfb.2013.01.030. PMid:23434691. 30. Liao, B., Sun, W., Guo, N., Ding, S., & Su, S. (2016). Equilibriums and kinetics studies for adsorption of Ni(II) ion onchitosan and its triethylenetetramine derivative. Colloids and Surfaces. A, Physicochemical and Engineering Aspects, 501, 32-41. http:// dx.doi.org/10.1016/j.colsurfa.2016.04.043. 31. Plazinski, W., Rudzinski, W., & Plazinska, A. (2009). Theoretical models of sorption kinetics including a surface reaction mechanism: A review. Advances in Colloid and Interface Science, 152(1-2), 2-13. http://dx.doi.org/10.1016/j. cis.2009.07.009. PMid:19735907. 32. Itodo, A. U., Itodo, H. U., & Gafar, M. K. (2010). Estimation of specific surface area using langmuir isotherm method. Journal of Environmental Management, 14(4), 141-146. http://dx.doi. org/10.4314/jasem.v14i4.63287. 33. Dubey, R., Bajpai, J., & Bajpai, A. K. (2016). Chitosan-alginate nanoparticles (CANPs) as potential nanosorbent for removal of Hg (II) ions. Journal of Environmental Management, 6, 32-44. http://dx.doi.org/10.1016/j.enmm.2016.06.008. 34. Chen, A. H., Liu, S. C., Chen, C. Y., & Chen, C. Y. (2008). Comparative adsorption of Cu(II), Zn (II), and Pb(II) ions in aqueous solution on the crosslinked chitosan with epichlorohydrin. Journal of Hazardous Materials, 154(1-3), 184-191. http:// dx.doi.org/10.1016/j.jhazmat.2007.10.009. PMid:18031930. 35. Azzam, E. M., Eshaq, G., Rabie, A. M., Bakr, A. A., Abd-Elaal, A. A., El Metwally, A. E., & Tawfik, S. M. (2016). Preparation and characterization of chitosan-clay nanocomposites for the removal of Cu(II) from aqueous solution. International Journal of Biological Macromolecules, 89, 507-517. http:// dx.doi.org/10.1016/j.ijbiomac.2016.05.004. PMid:27151669. 36. Wan Ngah, W. S., Teong, L. C., Toh, R. H., & Hanafiah, M. A. K. M. (2013). Comparative study on adsorption and desorption of Cu(II) ions by three types of chitosan-zeolite composites. Chemical Engineering Journal, 223, 231-238. http://dx.doi. org/10.1016/j.cej.2013.02.090. 37. Zhu, Y., Hu, J., & Wang, J. (2012). Competitive adsorption of Pb(II), Cu(II) and Zn(II) onto xanthate-modified magnetic chitosan. Journal of Hazardous Materials, 221-222, 155-161. http://dx.doi.org/10.1016/j.jhazmat.2012.04.026. PMid:22564487. 38. Vasconcelos, H. L., Guibal, E., Laus, R., Vitali, L., & Fávere, V. T. (2009). Competitive adsorption of Cu(II) and Cd(II) ions on spray-dried chitosan loaded with Reactive Orange 16. Materials Science and Engineering C, 29(2), 613-618. http:// dx.doi.org/10.1016/j.msec.2008.10.022. 39. Liu, D., Li, Z., Zhu, Y., Li, Z., & Kumar, R. (2014). Recycled chitosan nanofibril as an effective Cu(II), Pb (II) and Cd(II) ionic chelating agent: adsorption and desorption performance. Carbohydrate Polymers, 111, 469-476. http://dx.doi.org/10.1016/j. carbpol.2014.04.018. PMid:25037377. Received: Mar. 29, 2017 Revised: Aug. 04, 2017 Accepted: Sept. 04, 2017
Polímeros, 28(3), 231-238, 2018
ISSN 1678-5169 (Online)
http://dx.doi.org/10.1590/0104-1428.16816
UATR and NIRA evaluation in the quantification of ATBC in NC blends Talita de Souza Dias Mello1, Milton Faria Diniz2 and Rita de Cássia Lazzarini Dutra1* Instituto Tecnológico de Aeronáutica – ITA, São José dos Campos, SP, Brasil Divisão de Química – AQI, Instituto de Aeronáutica e Espaço – IAE, São José dos Campos, SP, Brasil 1
2
*ritacld@ita.br
Abstract The paint industry requires rapid and accurate methodologies of raw materials qualitiy controls. For example, the evaluation of the suitable ratio of binary mixtures of polymer/plasticizer such as nitrocellulose (NC)/acetyl tributyl citrate (ATBC) must be done, since this ratio is directly related to the performance of the final product. However, there is a small number of quantitative methodologies of such control in the literature. In this context, in this paper, the applicability of Infrared Fourier Transform Spectroscopy (FT-IR) techniques by using universal total attenuated reflection (UATR), in the middle infrared (MIR), and the reflectance analysis in the near infrared region (NIRA) is evaluated for the quantification of plasticizer ATBC in mixtures with NC. MIR and NIR methodologies presented good results such as: practicality of not requiring refined sample preparation, analysis time about 30 min and good accuracy, suitable data for using in the quality control laboratories of paint industry. Keywords: ATBC, NC, NIRA, paints, UATR.
1. Introduction Paints find applications in different industrial scenarios that are important for the economy and national sovereignty, such as naval, aeronautical, aerospace, microelectronic, automotive, graphic, and construction, among others[1-4]. It is known that the resins used in paints govern important properties, such as flexibility, hardness, and resistance to both alkalis and abrasion. Plasticisers also improve flexibility and resistance to wrinkling[1]. Alkyd resins and polyurethanes based in NC, among others, are used in the formulations of paints. Recently, a polymer based on oligopiperylene styrene modified with an alkoxysilane was tested as protection against adverse factors for construction, being considered as a new ecological coating[4]. As is known, the polymer used in this paper, NC, is a nitrate carbohydrate based on cellulose nitration. Specifically, one to three hydroxyl groups of D-glucopyranose may be substituted by the nitro groups thus producing various types of NC. These compounds are present in smokeless gunpowder as well as in a wide variety of everyday products. The different types of applications depend on the amount of nitrogen. Low nitrogen presence confers coverage and protection to the properties of NC, while high nitrogen provides explosive properties to this compound, making it an energetic material. In practice, NC with low nitration can be found in common products such as cigarettes, nail varnishes and varnishes, while NC with high nitration are parts of explosive components such as smokeless powder and dynamites[5]. These characteristics make NC a versatile polymer for different applications, and studies involving this polymer are welcomed in the scientific and technological communities.
Polímeros, 28(3), 239-245, 2018
The degree of nitration of the NC determines properties such as the solubility and flammability of the final product. NC with a high nitrogen content (≥ 12.5%), forms a soft substance known as powder cotton which is unstable to heat, and even a carefully prepared sample can decompose rapidly at temperatures above 150 °C. Powder cotton is used in smoke-free gunpowder, rocket propellants, and explosives. NC with a median nitrogen content (between 10.5% and 12.5%) was employed in this study and is thermally unstable, but it decomposes less violently than powder cotton and is soluble in alcohols and ethers. NC of this type is known by different names such as pyroxylin and cotton collodion, and is employed as a solvent-based paint-forming agent and in protective coatings[6]. NC is responsible for the adhesion of the paint to the substrate and the properties of the formed film. It is widely used in automotive refinishing, wood finishing and sealants, rotogravure and flexography printing inks, nail enamels, and leather finishes[7]. On the other hand, the main constituent of single base (SB) and double base (DB) propellants is NC[8]. It is also known that additives are added in polymer compositions, such as those used in paints, in order to impart specific properties to the final product. Plasticisers for example, are organic compounds added to polymeric materials in order to facilitate production and increase the flexibility and strength of the artefact. Among the families of plasticisers, phthalates are the most commonly used. Diethylphthalate (DEP) and dibutylphthalate (DBP) are low molecular weight phthalates and can be found in inks for printers and adhesives, among others.
239/245 239
O O O O O O O O O O O O O O O O
Mello, T. S. D., Diniz, M. F., & Dutra, R. C. L. Phthalates are also found in thermal shielding formulations of rocket motors based on butadiene copolymer and acrylonitrile (NBR)[9]. However, due to their potential to interfere negatively with the hormonal systems of animals and humans since belonging to endocrine disruptors, exogenous substances that alter the functions of the endocrine system and cause adverse effects on health[10], special attention has been given to these compounds in recent years. The migration of these compounds into packaging materials has become an important source of contamination for food[11]. Although citrates have higher production costs, act as plasticizers of great acceptance, since they are considered natural products without health risks. ATBC is the best known, compatible with a large number of resins, such as vinyls and cellulosics. In Brazil, citrates are ingredients in polymer films for packaging, notedly when they come into direct contact with food. With a constant concern for healthy sources of raw materials from the chemical industry, there are companies that prohibit the use of phthalates, and ATBC is a favorable option. Therefore, the development of methods that characterize and/or quantify plasticizers in a composition is of paramount importance[11]. In some market segments where public oversight is effective, it is common to use ATBC together with NC resin, such as in package printing inks. The NC / plasticiser ratio is directly related to the performance of the final product, including the steps of application and drying of the paint. In order to have a better product performance the NC/ATBC ratio must be explored in order to achieve the most satisfactory relation. Therefore, characterisation techniques that attest to the presence of ATBC and/or quantify it in mixtures with resins, such as NC, are therefore important, and methodologies should be developed to achieve this. FT-IR spectroscopy, coupled with other techniques, either on-line or off-line, has shown good results for the qualitative and quantitative analysis of resins and additives used in paints[11-15]. Souza et al.[16] applied Raman and infrared spectroscopy techniques for the identification and quantification of plasticisers in commercial stretchable PVC films. Calibration curves were constructed, by means of the addition of a standard, obtaining linearity and a high coefficient of correlation, resulting in the practicality and low cost of analysis using the FT-IR technique and the results obtained indicating that this technique can be used for the first evaluation of existing plasticisers in commercial stretchable PVC films. Rodrigues et al.[11] developed a methodology to evaluate the applicability of the coupling (indirect or off-line) of thin-layer chromatography (TLC) and FT-IR techniques called TLC-IR, and the selective extraction in the analysis of additives in polyurethane resin-based paint (PU) and NC. A formulation was used, evaluating three eluent systems and one type of developer, Gibbs. The FT-IR spectra were obtained by reflection, using the last generation reflection accessory, UATR. The main results showed that the ATBC, the major additive in the analysed composition, was easily identified by TLC-IR. The methodology allowed the detection and differentiation of plasticisers such as dioctylphthalate (DOP) and DBP, even in a small sample, and to identify the 240 240/245
oleamide (sliding promoter additive) by means of selective extraction and UATR. FT-IR and high performance liquid chromatography (HPLC) techniques were also used to develop a methodology for the separation and characterisation of the main biocides used in acrylic paints. It was observed that the direct FT-IR analysis of the biocides by means of reflection techniques was not possible, because the loads and the resins interfered in the analysis, due to the presence of intense bands and high concentrations in the inks. The solvent extraction technique, especially with carbon tetrachloride, eliminated and allowed the characterisation of the different biocides used[13]. Another methodology was developed by the same group[14] using the FT-IR technique, DRIFT / NIR, that is, obtaining diffuse reflectance spectra (DRIFT) in the near infrared (NIR) region for the characterisation of n-octylisothiazolinone (OIT), carbendazim and diuron in compositions of acrylic paints containing these milled microbicides reduced to particle sizes in the order of 5 μm. The DRIFT / NIR analysis indicated analytical bands (a term used in infrared spectroscopy to refer to the characteristic absorption of the group whose intensity varies with the concentration, thus obeying the Lambert-Beer Law) for the said microbicides. The presence of these compounds in the dry paint film was also confirmed by HPLC and microbiological analyses. Although the quantification of FT-IR data is smaller than the qualitative ones, especially in the analysis of paints, it is possible to highlight a methodology[15], in the medium infrared (MIR) region and the least explored, NIR, (UATR and DRIFT) and / or Transmission for the determination of the PU resin content in binary mixtures with NC, used in formulations of paints. The Reflection-UATR technique in the MIR region, as well as the DRIFT, Transmission and Reflection techniques in the NIR region, proved to be useful for quantifying the PU Resin content in NC binary systems with good precision. It is understood that the developed methodology, with adaptations relative to the analytical bands, can be used to quantify other binary mixtures. Although the aforementioned studies analyse polymers used in paints, including NC, by means of different techniques, it can be concluded that they still allow some gaps such as the application of unconventional FT-IR techniques for quantitative analysis of plasticisers in blends with resins, in particular NC. Thus, in this paper a methodology was proposed and evaluated with the use of FT-IR reflection techniques (usual and last generation) in a wide spectral range, including the near infrared region (NIR), with reflectance accessories (NIRA) and therefore the applicability of the UATR and NIRA techniques for the characterisation and quantification of the base polymer / plasticiser used in paint formulations.
2. Materials and Methods Low nitration (10.8-11.2% of nitrogen) NC samples and the plasticiser ATBC (Sandiflex) were assigned by the company Nitroquímica. Analytical grade methanol (MERCK) was the solvent employed in the study. The proportions of ATBC / NC were: 10/90, 15/85, 18/82, 19/81, 20/80, 22/78, 30/70. Polímeros, 28(3), 239-245, 2018
UATR and NIRA evaluation in the quantification of ATBC in NC blends The FT-IR quantitative analysis was performed using a PerkinElmer FT-IR spectrometer, Spectrum One model (spectral range 4000 to 550 cm-1 - medium infrared region - MIR and NIR region, from 10000 to 4000 cm-1, Resolution 4 cm-1, 20 scans). For the MIR analysis, samples with known concentrations were prepared with NC and the plasticiser and subsequently dissolved in methanol. The samples were analysed by the reflection mode, via universal attenuated total reflection accessory (UATR). For the NIR analysis, the samples with concentrations of 10, 15, 20 and 30% of ATBC were analysed as received by transflectance with the use of the NIRA accessory. A calibration or analytical MIR curve was developed, relating the relative band (A1740 / A1651) and the relative ratio of ATBC and NC. The analytical bands 1740 cm-1 (υ C=O) and 1651 cm-1 (υa NO2) were selected, respectively, for ATBC and NC. The baseline ranged from 1800 to 1590 cm-1. The data of the calibration curve represents the median of 5 values of the relative band A1740/A1651. The NIR calibration curve was computed by relating A5840 (ATBC) and the ratio of ATBC. A5840 is probably attributed to the first overtone of υ C = O band or the combination band of υ C = O and υs CO band around 1270 cm-1. The baseline ranged from 6100 to 5360 cm-1. The data represent the median of 5 values of the band intensity A5840.
2.1 Formulas used in calculations of deviations by FT‑IR[17] The deviation calculations were done according to Horák and Vítek[17] and with the methodology adopted successfully from the previous paper of the group, involving quantitative IR analyses of blends or polymer blends[15,18]. Thus, five aliquots for each sample were analysed, being calculated the particular relative band and the median (µ) of of these values was calculated. The mean standard deviation (σˆ µ ) of the relative band median value was calculated according to
Equation 1. For the calculation of the error of the methodology, the median value of the relative errors found was adopted according to previous studies[15,18]. σˆ µ σˆ µ = n
(1)
where, the standard deviation for the number of measurements realized is given by Equation 2: σˆ µ = K R .R
(2)
R is the difference between the highest and lowest absorbance values (Xn - X1). KR is the coefficient for calculating the standard deviation of a range of values (for 5 experiments[17], KR = 0.430). The relative error for each analysed sample was determined by Equation 3:
2.2 Relative error σˆ (%) = µ µ
x100
(3)
3. Results and Discussion 3.1 MIR region: spectra obtained by reflection, with UATR accessory 3.1.1 Characterization (qualitative analysis) FT-MIR of the NC resin and plasticiser ATBC - The most characteristic functional groups for the NC resin are the nitro groups which absorb in approximately (cm-1): 1650 (υa NO2), 1270 (υs NO2) and 860 (υNO). The most characteristic absorptions of ATBC are in approximately (cm-1): 1740 (υC= O), 1290-1000 (υsC-O)[19]. 3.1.2 FT-MIR analysis (quantitative) In the case of the studied system, NC / ATBC (1), the bands considered as analytical are at 1650 cm-1 (NC) and 1740 cm-1 (ATBC). In Figure 1, only some spectra of the
Figure 1. FT-IR (MIR-Reflection-UATR) spectra of NC and ATBC and their binary mixtures (NC / ATBC) (methanol solutions): (A) 90:10; (B) 82:18; (C) 80:20; (D) 78:22; (E) 70:30; (F) ATBC; (G) NC; *ATBC Band-target; + NC Band-target. Polímeros, 28(3), 239-245, 2018
241/245 241
Mello, T. S. D., Diniz, M. F., & Dutra, R. C. L. studied system were inserted, with the intention of not filling the chart with too much information and making it difficult to visualise the bands. However, the data of all the samples is in Table 1. They were used for the development of the calibration curve (Equation 4, Figure 2), with good linear correlation (R = 0.974) for the determination of ATBC in NC. The error of the methodology (0.73%), that is, the median of the relative errors is within the accuracy limits of the FT-IR spectrometer (≤2%). To find the percentages of ATBC / NC, for unknown samples (methodology evaluation), Equations 4 and 5 were used. = y 0.834 x − 0.0227
(4)
where y = (A1740/A1651) e x = [ATBC]/ [NC]. 100 [ ATBC ] + [ NC ] =
(5)
In order to verify the effectiveness of the methodology, three new ATBC / NC samples were prepared. The results were close to the nominal values, with relative errors, below 2%, that is, with good precision (Table 2). The analysis time was 30 minutes, demonstrating that the methodology can be used in industrial sectors, where time is an important factor in the emission of a result, for the proper interaction between laboratory and process.
3.2 NIR region: spectra obtained by reflection, with NIRA accessory 3.2.1 Characterisation (qualitative analysis) FT-NIR of ATBC plasticiser To validate the FT-MIR methodology, another methodology was developed by FT-NIR based on the band observed at 5840 cm-1. Only this band associated with the carbonyl of the ATBC was used because it is easier to be visualised and determined, due to its intensity and location in the NIR region, where the intensity of the bands is usually smaller, making it difficult to observe the weaker absorptions and implying the use of concentration increasing resources, sample thickness, etc.[20].
Table 1. FT-MIR data, including errors inherent in the methodology, for the preparation of the calibration curve A1740 / A1651 versus [ATBC] / [NC]. Samples ATBC/NC A1740/A1651 (relative concentration) 10/90 (0.11) 0.082 0.082 0.083 0.082 0.087 15/85 (0.18) 0.135 0.139 0.137 0.135 0.137 18/82 (0.22) 0.158 0.151 0.153 0.143 0.143 0.162 0.170 19/81 (0.23) 0.167 0.169 0.162 0.186 0.184 20/80 (0.25) 0.181 0.189 0.184 0.186 0.185 22/78 (0.28) 0.184 0.186 0.187 0.337 0.342 30/70 (0.43) 0.352 0.354 0.360
Median A1740/A1651 (µ)
(σˆ µ ) Standard deviation
Deviation or relative error (%)
0.082
0.001
1.22
0.137
0.001
0.73
0.151
0.003
1.99
0.167
0.001
0.60
0.184
0.001
0.54
0.186
0.001
0.54
0.352
0.004
1.14
Figure 2. FT-MIR calibration curve (A1740 / A1651) versus [ATBC] / [NC]. 242 242/245
Polímeros, 28(3), 239-245, 2018
UATR and NIRA evaluation in the quantification of ATBC in NC blends 3.2.2 FT-NIR Analysis (quantitative) In Figure 3, the NIR spectra of the studied system were inserted. The data used for the development of the calibration curve (Equation 6) is in Table 3 and Figure 4.
The sample, in solution, did not respond adequately to the NIR analysis, so an attempt was made to analyse it directly, as received, which is also not a simple task since the homogeneity of the sample and the morphology of the fibers hinder the repeatability of the tests due to the
Table 2. Data regarding the application of the MIR methodology to the ATBC/NC samples. SAMPLES ATBC/NC (nominal relative concentration) 14/86 (0.16)
16/84 (0.19)
17/83 (0.20)
A1740/A1651 0.109 0.104 0.107 0.109 0.104 0.139 0.136 0.137 0.136 0.142 0.154 0.155 0.156 0.153 0.162
0.107
0.001
0.93
Calculated sample ATBC/NC (relative concentration) 13.4/86.6 (0.15)
0.137
0.001
0.73
16.04/83.96 (0.19)
0.155
0.002
1.29
17.6/82.4 (0.21)
Median A1740/A1651 (µ)
(σˆ µ ) Standard deviation
Deviation or relative error (%)
Figure 3. FT-NIR spectra of NC / ATBC samples, analysed as received, with different ratios of ATBC: (A) 10; (B) 15; (C) 20; and (D) 30.
Figure 4. Calibration curve FT-NIR (A5840) versus [ATBC]. Polímeros, 28(3), 239-245, 2018
243/245 243
Mello, T. S. D., Diniz, M. F., & Dutra, R. C. L. Table 3. FT-NIR data, including errors inherent in the methodology, for the elaboration of the calibration curve A5840 versus [ATBC]. Proportion of ATBC in sample 10
15
20
30
A5840 0.088 0.091 0.094 0.095 0.092 0.112 0.119 0.123 0.110 0.116 0.136 0.120 0.127 0.134 0.126 0.178 0.175 0.178 0.163 0.179
Median A5840
Standard deviation
(µ)
(σˆ µ )
Deviation or relative error (%)
0.092
0.001
1.09
0.116
0.002
1.72
0.127
0.003
2.34
0.178
0.003
1.68
(6)
where y = A5840 e x = [ATBC].
Comparatively, the analysis time spent for the preparation of samples aiming at the UATR reflection technique (MIR region) was approximately 20 minutes, since the dissolution of NC/ ATBC in methanol was necessary. The NIRA technique (NIR region) is faster, around 5 minutes, because it discards the dissolution step, by analysing the samples as received. This shows that both methodologies, NIR and MIR, can be used in industrial sectors that have one or another spectrophotometer, and that need fast ATBC control techniques in NC.
4. Conclusion The Reflection techniques in the MIR and NIR regions were useful for quantifying the ATBC content in binary systems with NC, demonstrating practicality when using unconventional techniques such as UATR and NIR‑transflectance, which do not require sophisticated preparation techniques. Samples present reduced time of analysis and good precision, essential factors in industries or Research Centers for the evaluation of the studied system, 244 244/245
5. Acknowledgements This paper was supported in part by PVNS (National Senior Visiting Professor Program) and PROAP (Postgraduate Support Program) from CAPES and ADC/DCTA (Classical sports association of civil and military servants of the Aerospace Technical Center).
6. References
complexity of compaction and preparation of the samples. Thus, four samples were analyzed, representing lower, intermediate, and high levels. However, the methodology presented a good linear correlation (R = 0.989) for the determination of ATBC in NC binary mixtures by NIR according to Equation 6. The methodology error (1.70%) is within the spectrometer accuracy limits FT-IR (≤2%). = y 0.2632 x + 0.0647
since the proportion of plasticiser/NC is directly related to the performance of the final product.
1. Fazenda, J. M. R. (2005). Tintas e vernizes: ciência e tecnologia. São Paulo: Edgard Blücher. 2. Biscaro, R. S., Botelho, E. C., Takahashi, M. F. K., Faez, R., & Rezende, M. C. (2002). Estudo reológico de tintas de poliuretano contendo pani-DBSA aplicadas como materiais absorvedores de microondas (8-12 GHz). Polímeros: Ciência e Tecnologia, 12(4), 318-327. http://dx.doi.org/10.1590/S010414282002000400016. 3. Coutinho, F. M. B., & Delpech, M. C. (1999). Poliuretanos como materiais de revestimento de superfície. Polímeros: Ciência e Tecnologia, 9(1), 41-48. http://dx.doi.org/10.1590/ S0104-14281999000100006. 4. Trifonova, T., Selivanov, O., Chukhlanova, N., & Selivanova, N. (2013). Polymeric coatings composition based on modified oligopiperylene styrene binders with galvanic sludge as a filler. Chemical Engineering Science, 1(4), 75-78. http://dx.doi. org/10.12691/ces-1-4-5. 5. Fernández de la Ossa, M. Á., Ortega-Ojeda, F., & García-Ruiz, C. (2013). Discrimination of non-explosive and explosive samples through nitrocellulose fingerprints obtained by capillary electrophoresis. Journal of Chromatography. A, 1302, 197-204. http://dx.doi.org/10.1016/j.chroma.2013.06.034. PMid:23845757. 6. Química Nova Interativa. (2016). Retrieved in 2016, April 20, from http://qnint.sbq.org.br/qni/popup_visualizarMolecula. php 7. Mundocor. (2016). Retrieved in 2016, April 15, from http:// www.mundocor.com.br/tintas/nitrocelulose.asp 8. Andrade, J., Iha, K., Rocco, J. A. F. F., Franco, G. P., Suzuki, N., & Suárez-Iha, M. E. V. (2007). Determinação dos parâmetros cinéticos de decomposição térmica para propelentes BS e BD. Eclética Química, 32(3), 45-50. http://dx.doi.org/10.1590/ S0100-46702007000300007. 9. Sanches, N. B., Diniz, M. F., Alves, L. C., Dutra, J. C. N., Cassu, S. N., Azevedo, M. F. P., Mattos, E. C., & Dutra, R. C. L. (2008). Avaliação da aplicabilidade de técnicas FT-IR de reflexão (UATR) e de transmissão para a determinação do teor de acrilonitrila (AN) em NBR. Polímeros: Ciência e Tecnologia, 18(3), 249-255. http://dx.doi.org/10.1590/S010414282008000300011. 10. Souza, R. R., Martins, E. A. J., Otomo, J. I., Furusawa, H. A., & Pires, M. A. F. (2012). Determinação de plastificantes em água potável utilizando cromatografia gasosa e espectrometria de massas. Química Nova, 35(7), 1453-1458. http://dx.doi. org/10.1590/S0100-40422012000700028. 11. Rodrigues, V. C., Diniz, M. F., Mattos, E. C., & Dutra, R. C. L. (2016). Separação e identificação de aditivos em tinta por TLC-IR/ UATR e extração seletiva. Polímeros: Ciência e Tecnologia, 26, 68-74. http://dx.doi.org/10.1590/01041428.1887. 12. Germinario, G., Van der Werf, I. D., & Sabbatini, L. (2015). Chemical characterisation of spray paints by a multi-analytical Polímeros, 28(3), 239-245, 2018
UATR and NIRA evaluation in the quantification of ATBC in NC blends (Py/GC–MS, FTIR, μ-Raman) approach. Microchemical Journal, 124, 929-939. http://dx.doi.org/10.1016/j.microc.2015.04.016. 13. Pedro, R., Moraes, J. J., Diniz, M. F., Mattos, E. C., & Dutra, R. C. L. (2014). Análise por FT-IR (UATR e PAS) de microbicidas em filmes poliméricos de tintas comerciais. Polímeros: Ciência e Tecnologia, 24(2), 214-221. http://dx.doi. org/10.4322/polimeros.2014.041. 14. Pedro, R., Diniz, M. F., Mattos, E. C., & Dutra, R. C. L. (2014). Avaliação do efeito da moagem no desempenho e na caracterização DRIFT-NIR de microbicidas na superfície de tintas comerciais. Polímeros: Ciência e Tecnologia, 24(2), 250-258. http://dx.doi.org/10.4322/polimeros.2014.029. 15. Rodrigues, V. C., Diniz, M. F., Mattos, E. C., & Dutra, R. C. L. (2014). Quantificação por NIR/MIR de resina poliuretânica em misturas binárias com nitrocelulose utilizadas em Tintas. Polímeros: Ciência e Tecnologia, 24(3), 367-372. http://dx.doi. org/10.4322/polimeros.2014.027. 16. Souza, M. L., Corio, P., Temperini, M. L. A., & Temperini, J. A. (2009). Aplicação de espectroscopias raman e infravermelho
Polímeros, 28(3), 239-245, 2018
na identificação e quantificação de plastificantes em filmes comerciais de PVC esticável. Quimica Nova, 32(6), 1452-1456. http://dx.doi.org/10.1590/S0100-40422009000600017. 17. Horák, V. M., & Vítek, A. (1978). Interpretation and processing of vibrational spectra. Chichester: John Wiley & Sons. 18. Siqueira, S. H. S., Dutra, R. C. L., & Diniz, M. F. (2008). Determinação por espectroscopia nas regiões MIR/NIR do teor de NCO em adesivos poliuretânicos. Polímeros: Ciência e Tecnologia, 18(1), 57-62. http://dx.doi.org/10.1590/S010414282008000100012. 19. Smith, A. L. (1979). Applied infrared spectroscopy. New York: John Wiley & Sons. 20. Goddu, R. F. (1960). Near-infrared spectrophotometry. In C. N. Reilly (Ed.), Advances in analytical chemistry and instrumentation (pp. 347-425). New York: Interscience. Received: Dec. 20, 2016 Revised: Apr. 26, 2017 Accepted: May 28, 2017
245/245 245
ISSN 1678-5169 (Online)
http://dx.doi.org/10.1590/0104-1428.03617
O O O O O O O O O O O O O O O O
Polyurethane derived from Ricinus Communis as graft for bone defect treatments Tatiana Peixoto Telles de Sousa1, Maria Silvana Totti da Costa1, Renata Guilherme1, Wilson Orcini1, Leandro de Andrade Holgado1, Elcia Maria Varize Silveira1, Orivaldo Tavano2, Aroldo Geraldo Magdalena3, Sérgio Augusto Catanzaro-Guimarães1 and Angela Kinoshita1* Pró-reitoria de Pesquisa e Pós-graduação – PRPPG, Universidade do Sagrado Coração – USC, Bauru, SP, Brasil 2 Faculdade de Odontologia de Bauru, Universidade de São Paulo – USP, Bauru, SP, Brasil 3 Faculdade de Ciências, Universidade Estadual Paulista – UNESP, Bauru, SP, Brasil
1
*angelamitie@gmail.com; angela.kinoshita@usc.br
Abstract This work evaluated polyurethane (Polyquil) as a graft for treatment of bone defects. Bone defects of 1.5 × 0.5 cm were made in the calvaria of 16 rabbits. Eight animals had their defects treated with Polyurethane (Treated) and 8 of them had their defects filled with blood clot (Control). In the second experiment, segmental defects of 0.5 cm were performed at the zygomatic arch of 16 rabbits. Eight animals were treated by guided bone regeneration, using a latex membrane, associated to grafting of polyurethane while the others were not treated (Control). The bone tissue morphometry in the craniotomy experiment resulted in a higher bone volume in the Treated group at 60 days (p <0.05, t student test). Microscopic and radiographic images demonstrate the formation of a bone bridge in the segmental defect, 60 and 120 days after surgery in the Treated group, different from the Control group with incomplete healing. Keywords: biomaterial, bone defect, graft, polyurethane.
1. Introduction During the last decade, several segments of medicine, dentistry, among others, have sought biocompatible materials, harmless to the body, with the aim of replacing tissue. Biocompatible materials, natural or synthetic, can be prepared by different methods. There are many polymers used in dentistry, such as silicone, polyurethane and methyl methacrylate. The vegetable polyurethane, which combines the versatility of polymer formulation with the global concern of new biomaterials production with sustainable resources, has become one of the most studied biomaterials[1,2]. The polyurethane used in the present study - Poliquil - is a material derived from the Ricinus communis oil, plant shrubs very disseminated in Brazil. It is a plant known in Brazil and throughout the world by the synonyms of palma Christi, carrapateira, castor bean, tartago, “castor oil”, “castor bean”, probably originating in India[3]. This biopolymer has high capacity for interaction with human body cells in addition to not causing rejection. The success of the biopolymer can be explained by its compatibility with the human body[2,4-6]. The chemical composition of this material is formed by a chain of fatty acids whose molecular structure is present in human body fat, which facilitates its recognition. In addition, other advantages are observed which include a molecular formula with favorable aspects of processability; formulation flexibility; no emission of toxic vapors; good cell adhesion ability; non-release of toxic radicals when implanted, besides
246 246/255
low cost[4]. The biodegradation of this polymer by fungal and bacterial attack were demonstrated through Scanning Electron Microscopy (SEM), Thermogravimetry (TGA) and Infrared Spectroscopy (FTIR). The authors demonstrated that the degradation mechanisms are the same used by the microorganisms in fats[7]. Trovati et al.[8] used FTIR, TGA and X-ray Diffractometry (DRX) to investigate the rigid, semi rigid, and soft polyurethane obtained through different ratios between pre-polymer and polyol, showing that different ratios cause differences in thermal behavior and crystalline structure of the synthesized polyurethane. Jena and Gupta[9] reported that traditionally, the plant is used as a laxative, fertilizer and fungicide. It also has beneficial effects such as an anti-oxidant, antihistamic, antinociceptive, antiasthmatic, antiulcer aid, oral immunomodulator, anti‑inflammatory, antimicrobial, insecticide and larvicide, a hepatoprotective, fertility aid, central nervous system stimulant, lipolytic, wound healer, and having many other medicinal properties due to phytochemical components such as flavonoids, saponins, glycosides, alkaloids and steroids, among others. The polymer from Ricinus communis was studied due its biocompatibility and ability to stimulate bone regeneration. Leite and Ramalho[10] developed a study to compare the differences in the reaction of the connective tissue and the jaw bone of rats when exposed to this resin and demineralized bovine bone matrix. The results
Polímeros, 28(3), 246-255, 2018
Polyurethane derived from Ricinus Communis as graft for bone defect treatments indicate biocompatibility of both materials, considering their integration into the rat jaw, and demonstrating that polyurethane is an alternative in bone reconstruction with the advantage of being an inexhaustible source of biomaterial. In the form of resin, after polymerization, it has pores with a diameter of 120 to 500 μm, an important characteristic for osteoconductivity, since it allows the growth of the bone tissue in the interior, optimizing the integration between the material and the host bone tissue[11,12]. Other studies in animal models demonstrate good results of polymer applied as prosthesis for surgical correction of medial patellar luxation[13] and in the regeneration of segmental bone defects. The granular form showed to be biocompatible and osteointegrated[14]. It was demonstrated that when polyurethane is associated to calcium carbonate or calcium phosphate it can promote bone mass gain by bone matrix mineralization[15,16]. It was also observed that upon the incorporation of alkaline phosphatase to the polymer and subsequent incubation in synthetic body fluid, the biological properties of this polymer is improved[17]. When compared to the demineralized bone, it has the advantage of being reabsorbed more slowly[16]. The literature reports the association of polyurethane with other components with the objective of improving their properties as a biomaterial. Nacer et al.[18] describe the biological behavior of the polymer in combination with silica nanoparticles as a bone substitute, in a study on bone defects in rats, showing that the polyurethane is biocompatible and allows osteoconduction. In addition, they reported that the presence of osteoprogenitor cells suggests silica osteoinduction capacity. Barros et al.[19] evaluated the biocompatibility of three different Ricinus communis Polyurethane chemical compositions; pure, with calcium carbonate and calcium phosphate. They found that the calcium phosphate composite improves biocompatibility and osseointegration speed. In another experimental study on the biocompatibility of the Ricinus communis polymer with addition of calcium carbonate in comparison to titanium, a recognized biocompatible material, it was evidenced that composite was not statistically different from the titanium implant regarding tissue inflammatory reaction[20]. Another biomaterial used in this work is the latex derived from the rubber tree Hevea brasilienses. After the polymerization, a membrane is formed, presenting certain advantages such as: elasticity, flexibility, strength, ability to induce angiogenesis and low cost[21,22]. Its use as an occlusive membrane for bone regeneration has already been tested in animal models, with satisfactory results[23,24]. Other literature data indicate its biocompatibility[25,26], its angiogenic potential[21,27,28] and its ability to act as a drug delivery system[29-31]. Thus, the objective of this work was to study the effect of polyurethane granules (Polyquil) as graft material in bone regeneration cases. In the first part of the study, surgical defects of 1.5 × 0.5 cm were created in the calvaria of 16 rabbits and the polyurethane granules (Poliquil) were used as a graft to assist in bone repair. The defect diameter of 1.5 × 0.5 cm was chosen because it is similar to the critical size, which it is not repaired without treatment[32]. Thus, there would be a difference in responses between periods of 60 and 120 days, Polímeros, 28(3), 246-255, 2018
which might not occur for defects of smaller size. In the second part of the study, the conditions for the regeneration and restructuring of the zygomatic arch through the guided bone regeneration procedure were observed, using the natural latex membrane as occlusive membrane and polyurethane covering the graft. It was evaluated quantitatively by bone tissue morphometry, the behavior of the polymer material as osteoconductive, compared to the Control group, in which the defects were filled by blood clot.
2. Materials and Methods This study was approved by the Ethics Committee of the Universidade do Sagrado Coração, according to protocol number CEP/USC 26/07 and follows the recommendations set forth by the National Institute of Health (NIH)[33]. Thirty-two adult male New Zealand white rabbits, with an average weight of 3 kg, mean age between four and five months were used. They were maintained under good environmental conditions of food, temperature, hygiene and lighting during the entire experimental period. Sixteen animals were used in the calvaria defect experiments, using polyurethane as an intraosseous graft and 16 in the zygomatic arch segmental defect repair study. The polyurethane granules were purchased from Poliquil (Araraquara, Brazil); granulation 450μm and commercialized in 5g doses. This granulation allows the metabolization and formation of new bone tissue through the osteogenic cells. Figure 1 shows the infrared spectrum (FTIR) of the material using a Vertex 70 spectrometer (Bruker instruments). The description of bands are described in Table 1 and in agreement with Trovati et al.[8].
Figure 1. FTIR spectrum of polyurethane granules.The main bands are indicated and their descriptions are in Table 1. Table 1. FTIR bands assignment of polyurethane spectrum showed in Figure 1. Bands 3323 2925 2854 1731 1700 1596; 1513
Description O-H stretching C-H symmetrical stretching C-H asymmetrical stretching C = O stretching C = O stretching N-H angular deformation
247/255 247
Sousa, T. P. T., Costa, M. S. T., Guilherme, R., Orcini, W., Holgado, L. A., Silveira, E. M. V., Tavano, O., Magdalena, A. G., Catanzaro-Guimarães, S. A., & Kinoshita, A. The latex membrane was prepared as described by Guidelli et al.[30]. The colloidal solution of latex was provided by BDF Com Prod Agrícolas LTDA, Brazil and consisted of a mix of an extract of several H. brasiliensis clones. After extraction, the pH was adjusted to 10 with ammonium hydroxide (NH4OH) in order to avoid coagulation and the solution centrifuged at 8,000g, to reduce the amount of allergenic proteins[34]. The solution was then polymerized at ambient temperature in 15 cm-diameter Petri plates to produce a 2mm-thick membrane. After this procedure, the membranes were sterilized by gamma radiation (25kGy) for use in surgical procedures. During the procedure, the membranes were cut to a size which was slightly larger than the surgical defect (2 cm × 1cm), to facilitate their fixation. Guidelli et al.[30] described the FTIR spectrum of a latex membrane that is the same as that used in this present work.
2.1 Polyurethane as intraosseous graft The 16 animals were submitted to a surgical procedure for craniotomy under deep sedation (xylazine hydrochloride 10 mg/kg (Bayer, Brazil), followed by Ketamine hydrochloride 90 mg/kg (Vetbrands, Brazil) administration, also intramuscular, complemented with mepivacaine hydrochloride 2% with epinephrine 1:100,000 as local anesthetic, for purposes of ischemia in the preparation of surgical defects. The frontal and parietal bone were submitted to trichotomy, followed by antisepsis with PVPI (Polyvinylpyrrolidone iodine solution) topical application. A mucoperiostal linear incision, in the median line, in the frontal bone, with No. 15 scalpel blade, followed by muscle divulsion and periostal were performed. The separation of soft tissues was carried out in layers, exposing the parietal bones. A trephine bur, 0.5 cm in diameter, driven by a low speed rotation motor was used to make three perforations, under abundant irrigation with saline solution (sodium chloride 0.9%), removing all the cortical bone and cancellous bone, exposing the dura mater, configuring a bone defect of elliptical shape, 1.5 × 0.5 cm and depth equal to the thickness of the cortical bone. Whole bone within the defect was removed with caution to expose the dura mater, because there was a risk of dura mater damage. All bone was removed without
leaving bone spicules, to observed and compare, with fidelity, the bone growth from the blood clot and with the implanted materials. Technically, there is no way to leave a thin layer of bone on the dura mater, as there is no way to control whether all the animals are left with exactly the same thickness of this bone layer, resulting in errors in the results. Complete removal ensures uniformity of all defects. The animals belonging to the Control group (n=8) had the defects filled only by a blood clot (Figure 2A), followed by suturing in layers, first of the periosteum, followed by the skin suture. In the Treated group (n=8), the defects were filled with Polyurethane granules mixed in a blood clot (Figure 2B). The tissue was also immediately repositioned and sutured in layers, first of the periosteum followed by the skin suture. The animals received single dose of sodium dipyrone analgesic (25 mg/kg Fort Dodge, Brazil). Four animals of the Treated group and 4 of the Control group were submitted to euthanasia with an overdose of Sodium pentobarbitone (200 mg/kg IP), 60 and 120 days postoperatively. The specimens containing the bone defect were removed and preserved in 10% formalin and subsequently submitted to radiological and microscopic analysis.
2.2 Repair of segmental defect in the zygomatic arch Induction of deep sedation was done the same way as in the previous experiment. After shaving the area to be operated, a skin incision, 4 cm in length was performed with a Number 15 scalpel blade following the zygomatic arch bones. After the skin incision and divulsion of the musculature of the region, a new incision was made in the periosteum for exposure of whole bone tissue in the region of the zygomatic complex. The demarcation of the size of the defect was made using a trephine drill with diameter of 0.5 cm, driven by a low speed rotation motor with abundant irrigation using saline solution (sodium chloride 0.9%), in the central area of the zygomatic arch. Figure 3A illustrates the region where the defect was made. After the demarcation, the delimited bone segment was removed using Rongeur forceps. The edges of the defect
Figure 2. Bone defect created in the skull cap with elliptical shape and dimensions of 1.5 cm × 0.5 cm. (A) Defect filled by blood clot; (B) Defect filled with granulated polyurethane and blood clot. 248 248/255
Polímeros, 28(3), 246-255, 2018
Polyurethane derived from Ricinus Communis as graft for bone defect treatments
Figure 3. (A) Photograph of the bony structure of the rabbit skull showing the zygomatic arch and the region where the defect was created; (B) Segmental bone defect created in Control group, without treatment; (C) Segmental bone defect of the Treated group, enveloped by Latex membrane and filled with granulated polyurethane.
were conveniently regularized with an osteotome, taking care to remove all cortical bone without leaving bone spicules. In the Control group (Figure 3B), after the creation of segmental bone defect, the tissue was repositioned and simple sutures were placed in the periosteum and musculature and continuous in the skin with 3-0 silk suture (Ethicon). In the Treated group, the latex membrane was molded into cylindrical form to join the stumps of the defects. The inside of the membrane was filled with polyurethane granules mixed with blood clot (Figure 3C). Ethyl-cyanoacrylate adhesive was used for fixing the membrane on the stumps. The tissues were repositioned and sutured as described. Immediately after the surgical procedure the animals received single dose sodium dipyrone analgesics (25 mg/kg Fort Dodge, Brazil) intramuscularly. Four animals of the Treated group and 4 of the Control group were submitted to euthanasia with an overdose of Sodium pentobarbitone (200 mg/kg IP), 60 and 120 days postoperatively. The specimens containing the bone defect were removed and preserved in 10% formalin and subsequently submitted to radiological and microscopic analysis.
2.3 Radiographic analysis Digital radiographic images were taken using the Digora System in both experiments. Initially, the excessive humidity of the samples of the skull cap and the zygomatic arch was removed with a tissue. The positioning of the pieces was standardized: horizontally and parallel along the axis of the specific image plate of the Digora system, placed on a flat surface, covered by a Styrofoam piece. The dental X-ray equipment was regulated to 70kV and 8mA, and total filtration equivalent to 2mm of aluminum was used. The cylinder was positioned so that the beam was incident perpendicularly on the film plane with 40 cm focal distance and 2mAs exposure, previously selected. The sensitized image plate was positioned in the optical reader of the Digora System for later capture of the latent image contained in the active portion of the plate, through laser scanning. The software “Digora for Windows” was used for image analyses. Polímeros, 28(3), 246-255, 2018
2.4 Scanning electron microscopy A sample from the 60-day group was analyzed by scanning electron microscopy. Briefly, the sample was dehydrated with solutions of increasing concentrations of Ethanol (35%, 50%, 75%, 95% and absolute), dried in an oven at 37 °C and subsequently covered with a layer of gold (Sputter Coater SCD 050). The sample image was performed using microscope Leica-Steroscan 440.
2.5 Microscopic analysis After fixation, the pieces were rinsed in tap water and decalcified following the Morse method (50% formic acid and 20% sodium citrate aqueous solution). Subsequently, the microscopic preparation of usual procedures were performed. Three sections with 6μm thick were performed in the region of greater amplitude of the bone defect in the skull. Two of these sections were stained with Masson´s trichrome for histomorphometry, and one, stained with hematoxylin‑eosin (HE). For zygomatic arch experiment, two slices with 6μm thick, one for each stain.
2.6 Morphometric analysis The morphometric analysis was performed on the samples containing bone defect obtained by craniotomy using the Image-Pro Plus Software (Media Cybernetics), installed on a microcomputer coupled to a Nikon Eclipse 80i photomicroscope. Quantification of immature bone tissue, mature fibrovascular stroma in the region of the defect, was performed.
3. Results and Discussions 3.1 Polyurethane as intraosseous graft After the observation periods of 60 and 120 days, the presence of newly formed bone was macroscopically observed in the region of the defect in the pieces of the Treated group. In the Control group, the region of the defect presented scar tissue covering the defect, this tissue was thicker but not rigid like the bone tissue found in the Treated group. Radiographic analysis at 60 days in the Control group samples shows that most of the defect region is filled by a radiolucent area, with small radiopaque areas near the edges of the defect (Figure 4A, red arrows). In the Treated group, 249/255 249
Sousa, T. P. T., Costa, M. S. T., Guilherme, R., Orcini, W., Holgado, L. A., Silveira, E. M. V., Tavano, O., Magdalena, A. G., Catanzaro-Guimarães, S. A., & Kinoshita, A.
Figure 4. Radiograph at 60 days (A and B) and 120 days (C and D) of Control (A and C) and Treated (B and D) groups. (A) Control Group 60 days - there are small radiopaque areas near the edge of the defect (red arrows) and radiolucency in most of the region of the defect; (B) Treated group 60 days - radiopaque areas accompanying the entire extent of the defect (red arrow); (C) Control Group 120 days - areas of radiopacity located on the margin of the defect and radiolucency in most of the central region (yellow arrow); (D) Treated group 120 days - areas of radiopacity at the margins and central region of the defect (red arrows).
most of the region of the defect is filled by a radiolucent area, with radiopaque areas accompanying the edges of the defect and dispersed as isolated areas throughout the defect (Figure 4B, red arrows). At 120 days, digital radiography of the Control group shows a radiopaque area located specifically on the defect margin as in this example, in the right margin region. There are regions of radiolucency at the left end and at the center of the defect (Figure 4C, yellow arrow). In the Treated group, at 120 days, regions of radiopacity are noted in much of the central region of the defect (Figure 4D, red arrows). Figure 5A shows the photomicrography of the Control group bone defect at 60 days. The main features presented are: intramembranous ossification with absence of cartilage, presence of areas of immature bone tissue (black arrow) in the internal and external bone plates, and intertrabecular bone in formation. Figure 5B shows the photomicrograph at 60 days of the Treated group, which received the Polyurethane that was mixed with the clot. There is a repair process by intramembranous ossification with absence of cartilage. Bone regeneration observed around the polyurethane particles is observed throughout the bone defect (black rectangle). Figure 5C shows in higher magnification bone marrow tissue (*), mature bone tissue (+) and immature bone tissue (#). Figure 5D shows the photomicrograph 250 250/255
of the defect of the Control group at 120 days. The main features presented are: bone neofornation in the interparietal suture region (black rectangle) and poor definition of the external and internal bone plates, besides the presence of immature reticular bone tissue interspersed with trabeculae of lamellar bone. Osteogenic connective tissue was still visible in some areas. In the Treated group at 120 days (Figure 5E) the main features seen are: newly formed bone, particles of granulated polyurethane implanted in the surgical bone bed (green arrow), formation of mineralized bone matrix on the surfaces of the osseointegrated particles. In some areas, the remodeling of the bone/particle ensemble shows a mosaic composed of immature bone tissue, lamellar mature bone and osteogenic connective tissue in small proportions (red arrow). In a higher magnification image (Figure 5F), there was no foreign body type inflammatory reaction and areas where remodeling of the bone/particle occurred (green arrow). The material proved to be biocompatible and osteointegratable. Figure 6 shows SEM images of samples retrieved at 60 days post surgery. The images show the integration of polyurethane particles with bone tissue confirming the histological findings. Some particles are indicated by red arrows. Polímeros, 28(3), 246-255, 2018
Polyurethane derived from Ricinus Communis as graft for bone defect treatments Figure 7 shows the histomorphometry of the newly formed bone and fibrovascular stroma (FS) for periods of 60 (Figure 7A) and 120 days (Figure 7B). At 60 days there is a greater amount of newly formed bone (NFB) in the Treated group compared to the Control (p<0.05, t test). Also, the fibrovascular stroma volume is higher in the Control group (p<0.05, t test). However, after 120 days, the amount of bone tissue is the same in both groups (Figure 7B),
despite the amount of fibrovascular stroma being lower in the Treated group. As such, we can note that the presence of the Polyurethane sped up the bone repair process. In a similar study, Boeck-Neto et al.[15] found satisfactory results in facial bone repair. The authors compared the use of calcium phosphate and Ricinus communis, both associated with autogenous bone, to increase the maxillary sinus floor, resulting in a bone mass gain in both groups, but emphasized
Figure 5. Histological section of the bone defect. Masson’s trichrome. (A) Control Group - 60 days (2X). Areas of immature bone tissue are verified (black arrow); (B) Treated group - 60 days (2X). In the region marked by a rectangle, some particles covered by a thin layer of osteogenic connective tissue and immature bone (black arrow) are noticed. At higher magnification (10X) (C) bone marrow tissue (*), mature bone tissue (+) and immature bone tissue (#) are observed; (D) Control Group -120 days (2X). In the region delimited by a rectangle there is a large volume of newly formed bone tissue in the region of the interparietal suture and little definition of external and internal bone plates; (E) Treated group - 120 days (2X) showing advanced bone repair process. At higher magnification (F) (20X) some areas (green arrows) present remodeling of the bone / particle.
Figure 6. SEM images of section of the bone defect 60 days post surgery showing the integration between polyurethane particles (red arrow) and bone tissue. Polímeros, 28(3), 246-255, 2018
251/255 251
Sousa, T. P. T., Costa, M. S. T., Guilherme, R., Orcini, W., Holgado, L. A., Silveira, E. M. V., Tavano, O., Magdalena, A. G., Catanzaro-Guimarães, S. A., & Kinoshita, A. that the graft materials, that were biocompatible, were not completely reabsorbed after 10 months, but integrated into the bone.
3.1 Repair of segmental defect in the zygomatic arch Macroscopically, the pieces obtained from the Treated group with polyurethane and latex membrane were analyzed and showed growth of mineralized bone joining the stumps
of the zygomatic arch. In the Control group, the pieces obtained in periods of 60 and 120 days showed scar tissue in the region of the defect. Figure 8 presents X-ray images of Control and Treated groups in both observation periods, 60 and 120 days. In the images of the Control group at 60 days (Figure 8A), there is a radiopaque area in the stumps of the zygomatic arch (white arrow) and a radiotransparent area in the bone
Figure 7. Mean and standard deviation of volume fraction values of newly formed bone tissue (NFB) and fibrovascular stroma (FS) in bone defects obtained by craniotomy and treated with Polyurethane as a function of observation periods compared to Control group (A) 60 days and (B) 120 days. Statistically significant differences are indicated by (*) and (**) (p <0.05 t-test).
Figure 8. Digital radiography of the rabbit zygomatic arch of the Control and Treated groups at 60 days (A and B) and 120 days (C and D). (A) In the image of the Control group at 60 days the zygomatic arch stump region (red arrows) can be seen and the entire bone defect region with radiolucency (wide red arrow); (B) In the Treated group - 60 days there is an area of radiopacity connecting the stumps of the bony defect forming a bridge between the stumps (green arrow); (C) In the image of the Control group - 120 days radiolucency (wide arrow) in the region of the defect can also be seen; (D) In the Treated group at 120 days, a radiopaque area can be seen connecting the bony defect stumps, forming a bony bridge between them. 252 252/255
Polímeros, 28(3), 246-255, 2018
Polyurethane derived from Ricinus Communis as graft for bone defect treatments defect region (red arrow). In the Treated group (Figure 8B) there is a narrow region of radiopacity joining the stumps, forming a bridge between them (green arrow). In the images of the Control group - 120 days (Figure 8C) there is a radiotransparency in the center of the bone defect (red arrow). In this example, there is a small region of greater radiopacity in an area in the top margin to the right of the defect. The radiographic image of the Treated group at 120 days (Figure 8D) there is a homogeneous and greater radiopacity area joining the stumps, going toward the center of the defect, forming a thicker bone bridge compared to the 60-day period. In this example there is a thin radiotransparent line in the center of the defect in an s shape (green arrow). Figure 9A shows a photomicrograph of the region of the bone defect created in the zygomatic arch of the Control group 60 days post-surgery. The main structures presented are: incomplete bone repair process, with the presence of immature bone tissue and fibrous connective tissue (black arrow). In contrast, in the Treated group with polyurethane and Latex membrane (Figure 9B) there is a repair process via intramembranous ossification, with absence of cartilage. The complete covering of the bone defect by guided bone regeneration through the polyurethane
particles associated to latex membrane can also seen. In addition, the bone trabeculae under growth (green arrow) are observed along the edges of the bone surgical cavity and organized bony structure in the margin of the bone surgery (black arrow). At higher magnification, osteogenic connective tissue involving particles of material implanted in metabolization process can be seen. A cement line on the surface of the particles is also present. (Figure 9E, black arrow). Figure 9C presents a photomicrograph of the bone defect in the zygomatic arch for the 120-day period of the Control group. The main characteristics presented are: still incomplete bone repair process, with the presence of immature bone tissue and fibrous connective tissue (black arrow). In the Treated group at 120 days (Figure 9D) the main characteristics present are: a repair process via intramembranous ossification, with absence of cartilage, the covering of the bone defect by bone regeneration guided through the particles from the implanted material. At higher magnification (Figures 9F and G) the presence of newly formed bone tissue involving the polyurethane particles is notice, also areas under an active remodeling process (Figure 9F marked with *). Bone trabeculae that separated the particles among themselves, but integrated bone/particles
Figure 9. Photomicrographs of the histological sections of the bone defect in the zygomatic arch region (Masson’s trichrome stain) (A) Control group - 60 days (2X) there is immature bone tissue presence and incomplete bone repair; (B) 60-days treated group (2X) the particles present in the region of the defect (green arrow) and growing bone trabeculae (black arrow) and organized bone structure on the margin of the surgical bone defect (blue arrow) are observed. At higher magnification (10X) (E) it is possible to observe the deposition of a cement line on the surface of the particles (black arrows,10X); (C) The Control group at 120 days (2X) immature bone tissue (black arrow) and incomplete bone repair process (green arrow) are observed; (D) Treated group - 120 days (2X)presents the formation of the bone bridge attached to the stumps, and presence of newly formed bone on the border of the defect (black arrow). At higher magnification (F) (20X) particles implanted in areas under active remodeling process (*) can be seen. In (G), (20X) polyurethane particle with metabolic signs (*) becomes rarefied and leaves empty spaces between the growing bone trabeculae. Polímeros, 28(3), 246-255, 2018
253/255 253
Sousa, T. P. T., Costa, M. S. T., Guilherme, R., Orcini, W., Holgado, L. A., Silveira, E. M. V., Tavano, O., Magdalena, A. G., Catanzaro-Guimarães, S. A., & Kinoshita, A. with the bone cavity walls is also noticed (Figure 9F, G). Polyurethane particles with signs of metabolization becoming rarefied (Figure 9G marked with *) and leaving empty spaces between growing bone trabeculae. These results are in agreement with a previous study conducted by Pereira[14], who compared the polyurethanes containing castor oil in granular form with a spongy autogenous bone graft applied to a segmental bone defect in rabbit and concluded that the regeneration process was more evident and accelerated in the bone defects treated with spongy bone autotransplantation, but the polyurethane also induced bone regeneration. The authors described that the polyurethane acts as a filling material, minimizing the local production of fibrous tissue, similar to that found in this present work. In addition, in our work we also noted the absence of inflammatory reaction type foreign body and that the material is biocompatible and osteointegrable. Histological results in both experiments (Figures 5 and 9) demonstrated more pronounced bone formation in defects with granulated polyurethane graft as compared to their respective controls which can also be seen in x-ray images. In addition, the osseointegration of the polyurethane particles in contact with immature bone tissue and osteogenic connective tissue can be observed both in optical and scanning electron microscopy images.
4. Conclusions Polyurethane is a biocompatible, osteoconductive and osteointegrable material, which promotes new bone formation in both bone defects studied, it thus being an interesting option in bone defect treatments. The association with a latex membrane produced regeneration of segmental bone defects.
5. Acknowledgements The authors are grateful to Maira Couto for the technical assistance and FAPESP (São Paulo Research Foundation) for partial financial support.
6. References 1. Derceli, J. R., Fais, L. M., & Pinelli, L. A. (2014). A castor oil-containing dental luting agent: effects of cyclic loading and storage time on flexural strenght. Journal of Applied Oral Science, 22(6), 496-501. http://dx.doi.org/10.1590/1678775720140069. PMid:25591018. 2. Monteiro, A. S., Macedo, L. G., Macedo, N.-L., & Balducci, I. (2010). Polyurethane and PTFE membranes for guided bone regeneration: histopathological and ultrastructural evaluation. Medicina Oral, Patologia Oral y Cirugia Bucal, 15(2), e401-e406. http://dx.doi.org/10.4317/medoral.15.e401. PMid:19767699. 3. Lim, T. K. (2012). Ricinus communis. In T. K. Lim (Ed.), Edible medicinal and non-medicinal plants. Netherlands: Springer. http://dx.doi.org/10.1007/978-94-007-1764-0_64. 4. Eglin, D., Grad, S., Gogolewski, S., & Alini, M. (2010). Farsenol modified biodegradable polyurethanes for cartilage tissue engineering. Journal of Biomedical Materials Research. Part A, 92(1), 393-408. http://dx.doi.org/10.1002/jbm.a.32385. PMid:19191318. 254 254/255
5. Laureano, J. R., Fo., Andrade, E. S. S., Albergaria-Barbosa, J. R., Camargo, I. B., & Garcia, R. R. (2009). Effects of demineralized bone matrix and a ‘Ricinus communis’ polymer on bone regeneration: a histological study in rabbit calvaria. Journal of Oral Science, 51(3), 451-456. http://dx.doi. org/10.2334/josnusd.51.451. PMid:19776514. 6. Barros, V. M. R., Rosa, A. L., Beloti, M. M., & Chierice, G. (2003). In vivo biocompatibility of three different chemical compositions of Ricinus communis polyurethane. Journal of Biomedical Materials Research. Part A, 67(1), 235-239. http:// dx.doi.org/10.1002/jbm.a.10105. PMid:14517881. 7. Cangemi, J. M., Claro, S., No., Chierice, G. O., & Santos, A. M. (2006). Study of the biodegradation of a polymer derived from castor oil by scanning electron microscopy, thermogravimetry and infrared spectroscopy. Polímeros: Ciência e Tecnologia, 16(2), 129-135. http://dx.doi.org/10.1590/ S0104-14282006000200013. 8. Trovati, G., Sanches, E. A., Claro, S., No., Mascarenhas, Y. P., & Chierice, G. O. (2009). Characterization of polyurethane resins by FTIR, TGA, and XRD. Journal of Applied Polymer Science, 115(1), 263-268. http://dx.doi.org/10.1002/app.31096. 9. Jena, J., & Gupta, A. K. (2012). Ricinus communis linn: a phytopharmacological review. International Journal of Pharmacy and Pharmaceutical Sciences, 4(4), 25-29. Retrieved in 2017, May 2, from http://www.ijppsjournal.com/Vol4Issue4/4695. pdf 10. Leite, F. R. M., & Ramalho, L. T. O. (2008). Bone regeneration after demineralized bone matrix and castor oil (Ricinus communis). Journal of Applied Oral Science, 16(2), 122-126. http://dx.doi. org/10.1590/S1678-77572008000200008. PMid:19089203. 11. Belmonte, G. C., Catanzaro-Guimarães, S. A., Sousa, T. P. T., Carvalho, R. S., Kinoshita, A., & Chierici, G. O. (2013). Qualitative histologic evaluation of the tissue reaction to the polyurethane resin (ricinus communis-based biopolymer) implantation assessed by light and scanning electron microscopy. Polímeros: Ciência e Tecnologia, 23(4), 462-467. http://dx.doi. org/10.4322/polimeros.2013.063. 12. Saran, W. R., Chierice, G. O., Silva, R. A. B., Queiroz, A. M., Paula-Silva, F. W. G., & Silva, L. A. B. (2014). Castor oil polymer induces bone formation with high matrix metalloproteinase‐2 expression. Journal of Biomedical Materials Research. Part A, 102(2), 324-331. http://dx.doi.org/10.1002/jbm.a.34696. PMid:23670892. 13. Frazilio, F. O., Rossi, R., Negrini, J. M., No., Facco, G. G., Ovando, T. M., & Fialho, M. P. F. (2006). Use of castor oil polyurethane in an alternative technique for medial patella surgical correction in dogs. Acta Cirurgica Brasileira, 21(Suppl 4), 74-79. http://dx.doi.org/10.1590/S0102-86502006001000016. PMid:17293971. 14. Pereira-Júnior, O. C. M., Rahal, S. C., Iamaguti, P., Felisbino, S. L., Pavan, P. T., & Vulcano, L. C. (2007). Comparison between polyurethanes containing castor oil (soft segment) and cancellous bone autograft in the treatment of segmental bone defect induced in rabbits. Journal of Biomaterials Applications, 21(3), 283-297. http://dx.doi.org/10.1177/0885328206063526. PMid:16543284. 15. Boeck-Neto, R., Gabrielli, M., Shibli, J., Marcantonio, E., Lia, R. C. C., & Marcantonio, E., Jr. (2005). Histomorphometric evaluation of human sinus floor augmentation healing responses to placement of calcium phosphate or ricinus communis polymer associated with autogenous bone. Clinical Implant Dentistry and Related Research, 7(4), 181-188. http://dx.doi. org/10.1111/j.1708-8208.2005.tb00063.x. PMid:16336909. 16. Rana, M., Dhamija, H., Prashar, B., & Sharma, S. (2012). Ricinus communis L.: a review. International Journal of Chemtech Research, 4(4), 706-711. Retrieved in 2017, May 2, from http:// Polímeros, 28(3), 246-255, 2018
Polyurethane derived from Ricinus Communis as graft for bone defect treatments sphinxsai.com/2012/oct-dec/Pharmpdf/PT=48(1706-1711) OD12.pdf 17. Beloti, M. M., Oliveira, P. T., Tagliani, M. M., & Rosa, A. L. (2008). Bone cell responses to the composite of Ricinus communis polyurethane and alkaline phosphatase. Journal of Biomedical Materials Research. Part A, 84(2), 435-441. http:// dx.doi.org/10.1002/jbm.a.31344. PMid:17618485. 18. Nacer, R. S., Poppi, R. R., Carvalho, P. D. T. C., Silva, B. A. K., Odashiro, A. N., Silva, I. S., Delben, J. R. J., & Delben, A. A. S. T. (2012). Castor oil polyurethane containing silica nanoparticles as filling material of bone defect in rats. Acta Cirurgica Brasileira, 27(1), 56-62. http://dx.doi.org/10.1590/ S0102-86502012000100010. PMid:22159440. 19. Barros, V. M., Rosa, A. L., Beloti, M. M., & Chierice, G. (2003). In vivo biocompatibility of three different chemical compositions of Ricinus communis polyurethane. Journal of Biomedical Materials Research. Part A, 67(1), 235-239. http:// dx.doi.org/10.1002/jbm.a.10105. PMid:14517881. 20. Graça, Y. L. S. S., Opolski, A. C., Barboza, B. E. G., Erbano, B. O., Mazzaro, C. C., Klostermann, F. C., Sucharski, E. E., & Kubrusly, L. F. (2014). Biocompatibility of Ricinus communis polymer with addition of calcium carbonate compared to titanium: experimental study in guinea pigs. Revista Brasileira de Cirurgia Cardiovascular, 29(2), 272-278. http://dx.doi. org/10.5935/1678-9741.20140030. PMid:25140479. 21. Mendonça, R. J., Maurício, V. B., Teixeira, Lde. B., Lachat, J. J., & Coutinho-Netto, J. (2010). Increased vascular permeability, angiogenesis and wound healing induced by the serum of natural latex of the rubber tree Hevea brasiliensis. Phytotherapy Research, 24(5), 764-768. http://dx.doi.org/10.1002/ptr.3043. PMid:19943314. 22. Herculano, R. D., Silva, C. P., Ereno, C., Guimaraes, S. A. C., Kinoshita, A., & Graeff, C. F. O. (2009). Natural rubber latex used as drug delivery system in guided bone regeneration (GBR). Materials Research, 12(2), 253-256. http://dx.doi. org/10.1590/S1516-14392009000200023. 23. Ereno, C., Guimarães, S. A. C., Pasetto, S., Herculano, R. D., Silva, C. P., Graeff, C. F. O., Tavano, O., Baffa, O., & Kinoshita, A. (2010). Latex use as an occlusive membrane for guided bone regeneration. Journal of Biomedical Materials Research. Part A, 95(3), 932-939. http://dx.doi.org/10.1002/ jbm.a.32919. PMid:20845492. 24. Moura, J. M. L., Ferreira, J. F., Marques, L., Holgado, L., Graeff, C. F. O., & Kinoshita, A. (2014). Comparison of the performance of natural latex membranes prepared with different procedures and PTFE membrane in guided bone regeneration (GBR) in rabbits. Journal of Materials Science: Materials in Medicine, 25(9), 2111-2120. http://dx.doi.org/10.1007/s10856014-5241-1. PMid:24849612. 25. Floriano, J., Mota, L., Furtado, E., Rossetto, V., & Graeff, C. O. (2013). Biocompatibility studies of natural rubber latex from different tree clones and collection methods. Journal
Polímeros, 28(3), 246-255, 2018
of Materials Science. Materials in Medicine, 25(2), 461-470. http://dx.doi.org/10.1007/s10856-013-5089-9. PMid:24202915. 26. Balabanian, C. A. C. A., Coutinho-Netto, J., Lamano-Carvalho, T. L., Lacerda, S. A., & Brentegani, L. G. (2006). Biocompatibility of natural latex implanted into dental alveolys of rats. Journal of Oral Science, 48(4), 201-205. http://dx.doi.org/10.2334/ josnusd.48.201. PMid:17220617. 27. Paula, J. S., Ribeiro, V. R. C., Sampaio, R. B., Mendonca, R. J., Haddad, A., Tedesco, A. C., Coutinho-Netto, J., Haendchen, H. A., & Jorge, R. (2011). Rabbit Rubeosis Iridis Induced by Intravitreal Latex-derived Angiogenic Fraction. Current Eye Research, 36(9), 857-859. http://dx.doi.org/10.3109/027136 83.2011.576797. PMid:21599469. 28. Ferreira, M., Mendonça, R. J., Coutinho-Netto, J., & Mulato, M. (2009). Angiogenic properties of natural rubber latex biomembranes and the serum fraction of Hevea brasiliensis. Brazilian Journal of Physics, 39(3), 564-569. http://dx.doi. org/10.1590/S0103-97332009000500010. 29. Herculano, R. D., Silva, C. P., Ereno, C., Guimarães, S. A. C., Kinoshita, A., & Graeff, C. F. O. (2009). Natural rubber latex used as drug delivery system in guided bone regeneration (GBR). Materials Research, 12(2), 253-256. http://dx.doi. org/10.1590/S1516-14392009000200023. 30. Guidelli, É. J., Kinoshita, A., Ramos, A. P., & Baffa, O. (2013). Silver nanoparticles delivery system based on natural rubber latex membranes. Journal of Nanoparticle Research, 15(4), 1536. http://dx.doi.org/10.1007/s11051-013-1536-2. 31. Herculano, R. D., Tzu, L. C., Silva, C. P., Brunello, C. A., Queiroz, Á. A. A., Kinoshita, A., & Graeff, C. F. O. (2011). Nitric oxide release using natural rubber latex as matrix. Materials Research, 14(3), 355-359. http://dx.doi.org/10.1590/ S1516-14392011005000055. 32. Hollinger, J. O., & Kleinschmidt, J. C. (1990). The critical size defect as an experimental model to test bone repair materials. The Journal of Craniofacial Surgery, 1(1), 60-68. http://dx.doi. org/10.1097/00001665-199001000-00011. PMid:1965154. 33. Garber, J. C. (2011). Guide for the care and use of laboratory animals. Washington: National Academies Press. Retrieved in 2017, May 2, from https://grants.nih.gov/grants/olaw/guidefor-the-care-and-use-of-laboratory-animals.pdf 34. Neves-Junior, W. F. P., Ferreira, M., Alves, M. C. O., Graeff, C. F. O., Mulato, M., Coutinho-Netto, J., & Bernardes, M. S. (2006). Influence of fabrication process on the final properties of natural-rubber latex tubes for vascular prosthesis. Brazilian Journal of Physics, 36(2B), 586-591. http://dx.doi.org/10.1590/ S0103-97332006000400021. Received: May 02, 2017 Revised: Sept. 25, 2017 Accepted: Oct. 22, 2017
255/255 255
ISSN 1678-5169 (Online)
http://dx.doi.org/10.1590/0104-1428.03417
O O O O O O O O O O O O O O O O
Polyvinyl alcohol (PVA) molecular weight and extrusion temperature in starch/PVA biodegradable sheets Juliano Zanela1,2*, Ana Paula Bilck1, Maira Casagrande2, Maria Victória Eiras Grossmann1 and Fabio Yamashita1 Departamento de Ciência e Tecnologia de Alimentos – DCTA, Centro de Ciências Agrárias – CCA, Universidade Estadual de Londrina – UEL, Londrina, PR, Brasil 2 Universidade Tecnológica Federal do Paraná – UTFPR, Dois Vizinhos, PR, Brasil
1
*julianozanela@gmail.com
Abstract The aim of this work was to study the relationship of chain size of partially hydrolyzed PVA blended with starch in properties of biodegradable sheets produced by thermoplastic extrusion. It was also studied the effect of extrusion temperature profile to determine the better PVA chain size and temperature profile to produce biodegradable sheets through a factorial design. The processability and the mechanical, thermal, optical, and microstructural properties of the biodegradable sheets were adequate, indicating that PVA/cassava starch blends have potential to replace conventional non-biodegradable polymers. Tensile strength and Young’s modulus ranges from 1.0 to 2.6 and 3.0 to 6.9 MPa respectively, the elongation at break ranges from 42 to 421%. It was not possible to state a conclusive relationship between PVA molecular weight and the materials properties, but in general, PVA with medium molecular weight and high extrusion temperature profile promote an increase of mechanical properties of the sheets. Keywords: calendering, experimental design, mechanical properties.
1. Introduction Petroleum based plastics are used worldwide in a crescent number of application areas, and the disposal of plastics residues is an environmental problem. In the last years, several researchers have studied new eco-friendly materials preferentially from renewable resources to replace conventional petrochemical polymers[1,2]. Starch is one of most studied biopolymer for biodegradable materials production, due to its low cost, biodegradability, and from renewable sources[3], but pure starch presents some drawbacks to replace conventional polymers due to its inherent brittleness, poor mechanical properties, and the hydrophilic character. These drawbacks can be overcome by blending starch with other biodegradable polymers like polyvinyl alcohol - PVA[4]. PVA is an odorless, nontoxic, water-soluble, and fully biodegradable polymer that presents good film-forming capacity, resistance to greases and oil, good mechanical properties, and good barrier to oxygen and aroma[3]. According to Majdzadeh-Ardakani and Nazari[5], the coprocessing of starch with polar polymers, like PVA, can improve the mechanical properties when compared to pure starch materials, and several studies reported a good compatibility of these polymers in starch/PVA blends. Mao et al.[6] produced sheets by extrusion using cornstarch plasticized with glycerol (30% w/w), with tensile strength of 1.8 MPa and elongation at break of 113%, and adding PVA (9.1% w/w) the tensile strength and elongation at break were enhanced to 4 MPa and 150% respectively. According to the authors, the material with PVA had no cracks, unlike that
256 256/265
observed by SEM in materials without PVA. Similar results was observed by Ray et al.[7], that produced casting films with starch:PVA:glycerol of 60:40:30 and 50:50:30 (wt%). Films with higher PVA content had better mechanical properties, and the polymers dispersion in the blend was improved, creating a more homogeneous network as observed by SEM and FT-IR. Therefore, PVA and starch have a good compatibility for the production of biodegradable materials. Zanela et al.[8] produced extruded sheets based on different proportions of starch, PVA and glycerol through a mixture design. The authors observed that all formulations were homogeneous, without visible cracks by SEM, demonstrating a good miscibility between both polymers, and with the increasing, the level of PVA present in the blends, the mechanical and barrier properties of the sheets were increased. There are several PVA grades, with different molecular weights and chain sizes, due to their large field of application, so it is important to study how these characteristics affect the properties of the films. Limpan et al.[9] evaluate the influence of PVA with different hydrolysis degree and molecular weight in properties of fish myofibrillar protein/PVA blends with glycerol as plasticizer. The author observed that PVA with higher molecular weight improves tensile strength and elongation at break, and PVA with higher hydrolysis degree led to films that were more rigid. Silva et al.[10] produced films by casting using PVA with different hydrolysis degree and pigskin gelatin, and they observed that the hydrolysis degree influenced the properties
Polímeros, 28(3), 256-265, 2018
Polyvinyl alcohol (PVA) molecular weight and extrusion temperature in starch/PVA biodegradable sheets of the films, but they were unable to find a relationship of the hydrolysis degree with the physical properties of the films.
desirability of responses, was determined using the Experimental Design proceeding of Statistica 7.0 (Statsoft, USA) software.
The goal of this work was to study the relationship of chain size of partially hydrolyzed PVA blended with starch in properties of biodegradable sheets produced by thermoplastic extrusion. It was also studied the effect of extrusion temperature profile to determine the better PVA concentration and temperature profile to produce biodegradable sheets through a factorial design.
2.3 Sheet production All the formulations were composed of 38 wt% of starch, 27 wt% of PVA and 35 wt% of glycerol. After manual homogenization, the samples were placed in a vacuum oven (model Q819V2, Quimis, Brazil) with a vacuum pressure of 0.085 MPa for 90 minutes at 85 °C to incorporate the glycerol using the methodology adapted from Jang and Lee[11]. After this step, the blends were extruded in a co-rotating twin screw extruder (model D-20, BGM, Brazil) with a screw diameter of 20 mm (L/D = 35), a screw speed of 100 RPM and a temperature profile according to the experimental design based in Table 1. The samples were extruded using a flat die with 0.8 mm height and 320 mm length, coupled with a 3-roll water-cooled calender (AX Plásticos, Brazil) for sheets production.
2. Materials and Methods 2.1 Materials It was used three PVA grades (Sekisui Chemical, Japan) with different degrees of hydrolysis (DH) and chain sizes (based on their viscosity in 4% aqueous solution): Selvol™ 203 (DH: 88.14%, viscosity 4.10 cP); Selvol™ 523 (DH: 87.84%, viscosity: 24.50 cP) and Selvol™ 540 (DH: 88.04%, viscosity: 49.40 cP); native cassava starch (Indemil, Brazil) and glycerol (Dinamica, Brazil).
2.4 Mechanical properties The tensile strength, Young’s modulus, and elongation at break were analyzed in a texture analyzer (model TA.XT2i, Stable Micro Systems, England) with an initial distance between the grips of 30 mm and a crosshead speed of 0.8 mm.s-1, according to ASTM D882-02[12] method, with some modifications. Ten samples from each treatment (50 mm in length and 20 mm in width) were conditioned in a desiccator with controlled relative humidity and temperature (53 ± 2% and 23 ± 2 °C respectively) for 72 hours before analysis. For puncture analysis, ten specimens from each treatment were conditioned as described above and punctured perpendicularly with a 6.35 mm diameter cylindrical probe at a velocity of 2.0 mm.s-1. The puncture elongation (mm) was characterized as the maximum elongation supported by the sheet. The puncture strength (N/mm) was obtained by dividing the maximum force by the sheet thickness.
2.2 Factorial design It was used a 32 factorial design with replicate in the central point (total of 10 runs), the independent variables were PVA grade and barrel temperature profile of the five heating zones of the extruder, and the coded and real values for all runs are shown in Table 1. The factorial designs were analyzed using the Experimental Design proceeding of Statistica 7.0 (Statsoft, USA) software. The 2-way interaction equation (Equation 1) was used to modeling the responses: y =β0 +β1x1 +β11x12 +β2 x2 +β22 x22 + β12 x1x2 +β122 x1x22 +β112 x12 x2 +β1122 x12 x22
(1)
where: y is the dependent variable (response); β is the regression coefficient of each term; x1 is the PVA grade; x2 is the extrusion temperature profile.
2.5 Water Vapor Permeability (WVP) Water vapor permeability was determined gravimetrically according to the ASTM E96-009[13] standard. The measurements were performed using a relative humidity gradient of 33-64%.
The desirability function, the relationship between predicted responses on one or more dependent variables and the Table 1. Coded and real values for 32 factorial design. Formulation
PVA Grade X1
1 2 3 4 5 6 7 8 9 10
-1 -1 -1 0 0 0 1 1 1 0
Coded value Temperature Profile X2 -1 0 1 -1 0 1 -1 0 1 0
PVA Grade* X1 S 203 S 203 S 203 S 523 S 523 S 523 S 540 S 540 S 540 S 523
Real value Temperature Profile**X2 170 °C 190 °C 210 °C 170 °C 190 °C 210 °C 170 °C 190 °C 210 °C 190 °C
* S 203: Selvol™ 203; S 523: Selvol™ 523; S 540: Selvol™ 540; ** 170 °C: 90/170/170/170/170 °C; 190 °C: 90/170/190/190/190 °C; 210 °C: 90/170/210/210/210 °C.
Polímeros, 28(3), 256-265, 2018
257/265 257
Zanela, J., Bilck, A. P., Casagrande, M., Grossmann, M. V. E., & Yamashita, F. 2.6 Weight Loss in Water (WLW) The WLW analysis was performed according to Olivato et al.[14], in triplicate and expressed as the percentage of the original mass (Mi) and the final mass (Mf) of the film after immersion in water for 48 hours at 25 °C, according to Equation 2. WLW = ( Mi − M f ) / Mi ×100
(2)
2.7 Apparent Opacity (Op) The Op values of the films were measured according to the method described by Maria et al.[15], using a colorimeter (BYK Gardner, Germany) with illuminant D65 (daylight) and visual angle of 10°. Opacity (Op) was determined as the ratio of the opacity of the sample over a black standard Opb and the opacity over a white standard Opw being represented on an arbitrary scale (0-100%), and the analyses were performed in triplicate according to Equation 3. Op = (Op b /Op w ) × 100
(3)
2.8 X-ray Diffraction (XRD) XRD analysis was performed using a diffractometer (Panalytical X´Pert PRO MPD, Netherlands), emitting copper Kα radiation (λ = 1.5418 angstrom). The anode radiation was generated at 40 kV and 50 mA and was monochromatized using a 20 mA current. Diffraction intensity measurements were performed between 2θ = 2° to 60° at room temperature. The relative crystallinity of each film was calculated by dividing the area of the strongest peaks by the total area of the crystalline region (the total area under the curve minus the baseline).
2.9 Scanning Electron Microscopy (SEM) SEM was recorded using a scanning electron microscope (FEI Quanta 200, USA). The films were fractured in liquid nitrogen, attached to aluminum supports and coated with gold
(BAL-TEC SCD 050 sputter coater, Leica Microsystems, Germany) (40-50 nm in thickness) at 25 °C and a pressure of 2.105 Torr for 180 seconds. The surface and the fracture surface of the films were analyzed.
2.10 Fourier Transform Infrared Spectroscopy (FT-IR) The samples were dried over anhydrous calcium chloride salt for one week and analyzed in a Fourier transform infrared spectrophotometer (FT-IR) (IR Prestige 21, Shimadzu, Japan) using a horizontal attenuated total reflection (ATR) module operating over the spectral range of 4000-750 cm-1.
2.11 Thermogravimetric Analysis (TGA) Thermogravimetric analysis was performed using a TGA – 50 (Shimadzu, Japan). The samples were dried over anhydrous calcium chloride salt and analyzed from 25 °C to 600 °C with a 10 °C.min-1 heating rate under a nitrogen atmosphere (20 mL.min-1).
3. Results and Discussions The sheets of all formulation were continuous and visually homogenous, with a medium thickness of 850±191 µm. Most of the equations generated by the mathematical model had coefficients of determination (R2) higher than 0.70, indicating that the models fitted the experimental data well.
3.1 Mechanical properties Table 2 presents the factorial design models (Equation 1) for the mechanical properties of the sheets, Table 3 presents the experimental data, and the values predicted by the model, and Figure 1 presents the response surface plots. The tensile strength of the sheets ranged from 1.0 to 2.6 MPa, and according to the factorial design model the tensile strength was influenced mainly by the PVA grade (Table 2 and Figure 1A), in that the medium chain size PVA (S503) promoted the more resistant material.
Table 2. Factorial design models for the mechanical properties of the biodegradable sheets. Tensile strength
Young’s modulus
Elongation at break
Puncture strength
Puncture elongation
β0
(MPa) 1.68
(MPa) 4.80
(%) 184
(N/mm) 68.54
(mm) 12.75
β1
0.23
– 0.48
68
11.26
4.02
β11
0.43
ns
91
13.43
3.35
β2
0.10
– 0.95
94
ns
3.78
β 22
-0.06
– 1.04
29
-2.95
1.0
β12
0.20
– 0.54
60
9.24
3.18
Coefficient
β122
ns
ns
ns
ns
ns
β112
0.16
– 0.26
46
ns
ns
β1122
0.08
ns
26
ns
0.87
R2
0.85
0.92
0.93
0.65
0.94
a y =β 0 + β1x1 + β11x12 + β 2 x2 + β 22 x22 + β12 x1x2 + β122 x1x22 + β112 x12 x2 + β1122 x12 x22 ; y = response; x1 = PVA grade; x2 = extrusion temperature profile; ns = not significant; R² = Coefficient of determination.
258 258/265
Polímeros, 28(3), 256-265, 2018
Polyvinyl alcohol (PVA) molecular weight and extrusion temperature in starch/PVA biodegradable sheets Table 3. Experimental and predicted data for mechanical properties of the sheets. Formulation 1 2 3 4 5 6 7 8 9 10
Tensile strength (MPa) Exp* Pred 1.5±0.2 1.4 1.0±0.1 1.0 1.0±0.1 1.0 1.9±0.2 1.9 2.0±0.3 2.3 2.5±0.2 2.5 1.5±0.1 1.5 1.5±0.1 1.5 1.9±0.1 1.9 2.6±0.2 2.3
Young’s modulus (MPa) Exp Pred 6.2±0.9 6.2 3.6±0.2 3.9 5.8±0.3 5.7 6.9±0.3 6.8 3.2±0.2 3.4 4.3±0.3 4.2 6.2±0.3 6.3 3.0±0.2 2.9 3.6±0.1 3.7 3.9±0.3 3.4
Elongation at break (%) Exp Pred 42±5 44 74±7 70 49±5 51 107±43 107 358±76 389 418±51 418 62±10 60 203±34 207 310±55 308 421±51 389
Puncture strength (N/mm) Exp Pred 64±6 59 41±1 44 40±3 41 85±15 88 82±12 82 92±7 88 64±10 63 70±28 67 78±12 82 84±6 82
Puncture elongation (mm) Exp Pred 5.9±0.3 5.6 6.8±0.2 7.0 6.8±0.1 6.8 11.7±3.0 12.0 18.7±1.7 20.1 19.9±0.6 19.6 7.3±0.7 7.3 15.4±2.0 15.1 20.9±1.1 21.2 21.5±1.0 20.1
*Exp – experimental data; Pred –predicted by the model.
Figure 1. Surface response plot for tensile strength (A); Young’s modulus (B); elongation at break (C); puncture strength (D); puncture elongation (E) and water vapor permeability - WVP (F) of the biodegradable sheets. Polímeros, 28(3), 256-265, 2018
259/265 259
Zanela, J., Bilck, A. P., Casagrande, M., Grossmann, M. V. E., & Yamashita, F. The Young’s modulus ranged from 3.0 to 6.9 MPa, and according to the factorial design model, the modulus was negatively influenced by both the PVA grade and the temperature profile (Table 2 and Figure 1B). According to the model the interaction between PVA and temperature profile of extrusion was negative, i.e., lower temperature profiles increased the values of Young’s modulus independent of the PVA grade. The elongation at break ranged from 42 to 421%, and according to the factorial design model, the elongation was influenced by both the PVA grade and the temperature profile (Table 2 and Figure 1C), and there was a positive interaction between PVA and temperature profile of extrusion. The materials produced with PVA S503 (medial DH) and the higher extrusion temperature profile (90/170/190/190/190 °C) presented the higher elongation values. Mao et al.[6] extruded films of cornstarch (61 wt%), PVA (9 wt%), and glycerol (30 wt%), and the films had a tensile strength of 4 MPa and elongation at break of 150% at conditioning humidity of 50%. The PVA used by the authors was similar to S540, and the tensile strength was higher, and the elongation was lower than those obtained in this study. These differences can be attributed to the PVA concentration in the films, because according to Zanela et al.[8] the concentration of PVA was the main factor for increasing the tensile strength, elongation at break and Young modulus of cassava starch/PVA biodegradable sheets. According to Follain et al.[16], the PVA molecules are stretched and aligned during the extrusion, promoting a good interaction with starch, and higher temperatures enhance these interactions because the PVA reaches the melting temperature. According to Sin et al.[17], the neat PVA (fully hydrolyzed and with medium chain size) has a melting temperature of 207 °C, and when plasticized with 40 parts per hundred parts of resin (phr) of glycerol, the melting point reduces to 177 °C. Therefore, higher temperatures can melt the PVA completely, and the shear forces in extrusion process promoted a better interaction with polymer chains. Thus, these facts explain the tendency of increasing tensile strength and elongation at break of the materials with increasing the extrusion temperature profile.
puncture elongation was produced with PVA S540 (+1) and temperature profile of 90/170/210/210/210 °C (+1).
3.2 Water Vapor Permeability (WVP) The WVP of the sheets ranged from 3.4 to 8.2 x10-10 g.m-1.s-1.kPa-1, and according to the factorial design model the WVP were influenced mainly by the PVA grade (Table 4 and Figure 1F), and there was a positive interaction between quadratic terms of PVA and temperature profile of extrusion. The sheets with the lower WVP was produced with PVA S203 (-1) and temperature profile of 90/170/210/210/210 °C (+1). Sheets produced with lower molecular weight PVA had lower WVP, probably because the PVA with lower MW can originate a more compact network with starch, that difficult the transport of water molecules through the polymeric matrix. Limpan et al.[9] had similar results in films produced with fish myofibrillar protein blended with different PVA grades and according to the authors, PVA with higher molecular weight increase the disorder in the amorphous region, promoting an increase in the free volume between the polymer chains. Zanela et al.[8] obtained WVP values ranging from 3.0 to 8.56 x10-10 g.m-1.s-1.kPa-1 for cassava starch/PVA (S203) sheets using glycerol as plasticizer and PVA ranging from 7.5 to 22.5 wt%. The authors observed that glycerol was the main factor for WVP increasing due to its plasticizer properties. Wang et al.[18] also observed an increase of WVP in casting films of PVA/xylan with high plasticizer content, because glycerol is a small hydrophilic molecule that allows its inclusion between polymers chains, increasing the free volume and so permitting water mobility through the material. Table 4. Regression coefficients of the factorial design models for water vapor permeability, weight loss in water, and apparent opacity of the biodegradable sheets. Coefficienta
β0
The puncture strength of the sheets ranged from 40 to 92 N/mm, and according to the factorial design model, the puncture strength was influenced mainly by the PVA grade (Table 2 and Figure 1D). There was a positive interaction between PVA and temperature profile of extrusion, and the materials produced with intermediate PVA grade level (S503) had the higher puncture strength values. The puncture elongation ranged from 5.9 to 21.5 mm, and according to the factorial design model the puncture elongation were influenced by both the PVA grade and the temperature profile (Table 2 and Figure 1E), and there was a positive interaction between PVA and temperature profile of extrusion. The material with the higher 260 260/265
a
Water vapor Weight loss Apparent permeability in water opacity (%) -1 -1 -1 10 (g.m .s .kPa ) (x10 ) (%) 4.99 46.66 39.52
β1
1.32
– 6.39
– 4.31
β11
ns
ns
– 3.20
β2
ns
ns
3.44
β 22
ns
ns
– 1.06
β12
0.53
ns
– 3.38
β122
ns
ns
ns
ns
– 3.31 – 1.35
β112
ns
β1122
0.84
ns
R2
0.71
0.62
y =β 0 + β1x1 +
β11x12
+
β 2 x2 + β 22 x22
+ β12 x1x2 + β122 x1x22
+ β112 x12 x2
0.82 + β1122 x12 x22,
y = response; x1 = PVA grade; x2 = extrusion temperature profile;
ns = not significant; R² = Coefficient of determination.
Polímeros, 28(3), 256-265, 2018
Polyvinyl alcohol (PVA) molecular weight and extrusion temperature in starch/PVA biodegradable sheets 3.3 Weight Loss in Water (WLW) The WLW of the sheets ranged from 36.2 to 55.4%, and according to the factorial design model, the WLW was influenced negatively only by the PVA grade (Table 4). The PVA with higher chain size reduced the material solubility, and according to the manufacturer[19], this behavior is typical of PVA. Limpan et al.[9] observed the same behavior, but the factor of major influence in films solubility was the PVA hydrolysis degree. The surface plot of WLW is shown in Figure 2A. According to Zanela et al.[20], the high WLW can be important for biodegradable sheets, accelerating the biodegradation process when these materials are discarded in the aquatic environment for example.
3.4 Apparent opacity (Op) The Op analysis ranged from 29.1 to 55.3% (Table 5), and according to the factorial design model the Op was influenced negatively by PVA grade and positively by the linear coefficient of temperature profile (Table 4 and Figure 2B), and the interaction between PVA and temperature profile of extrusion was negative. The sheets produced with
PVA S203 were more opaque than those with PVA S523, for all extrusion temperatures. Maria et al. [15] produced casting films with gelatin/PVA/glycerol and they did not find a relationship with Op and PVA grade (hydrolysis degree and molecular weight). The values of Op reported were lower than those obtained in this study, probably because films are thinner than sheets, and the casting process leads to more transparent materials than extrusion. Besides that, the high temperatures in extrusion can degrade some compounds and/or induce non-enzymatic reactions, resulting in yellowish sheets that present greater difference when compared to casting films.
3.5 X-Ray Diffraction (XRD) The biodegradables sheets of all formulations presented similar diffractograms (Figure 3), with a sharp peak at 19.9°, and an overlapped peaks at 17.7° and 23.5°, similar to observed by Zanela et al.[8], and a diffraction peak at 40.6°. Das et al.[21] produced starch:PVA film and they attributed the 19.6° peak to the ordered arrangement of PVA chains in the material, and the 17.7° peak was attributed to plasticized starch, but this peak had little intensity, and it was overlapped by the peak at 19.9°.
Figure 2. Surface response plot for weight loss in water – WLW (A) and apparent opacity (B) of the biodegradable sheets. Table 5. Experimental data and predicted values by the factorial design model for water vapor permeability, weight loss in water, apparent opacity and crystallinity index of the biodegradable sheets. Formulation 1 2 3 4 5 6 7 8 9 10 a
WVP (x1010)a Obsb Pred 3.4±0.1 4.3 3.4±0.1 3.8 3.5±0.3 3.3 4.2±0.3 5.2 5.2±0.6 5.2 4.5±0.1 5.2 5.7±0.2 5.9 5.0±0.2 6.5 7.9±0.1 7.0 8.2±0.1 5.2
Weight loss in water (%) Obs Pred 55.4±0.2 53.0 48.7±2.6 53.0 53.7±1.2 53.0 48.2±1.5 46.7 46.8±3.7 46.7 46.3±0.5 46.7 36.2±9.1 40.3 41.0±2.7 40.3 42.3±4.4 40.3 48.0±3.2 46.7
Apparent opacity (%) Obs Pred 37.2±2.1 37.0 45.3±2.6 45.8 55.3±1.1 55.1 38.1±6.4 38.1 33.7±3.7 31.4 36.2±5.7 36.2 35.0±2.9 35.2 37.6±1.2 37.1 39.5±1.2 39.7 29.1±0.8 31.4
Crystallinity Index (%) 21.2 21.4 21.7 20.0 20.6 20.8 20.7 20.4 21.5 21.9
WVP – Water vapor permeability (g.m-1.s-1.kPa-1); b Obs – experimental data; Pred –predicted by the model.
Polímeros, 28(3), 256-265, 2018
261/265 261
Zanela, J., Bilck, A. P., Casagrande, M., Grossmann, M. V. E., & Yamashita, F. According to Moorthy[22], cassava starch presents diffraction pattern A, C or a mixture of them, and three major peaks were observed in 15.3, 17.1 and 23.5°, but these peaks were not seen in our diffractograms, and probably they were overlapped by the 19.9° sharp peak. The crystallinity index ranges from 20.0 to 21.9% according to Table 5. The starch crystallinity could be originated from the retrogradation of starch after the extrusion process or, according to Li et al.[23] the crystallinity structure could be due to the native crystallinity that remains after the extrusion, but the crystallinity of the sheets can be attributed mainly to PVA.
3.6 Scanning Electron Microscopy (SEM) Figure 4 shows the SEM analysis of the fracture of the biodegradable sheets, and it is possible to observe a smooth surface, without cracks and domains, due to the good compatibility of both polymers. Similar results was reported by Priya et al.[24] for PVA:cornstarch casting film, and by Maiti et al.[25], for casting starch:PVA films. It was not possible to observe differences in microstructure promoted by temperature or PVA grade used.
Figure 3. Diffractograms of the starch-PVA biodegradable sheets (F1-F10, formulations according to Table 1).
3.7 Fourier Transform Infrared Spectroscopy (FT-IR) The FT-IR spectra of the biodegradable sheets are shown in Figure 5, and it is possible to observe that all sheets presented similar spectra. The region near 3300 cm-1 is related to the hydroxyl (O-H) stretching assigned to molecular and intermolecular hydrogen bonding. The vibration region between 3000-2800 cm-1 is due to the (C-H) stretching, and it is in agreement with observed by Singha and Kapoor[2], in potato starch:PVA blends produced by casting. According to Mansur et al.[26], the spectral range between 1750-1735 cm-1 is assigned to the stretching of C-O and C=O groups present in residual acetate groups of PVA molecules. The band at 1275 cm-1 is due to the secondary alcohol present in PVA structure. Similar results were observed by Brandelero et al.[27], and Imam et al.[28] in films based on cassava starch:PVA and cornstarch:PVA respectively.
3.8 Thermogravimetric Analysis (TGA) Figure 6 presents the weight loss versus temperature curves of the biodegradable sheets, and the weight loss occurred between 150-450 °C and the main weight loss was around 300 °C (61.8 to 53.5%), and these results are similar to those reported by Zanela et al.[20]. The DTG curves (Figure 6) show three main steps of degradation, occurring around 250, 320 and 440 °C, respectively. According to Rahman et al.[29], the degradation of starch:PVA blends plasticized with glycerol can be divided into three phases. The first phase is below 200 °C, that involves the vaporization of volatiles substances, for example, water. The second phase occurs between 200-500 °C and is related to boiling of glycerol (290 °C), and severe degradation of the main chain, lateral groups, and depolymerization producing small hydrocarbon degradation products, like alkenes, alkanes, and aromatics with posterior vaporization of degradation products. The third phase occurs above 500 °C and corresponds to the carbonization of the organic matter. According to the TGA analysis, it was not possible to observe a distinct pattern between each PVA grade and extrusion temperature profile, because all the curves were similar, indicating that neither the PVA grade nor the temperature profile of the extruder influenced the thermal behavior of the biodegradable sheets.
Figure 4. Scanning electron microscopy micrographs of the fracture of the sheets with magnification at 800 x (F1-F10, formulations according to Table 1). 262 262/265
Polímeros, 28(3), 256-265, 2018
Polyvinyl alcohol (PVA) molecular weight and extrusion temperature in starch/PVA biodegradable sheets
Figure 5. FT-IR analysis of the starch-PVA biodegradable sheets (F1-F10, formulations according to Table 1).
Figure 6. TGA and DTG analysis of the starch-PVA biodegradable sheets (F1-F10, formulations according to Table 1).
4. Conclusions The biodegradable sheets presented good processability, mechanical, thermal, optical, and microstructural properties; characterizing the interaction between the PVA and the cassava starch and indicating the potential for replacement of conventional non-biodegradable polymers for specific uses, e.g., when water resistance or high transparency was not necessary. It was not possible to state a conclusive relationship between PVA molecular weight and the materials properties, but in general, PVA with medium molecular weight and Polímeros, 28(3), 256-265, 2018
high extrusion temperature profile promote an increase of mechanical properties of the sheets in the conditions tested.
5. Acknowledgements The authors thank the multi-user Laboratories at Londrina State University (UEL) for the X-ray diffraction analysis (LARX), FT-IR and thermal analysis (ESPEC), and scanning electron microscopy (LMEM). The authors also thank the Federal University of Technology - Parana (UTFPR), the National Council for Scientific and Technological Development (CNPq) and the Araucaria Foundation (Fundação Araucaria) for their financial support. 263/265 263
Zanela, J., Bilck, A. P., Casagrande, M., Grossmann, M. V. E., & Yamashita, F.
6. References 1. Park, H. R., Chough, S. H., Yun, Y. H., & Yoon, S. D. (2005). Properties of starch/PVA blend films containing citric acid as additive. Journal of Polymers and the Environment, 13(4), 375-382. http://dx.doi.org/10.1007/s10924-005-5532-1. 2. Singha, A. S., & Kapoor, H. (2014). Effects of plasticizer/ cross-linker on the mechanical and thermal properties of starch/ PVA blends. Iranian Polymer Journal, 23(8), 655-662. http:// dx.doi.org/10.1007/s13726-014-0260-9. 3. Tang, X., & Alavi, S. (2011). Recent advances in starch, polyvinyl alcohol based polymer blends, nanocomposites and their biodegradability. Carbohydrate Polymers, 85(1), 7-16. http://dx.doi.org/10.1016/j.carbpol.2011.01.030. 4. Aydin, A. A., & Ilberg, V. (2016). Effect of different polyol-based plasticizers on thermal properties of polyvinyl alcohol:starch blends. Carbohydrate Polymers, 136, 441-448. http://dx.doi. org/10.1016/j.carbpol.2015.08.093. PMid:26572374. 5. Majdzadeh-Ardakani, K., & Nazari, B. (2010). Improving the mechanical properties of thermoplastic starch/poly(vinyl alcohol)/clay nanocomposites. Composites Science and Technology, 70(10), 1557-1563. http://dx.doi.org/10.1016/j. compscitech.2010.05.022. 6. Mao, L., Imam, S., Gordon, S., Cinelli, P., & Chiellini, E. (2000). Extruded cornstarch - glycerol - polyvinyl alcohol blends: mechanical properties, morphology, and biodegradability. Journal of Polymers and the Environment, 8(4), 205-211. http://dx.doi.org/10.1023/A:1015201928153. 7. Ray, D., Roy, P., Sengupta, S., Sengupta, S. P., Mohanty, A. K., & Misra, M. (2009). A study of physicomechanical and morphological properties of starch/poly(vinylalcohol) based films. Journal of Polymers and the Environment, 17(1), 56-63. http://dx.doi.org/10.1007/s10924-009-0117-z. 8. Zanela, J., Shirai, M. A., Reis, M. O., Mali, S., Grossmann, M. V. E., & Yamashita, F. (2015). Mixture design to develop biodegradable sheets with high levels of starch and polyvinyl alcohol. Starch, 67(11-12), 1011-1019. http://dx.doi.org/10.1002/ star.201500094. 9. Limpan, N., Prodpran, T., Benjakul, S., & Prasarpran, S. (2012). Influences of degree of hydrolysis and molecular weight of poly(vinyl alcohol) (PVA) on properties of fish myofibrillar protein/PVA blend films. Food Hydrocolloids, 29(1), 226-233. http://dx.doi.org/10.1016/j.foodhyd.2012.03.007. 10. Silva, G. G. D., Sobral, P. J. A., Carvalho, R. A., Bergo, P. V. A., Mendieta-Taboada, O., & Habitante, A. M. Q. B. (2008). Biodegradable films based on blends of gelatin and poly (vinyl alcohol): effect of PVA type or concentration on some physical properties of films. Journal of Polymers and the Environment, 16(4), 276-285. http://dx.doi.org/10.1007/s10924-008-0112-9. 11. Jang, J., & Lee, D. K. (2003). Plasticizer effect on the melting and crystallization behavior of polyvinyl alcohol. Polymer, 44(26), 8139-8146. http://dx.doi.org/10.1016/j.polymer.2003.10.015. 12. American Society for Testing and Materials – ASTM. (2002). ASTM D882-02: standard test methods for tensile properties of thin plastic sheeting. West Conshohocken: ASTM. http:// dx.doi.org/10.1520/D0882-12. 13. American Society for Testing and Materials – ASTM. (2000). ASTM E96: standard test methods for water vapor transmission of materials. West Conshohocken: ASTM. http://dx.doi. org/10.1520/E0096_E0096M-16. 14. Olivato, J. B., Grossmann, M. V. E., Bilck, A. P., & Yamashita, F. (2012). Effect of organic acids as additives on the performance of thermoplastic starch/polyester blown films. Carbohydrate Polymers, 90(1), 159-164. http://dx.doi.org/10.1016/j. carbpol.2012.05.009. PMid:24751025. 264 264/265
15. Maria, T. M. C., Carvalho, R. A., Sobral, P. J. A., Habitante, A. M. B. Q., & Solorza-Feria, J. (2008). The effect of the degree of hydrolysis of the PVA and the plasticizer concentration on the color, opacity, and thermal and mechanical properties of films based on PVA and gelatin blends. Journal of Food Engineering, 87(2), 191-199. http://dx.doi.org/10.1016/j. jfoodeng.2007.11.026. 16. Follain, N., Joly, C., Dole, P., & Bliard, C. (2005). Properties of starch based blends. Part 2. Influence of poly vinyl alcohol addition and photocrosslinking on starch based materials mechanical properties. Carbohydrate Polymers, 60(2), 185192. http://dx.doi.org/10.1016/j.carbpol.2004.12.003. 17. Sin, L. T., Aizan, W., Abdul, W., & Rahmat, A. R. (2010). Specific heats of neat and glycerol plasticized polyvinyl alcohol. Pertanika Journal of Science & Technology, 18(2), 387-391. Retrieved in 2017, May 31, from http://www.pertanika.upm. edu.my/Pertanika%20PAPERS/JST%20Vol.%2018%20(2)%20 Jul.%202010/17%20Pg%20387-391.pdf 18. Wang, S., Ren, J., Kong, W., Gao, C., Liu, C., Peng, F., & Sun, R. (2014). Influence of urea and glycerol on functional properties of biodegradable PVA / xylan composite films. Cellulose, 21(1), 495-505. http://dx.doi.org/10.1007/s10570013-0091-4. 19. Sekisui Chemical Co. (2016, november 22). Selvol™ polyvinyl alcohol. Calvert City. Retrieved in 2017, May 31, from http:// www.sekisui-sc.com/products/polyvinyl-alcohol 20. Zanela, J., Olivato, J. B., Dias, A. P., Grossmann, M. V. E., & Yamashita, F. (2015). Mixture design applied for the development of films based on starch, polyvinyl alcohol, and glycerol. Journal of Applied Polymer Science, 132(43), 42697. http://dx.doi.org/10.1002/app.42697. 21. Das, K., Ray, D., Bandyopadhyay, N. R., Gupta, A., Sengupta, S., Sahoo, S., Mohanty, A., & Misra, M. (2010). Preparation and characterization of cross-linked starch/poly(vinyl alcohol) green films with low moisture absorption. Industrial & Engineering Chemistry Research, 49(5), 2176-2185. http:// dx.doi.org/10.1021/ie901092n. 22. Moorthy, S. N. (2002). Physicochemical and functional properties of tropical tuber starches: a review. Stärke, 54(12), 559-596. http://dx.doi.org/10.1002/1521-379X(200212)54:12<559::AIDSTAR2222559>3.0.CO;2-F. 23. Li, M., Hasjim, J., Xie, F., Halley, P. J., & Gilbert, R. G. (2014). Shear degradation of molecular, crystalline, and granular structures of starch during extrusion. Starch, 66(7-8), 595-605. http://dx.doi.org/10.1002/star.201300201. 24. Priya, B., Gupta, V. K., Pathania, D., & Singha, A. S. (2014). Synthesis, characterization and antibacterial activity of biodegradable starch/PVA composite films reinforced with cellulosic fibre. Carbohydrate Polymers, 109, 171-179. http:// dx.doi.org/10.1016/j.carbpol.2014.03.044. PMid:24815414. 25. Maiti, S., Ray, D., & Mitra, D. (2012). Role of Crosslinker on the Biodegradation Behavior of Starch/Polyvinylalcohol Blend Films. Journal of Polymers and the Environment, 20(3), 749-759. http://dx.doi.org/10.1007/s10924-012-0433-6. 26. Mansur, H. S., Sadahira, C. M., Souza, A. N., & Mansur, A. A. P. (2008). FTIR spectroscopy characterization of poly (vinyl alcohol) hydrogel with different hydrolysis degree and chemically crosslinked with glutaraldehyde. Materials Science and Engineering C, 28(4), 539-548. http://dx.doi.org/10.1016/j. msec.2007.10.088. 27. Brandelero, R. P. H., Yamashita, F., Zanela, J., Brandelero, E. M., & Caetano, J. G. (2015). Mixture design applied to evaluating the effects of polyvinyl alcohol (PVOH) and alginate on the properties of starch-based films. Stärke, 67(1-2), 191-199. http://dx.doi.org/10.1002/star.201400119. Polímeros, 28(3), 256-265, 2018
Polyvinyl alcohol (PVA) molecular weight and extrusion temperature in starch/PVA biodegradable sheets 28. Imam, S. H., Cinelli, P., Gordon, S. H., & Chiellini, E. (2005). Characterization of biodegradable composite films prepared from blends of poly(vinyl alcohol), cornstarch, and lignocellulosic fiber. Journal of Polymers and the Environment, 13(1), 47-55. http://dx.doi.org/10.1007/s10924-004-1215-6. 29. Rahman, W. A. W. A., Sin, L. T., Rahmat, A. R., & Samad, A. A. (2010). Thermal behaviour and interactions of cassava
PolĂmeros, 28(3), 256-265, 2018
starch filled with glycerol plasticized polyvinyl alcohol blends. Carbohydrate Polymers, 81(4), 805-810. http://dx.doi. org/10.1016/j.carbpol.2010.03.052. Received: May 31, 2017 Revised: Sept. 07, 2017 Accepted: Oct. 18, 2017
265/265 265
ISSN 1678-5169 (Online)
http://dx.doi.org/10.1590/0104-1428.09917
O O O O O O O O O O O O O O O O
Molecular dynamics studies of amylose plasticized with Brazilian Cerrado oils: part I Felipe Azevedo Rios Silva1, Maria José Araújo Sales2, Leonardo Giordano Paterno2, Mohamed Ghoul3, Latifa Chebil3 and Elaine Rose Maia1* Laboratório de Estudos Estruturais Moleculares – LEEM, Instituto de Química – IQ, Universidade de Brasília – UnB, Campus Darcy Ribeiro, Brasília, DF, Brasil 2 Laboratório de Pesquisa em Polímeros e Nanomateriais – LabPolN, Instituto de Química – IQ, Universidade de Brasília – UnB, Campus Darcy Ribeiro, Brasília, DF, Brasil 3 Laboratoire d’Ingénierie des Biomolécules – LIBio, Ecole Nationale Supérieure d’Agronomie et des Industries Alimentaires – ENSAIA, Institut National Polytechnique de Lorraine – INPL, Université de Lorraine – UL, Vandœuvre-lès-Nancy, France 1
*elaine.rose.maia@gmail.com
Abstract Biodegradable polymers have become part of the realm of polymer science with specially when associated to renewable sources. Unraveling the plasticizer effect of natural occurring fatty acids in the Brazilian Cerrado on amylose oligomers was aimed in this work in an aqueous environment. Since the interactions within a material are of extreme importance to its molecular behavior, the main focus was directed to the molecular interactions whether intra or intermolecular type. Molecular Mechanics and Dynamics were carried out to shed light on this issue. The simulation results suggest the fatty acids could perform as efficient plasticizers for more complex polysaccharides such as starch. It also highlights the importance the solvation on the system stabilization, thus contributing to a clearer understanding of the chemical interactions role on plasticization. Our results provide a basis for simulating more complex systems such as a clay‑mineral which will culminate in the parameterization for mesoscale studies. Keywords: amylose, Cerrado oils, molecular mechanics and dynamics, plasticization, polymer consistent force field.
1. Introduction The use of starch-based polymers in films and wrapping, bags, personal hygiene items, cups, straws, strings, and many other materials is recognized for being a cost-effective green technology in comparison to synthetic polymeric materials derived from petroleum[1]. However, all these products demand for starch with some level of chemical manipulation, because in its natural form starch presents low film processability and poor mechanical properties[2-5]. Starch is a semi-crystalline biopolymer[5] mainly formed by two components with repeating α-D-glucopyranosyl units, although other components such as proteins and lipids are present in small ammouts. These two main components are amylose, a mostly linear polysaccharide and amylopectin, a highly branched one. In the amylose linear main chain, the repeating units are connected by α(1-4)-glycosidic bonds (Figure 1), while in longer chains some branches may be present. It has a tendency to acquire an stable helical shape when complexed with several ligands[5]. The quantity of amylose in a starch can make it form stronger films, the amount of amylose in normal starches (i.e. maize, potato, rice, etc) is about 20-30%[5,6]. The amylopectin main chain is formed by α(1-4)‑glycosidic bonds with about 5% of α(1-6)-glycosidic branches[3,5]. This macromolecule has a much more complex structure than the latter, which is accepted to be represented by a
266 266/274
cluster model[5]. The amount of amylopectin found in normal starches is around 70-80%, its quantity can influence in the strength of starch films, weakening and diminishing its processability[1,3,6-8]. A common approach for improving starch film ability is plasticization, which can be efficiently and environmentally friendly achieved with small amounts of vegetable oils[9] and its fatty acids[2,7,10-12]. Our group has developed starch films produced by casting of aqueous starch suspensions plasticized with vegetable oils from Cerrado, the second largest biome in South America[13], as well as from Amazonia. More specifically, with pequi (Caryocar brasiliense) and buriti (Mauritia flexuosa L.) fruits, which are used in the region with different purposes such as human and animal feeding[14,15], cosmetic and medicinal uses[16]. The fundamental importance in its composition is the abundance in unsaturated fatty acids, with the predominance of oleic acid, between 50-80% in mass, and also, the presence of tocopherols and carothenoids[11,14,15]. They have proved to efficiently plasticize starch and enable fabrication of films by casting[2,3,7,11]. A step further consisted on the preparation of bionanocomposites of plasticized starch and lamellar oxides, such as montimorillonite clay, for improved thermal and barrier properties[1,2,7,8,10,12]. Therefore, plasticization
Polímeros, 28(3), 266-274, 2018
Molecular dynamics studies of amylose plasticized with Brazilian Cerrado oils: part I
Figure 1. (Top) Structural representation of a α(1,4)-glycosidic bond between two D-glucose residues; (Bottom) amylose oligomer with 10 monomer units. Color code: C = gray; H = white; O = red.
is an essential step to allow the use of starch in advanced biodegradable materials[2,3,7,12]. The plasticization of starch with vegetable oils opens‑up a venue for new biomaterials and processing as well as for theoretical and molecular models of chemical interactions. In particular, molecular dynamics simulation (MD) has been performed to understand, at the molecular level, the interaction between starch components, such as amylose, and common solvents (water and DMSO) as well as to evaluate current and future candidates for possible plasticization. This approach should be advantageous before any expensive and time‑consuming real processing is carried out. For example, Yang et al.[17] performed MD of a single amylose chain (36 monomeric units) combined with glycerol as plasticizer. They found that strong hydrogen bonding was essential for cohesion of the amylose/glycerol model, which was further corroborated by experimental data (X-ray diffraction, tensile strength and water uptake), probed with starch/glycerol cast films. Tusch et al.[18] observed that at certain simulation times the helix character of a single helical amylose chain (55 monomeric units) in water/DMSO mixtures was stabilized after increasing the DMSO concentration, since DMSO helps to preserve intramolecular hydrogen bonds in starch. Their findings were in consonance with NMR data. López et al.[19] verified by MD that the addition of lipids, such as dipalmitoyl‑phosphatidylcholine and glycerol monooleate, induces amylose fragments (13 monomeric units) to folding into a helical structure. The latter matches the V-amylose structure, which is a type of amylose polymorph[5,20], observed in X-ray diffraction. Finally, Feng et al.[21] observed by MD similar V-amylose structures when in the presence of linoleic acid. As observed in experimental data and corroborated further by MD studies, inter- and intra-molecular interactions play a major role on the molecular behavior of complex systems comprised by natural plasticizers and polysaccharides[12,17-19,21]. In fact, they are essential for the Polímeros, 28(3), 266-274, 2018
rational design of high performance biomaterials as well as to predict its properties. Most of MD studies were limited to a single and short poly‑glucose chains, mainly because larger and more complex systems would demand for longer simulation times and even more expensive computational instrumentation[17-19,21]. In this regard, the present contribution provides a systematic study performed by Molecular Mechanics (MM) and Molecular Dynamics methods with the Polymer Consistent Force Field (PCFF) on the assembly of multiple amylose oligomers (25 to 120 monomeric units) in aqueous medium. The oligomer is complexed with a mixture of fatty acids (oleic, palmitic, and stearic) set in proportions that mimic those found in pequi oil. The molecular assemblies were confined into cells and studied under periodic boundary condition (PBC) and constant number of particles, temperature and volume (NVT) ensemble, at 363 K, at variable elapsed times. Its main goal was directed toward observation of the molecules’ motion when evolving together, to the bonded and non-bonded intermolecular forces, and to the respective structural and behavioral correlations. These changes are known for reflecting the strength of intra- and intermolecular interactions that act on the molecular system.
2. Materials and Methods All calculations were performed with BIOVIA Materials StudioTM suite[22]. The atomistic coordinates for all molecules and simulated cells were built using the Visualizer, Builder and Amorphous Cell modules[22]. The MM and MD calculations were carried out with PCFF[23,24] interfaced to the Forcite module[22]. The PCFF is a member of the consistent force field family. It was developed with a base on CFF91 and is intended for application to polymers, organic materials, metals, and zeolites[22-26], also being suitable for carbohydrates[3,27] and water solvation[28]. The dynamic 267/274 267
Silva, F. A. R., Sales, M. J. A., Paterno, L. G., Ghoul, M., Chebil, L., & Maia, E. R. trajectories were carefully analyzed using data correlation graphs implemented in Analysis/Forcite[22].
2.1 Specificities for the calculation protocol of amylose fatty acid - water systems: studies by NVT ensemble The atomistic simulations essentially followed the calculation protocols hereafter described. (i) The molecular systems were composed for a better understanding about the intra- and intermolecular interactions and the structural behavior when the three chemical species evolve together. These simulations were carefully made with different systems, which varied, fundamentally, in the proportion of fatty acids; in the initial form and number of the amylose oligomers, in which the sizes varied from 25 to 120 monomers. These two chemical species were created from features implemented in Visualizer and Builder programs, such as Fragment browser. The amylose chains were built by “Build Polymers” feature as isotactic homopolymers from the carbohydrates database of the BIOVIA Materials StudioTM program, using 1,4-α-D-glucose repeat units, with chains of variable lengths. After each construction, geometries and energies were fully optimized by MM and the results stored for future usage. Periodic cells were created with the Build Crystals functionality. The unit cells dimensions varied according to the number and length of amylose chains. The fatty acids were manually inserted into the cells, near the amylose oligomers, in the proportions as close as possible to those found in the buriti and pequi oils. The unit cells were filled by water molecules ranging around 2000 to 4000 molecules in explicit solvent model, to simulate the experimental conditions carried out in previous studies[2,7,10], where the components of the system were dispersed in distilled water. (i.1) Intermediate energy minimizations allowed corrections and eliminated steric hindrance with the steepest descent algorithm. The number of iterations limited to coarse accuracy of the gradient was set to 2.0 x 10-3 kcal mol-1. Optimizations continued throughout the conjugate gradient algorithm, with the convergence of the gradient limited to 1.0 x 10-3 kcal mol-1 Å-1. In general terms, these limit values indicate medium precision. The molecular dynamics method of the Forcite program follows the classical Newtonian equations of motion for the integrated Verlet algorithm[29]. The initial injected speeds were random and assigned to the atoms according to the Maxwell-Boltzmann distribution. The observation times ranged from 100 ps to 1 ns, according to the systems’ sizes. For all simulations, the time step was set to 1 fs. The coordinates of the molecular systems corresponding to the dynamics fluctuations (frames) were saved in periodic intervals of at least 1000 frames, which allowed posterior analysis via control graphs and molecular coordinates, across the Analysis program features. All dynamic simulations ran under the canonical ensemble NVT. The temperature control in Forcite is based on the Nosé-Hoover chain (NHC) thermostat[30-32]. All simulations were conducted under PBC. 268 268/274
After a manual and progressive addition of significant amounts of water, while being intermediated by some energy optimization steps, systems were submitted to fast dynamic trajectories at 500 K, during 40 ps, for their rearrangement. In this case, all other molecules had their atomic coordinates fixed under constraints. This procedure allowed for rapid movement of the solvent around other molecules and prevents water molecules, initially misplaced, to assume unrealistic positions and, consequently, to conduce for not productive results. Solvent molecules out of the unit cell were erased or manually repositioned. The procedure restarted until coarse system stabilization has been reached. (i.2) After system optimization, constraints were removed and a new optimization was performed. Then, the system was submitted to a dynamic trajectory, at 363 K until reorganization. This is the temperature at which systems were experimentally studied.[2,7,10] Simulation times depend mainly on the size of the system under observation. When equilibrium state was not critical, i.e., when systems were designed primarily to motion analysis and conformational modification, tolerances of gradient convergence (rms) were set to 1.0 x 10-3 kcal mol-1, for energy; 5.0 x 10-1 kcal mol-1 Å-1, for maximum strength; and the precision of the electrostatic and van der Waals terms to 1.0 x 10-3 kcal mol-1 (medium accuracy). When the system reached situations in which the organization had already occurred, they were used as an input file for subsequent simulations; the parameters for the convergence tolerance were limited to 1.0 x 10-4 kcal mol-1 for energy; 5.0 x10-3 kcal mol-1 Å-1, for force; 5.0 x 10-5 Å, for displacement; and the precision of the nonbonding interactions terms, 1.0x10-4 kcal mol-1 (fine precision). The nonbonding interactions of those periodic systems were computed by the Ewald summation method[33,34]. Under NVT ensemble, the automatic correction of the simulated cells dimensions was not used.
In addition to the general understanding about the behavior of these different chemical species working together, these models allowed one to verify the permissible size of the oligomeric chain and the number fatty acids and water molecules that could be added to the system. All together, they enabled the calculations in PBC, according to our computational capacity; a CPU time that would be logical and reliable to make the dynamic trajectories in different situations; and if the number and the length of the amylose chains participating to the systems could lead to different behavior. All calculations were carefully performed with fatty acids set in the proportion found in pequi oil (54% of oleic acid, 40%, palmitic acid and 2%, stearic)[2,7,10].
3. Results and Discussions 3.1 First system modeled from four amylose short chains of 40 monomeric units and 49 fatty acid molecules In the first simulated system, four amylose oligomers with 40 monomeric units each (842 atoms/oligomer; 3,368 atoms) were confined into an orthorhombic cell with dimensions of Polímeros, 28(3), 266-274, 2018
Molecular dynamics studies of amylose plasticized with Brazilian Cerrado oils: part I a = 100 Å, b = 200 Å and c = 90 Å; α = β = γ = 900, which enabled the study in PBC. The fatty acids were placed perpendicular to the amylose chains’ medial plane, in a proportion of 28 oleic acids, 20 palmitic acids and 1 stearic acid molecules (2,568 atoms). Finally, 2,668 molecules of water were gradually added that resulted in a system composed of 13,940 atoms in the asymmetric unit. The starting (t = 0 s) and subsequent molecular assembly attained after different simulated elapsed times as well as the time dependence of dynamics energies (kinetic, potential, non-bond, and total energies) and temperature are depicted in Figure 2. As seen in the first image from Figure 2 (clockwise), the frontal view of the initial system comprises four elongated amylose chains lined two by two with fatty acids, while water molecules are randomly distributed around them. The subsequent image provides side view of the initial system. In analogy to two comets, heads and tails are composed of amylose and fatty acids, respectively, in which two are face to face while other two are distant of about 180 degrees. Then, solvent molecules were left moving around during a short dynamic process at 500 K, while the main molecules are kept fixed, under constraints. After water reorganization and energetic optimization, the constraints were removed and the free
system was optimized again, before being submitted to a dynamic process at 363 K, for 200 ps (middle images). The thermalization period took about 5 ps. At 10 ps, amylose chains remained elongated. Between 20 to 80 ps, amylose chains progressively coiled. The two “comets” (chains in lilac and light orange) that were farther from the central assemblies (chains in purple and dark orange) were strongly attracted and then distorted until they approached the core, leading to a new assembly in which the amylose chains were strongly aggregated (middle images). The nonpolar fatty acids tails were driven away from amylose while their polar heads remained near to each other, probably closer to the electronegative amylose groups via hydrogen bonds. Meanwhile, water molecules moved out of the cavities formed by amylose chains which, in turn, approached and coiled. The elongated fatty acids covered the amylose coil in an unusual rearrangement. This behavior can be monitored throughout frames from 40 to 200 ps. This simulation allowed us to observe that the key factor is not the stabilization time. The complete system folding was reached at 80 ps, and during the remaining 120 ps, only minor positions were changed. The last trajectory frame is subjected to geometric and energy correction (Figure 3), after repositioning of the few water molecules that may
Figure 2. Two different perspectives of the starting system, in which fatty acids interact with four amylose oligomers, surrounded by water. The system had not yet been led to equilibrium (0 ps). The top and side views of the cell (left and right) show the spatial arrangement of key molecules, such as four “heads and tails of comets”. (Middle) Snapshots of dynamics trajectory identifying fluctuations at 40, 74 and 200 ps. The oligomer chains are highlighted by spheres (CPK) and colored in lilac and purple, light and dark orange. Fatty acids are highlighted by thick lines (stick) and water molecules are visible because of the red color of the oxygen atoms. (Bottom) Graphics of dynamic process control (Energy (kcal mol-1) vs. time (ps) and temperature (K) vs. time (ps)) to a 200 ps trajectory at 363 K. Polímeros, 28(3), 266-274, 2018
269/274 269
Silva, F. A. R., Sales, M. J. A., Paterno, L. G., Ghoul, M., Chebil, L., & Maia, E. R.
Figure 3. (Top) Zoom of the system segment already energetically optimized. It indicates the elongated fatty acids (in CPK) and water molecules (in stick) around amylose chains. (Bottom) View of segment highlighting the water molecules (in ball-and-stick) involving densely amylose oligomers (in CPK) in the regions without interactions with the amylose oligomers.
have been dispersed into the cell. The computation time (CPU) required to perform the dynamic path detailed above, at 363 K, in PBC, during 200 ps, was 88 h. This is quite reasonable to treat the 13,940 atoms that make up the system, in an 8 Gb RAM PC and 4 processors.
3.2 Second system modeled from one amylose chain of 120 monomeric units and 32 fatty acid molecules In the second system, one amylose oligomeric chain containing 120 monomeric units (2,522 atoms) was built with the Amorphous Cell program. Fatty acid molecules were randomly distributed around the amylose chain, in a proportion of 18 oleic acids, 13 palmitic acids and 1 stearic acid molecules (1,678 atoms), in a total of 4,200 atoms. The original cell containing the oligosaccharide chain was enlarged to a cubic cell of a = b = c = 100 Å; α = β = γ = 90o (Figure 4). Water molecules were gradually added, resulting in a system composed by 8,043 atoms into the cell. Energetic optimizations and dynamics trajectories followed protocols described above. Solvent molecules were progressively 270 270/274
added and, after each significant quantity, the system was reorganized by fast molecular dynamics trajectories of 20 ps at 500 K, reoptimized to avoid steric hindrance, and the atomic coordinates of all organic molecules were placed under constraints. Water molecules dispersed into the cell were repositioned or erased, and another ensemble was added to the system. Then the constraints were deleted and dynamic trajectories by 30 ps, at 363 K were carried out until system stabilization was reached. It is important to note that during dynamics, some water molecules were always dispersed. Those water molecules were then deleted, the system was reoptimized, and the procedure was restarted, as frequently as necessary. The analyses of those results are essentially the same, regardless the shape and length of the amylose chain. The starting radius of gyration and volume varied from 27.80 Å and 17,941.67 Å3 to 18.14 Å and 18,620.02 Å3, respectively, at the end of the process. The amylose chain, progressively, became compacted until being coiled. The polar head of fatty acids stabilized closer to the electronegative Polímeros, 28(3), 266-274, 2018
Molecular dynamics studies of amylose plasticized with Brazilian Cerrado oils: part I
Figure 4. (Top) Initial system structure submitted for dynamics. Waters molecules were undisplayed for clarity. On the left, atoms are represented in stick and colored by elements colors - C, in grey; O, in red; H, in white. On the center, the similar image highlight compounds in CPK and different colors (amylose, in light orange; oleic, in medium blue; palmitic, in green, stearic, in light blue). (Top, right; down, left) Snapshots of dynamics trajectory, intermediate frame corresponding to 40 ps; last frame, 100 ps, when system was already coiled and water molecules were fully stabilized. Image shows the energetic optimized system to highlight the positions of fatty acids covering amylose chain and the solvent molecules distribution. Oxygen atoms of water molecules are colored in red. (Down, right) Snapshots of control graphics for Energy (kcal mol-1) vs. time (ps) and temperature (K) vs. time (ps).
amylose groups and formed a hydrogen bond network. Their nonpolar portions rejected water molecules and covered the oligomer “protecting” it. The water molecules covered every other surface of the oligomer with no fatty acids and stabilized by a very dense hydrogen bond network. The unexpected rearrangement comprised by elongated fatty acids encapsulating the amylose chains occurred again. Those results permit one to suppose that once the number of fatty acid molecules increases they will encapsulate the amylose chain and most of water molecules would be rejected. Only few water molecules would stay into the ensemble, due to their orientation close to oxygenated groups of the organic compounds.
3.3 Third system modeled from one amylose chain of 120 monomeric units and 96 fatty acid molecules In order to verify those considerations, a third and last system among the many different systems studied will be described. A different conformer of amylose oligomer comprised by 120 monomeric units as built with the Amorphous Cells program was involved by randomly distributed fatty acid molecules. A higher concentration of Polímeros, 28(3), 266-274, 2018
fatty acids was considered, in a proportion of 54 oleic acid, 40 palmitic acid and 2 stearic acid molecules (5,028 atoms). Compounds were confined in a starting cell of a = 140 Å, b =120 Å, c = 100 Å; α = β = γ = 90o (Figure 5). Since a fast optimization was accomplished, an amount of 2,207 water molecules were distributed into the asymmetric unit around the organic ensemble. The entire system had 13,342 atoms. Energetic optimizations and dynamics trajectories following the described protocols were carried out, firstly to correct water positions, leaving the atomic coordinates of organic molecules constrained. The misplaced water molecules were progressively deleted or correctly repositioned into the cell. The asymmetric unit axis were increased to a = 140 Å, b =140 Å, c = 130 Å and then, restrictions on the molecular freedom were withdrawn. The full procedure involved many gradient optimizations, intermediated by a few and fast dynamics trajectories at 363 K. When the system was considered stabilized enough and reached its final form, 1,406 water molecules were conserved inside it. The amylose radius of gyration and volume in the starting conformation were 32.37 Å and 17,945.86 Å3, while the final conformation reached 20.52 Å and 18,326.74 Å3, respectively. 271/274 271
Silva, F. A. R., Sales, M. J. A., Paterno, L. G., Ghoul, M., Chebil, L., & Maia, E. R. It was possible to confirm the precedent analysis and interpretations. Even randomly distributed, the majority of water molecules flow thru the organic system. The molecules
farther from the central system forces stabilized among themselves. As the acids cover the most part of amylose surface, a remaining small portion could be covered by water. The electrostatic force of water was not strong enough to overcome the repulsion force imposed by nonpolar tails of fatty acids. A significant number of water molecules remain into the cell since they were stabilized by hydrogen bonds (Figure 5). However, if dynamics trajectories were longer, these water clusters would be progressively driven away from the organic assembly and, then, out of the unit cell. Among the 1,406 water molecules inside the cell, as shown in Figure 5, only 510 were estimated to be located around amylose and fatty acids polar heads to contribute for the system stabilization. Also, it should be noted that the gradient convergence is limited because of the water dispersion. The system converges following medium precision, because the water movement is slow but continuous. In spite of the strong water repulsion by the amylose-fatty acid complex, this solvent is absolutely essential to have a consistent system. As fatty acids easily cover the polysaccharide chain we could interpret the behavior of those promising compounds when acting as plasticizers, which was already stablished based in results achieved by our research group[7,11].
4. Conclusions
Figure 5. (Top) Initial system structure submitted for dynamics Starting form and molecular distribution for the last model which is composed by 54 oleic acids; 40 palmitic acids and 2 stearic acids disposed around 120 monomers of amylose chain. Waters molecules were undisplayed for clarity. Atoms are represented in stick and colored by elements colors - C, in grey; O, in red; H, in white. In the center, the similar image highlight compounds in CPK and different colors (amylose, in purple; oleic, in medium blue; palmitic, in green, stearic, in light blue). (Down) - Final result’s snapshots of the third model. Fatty acids cover amylose chain and there is no flown of water to amylose. Only few solvent molecules stay stabilized over amylose surface, when that surface was enough exposed. When concentration of fatty acid is dense, water molecules are strongly reject far from organic molecules. 272 272/274
A systematic study was performed by Molecular Mechanics and Molecular Dynamics methods with PCFF on the assembly of multiple amylose oligomers (25 to 120 monomeric units) in aqueous medium while complexed with a mixture of fatty acids (oleic, palmitic, and stearic) set in proportions that mimic those found in pequi oil. The systems were assembled in simulated boxes, composed by helix amylose chains, lined with fatty acids and immersed in water molecules randomly distributed. All these calculations series were carried in NVT ensemble and PBC conditions. For all of configuration simulated, the behavior was essentially convergent. As the simulation time elapsed, the non-polar tails of fatty acid molecules encapsulated amylose chains while most of water molecules were driven away from the amylose-fatty acid complex. Nonetheless, remaining water molecules were located at amylose sites uncoated by fatty acids and formed a dense network of hydrogen bonds, which was responsible for the entire system stabilization. The rapid and strong association of fatty to amylose chains found in the simulations suggests the behavior of those plasticizers with amylose. These theoretical results are in good agreement with what was experimentally observed for the interaction between fatty acids and starch. For this reason, we think that the molecular dynamics method presented in this work is useful for predicting the behavior of the interaction between fatty acids and more complex polysaccharides, more similar to starch, as well as systems enriched by inclusion of clay-minerals.
5. Acknowledgements Felipe Silva thanks the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), for the doctoral fellowship in Brazil, and the Polímeros, 28(3), 266-274, 2018
Molecular dynamics studies of amylose plasticized with Brazilian Cerrado oils: part I Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), via the Science without Borders Program (CSF), for the year-long doctoral fellowship in France. He would, also, like to thank the Laboratoire d´Ingénierie des Biomolécules (LIBio/ENSAIA), from the University of Lorraine (FR) and its team for the structural and academic support he received during the doctoral internship in France.
6. References 1. Halley, P. J. (2005). Themoplastic starch biodegradable polymers. In R. Smith (Ed.), Biodegradable polymers for industrial applications (p. 140-162). Cambridge: Woodhead Publishing. http://dx.doi.org/10.1533/9781845690762.1.140. 2. Schlemmer, D., Angélica, R. S., & Sales, M. J. A. (2010). Morphological and thermomechanical characterization of thermoplastic starch/montmorillonite nanocomposites. Composite Structures, 92(9), 2066-2070. http://dx.doi.org/10.1016/j. compstruct.2009.10.034. 3. Pérez, S., Kouwijzer, M., Mazeau, K., & Engelsen, S. B. (1996). Modeling polysaccharides: present status and challenges. Journal of Molecular Graphics, 14(6), 307-321. http://dx.doi. org/10.1016/S0263-7855(97)00011-8. PMid:9195482. 4. Angellier, H., Molina-Boisseau, S., Dole, P., & Dufresne, A. (2006). Thermoplastic starch-waxy maize starch nanocrystals nanocomposites. Biomacromolecules, 7(2), 531-539. http:// dx.doi.org/10.1021/bm050797s. PMid:16471926. 5. Pérez, S., & Bertoft, E. (2010). The molecular structures of starch components and their contribution to the architecture of starch granules: a comprehensive review. Starch, 62(8), 389-420. http://dx.doi.org/10.1002/star.201000013. 6. Lu, D. R., Xiao, C. M., & Xu, S. J. (2009). Starch-based completely biodegradable polymer materials. Express Polymer Letters, 3(6), 366-375. http://dx.doi.org/10.3144/ expresspolymlett.2009.46. 7. Schlemmer, D., & Sales, M. J. A. (2010). Thermoplastic starch films with vegetable oils of Brazilian Cerrado. Journal of Thermal Analysis and Calorimetry, 99(2), 675-679. http:// dx.doi.org/10.1007/s10973-009-0352-5. 8. Ray, S. S. (2014). Recent trends and future outlooks in the field of clay-containing polymer nanocomposites. Macromolecular Chemistry and Physics, 215(12), 1162-1179. http://dx.doi. org/10.1002/macp.201400069. 9. Castro, D. O., Frollini, E., Ruvolo-Filho, A., & Dufresne, A. (2015). “Green polyethylene” and curauá cellulose nanocrystal based nanocomposites: effect of vegetable oils as coupling agent and processing technique. Journal of Polymer Science. Part B, Polymer Physics, 53(14), 1010-1019. http://dx.doi. org/10.1002/polb.23729. 10. Schlemmer, D., Oliveira, E. R., & Sales, M. J. A. (2007). Polystyrene/thermoplastic starch blends with different plasticizers. Journal of Thermal Analysis and Calorimetry, 87(3), 635-638. http://dx.doi.org/10.1007/s10973-006-7776-y. 11. Pimentel, T. A. P. F., Durães, J. A., Drummond, A. L., Schlemmer, D., Falcão, R., & Sales, M. J. A. (2007). Preparation and characterization of blends of recycled polystyrene with cassava starch. Journal of Materials Science, 42(17), 7530-7536. http:// dx.doi.org/10.1007/s10853-007-1622-x. 12. Yu, L., Dean, K., & Li, L. (2006). Polymer blends and composites from renewable resources. Progress in Polymer Science, 31(6), 576-602. http://dx.doi.org/10.1016/j.progpolymsci.2006.03.002. 13. Brasil. Ministério do Meio Ambiente. (2017). O bioma Cerrado. Brasília. Retrieved in 2017, October 19, from http://www.mma. gov.br/biomas/cerrado Polímeros, 28(3), 266-274, 2018
14. Traesel, G. K., de Araújo, F. H. S., Castro, L. H. A., de Lima, F. F., Menegati, S. E. L. T., Justi, P. N., Kassuya, C. A. L., Cardoso, C. A. L., Argandoña, E. J. S., & Oesterreich, S. A. (2017). Safety assessment of oil from pequi (Caryocar brasiliense Camb.): evaluation of the potential genotoxic and clastogenic effects. Journal of Medicinal Food, 20(8), 804-811. http://dx.doi.org/10.1089/jmf.2017.0021. PMid:28557544. 15. Guedes, A. M. M., Antoniassi, R., & de Faria-Machado, A. F. (2017). Pequi: a Brazilian fruit with potential uses for the fat industry. Oilseeds & Fats Crops and Lipids, 24(5), 1-4. http:// dx.doi.org/10.1051/ocl/2017040. 16. Barbosa, M. U., Silva, M. A., Barros, E. M. L., Barbosa, M. U., Sousa, R. C., Lopes, M. A. C., & Coelho, N. P. M. F. (2017). Topical action of Buriti oil (Mauritia flexuosa L. ) in myositis induced in rats. Acta Cirurgica Brasileira, 32(11), 956-963. http://dx.doi.org/10.1590/s0102-865020170110000007. PMid:29236800. 17. Yang, J., Tang, K., Qin, G., Chen, Y., Peng, L., Wan, X., Xiao, H., & Xia, Q. (2017). Hydrogen bonding energy determined by molecular dynamics simulation and correlation to properties of thermoplastic starch films. Carbohydrate Polymers, 166(15), 256-263. http://dx.doi.org/10.1016/j.carbpol.2017.03.001. PMid:28385231. 18. Tusch, M., Krüger, J., & Fels, G. (2011). Structural stability of V-amylose helices in water-DMSO mixtures analyzed by molecular dynamics. Journal of Chemical Theory and Computation, 7(9), 2919-2928. http://dx.doi.org/10.1021/ ct2005159. PMid:26605481. 19. López, C. A., Vries, A. H., & Marrink, S. J. (2012). Amylose folding under the influence of lipids. Carbohydrate Research, 364(15), 1-7. http://dx.doi.org/10.1016/j.carres.2012.10.007. PMid:23128420. 20. Dufresne, A. (2015) Starch and nanoparticle. In K. G. Ramawat, J. M. Mérillon (Eds.), Polysaccharides: bioactivity and biotechnology (p. 417-449). Cham: Springer International Publishing. http://dx.doi.org/10.1007/978-3-319-16298-0_72. 21. Feng, T., Wang, K., Zhuang, H., Bhopatkar, D., Carignano, M. A., Park, S. H., & Bing, F. (2017). Molecular dynamics simulation of amylose-linoleic acid complex behavior in water. Journal of Nanoscience and Nanotechnology, 17(7), 4724-4732. http://dx.doi.org/10.1166/jnn.2017.13444. 22. BIOVIA. (2012). Dassault systèmes BIOVIA: materials studio, release 6.0. San Diego: Dassault Systèmes BIOVIA. 23. Sun, H., Mumby, S. J., Maple, J. R., & Hagler, A. T. (1994). An ab Initio CFF93 all-atom force field for polycarbonates. Journal of the American Chemical Society, 116(7), 2978-2987. http://dx.doi.org/10.1021/ja00086a030. 24. Sun, H. (1995). Ab initio calculations and force field development for computer simulation of polysilanes. Macromolecules, 28(3), 701-712. http://dx.doi.org/10.1021/ma00107a006. 25. Dauber-Osguthorpe, P., Roberts, V. A., Osguthorpe, D. J., Wolff, J., Genest, M., & Hagler, A. T. (1988). Structure and energetics of ligand binding to proteins: Escherichia coli dihydrofolate reductase-trimethoprim, a drug-receptor system. Proteins, 4(1), 31-47. http://dx.doi.org/10.1002/prot.340040106. PMid:3054871. 26. Hill, J. R., & Sauer, J. (1994). Molecular mechanics potential for silica and zeolite catalysts based on ab initio calculations. 1. Dense and microporous silica. Journal of Physical Chemistry, 98(4), 1238-1244. http://dx.doi.org/10.1021/j100055a032. 27. Tang, C., Zhang, S., Wang, Q., Wang, X., & Hao, J. (2017). Thermal stability of modified insulation paper cellulose based on molecular dynamics simulation. Energies, 10(3), 397-408. http://dx.doi.org/10.3390/en10030397. 28. Min, S. H., Kwak, S. K., & Kim, B.-S. (2015). Atomistic simulation for coil-to-globule transition of poly(2-dimethylaminoethyl 273/274 273
Silva, F. A. R., Sales, M. J. A., Paterno, L. G., Ghoul, M., Chebil, L., & Maia, E. R. methacrylate). Soft Matter, 11(12), 2423-2433. http://dx.doi. org/10.1039/C4SM02242D. PMid:25662300. 29. Verlet, L. (1967). Computer “experiments” on classical fluids. I. Thermodynamical properties of lennard-jones molecules. Physical Review, 159(1), 98-103. http://dx.doi.org/10.1103/ PhysRev.159.98. 30. Hoover, W. G. (1985). Canonical dynamics: equilibrium phasespace distributions. Physical Review A., 31(3), 1695-1697. http://dx.doi.org/10.1103/PhysRevA.31.1695. PMid:9895674. 31. Allen, M. P., & Tildesley, D. J. (1989). Computer simulation of liquids. New York: Clarendon Press. 32. Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A., & Haak, J. R. (1984). Molecular dynamics with
274 274/274
coupling to an external bath. The Journal of Chemical Physics, 81(8), 3684-3690. http://dx.doi.org/10.1063/1.448118. 33. Ewald, P. P. (1921). Die Berechnung optischer und elektrostatischer Gitterpotentiale. Annalen der Physik, 369(3), 253-287. http:// dx.doi.org/10.1002/andp.19213690304. 34. Tosi, M. P. (1964) Cohesion of Ionic Solids in the Born Model Based on work performed under the auspices of the U.S. Atomic Energy Commission. In F. Seitz, & D. Turnbull (Eds.), Solid state physics (p. 1-120), New York: Academic Press. Received: Oct. 19, 2017 Revised: Jan. 30, 2018 Accepted: Jan. 31, 2018
Polímeros, 28(3), 266-274, 2018
ISSN 1678-5169 (Online)
http://dx.doi.org/10.1590/0104-1428.08316
Review of fungal chitosan: past, present and perspectives in Brazil Anabelle Camarotti de Lima Batista1,2*, Francisco Ernesto de Souza Neto2 and Weslley de Souza Paiva2 Departamento de Agricultura – DA, Centro de Ciências Humanas, Sociais e Agrárias – CCHSA, Universidade Federal da Paraíba – UFPB, Bananeiras, PB, Brasil 2 Programa de Pós-graduação em Ciência e Engenharia de Materiais – PPGCEM, Universidade Federal Rural do Semi-Árido – UFERSA, Mossoró, RN, Brasil 1
*bellecamarotti@gmail.com
Abstract Fungal chitosan is a polymer that has been discussed and studied since 1859 in the world with great advances occurring over the years. Due to its global importance, this review aims to expose the history of the production and application of fungal chitosan in Brazil. Data collection was done at the Scielo, Sciencedirect and Pubmed databases, considering the period of the last 50 years. The inclusion criteria were articles on pure or associated chitosan and, in particular, fungal chitosan produced or applied by Brazilian research groups. At the end of the review, it was noticed a fungal chitosan very studied in different continents, and in Brazil is still used in specific and small groups. With the present work, it is expected that the diffusion of the studies will be accelerated and that potential research groups for fungal chitosan may grow through interaction with the existing ones. Keywords: fungal chitosan, biopolymer, biomedical application, biotechnology industries.
1. Introduction The production of chitosan by the process of chemical deacetylation from the shell of crustaceans is financially advantageous for some industries. In Brazil, this process produces, in general, medium molecular weight chitosan and ~ 80% deacetylation degree. Chitosan with this standard is marketed nationally by the company Polymar Science and Nutrition S.A, located in Ceará and classified as food by the Ministry of Health. It is marketed only with weight reducer and cholesterol. This marketing line released by the Brazilian Ministry of Health takes into account literature reports on the purity of preparations containing chitosan obtained from α-chitin, which does not always meet the standards required in the areas related to pharmacy, medicine and food[1-4]. In order for the chitosan obtained from α-chitin to be used by the pharmaceutical, medical and food industries, it is necessary that several specific purification processes need to be done in order to correctly remove traces of proteins and pigments[5,6]. These processes raise production costs and decrease yield. These processes often depolymerize the resulting chitosan in a random manner[7,8], in addition to producing many chemical pollutants that are difficult to reuse or discard. This fact does not occur with chitosan originating from fungi, leaving its production potentially profitable financially. Another argument is the difficulty of adapting a standard in the production of chitosan from the crustacean shell inside an industrial plant, in order to maintain the physicochemical properties of the biopolymer[9].
Polímeros, 28(3), 275-283, 2018
With this in mind, the microbiological production of chitosan from submerged fungi cultures has the advantage of being easier to set a standard for desirable physicochemical characteristics and to implement in an industrial plant because it has the advantage of yielding greater production in smaller areas[10-14]. As well as contributing to the reduction of environmental waste generated by the production of chitosan from the deacetylation of α-chitin. Based on its biotechnological potential and global importance, this review intends to organize and catalog the researchers and research groups that work directly or indirectly with fungal chitosan in Brazil with a view to facilitating the exchange of knowledge between the groups. In addition to promoting the dissemination of what has been done about the production and application in the last 33 years, since it was published pioneering work in Brazil on the analysis of the cell wall of fungi belonging to the class of Zygomycetes, Mucorales order by Campos-Takaki et al.[15].
2. Fungal Chitosan The fungal cell wall is mainly composed of a network of interconnected molecules consisting of proteins, glucans, chitin, chitosan, lipid and polyphosphates which may have the quantity and/or quality changed due to environmental conditions and intrinsic characteristics of their own species[15,16]. Among the compounds found in the cell wall of fungi, chitosan stands out for being associated to increased
275/283 275
R R R R R R R R R R R R R R
Batista, A. C. L., Souza Neto, F. E., & Paiva, W. S. integrity, favoring protection against high temperatures and cell inhibitors to which the fungal strain may be subjected[17-19]. The production of chitosan in the cell wall of fungi occurs due to the activity of the chitin deacetylase enzyme (EC 3.5.1.41) on the chitin residues present in the cell wall itself. This synthesis was first described in 1974 by Araki and Ito in Mucor rouxii (Zygomycetes) showing that about 14% of chitin deacetylase directly involved in the natural synthesis of chitosan in fungi is present and associated with particles of cell fractions, 49% is associated with a soluble fraction of the supernatant, and about 37% is extracellular[20]. Complementing the route of synthesis of chitosan, Davis and Bartnicki-Garcia[21] found strong indications that the activity of the chitin deacetylase is highly efficient on residues from a growing chain of chitin, and has low efficiency in the preformed chitin molecule suggesting that changes in the culture medium in which the fungi grow can directly influence the production of chitosan. In the following years, new researchers studied the line of analysis of the wall and the confirmation of the presence of chitosan in different fungal species[22-24].
3. History of Fungal Chitosan in the World The first chitin isolation reports are from 1811, when the French professor Henri Braconnot conducted tests with isolates from Agaricus volvaceus, A. acris, A. Cantarellus, A. piperatus, Hydnum repandum, H. hybridum and Boletus viscidus and obtained a crude extract formed by chitin-glucan complex, lacking proteins and pigments and presenting acetyl groups, which he called “fungine”. Odier, in 1823, named “chitin” the insoluble substance that he
isolated from the exoskeleton of insects. Odier also realized this same substance could be found in the shell of crabs, and speculated that this could be a basic material present in the exoskeleton of insects[25]. In the nineteenth century, the researchers Odier described the isolation of chitin from the cuticle of insects as well as from mushrooms. This process was done by performing multiple treatments with a concentrated sodium hydroxide solution. With this report in mind, it is reasonable to believe that the material which resulted from such process had been chitosan, because when chitin is exposed to concentrated alkaline media, the deacetylation of this biopolymer is stimulated, and chitosan is obtained[26]. The description of the presence of an amine grouping at carbon 2 of the residues, determining characteristic of the formation of chitosan, was initially done by Rouget in 1859, however the name “chitosan” was proposed by Hoppe-Seyler in 1984[27]. Since its description and designation, chitosan was for many years the subject of basic research only. However, this scenario has been changing since the mid-1970s, when its vast potential in different applications was noted (Table 1).
4. Fungal Chitosan in the Industry Companies that realized its marketable potential have used the fungal chitosan in industries worldwide. These companies have diverse uses for this biopolymer, more specifically in the medical field. Among the companies that promote bioproducts produced with fungal chitosan, we have:
Table 1. Overview of studies on the potential of chitosan in different applications regardless of the method used to obtain it and its physicochemical characteristics, from 1970 until now. Period 1970-79
Application potential Enzyme immobilization;
1980-89 1990-99
Healing activity Treatment of industrial residues; Heavy metals biosorption Muzzarelli et al.[35,36], Muzzarelli[37] Effluent treatment; Muzzarelli et al.[38], Muzzarelli[39], Dobetti and Delben[40], Hoagland and Parris[41] Enzyme immobilization;
2000-09
Film production Degradation of dyestuff; Enzyme immobilization; Biosorption and adsorption of Heavy metals;
References Muzzarelli[28,29], Balassa30], Leuba and Widmer31,32], Kasumi et al.[33, Bissett and Sternberg[34]
Jin and Bai[42], Amorim et al.[43], Chiou et al.[44], Franco et al.[45], Jeon and Ha Park[46], Rungsardthong[47], Zhao et al.[48], Crini and Badot[49], Baroni et al.[50], Perioli et al.[51], Cheung et al.[52]
Food conservation; 2010-now
Medical application Cosmetics; Medical application; Food conservation;
Abdull Rasad et al.[53], Tajdini et al.[54], Gomathi et al.[55], Harris et al.[56], Li et al.[57], Batista et al.[58], Yu et al.[59], Moussa et al.[60], Paiva et al.[61], Oliveira et al.[62], Tayel et al.[63], Taillandier et al.[64], Souza et al.[65]
Biocapsules production; Effluent treatment; Adsorption of heavy metals Antiparasitic
276 276/283
Polímeros, 28(3), 275-283, 2018
Review of fungal chitosan: past, present and perspectives in Brazil • Kitozyme: Belgian company pioneer in the production of highly pure non-animal chitosan. It holds some unique patents on the production processes of polymers, such as chitosan. Its products are KiOnutrime-CsG, which absorbs of fat in the human organism; KiObind, which is a combination of the previous with vitamin C; and KioCardio, which decreases cholesterol levels[66]; • InvivoGen: American company that uses chitosan as a vaccine adjuvant to promote protein adsorption on mucosal surfaces. Their commercial product is called Chitosan VaccGrade[67]; • nbiose: Belgian biotechnology company focused on providing carbohydrates with added value to their clients, aiming applications in various fields, such as nutrition, biomedical, pre-biotics, etc. They make use of a technology that yields good chitosan production using fermentation techniques. This allows reasonable costs and high productive yield[68].
5. History of Fungal Chitosan in Brazil 5.1 Past The pioneering studies to identify and quantify fungal chitosan in Brazil were the works published by Galba Maria Campos-Takaki et al.[15]. In 1983, this group analyzed the cell wall of fungi, from the Zygomycetes class and Mucorales order, using cytochemical, ultrastructural and X-ray microanalysis techniques. In the said work, they confirmed the presence of chitosan in the cell wall of those fungi and that noted that the polysaccharide values vary from 26-28% of dry weight of the species analyzed. From 2001, Amorim and colleagues, obtained for the first time chitosan from fungi, using strains of Mucorales. In addition, one of their results was the finding of a degree of N-acetylation of 49% for Mucor recemosus strain and 20% for Cunnigamella elegans, and quantities of D-glucosamine of 48% and 90%, respectively[10]. Continuing the research on chitosan extracted from strains of the Mucorales order, Amorim et al., (2003) obtained chitosan from Syncephalastrum racemosum with degree of deacetylation (DD) of 88.99%, and performed lipase immobilization using support film made from the chitosan they obtained[43]. The immobilization of the lipase with this biofilm reduced the catalytic activity of the enzyme in about 47%. These are similar results to those collected from studies that used the immobilization film made from crustacean chitosan, which demonstrates the potential of fungal chitosan in biotechnological production against similar products that are already commercialized for different applications, such as crustacean chitosan[40,42,44]. In 2004, Franco et al.[45] continued Brazilian research using fungal chitosan in bonds with metals, such as iron and copper. The work intended to use this polysaccharide as a bioremedy agent, assisting in the elimination of heavy metals from the environment. The experiment demonstrated good biotechnological potential for fungal chitosan obtained from Cunninghamella elegans in copper adsorption. Having potential producers in mind and always comparing their samples to chitosan produced from chitin Polímeros, 28(3), 275-283, 2018
obtained from the shell of crustaceans. The same research group, in 2005, published a study that aimed to assess the production yield from the extraction of chitin and chitosan from a strain of Cunninghamella elegans (IFM 46109) when compared to other strains of the Zygomycetes class, and to the chitosan extracted from the shell of crustaceans. As a result, they demonstrate that fungal chitosan compares to chitosan derived from chitin obtained from crustaceans as to their physicochemical characteristics[69]. Stamford et al, 2007 conducted tests on various culture media and different incubation periods to optimize the production of chitosan from the fungus Cunninghamella elegans, they obtained chitosan with a degree of deacetylation of 85%[70]. In 2008, Fai et al.[71] expanded studies regarding chitosan by applying it as an additive to better food conservation over time, proving its efficiency and turning it into a promising molecule for large-scale use in the food industry. In 2009, Bento et al.[72] investigated the production of chitosan from the fungus Mucor rouxxi UCP 064, obtaining deacetylation degree of 85% and the highest yield of the polymer with only 48 hours of cultivation. The same study applied the chitosan they obtained in an antimicrobial test against Listeria monocytogenes, finding good results on the inhibition of this microorganism. In the same year, Bento et al. [73] applied chitosan as an inhibitor of Listeria monocytogenes in meat products, and had satisfactory results on the inhibition of this microorganism.
5.2 Present At the present moment, the Mucorales order Zygomycetes class, is the most representative and studied in the biotechnology field for containing most species that produce biocompounds of great commercial interest (Table 2). Among many compounds of interest, chitosan should be highlighted, since these species have large quantities of this biopolymer in their cell wall when they are in their vegetative form[21,83]. Currently the work on fungal chitosan is divided into production, where studies concern more profitable and sustainable ways of obtaining this biopolymer; and application, in which one observes patents and marketable potential for this biotechnological product of international acceptance.
5.3 Production In the nineteenth century, the scientist White et al.[84] developed a methodology that allowed the isolation of chitosan from fungal mycelium. From this study, several other scientists have begun to develop adaptations to seek improving the process efficiency[24,85,86]. Based on these studies, the scientist HU and colleages (1999), in the late twentieth century, developed an extraction protocol that is currently one of the most used in experiments with fungal chitosan. This protocol describes each step to be performed from cultivation period to specific pH values, which are essential for the success of the extraction[87]. Using the extraction process described by Hu et al.[87], many Brazilian researchers aim to acquire higher yield of fungal chitosan as well as decrease production costs in order 277/283 277
Batista, A. C. L., Souza Neto, F. E., & Paiva, W. S. Table 2. Biopolymer production by species from the Mucorales order as depend of the cultivation conditions.The cultivation conditions interfere directly of the better biopolymer production. Species Syncephalastrum sp.
Biopolymer α-amylase;
Reference Yu et al.[59], Batista et al.[74], Freitas et al.[75]
Cunninghamella sp. Rhizopus sp.
chitosan; Chitosan chitin;
Oliveira et al.[62], Tayel et al.[63] Cardoso et al.[76], Berger et al.[77]
Rhizopus stolonifer Mucor spp.
chitosan Chitosan enzyme production;
Paiva et al.[78] Bento et al.[79], Fai et al.[80], Souza et al.[81]
Fusarium sp.
chitosan Chitosan
Berger et al.[82]
to compete commercially with chitosan obtained from chitin from crustacean’s shell. Cardoso et al.[76] performed the extraction of fungal chitosan by media comprised of by-products of the food agribusiness and were able to obtain 29.30 mg of chitosan per gram of dry mass of Rhizopus arrhizus; and Berger et al.[77] obtained 57.82 mg of chitosan per gram of dry mass of Cunnigamella ellegans UCP / WFCC 0542. Batista et al.[88], managed to increase yield working with a medium composed of the by-product of shrimp industry, reaching up to 89.95 mg of chitosan per 1g of biomass of Rhyzopus spp. The values aforementioned, when compared to animal chitosan, obtained from the chitin extracted from the shell of crustaceans, still reflect lower yield. However, when taking into account the physicochemical characteristics, the ease of obtaining, the low cost purification and the little chemical waste generated by the production of fungal chitosan when compared to animal chitosan production, this difference becomes minimal[89,90]. Based on the synthesis path of chitosan in fungi, many researchers have shown that several factors can affect the production of this biopolymer by submerged fungi culture. These factors have also been described as important for the production and manipulation of physicochemical characteristics of chitosan according to the literature[91-95].
5.4 Some characterizations and applications In Brazil, fungal chitosan has already been tested in different applications depending on its physicochemical characteristics. The most used characterizations in chitosan studies were infrared spectroscopy and molecular weight. The infrared technique is based on the Fourier Transform Infrared Spectroscopy, FT-IR, to obtain the degree of deacetylation as a function of Equation 1: A 100 =) 100 − 1655 x DD ( % A 3450 1.33
(1)
where: A1655 is the absorbance at 1655 cm−1 of the amide I band, A3450 is the absorbance at 3450 cm−1 of the hydroxyl band and 1.33 is the value of the A1655/A3450 proportion for a completely acetylated chitosan. Adapted from Martínez-Camacho et al.[96] and Khan et al.[97]. 278 278/283
The molecular weight characterization is closely related to the intrinsic viscosity, where chitosan solutions are passed through Ubbelohde capillary in a water bath and the determination of the molecular weight occurs as a function of the Mark-Houwink-Sakurada Equation 2:
[η] =kMV a
(2)
where [η] is the intrinsic viscosity, MV is the average viscometric molecular weight and both “κ” and “a” are empirical constants that depend of the polymer nature, the solvent and the temperature. “κ” and “a” values were 3.04 × 10 −5 and 1.26, respectively adapted from Martínez-Camacho et al.[96] and Rinaudo et al.[98]. Other techniques that are used to characterize fungal chitosan are thermogravimetry, where it is possible to analyze if there are contaminants in the sample; Scanning electron microscopy, which demonstrates the structure of the polymer; and X-ray diffraction, which indicates the crystallinity of the material. These characteristics are very important for choosing a good polymer for an efficient application[43,60,62]. 5.4.1 Antimicrobial activity application Currently, a major concern in the use of antibiotics is the accumulation of synthetic substances in the body, in addition to the high rate in which the selection of microorganisms resistant to commonly known drugs increases. Therefore, some groups of Brazilian researchers conducted studies in which they used fungal chitosan as a bactericidal agent[62,65]. For the antimicrobial action three mechanisms are proposed: (1) related to the formation of polyelectrolytic complexes, because chitosan has positive charges present in its chain, due to its amine group, that bind selectively to the cell surface of the microorganisms, changing its activity, Permeability of the membrane and reducing the cellular components, resulting, then, in the inhibition or cell death; (2) chitosan acts as a chelating agent, binding to the ions that are necessary for the functioning of certain enzymes; (3) when chitosan is also low in molecular weight, being able to insert into the nucleus of the microbial cell, interacting with the DNA, and interfering with the activities of messenger RNA, consequently affecting protein synthesis[54,60,63,96]. In order for the antimicrobial action to occur, some studies show a connection with the degree of deacetylation above 75%; And molecular weight, that the lower, the greater the action of chitosan against microorganisms[43,45,54,60,62]. Polímeros, 28(3), 275-283, 2018
Review of fungal chitosan: past, present and perspectives in Brazil Paiva and colleagues, in 2014, tested the chitosan extracted from Cunninghamella elegans, as an antibacterial agent against Escherichia coli and Staphylococcus aureus, the study was successful in inhibiting bacteria in all times tested, proving the effectiveness of chitosan as an antibacterial agent[61]. In addition to the bactericidal activity, fungal chitosan may also have fungicidal activity. Oliveira et al.[62] extracted chitosan from Cunninghamella elegans to used as coating for fruit (grape - Vitis labrusca L.) and prevented the growth of pathogenic fungi. In this work, a chitosan was used in film form with 81% degree of deacetylation and average molecular weight. For this application in the form of film, in particular, it was pointed out that if the polymer under study were of low molecular weight, there would be a decrease in the tensile strength and permeability of the film. Within this experiment, they also found that such alternative coating did not influence the sensory conditions of this fruit. 5.4.2 Application of chitosan as biofertilizers Stamford et al (2015) published a patent in Brazil describing the biofertilizer and bioprotector abilities of fungal chitosan. This chitosan was produced from mixed biofertilizer (phosphate and potassium rocks, along with sulfur and an inoculum of acidithiobacillus bacteria; organic matter inoculated with diazotrophs free life bacteria; and fungi that produce chitin and chitosan). This production was carried out in a purely biological way, and resulted in a product with a high fertilizer and protection effect against phytopathogenic microorganisms present in the soil, without the need to use external energy sources[99]. 5.4.3 Enzyme immobilization The enzyme immobilization technique is much valued in cosmetics, food and pharmaceutical industries. Having this in mind, Amorim et al.[43] used fungal chitosan as a film for perform enzyme immobilization. In their experiment, the degree of deacetylation of fungal chitosan was 88.9%. This characteristic was thought due to chitosan to provide as reactive points a smaller amount of acetamide clusters. In this way, there is a decrease in the density of the chitosan molecule and favoring its function as a support for the immobilization of enzymes. They aimed to immobilize an enzyme called lipase and watch its time of activity. As a result, they found 47% of initial catalytic activity after four reaction cycles. Therefore, these results are as efficient as those found in studies using animal chitosan (obtained from crustaceans shells).
6. Prospects After its discovery, chitosan has become a source of numerous studies around the world due to its versatility of use. Initially, the most commonly used type of chitosan was that obtained from the shell of shrimp, however, with the discovery of the presence of chitosan in the cell wall of fungi, which is extracted with less production of waste during purification and no presence of allergenic substances, the fungal or biotechnological chitosan gained ground in worldwide research groups[100]. Polímeros, 28(3), 275-283, 2018
In Brazil, the first reports date back to the early 80’s, with the research group of Professor Dr. Galba Maria de Campos-Takaki. After this first study, the others that followed tried to understand the structure of the fungus, how this chitosan behaves under different conditions, alternative culture media for growth of fungus and measuring the chitosan production, as well as physicochemical characterizations of this chitosan using techniques such as X-ray, infrared, thermogravimetry, zeta pontencial and others[15]. Currently, studies with fungal chitosan in Brazil seek a future biomedical application for this polymer. This can be in helping the body accept implants, such as dental, muscle or bone; using chitosan membranes to help with burn victims, establishing a relationship between the polymer and its reparative action on biological tissues; and in the antimicrobial activity of this polymer, helping to maintain aseptic conditions in hospital equipment. The versatility of this polymer has also been found effective in the food engineering area, where research is focused on the production of biofilms and membranes that protect food from microorganisms, increasing its shelf-life and reducing financial losses due to contamination[59,63,77,101,102]. Another way that fungal chitosan is being used in Brazil is in the material engineering field, where the polymer is being used as an insulating material to composites, resulting in decrease of plastic material, thus avoiding the serious environmental damage that the plastic accumulation causes in the environment. Concerning the companies in this sector, Brazil is still taking regular steps when compared to the reality of Europe and the United States. Even presenting characteristics and qualified personnel for the development of new technologies. What we can observe from the groups involved in this area are robust visions that contribute to making Brazil a leading producer of fungal chitosan for any feasible purpose. For this, further studies concerning fungal chitosan are relevant in order to be sure that its use is feasible and safe for humans. Even though there is some resistance from the industry to this polymer obtained from fungi due to its lower yield when compared to chitosan of animal origin, it is important to note that the cost of purification and the amount of waste generated are much lower. These facts gives the fungal chitosan a prominent place in scientific research in Brazil, as well as in the world.
7. Conclusion Given the knowledge acquired during this work, we see that the use of fungal chitosan in Brazil needs to be further explored. To date, Brazilian scientists have cataloged different biological activities of fungal chitosan. These studies, even if they are still on bench scale and with few deposited patents, are important because they demonstrate the biotechnological potential of some groups operating in the Brazilian Northeast, an area still with little financial investment for the research when compared to the Southeast region. In the world fungal chitosan has already been reported for different purposes and has already been used in different patented formulations. In Brazil, there are still studies of production and small applications that normally do not 279/283 279
Batista, A. C. L., Souza Neto, F. E., & Paiva, W. S. transgress the academic area. With the dissemination of the papers and exposition of the research groups it is expected that new agreements will be set up and that agreements with industrial sectors be signed for the purpose of patents and commercial projection of fungal chitosan, as it happens in Europe.
8. Acknowledgements To doctoral program of the Northeast Biotechnology Network – RENORBIO; Center for Research in Environmental Sciences – NPCIAMB, Catholic University of Pernambuco; Federal Rural University of the Semi Arid; CNPq and CAPES for financial support.
9. References 1. Khor, E., & Lim, L. Y. (2003). Implantable applications of chitin and chitosan. Biomaterials, 24(13), 2339-2349. http:// dx.doi.org/10.1016/S0142-9612(03)00026-7. PMid:12699672. 2. Campana-Filho, S. P., Britto, D., Curti, E., Abreu, F. R., Cardoso, M. B., Battisti, M. V., Sim, P. C., Goy, R. C., Signini, R., & Lavall, R. L. (2007). Extração, estruturas e propriedades de alfa- e beta-quitina. Química Nova, 30(3), 644-650. http:// dx.doi.org/10.1590/S0100-40422007000300026. 3. Queiroz, M. F., Melo, K. R. T., Sabry, D. A., Sassaki, G. L., & Rocha, H. A. O. (2014). Does the use of chitosan contribute to oxalate kidney stone formation? Marine Drugs, 13(1), 141-158. http://dx.doi.org/10.3390/md13010141. PMid:25551781. 4. Naghdi, M., Zamani, A., & Karimi, K. (2014). A sulfuric-lactic acid process for efficient purification of fungal chitosan with intact molecular weight. International Journal of Biological Macromolecules, 63, 158-162. http://dx.doi.org/10.1016/j. ijbiomac.2013.10.042. PMid:24211428. 5. Rinaudo, M. (2006). Chitin and chitosan: properties and applications. Progress in Polymer Science, 31(7), 603-632. http://dx.doi.org/10.1016/j.progpolymsci.2006.06.001. 6. Struszczyk, M. H. (2002). Chitin and chitosan - part I. Properties and production, Warsaw, Poland. Polimery, 1(5), 316-325. Retrieved in 2016, July 15, from http://en.www.ichp. pl/Chitin-and-Chitosan-Part-I-Properties-and-production 7. Choi, W.-S., Ahn, K.-J., Lee, D.-W., Byun, M.-W., & Park, H.-J. (2002). Preparation of chitosan oligomers by irradiation. Polymer Degradation & Stability, 78(3), 533-538. http://dx.doi. org/10.1016/S0141-3910(02)00226-4. 8. Yue, W. (2014). Prevention of browning of depolymerized chitosan obtained by gamma irradiation. Carbohydrate Polymers, 101, 857-863. http://dx.doi.org/10.1016/j.carbpol.2013.10.011. PMid:24299848. 9. Tayel, A. A., Moussa, S. H., El-Tras, W. F., Knittel, D., Opwis, K., & Schollmeyer, E. (2010). Anticandidal action of fungal chitosan against Candida albicans. International Journal of Biological Macromolecules, 47(4), 454-457. http://dx.doi. org/10.1016/j.ijbiomac.2010.06.011. PMid:20603144. 10. Amorim, R. V. S., Souza, W., Fukushima, K., & Campos-Takaki, G. M. (2001). Faster chitosan production by Mucorelean strains in submerged culture. Brazilian Journal of Microbiology, 32(1), 20-23. http://dx.doi.org/10.1590/S1517-83822001000100005. 11. Amorim, R. V. S., Ledingham, W. M., Kennedy, J. F., & Campos-Takaki, G. M. (2006). Chitosan from Syncephalastrum racemosum using sugar cane substrates as inexpensive carbon sources. Food Biotechnology, 20(1), 43-53. http://dx.doi. org/10.1080/08905430500524028. 12. Feng, Y. L., Li, W. Q., Wu, X. Q., Cheng, J. W., & Ma, S. Y. (2010). Statistical optimization of media for mycelial growth 280 280/283
and exo-polysaccharide production by Lentinus edodes and a kinetic model study of two growth morphologies. Biochemical Engineering Journal, 49(1), 104-112. http://dx.doi.org/10.1016/j. bej.2009.12.002. 13. Liu, R. S., Li, D. S., Li, H. M., & Tang, Y. J. (2008). Response surface modeling the significance of nitrogen source on the cell growth and Tuber polysaccharides production by submerged cultivation of Chinese truffle Tuber sinense. Process Biochemistry, 43(8), 868-876. http://dx.doi.org/10.1016/j. procbio.2008.04.009. 14. Pokhrel, C. P., & Ohga, S. (2007). Submerged culture conditions for mycelial yield and polysaccharides production by Lyophyllum decastes. Food Chemistry, 105(2), 641-646. http://dx.doi.org/10.1016/j.foodchem.2007.04.033. 15. Campos-Takaki, G. M., Beakes, G. W., & Dietrich, S. M. (1983). Electron microscopic X-ray microprobe and cytochemical study of isolated cell walls of mucoralean fungi. Transactions of the British Mycological Society, 80(3), 536-541. http://dx.doi. org/10.1016/S0007-1536(83)80053-9. 16. Ruiz-Herrera, J. (2012). Fungal cell wall: estructure, synthesis, and assembly. In J. Ruiz-Herrera (Ed.), Current topics in medical mycology (Vol. 3, pp. 168-217). New York: Springer. http:// dx.doi.org/10.1201/b11873. 17. Adams, D. J. (2004). Fungal cell wall chitinases and glucanases. Microbiology, 150(1), 2029-2035. http://dx.doi.org/10.1099/ mic.0.26980-0. PMid:15256547. 18. Baker, L. G., Specht, C. A., Donlin, M. J., & Lodge, J. K. (2007). Chitosan, the Deacetylated form of chitin, is necessary for cell wall integrity in Cryptococcus neoformans. Eukaryotic Cell, 6(5), 855-867. http://dx.doi.org/10.1128/EC.00399-06. PMid:17400891. 19. Banks, I. R., Specht, C. A., Donlin, M. J., Gerik, K. J., Levitz, S. M., & Lodge, J. K. (2005). A chitin synthase and its regulator protein are critical for chitosan production and growth of the fungal pathogen Cryptococcus neoformans. Eukaryotic Cell, 4(11), 1902-1912. http://dx.doi.org/10.1128/EC.4.11.19021912.2005. PMid:16278457. 20. Araki, Y., & Ito, E. (1974). A pathway of chitosan formation in Mucor rouxii: enzymatic deacetylation of chitin. Biochemical and Biophysical Research Communications, 56(3), 669-675. http:// dx.doi.org/10.1016/0006-291X(74)90657-3. PMid:4826874. 21. Davis, L. L., & Bartnicki-Garcia, S. (1984). Chitosan synthesis by the tandem action of chitin synthetase and chitin deacetylase from Mucor rouxii. Biochemistry, 23(6), 1065-1073. http:// dx.doi.org/10.1021/bi00301a005. 22. Kendra, D. F., Christian, D., & Hadwiger, L. A. (1989). Chitosan oligomers from Fusarium solani/pea interactions, chitinase/ßglucanase digestion of sporelings and from fungal wall chitin actively inhibit fungal growth and enhance disease resistance. Physiological and Molecular Plant Pathology, 35(3), 215-230. http://dx.doi.org/10.1016/0885-5765(89)90052-0. 23. Sudarshan, N. R., Hoover, D. G., & Knorr, D. (1992). Antibacterial action of chitosan. Food Biotechnology, 6(3), 257-272. http://dx.doi.org/10.1080/08905439209549838. 24. Synowiecki, J., & Al-Khateeb, N. A. A. Q. (1997). Mycelia of Mucor rouxii as a source of chitin and chitosan. Food Chemistry, 60(4), 605-610. http://dx.doi.org/10.1016/S03088146(97)00039-3. 25. Braconnot, H. (1811). Sur la nature des champignons. Annales de Chimie Physique, 79, 265-304. 26. Odier, A. (1823). Mémoir sur la composition chimique des parties cornées des insectes. Mémoirs de la Societé d’Histoire Naturelle, 1, 29-42. 27. Hoppe-Seyler F. (1894). Ueber chitosan und zellulose. Germany: Ber. Deutsche Chemische Gesellschaft. Polímeros, 28(3), 275-283, 2018
Review of fungal chitosan: past, present and perspectives in Brazil 28. Muzzarelli, R. A. A. (1973). Natural chelating polymers: alginic acid, chitin, and chitosan. Oxford: Pergamon Press. 29. Muzzarelli, R. A. A. (1976).Biochemical modifications of chitin. In H. R. Hepburn (Ed.), The insect integument (pp. 63-87). New York: Elsevier Scientific Publishing Company. 30. Balassa, L. L. (1975). US Patent No 3903268. Estados Unidos. Retrieved in 2016, July 15, from http://patft.uspto.gov/netacgi/ nph-Parser?Sect2=PTO1&Sect2=HITOFF&p=1&u=/netahtml/ PTO/searchbool.html&r=1&f=G&l=50&d=PALL&RefSrch= yes&Query=PN/3903268 31. Leuba, J. L., & Widmer, F. (1977). Immobilization of the β-galactosidase from Aspergillus niger on chitosan. Journal of Solid-Phase Biochemistry, 3(2), 257-271. http://dx.doi. org/10.1007/BF02996747. 32. Leuba, J. L., & Widmer, F. (1979). Immobilization of proteinases on chitosan. Biotechnology Letters, 1(3), 109-114. http://dx.doi. org/10.1007/BF01386708. 33. Kasumi, T., Tsuji, M., Hayashi, K., & Tsumura, N. (1977). Preparation and some properties of chitosan bounds enzymes. Agricultural and Biological Chemistry, 41(10), 1865-1872. http://dx.doi.org/10.1080/00021369.1977.10862778. 34. Bissett, F., & Sternberg, D. (1978). Immobilization of Aspergillus beta-glucosidase on chitosan. Applied and Environmental Microbiology, 35(4), 750-755. PMid:25624. 35. Muzzarelli, R. A. A., Tanfani, F., Emanuelli, M., & Mariotti, S. (1982). N-(carboxymethylidene) chitosans and n-(carboxymethyl)chitosans: novel chelating polyampholytes obtained from chitosan glyoxylate. Carbohydrate Research, 107(2), 199-214. http://dx.doi.org/10.1016/S0008-6215(00)80539-X. 36. Muzzarelli, R. A. A., Tanfani, F., & Emanuelli, M. (1984). Chelating derivatives of chitosan obtained by reaction with ascorbic acid. Carbohydrate Polymers, 4(2), 137-151. http:// dx.doi.org/10.1016/0144-8617(84)90020-1. 37. Muzzarelli, R. A. A. (1985). Removal of uranium from solutions and brines by a derivative of chitosan and ascorbic acid. Carbohydrate Polymers, 5(2), 85-89. http://dx.doi. org/10.1016/0144-8617(85)90026-8. 38. Muzzarelli, R. A. A., Weckx, M., Filippini, O., & Sigon, F. (1989). Removal of trace metal ions from industrial waters, nuclear effluents and drinking water, with the aid of cross-linked N-carboxymethyl chitosan. Carbohydrate Polymers, 11(4), 293-306. http://dx.doi.org/10.1016/0144-8617(89)90004-0. 39. Muzzarelli, R. A. A. (1990). Book review: methods in enzymology: lignin, pectin and chitin. Carbohydrate Polymers, 12(2), 242243. http://dx.doi.org/10.1016/0144-8617(90)90024-M. 40. Dobetti, L., & Delben, F. (1992). Binding of metal cations by N-carboxymethyl chitosans in water. Carbohydrate Polymers, 18(4), 273-282. http://dx.doi.org/10.1016/0144-8617(92)900925. 41. Hoagland, P. D., & Parris, N. (1996). Chitosan/pectin laminated films. Journal of Agricultural and Food Chemistry, 44(7), 1915-1919. http://dx.doi.org/10.1021/jf950162s. 42. Jin, L., & Bai, R. (2002). Mechanisms of lead adsorption on chitosan/PVA hydrogel beads. Langmuir, 18(25), 9765-9770. http://dx.doi.org/10.1021/la025917l. 43. Amorim, R. V. S., Melo, E. S., Carneiro-da-Cunha, M. G., Ledingham, W. M., & Campos-Takaki, G. M. (2003). Chitosan from Syncephalastrum racemosum used as a film support for lípase immobilization. Bioresource Technology, 89(1), 35-39. http://dx.doi.org/10.1016/S0960-8524(03)00035-X. PMid:12676498. 44. Chiou, M. S., Ho, P. Y., & Li, H. Y. (2004). Adsorption of anionic dyes in acid solutions using chemically cross-linked chitosan beads. Dyes and Pigments, 60(1), 69-84. http://dx.doi. org/10.1016/S0143-7208(03)00140-2. Polímeros, 28(3), 275-283, 2018
45. Franco, L. O., Maia, R. C. C., Porto, A. L. F., Messias, A. S., Fukushima, K., & Campos-Takaki, G. M. (2004). Heavy metal biosorption by chitin and chitosan isolated from Cunninghamella elegans (IFM 46109). Brazilian Journal of Microbiology, 35(3), 243-247. http://dx.doi.org/10.1590/ S1517-83822004000200013. 46. Jeon, C., & Ha Park, K. (2005). Adsorption and desorption characteristics of mercury (II) ions using aminated chitosan bead. Water Research, 39(16), 3938-3944. http://dx.doi. org/10.1016/j.watres.2005.07.020. PMid:16129473. 47. Rungsardthong, V., Wongvuttanakul, N., Kongpien, N., & Chotiwaranon, P. (2006). Application of fungal chitosan for clarification of apple juice. Process Biochemistry, 41(3), 589593. http://dx.doi.org/10.1016/j.procbio.2005.08.003. 48. Zhao, F., Yu, B., Yue, Z., Wang, T., Wen, X., Liu, Z., & Zhao, C. (2007). Preparation of porous chitosan gel beads for copper(II) ion adsorption. Journal of Hazardous Materials, 147(1-2), 67-73. http://dx.doi.org/10.1016/j.jhazmat.2006.12.045. PMid:17258856. 49. Crini, G., & Badot, P. M. (2008). Application of chitosan, a natural aminopolysaccharide, for dye removal from aqueous solutions by adsorption processes using batch studies: a review of recent literature. Progress in Polymer Science, 33(4), 399447. http://dx.doi.org/10.1016/j.progpolymsci.2007.11.001. 50. Baroni, P., Vieira, R. S., Meneghetti, E., da Silva, M. G., & Beppu, M. M. (2008). Evaluation of batch adsorption of chromium ions on natural and crosslinked chitosan membranes. Journal of Hazardous Materials, 152(3), 1155-1163. http:// dx.doi.org/10.1016/j.jhazmat.2007.07.099. PMid:17826905. 51. Perioli, L., Ambrogi, V., Pagano, C., Scuota, S., & Rossi, C. (2009). FG90 chitosan as a new polymer for metronidazole mucoadhesive tablets for vaginal administration. International Journal of Pharmaceutics, 377(1-2), 120-127. http://dx.doi. org/10.1016/j.ijpharm.2009.05.016. PMid:19454304. 52. Cheung, W. H., Szeto, Y. S., & Mckay, G. (2009). Enhancing the adsorption capacities of acid dyes by chitosan nano particles. Bioresource Technology, 100(3), 1143-1148. http:// dx.doi.org/10.1016/j.biortech.2008.07.071. PMid:18829305. 53. Abdull Rasad, M. S. B., Halim, A. S., Hashim, K., Rashid, A. H. A., Yusof, N., & Shamsuddin, S. (2010). In vitro evaluation of novel chitosan derivatives sheet and paste cytocompatibility on human dermal fibroblasts. Carbohydrate Polymers, 79(4), 1094-1100. http://dx.doi.org/10.1016/j.carbpol.2009.10.048. 54. Tajdini, F., Amini, M. A., Nafissi-Varcheh, N., & Faramarzi, M. A. (2010). Production, physiochemical and antimicrobial properties of fungal chitosan from Rhizomucor miehei and Mucor racemosus. International Journal of Biological Macromolecules, 47(2), 180-183. http://dx.doi.org/10.1016/j. ijbiomac.2010.05.002. PMid:20471417. 55. Gomathi, P., Ragupathy, D., Choi, J. H., Yeum, J. H., Lee, S. C., Kim, J. C., Lee, S. H., & Ghim, H. D. (2011). Fabrication of novel chitosan nanofiber/gold nanoparticles composite towards improved performance for a cholesterol sensor. Sensors and Actuators. B, Chemical, 153(1), 44-49. http:// dx.doi.org/10.1016/j.snb.2010.10.005. 56. Harris, R., Lecumberri, E., Mateos-Aparicio, I., Mengíbar, M., & Heras, M. (2011). Chitosan nanoparticles and microspheres for the encapsulation of natural antioxidants extracted from Ilex paraguariensis. Carbohydrate Polymers, 84(2), 803-806. http://dx.doi.org/10.1016/j.carbpol.2010.07.003. 57. Li, X., Shi, X., Wang, M., & Du, Y. (2011). Xylan chitosan conjugate: a potential food preservative. Food Chemistry, 126(2), 520-525. http://dx.doi.org/10.1016/j.foodchem.2010.11.037. PMid:25212164. 58. Batista, A. C. L., Villanueva, E. R., Amorim, R. V. S., Tavares, M. T., & Campos-Takaki, G. M. (2011). Chromium (VI) ion 281/283 281
Batista, A. C. L., Souza Neto, F. E., & Paiva, W. S. adsorption features of chitosan film and its chitosan/zeolite conjugate 13X film. Molecules (Basel, Switzerland), 16(5), 3569-3579. http://dx.doi.org/10.3390/molecules16053569. PMid:21527884. 59. Yu, T., Yu, C., Chen, F., Sheng, K., Zhou, T., Zunun, M., Abudu, O., Yang, S., & Zheng, X. (2012). Integrated control of blue mold in pear fruit by combined application of chitosan, a biocontrol yeast and calcium chloride. Postharvest Biology and Technology, 69, 49-53. http://dx.doi.org/10.1016/j. postharvbio.2012.02.007. 60. Moussa, S. H., Tayel, A. A., & Al-Turki, I. A. (2013). Evaluation of fungal chitosan as a biocontrol and antibacterial agent using fluorescence-labeling. International Journal of Biological Macromolecules, 54, 204-208. http://dx.doi.org/10.1016/j. ijbiomac.2012.12.029. PMid:23270832. 61. Paiva, W. S., Souza, F. E., No., & Batista, A. C. L. (2014). Avaliação da atividade antibacteriana da quitosana fúngica. Perspectivas Online: Biológicas e Saúde, 13, 37-43. Retrieved in 2016, July 15, from http://www.seer.perspectivasonline. com.br/index.php/biologicas_e_saude/article/view/495/442 62. Oliveira, C. E. V., Magnani, M., Sales, C. V., Pontes, A. L. S., Campos-Takaki, G. M., Stamford, T. C. M., & Souza, E. L. (2014). Effects of chitosan from Cunninghamella elegans on virulence of post-harvest pathogenic fungi in table grapes (Vitis labrusca L.). International Journal of Food Microbiology, 171, 54-61. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.11.006. PMid:24321603. 63. Tayel, A. A., Ibrahim, S. I. A., Al-Saman, M. A., & Moussa, S. H. (2014). Fungal chitosan from date wastes and its application as a biopreservative for minced meat. International Journal of Biological Macromolecules, 69, 471-475. http://dx.doi. org/10.1016/j.ijbiomac.2014.05.072. PMid:24942991. 64. Taillandier, P., Joannis-Cassan, C., Jentzer, J. B., Gautier, C. S. N., Sieczkowski, N., Granes, D., & Brandam, C. (2015). Effect of a fungal chitosan preparation on Brettanomyces bruxellensis, a wine contaminant. Journal of Applied Microbiology, 118(1), 123-131. http://dx.doi.org/10.1111/jam.12682. PMid:25363885. 65. Souza, F. E., No., Silva, H. C. A., Paiva, W. S., Torres, T. M., Rocha, A. C. P., Bezerra, A., & Batista, A. C. L. (2017). Quitosana fúngica sobre larvas de nematoides gastrintestinais de caprinos. Arquivos do Instituto Biológico, 84(0), 1-5. http:// dx.doi.org/10.1590/1808-1657000542015. 66. Kitozyme. (2017, June 26). Belgium. Retrieved in 2016, July 15, from http://kitozyme.com/eng/innovation/technologieswith-non-animal-chitosan-and-chitin-glucan/kionutrime-csg 67. InvivoGen. (2017, June 26). San Diego. Retrieved in 2016, July 15, from http://www.invivogen.com/chitosan-vaccigrade 68. Inbiose. (2017, June 26). Belgium. Retrieved in 2016, July 15, from http://inbiose.com/products.html 69. Franco, L. O., Stamford, T. C. M., Stamford, N. P., & CamposTakaki, G. M. (2005). Cunningamella elegans (IFM 46109) como fonte de quitina e quitosana. Reviews in Analgesia, 14, 40-44. Retrieved in 2016, July 15, from http://www.scielo.cl/ scielo.php?script=sci_nlinks&ref=2120900&pid=S0717345 8200700010000600009&lng=es 70. Stamford, T. C. M., Stamford, T. L. M., Stamford, N. P., Barros, B., No., & Campos-Takaki, G. M. (2007). Growth of Cunninghamella elegans UCP 542 and production of chitin and chitosan using yam bean medium. Electronic Journal of Biotechnology, 10(1), 1-6. http://dx.doi.org/10.2225/vol10issue5-fulltext-1. 71. Fai, A. E. C., Stamford, T. C. M., & Stamford, T. L. M. (2008). Potencial biotecnológico de quitosana em sistemas de conservação de alimentos. Revista Iberoamericana de Polímeros, 9(5), 435-451. Retrieved in 2016, July 15, from http://www.ehu.eus/reviberpol/pdf/JUL08/fai.pdf 282 282/283
72. Bento, R. A., Stamford, T. L. M., Campos-Takaki, G. M., Stamford, T. C. M., & Souza, E. L. (2009a). Potential of chitosan from Mucor rouxxi UCP064 as alternative natural compound to inhibit Listeria monocytogenes. Brazilian Journal of Microbiology, 40(3), 583-589. http://dx.doi.org/10.1590/ S1517-83822009000300022. PMid:24031403. 73. Bento, R. A., Souza, E. L., Stamford, T. L. M., & Stamford, T. C. M. (2009b). Perspectiva e potencial aplicação de quitosana como inibidor de Listeria monocytogenes em produtos cárneos. Revista Iberoamericana de Polímeros, 10(5), 260-274. Retrieved in 2016, July 15, from http://www.ehu.eus/reviberpol/pdf/ SEP09/bento.pdf 74. Batista, A. C. L., Silva, M. C. F., Batista, J. B., Nascimento, A. E., & Campos-Takaki, G. M. (2013). Eco-friendly chitosan production by Syncephalastrum racemosum and application to the removal of acid orange 7 (AO7) from wastewaters. Molecules, 18(7), 7646-7660. http://dx.doi.org/10.3390/ molecules18077646. PMid:23884118. 75. Freitas, L. S., Martins, E. S., & Ferreira, O. S. (2014). Produção e caracterização parcial de α-amilase de Syncephalastrum racemosum. Revista Brasileira de Biociências, 12(4), 226-232. Retrieved in 2016, July 15, from http://www.ufrgs.br/seerbio/ ojs/index.php/rbb/article/view/3120 76. Cardoso, A., Lins, C. I. M., Santos, E. R., Silva, M. C. F., & Campos-Takaki, G. M. (2012). Microbial enhance of chitosan production by Rhizopus arrhizus using agroindustrial substrates. Molecules, 17(5), 4904-4914. http://dx.doi.org/10.3390/ molecules17054904. PMid:22543505. 77. Berger, L. R. R., Stamford, T. C. M., Stamford-Arnaud, T. M., Alcântara, S. R. C., Silva, A. C., Silva, A. M., Nascimento, A. E., & Campos-Takaki, G. M. (2014). Green conversion of agroindustrial wastes into chitin and chitosan by Rhizopus arrhizus and Cunninghamella elegans strains. International Journal of Molecular Sciences, 15(5), 9082-9102. http://dx.doi. org/10.3390/ijms15059082. PMid:24853288. 78. Paiva, W. S., Souza, F. E., No., & Batista, A. C. L. (2017). Characterization of polymeric biomaterial chitosan extracted from Rhizopus stolonifer. Journal of Polymer Materials, 34(1), 115-121. Retrieved in 2016, July 15, from https://search. proquest.com/openview/80a89aa7245f255dd08dd6497c2ae 7af/1?pq-origsite=gscholar&cbl=506337 79. Bento, A. R., Stamford, T. L. M., Stamford, T. C. M., Andrade, S. A. C., & Souza, E. L. (2011). Sensory evaluation and inhibition of Listeria monocytogenes in bovine pâté added of chitosan from Mucor rouxii. Lebensmittel-Wissenschaft + Technologie, 44(2), 588-591. http://dx.doi.org/10.1016/j.lwt.2010.08.016. 80. Fai, A. E. C., Stamford, T. C. M., Stamford-Arnaud, T. M., Santa-Cruz, P. D. A., Silva, M. C. F., Campos-Takaki, G. M., & Stamford, T. L. M. (2011). Physico-chemical characteristics and functional properties of chitin and chitosan produced by Mucor circinelloides using yam bean as substrate. Molecules, 16(8), 7143-7154. http://dx.doi.org/10.3390/molecules16087143. PMid:21862956. 81. Souza, E. L., Sales, C. V., Oliveira, C. E. V., Lopes, L. A. A., Conceição, M. L., Berger, L. R. R., & Stamford, T. C. M. (2015). Efficacy of acoating composed of chitosan from Mucor circinelloides and carvacrol to control Aspergillus flavus and the quality of cherry tomato fruits. Frontiers in Microbiology, 6, 732. http://dx.doi.org/10.3389/fmicb.2015.00732. PMid:26257717. 82. Berger, L. R. R., Stamford, N. P., Willadino, L. G., Laranjeira, D., Lima, M. A. B., Malheiros, S. M. M., Oliveira, W. J., & Stamford, T. C. M. (2016). Cowpea resistance induced against Fusarium oxysporum f. sp. tracheiphilum by crustaceous chitosan and by biomass and chitosan obtained from Cunninghamella elegans. Biological Control, 92, 45-54. http://dx.doi.org/10.1016/j. biocontrol.2015.09.006. Polímeros, 28(3), 275-283, 2018
Review of fungal chitosan: past, present and perspectives in Brazil 83. Campos-Takaki, G. M. (2005). The fungal versatility on the copolymers chitin and chitosan production. In P. K. Dutta (Ed.), Chitin and chitosan opportunities and challenges (pp. 69-94). India: Dubey Printers and Graphics. 84. White, S. A., Farina, P. R., & Fulton, I. (1979). Production and isolation of chitosan from Mucor rouxii. Applied and Environmental Microbiology, 38(2), 323-328. PMid:518086. 85. Crestini, C., Kovac, B., & Giovannozzi-Sermanni, G. (1996). Production and isolation of chitosan by submerged and solidstate fermentation from Lentinus edodes. Biotechnology and Bioengineering, 50(2), 207-210. http://dx.doi.org/10.1002/ bit.260500202. PMid:18626937. 86. Rane, K. D., & Hoover, D. G. (1993). Production of chitosan by fungi. Food Biotechnology, 7(1), 11-33. http://dx.doi. org/10.1080/08905439309549843. 87. Hu, K. J., Yeung, K. W., Ho, K. P., & Hu, J. L. (1999). Rapid extraction of high-quality chitosan from mycelia of Absidia glauca. Journal of Food Biochemistry, 23(2), 187-196. http:// dx.doi.org/10.1111/j.1745-4514.1999.tb00013.x. 88. Batista, A. C. L., Bandeira, M. G. L., Souza, F. E., No., Paiva, W. S., Rodrigues, D. N. R., & Costa, A. C. A. A. (2014). Obtenção de quitosana fúngica crescida em meio alternativo constituído com farinha de carapaça de camarão. Revista Saúde e Ciência On Line, 3(3), 11-17. Retrieved in 2016, July 15, from http://www.ufcg.edu.br/revistasaudeeciencia/index.php/ RSC-UFCG/article/view/246 89. Bessa-Junior, A. P., & Gonçalves, A. A. (2013). Análises econômica e produtiva da quitosana extraída do exoesqueleto de camarão. Acta Of Fisheries and Aquatic Resources, 1(1), 13-28. Retrieved in 2016, July 15, from http://www.seer.ufs. br/index.php/ActaFish/article/view/1589/1488 90. Berger, L. R. R., Stamford, T. C. M., Stamford-Arnaud, T. M., Franco, L. O., Nascimento, A. E., Cavalcante, H. M. M., Macedo, R. O., & Campos-Takaki, G. M. (2014). Effect of corn steep liquor (CSL) and cassava wastewater (CW) on chitin and chitosan production by Cunninghamella elegans and their physicochemical characteristics and cytotoxicity. Molecules, 19(3), 2771-2792. http://dx.doi.org/10.3390/ molecules19032771. PMid:24590203. 91. Neves, A. C., Schaffner, R. A., Kugelmeier, C. L., Wiest, A. M., & Arantes, M. K. (2013). Otimização de processos de obtenção de quitosana a partir de resíduo da carcinicultura para aplicações ambientais. Revista Brasileira de Energias Renováveis, 2(1), 34-47. http://dx.doi.org/10.5380/rber. v2i1.33800. 92. Jaworska, M. M., & Konieczna, E. (2001). The influence of supplemental components in nutrient medium on chitosan formation by the fungus Absidia orchidis. Applied Microbiology and Biotechnology, 56(1-2), 220-224. http://dx.doi.org/10.1007/ s002530000591. PMid:11499934. 93. Tanaka, H. (2001). Scale-up for fermentative production. Japão: Kyoritu Shuppan.
Polímeros, 28(3), 275-283, 2018
94. Chatterjee, S., Adhya, M., Guha, A. K., & Chatterjee, B. P. (2005). Chitosan from Mucor rouxii: production and physicochemical characterization. Process Biochemistry, 40(1), 395400. http://dx.doi.org/10.1016/j.procbio.2004.01.025. 95. Chatterjee, S., Chatterjee, S., Chatterjee, B. P., & Guha, A. K. (2008). Enhancement of growth and chitosan production by Rhizopus oryzae in whey medium by plant growth hormones. International Journal of Biological Macromolecules, 42(2), 120-126. http://dx.doi.org/10.1016/j.ijbiomac.2007.10.006. PMid:18023862. 96. Martínez-Camacho, A. P., Cortez-Rocha, M. O., Ezquerra-Brauer, J. M., Graciano-Verdugo, A. Z., Rodriguez-Félix, F., CastilloOrtega, M. M., Yépiz-Gómez, M. S., & Plascencia-Jatomea, M. (2010). Chitosan composite films: Thermal, structural, mechanical and antifungal properties. Carbohydrate Polymers, 82(2), 305-315. http://dx.doi.org/10.1016/j.carbpol.2010.04.069. 97. Khan, T. A., Peh, K. K., & Ch’ng, H. S. (2002). Reporting degree of deacetylation values of chitosan: the influence of analytical methods. Journal of Pharmacy & Pharmaceutical Sciences, 5(3), 205-212. PMid:12553887. 98. Rinaudo, M., Milas, M., & Dung, P. L. (1993). Characterization of chitosan. Influence of ionic strength and degree of acetylation on chain expansion. International Journal of Biomacromolecules, 15(5), 281-285. http://dx.doi.org/10.1016/0141-8130(93)90027-J. PMid:8251442. 99. Stamford, N. P., Santos, C. E. R. S., Stamford, T. C. M., Franco, L. C., & Arnaud, T. M. S. (2015). BR 10 2013 003043 0. Rio de Janeiro: INPI. Retrieved in 2016, July 15, from https://gru. inpi.gov.br/pePI/servlet/PatenteServletController?Action=de tail&CodPedido=987310&SearchParameter=BIOFERTILIZ ANTE%20%20%20%20%20%20&Resumo=&Titulo= 100. Chien, R., Yen, M., & Mau, J. (2016). Antimicrobial and antitumor activities of chitosan from shiitak estipes, compared to commercial chitosan from crab shells. Carbohydrate Polymers, 138(1), 259-264. http://dx.doi.org/10.1016/j. carbpol.2015.11.061. PMid:26794761. 101. Tayel, A. A. (2016). Microbial chitosan as a biopreservative for fish sausages. International Journal of Biological Macromolecules, 93(1), 41-46. http://dx.doi.org/10.1016/j. ijbiomac.2016.08.061. PMid:27565293. 102. Tayel, A. A., El-Tras, W. F., & Elguindy, N. M. (2016). The potentiality of cross-linked fungal chitosan to control watercontamination through bioactive filtration. International Journal of Biological Macromolecules, 88(1), 59-65. http:// dx.doi.org/10.1016/j.ijbiomac.2016.03.018. PMid:26995612. Received: July 15, 2016 Revised: Aug. 22, 2017 Accepted: Oct. 10, 2017
283/283 283
40.00
35.00
Caracterização de Polímeros 30.00
µRIU
25.00
20.00
15.00
10.00
APC vs. GPC tradicional com padrão de poliestireno Mp = 510 5.00
0.00
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
4.00
Minutes
40.00
22.0
30.00
µ RIU
25.00
20.00
15.00
10.00
5.00
0.00
GPC
20.0 18.0 16.0 14.0
µ RIU
APC
35.00
12.0 10.0 8.0 6.0 4.0 2.0 0.0
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
4.00
4.00
4.20
4.40
4.60
4.80
5.00
5.20
5.40
5.60
5.80
6.00
6.20
6.40
Minutes
Minutes
22.0 20.0 18.0
Sistema Acquity Advanced Polymer Chromatography (APC) 16.0
µRIU
14.0
12.0 10.0 8.0
Muito mais informações em menos tempo.
6.0 4.0
Incomparável resolução para análises em ampla faixa de peso molecular.
2.0 0.0
Minutes De 5 a 20 vezes mais rápido quando comparado ao tradicional GPC/SEC 4.00
4.20
4.40
4.60
4.80
5.00
5.20
5.40
5.60
5.80
6.00
6.20
6.40
Redução no consumo de solvente e consequentemente na geração de resíduos Novas tecnologias de colunas que permitem rápida troca de solvente
waters.com
Visite nosso stand e saiba mais sobre essa técnica! Waters Brasil (11)4134-3788 vendas_brasil@waters.com
|
|
Pharmaceutical & Life Sciences
Food
|
Environmental
|
Clinical
Chemical Materials
©2017 Waters Corporation. Waters, ACQUITY, Advanced Polymer Chromatography , APC and The Science of What’s Possible are trademarks of Waters Corporation.
Polímeros VOLUME XXVIII - Issue II - Apr./May, 2018
São Paulo 994 St. São Carlos, SP, Brazil, 13560-340 Phone: +55 16 3374-3949 Email: abpol@abpol.com.br 2018