Preparation and characterization of carbon fibers coated by Fe3O4 nanoparticles

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中国艺术研究院博士生入学考试英语作文

中国艺术研究院博士生入学考试英语作文The Chinese Academy of Arts (CAA) is one of the most prestigious institutions for arts education in China. Every year, hundreds of students apply for the doctoral program at CAA, hoping to further their studies and research in various fields of art. The entrance exam for the doctoral program is known for its rigor and high standards, as the CAA seeks to enroll only the most talented and dedicated students.The entrance exam for the doctoral program at CAA is comprised of several components, including a written exam, an interview, and a portfolio review. The written exam tests students' knowledge of art history, theory, and criticism, as well as their ability to analyze and interpret works of art. The interview assesses students' research interests and goals, as well as their potential for contributing to the field of art.In order to prepare for the entrance exam, prospective doctoral students at CAA must complete a rigorous course of study, including coursework in art history, theory, and criticism, as well as studio practice. They must also develop a research proposal outlining their intended area of study and research questions. This proposal is critical to their application, as itdemonstrates their ability to think critically and creatively about their chosen field of study.Once admitted to the doctoral program at CAA, students have the opportunity to work closely with faculty mentors and other students in their cohort. They are encouraged to participate in seminars, workshops, and conferences, as well as to engage in independent research and creative work. The doctoral program at CAA is designed to help students develop their skills as artists, scholars, and critics, and to prepare them for careers in academia, museums, galleries, and other arts-related fields.Overall, the doctoral program at CAA is a challenging and rewarding experience for students who are passionate about art and committed to advancing the field through their research and creative work. By enrolling in the doctoral program at CAA, students have the opportunity to study with some of the leading artists and scholars in the field, and to make a valuable contribution to the world of art.。

甲基全氟丁基醚——韩

J. Ind. Eng. Chem.,Vol. 13, No. 4, (2007) 537-544Preparation of Methoxy and Ethoxy Nonafluorobutanes Sun-Hee Hwang, Ju-Ryun Kim, Sang Deuk Lee, Hyunjoo Lee, Hoon Sik Kim*, and Honggon Kim†Energy and Environment Technology Division, Korea Institute of Science and Technology, P.O. Box 131,Cheongryang, Seoul 130-650, Korea*Department of Chemistry, Kyunghee University, 1 Hoegy-dong, Dongdaemoon-gu, Seoul 130-701, KoreaReceived August 30, 2006; Accepted May 18, 2007Abstr act:Syntheses of methoxynonafluorobutane (C4F9OCH3) and ethoxynonafluorobutane (C4F9OC2H5), which are considered as prospective third generation alternative cleaning agents replacing CFC-113 (CF2ClCFCl2), CH3CCl3, and HCFC-141b (CH3CFCl2), were studied. Heptafluorobutyryl chloride (C3F7COCl) or hepta-fluorobutyryl fluoride (C3F7COF) was firstly reacted with alkali metal fluoride (KF or CsF) in an aprotic polar solvent to produce alkali metal nonafluorobutoxide. It was consecutively converted to C4F9OCH3 and C4F9OC2H5 through the reaction with alkylating agents such as dimethyl sulfate, methyl trifluoromethane sulfonate and di-ethyl sulfate. Identification and characterization of major products were carried out by GC-MS, FT-IR, 1H-NMR, and 19F-NMR spectroscopies.Keywords:methoxynonafluorobutane, ethoxynonafluorobutane, perfluoroacyl halide, heptafluorobutyryl chlor-ide, heptafluorobutyryl fluoride, nonafluorobutoxideIntroductionChlorofluorocarbons (CFCs) have been widely used for refrigerants, cleaning solvents, foam blowing agents, propellants, and fire extinguishing agents since early twenty centuries because of their excellent physical prop-erties, thermal and chemical stability and harmlessness in contact. However, chlorine (Cl) or bromine (Br) mole-cules in CFCs are recognized to react with ozone, thus destroying the stratospheric ozone layer when CFCs are released to the air and migrate to the stratosphere. Therefore, CFCs are requested to be phased out accord-ing to an international agreement, the Montreal Protocol on Substances that Deplete the Ozone Layer. Various HCFCs (hydrochlorofluorocarbons) and HFCs (hydrofl- uorocarbons), so-called the second-generation CFCs, have been proposed for replacing CFCs [1]. However, these are accepted only as interim alternatives because HCFC still contains chlorine and HFC generally has long atmospheric lifetime and high global warming potential.Hydrofluoroether (HFE) containing fluorine atoms in an ether molecule is currently considered as a third-gen-†To whom all correspondence should be addressed.(e-mail: hkim@kist.re.kr)eration CFC, which has zero ozone depletion potential (ODP) and low atmospheric lifetime, thus low global warming potential (GWP) [2,3]. Among HFEs, mole-cules of C4~C6 are considered as possible candidates for alternative cleaning agents due to their reasonable boil-ing points.Methoxynonafluorobutane (C4F9OCH3) and ethoxyno- nafluorobutane (C4F9OC2H5), which are clear, colorless, and low-odor solvents, have been developed and com-mercialized by 3M Chemicals [4]. Since both have low flammability, relatively low vapor pressure, low heat of vaporization, and low surface tension, they are consid-ered to replace the chlorine-containing cleaning solvents such as 1,1,1-trichloroethane, CFC-113 (CF2ClCFCl2), and HCFC-141b (CH3CFCl2). Table 1 compares physical and environmental properties of methoxy- and ethox-ynonafluorobutanes with those of major CFC and CFC alternative cleaning solvents formerly used [5,6]. Because methoxy- and ethoxynonafluorobutanes con-tain perfluoroalkyl (R f) group on one side and alkyl (R) group on the other side of ether linkage, they cannot be synthesized by selective partial fluorination of their hy-drocarbon precursors. Instead, they may be prepared by coupling reactions of two reactants having R f and R, respectively. One is a reaction of acyl halide with a fluo-Sun-Hee Hwang, Ju-Ryun Kim, Sang Deuk Lee, Hyunjoo Lee, Hoon Sik Kim, and Honggon Kim538Table 1. Physical and Environmental Properties of Cleaning Solvents [5,6]Properties ENFB6MNFB6HFC-4310HCFC-225 ca/cb HCFC-141b CFC-1131,1,1-TCE Formular C4F9OC2H5C4F9OCH3C5H2F10C3Cl2HF5C2Cl2H3F C2Cl3F3CH3CCl3 Boiling pt. (o C)78615454324874 Freezing pt. (o C)-138-135-80-131-103-35-39 Liquid density (g/ml)1 1.43 1.52 1.58 1.55 1.23 1.56 1.32 Surface tension (dyne/cm)113.613.614.116.219.317.325.1 Solubility in water (ppm)1< 2012140330210170700 Solubility of water (ppm)19295490310420110170 Vapor pressure (mmHg)1109202226290569331121 Viscosity (cps)10.610.610.670.590.430.680.83 Heat of vaporization (cal/g)230303134.653.33558 Specific heat (cal/g o C)10.290.280.270.240.300.220.25 Flammability3 2.4-12.4none none none7.1-18.6none none ODP40.00.00.00.030.100.800.1GWP5904801300170/5306305000110 Atmospheric lifetime (yrs)0.8 4.117.1 2.5-6.69.485 5.41.@ 25 o C2.@ boiling point3.vol% in air @ 100 o C4.referring to CFC-11 (=1.0)5.GWP-100 year integrated time horizon6.MNFB (methoxy nonafluorobutane), ENFB (ethoxy nonafluorobutane).rinating agent and an alkylating agent. For example, a batch reaction of heptafluorobutyryl fluoride (C3F7COF) with dimethy or diethylsulfate in the presence of KF can produce C4F9OCH3 or C4F9OC2H5 [4]. Another is a re-action of perfluoroalkyl iodide with metal alkoxide. In this work, plausible synthetic reactions and their optimal conditions for the former case were investigated, and proper reaction pathways and reaction conditions were proposed. Identification and characterization of the prod-ucts, C4F9OCH3 and C4F9OC2H5, were also carried out by GC-MS, FT-IR, 1H-NMR, and 19F-NMR spectroscopies.ExperimentalMaterialsAmong well-known alkylating agents, dimethyl sulfate or methyl triflate (methyl trifluoromethane sulfonate) for C4F9OCH3 and diethyl sulfate for C4F9OC2H5 were se-lected because of their monoalkyl sulfate ion (ROSO3-), an excellent leaving group for enhancing methylation of perfluoroalkoxide [7]. Reacting materials such as hepta-fluorobutyryl chloride (C3F7COCl, 98 %), dimethyl sul-fate ((CH3)2SO4, 99 %), diethyl sulfate ((C2H5)2SO4, 98 %), and methyl triflate (CF3SO3CH3, 96 %) were pur-chased from Aldrich Chemicals. Solvents, dimethylfor-amide (DMF, 99.8 %, Aldrich) and diethyleneglycol di-methylether (diglyme, 99 %, Acros), were used without further purification. Spray-dried KF (99 %, Sigma) and CsF (99 %, Acros) were dried for 12 h at 150 o C in a PyrexⓇ vacuum line just before being used. Heptafluo- robutyryl fluoride (C3F7COF) was prepared from hepta-fluorobutyryl chloride as described in the literature [8,9], and its purity was checked by GC.AnalysisProducts were confirmed by a GC-MS of HP 6890 GC and HP 5973 MSD, and their compositions were ana-lyzed by a Donam 6200 GC equipped with FID under operating conditions shown in Table 2. IR analysis of liq-uid products was conducted with a Galaxy series FT-IR 6030 Infrared spectrometer. Both 1H-NMR and 19F-NMR spectra were obtained from a Varian Unity 300 NMR spectrometer in CDCl3 solvent with (CH3)4Si and CFCl3 as internal references.The product solution was collected after the reaction, and the organic layer was carefully separated. The prod-uct yield was calculated based on the weight of separated organic layer and the peak area ratio of products in the gas chromatogram. The selectivity was calculated from GC peak areas of C4F9OR and C3F7COOR (R = CH3, C2H5).Preparation of C4F9OR (R = CH3, C2H5) with C3F7COCl or C3F7COFHeptafluorobutyryl chloride (C3F7COCl) or heptafluo- robutyryl fluoride (C3F7COF) and alkali metal fluoride (spray-dried KF, CsF) were reacted in an aprotic polarPreparation of Methoxy and Ethoxy Nonafluorobutanes539 Table 2. Operation Conditions of GC and GC-MSCondition GC with FID GC-MSColumnInjector temperature Detector temperature Oven temperature InitialIncrease rateFinalCarrier gas flow HP-1, capillary 50 m180 o C250 o C30 o C, 5 min+10 o C/min150 o C, 2 minHe, 0.8 mL/minPoraplot Q, 2 m200 o C250 o C100 o C, 3 min+10 o C/min200 o C, 5 minHe, 0.8 mL/minsolvent. Consecutive reaction with alkylating agents mi- ght produce corresponding heptafluorobutyryl alkyl ethers. Reactions were carried out in three ways: the one-pot reaction in which all reactants were charged to-gether and simultaneously reacted, the two-step reaction in which reactants were added and reacted step by step at different temperatures, and the two-step reaction with dropping reactants in which reactants were slowly added drop by drop during each step running at different tem- peratures. In the one-pot reaction, spray-dried KF (100 mmol, 5.81 g) or CsF (100 mmol, 15.19 g) was loaded into a cylindrical stainless steel reactor (75 mL) equipped with a magnetic stirring bar, a pressure gauge, and a ther-mocouple under nitrogen condition. The reactor was evacuated for 12 h at 150 o C in order to remove moisture in the alkali metal fluoride. A solvent, DMF or diglyme (25 mL), C3F7COCl (25 mmol, 2.33 g), and an alkylating agent, (CH3)2SO4 (50 mmol, 4.8 mL)or CF3SO3CH3 (50 mmol, 5.8 mL), were added into the reactor through well-dried syringes around 0 o C. N2 gas was charged to make the internal pressure 0.5 bar. Once the reactor was warmed up to a temperature designed, the reactants were stirred and started to react. In the two-step reaction, spray-dried KF or CsF, a solvent (DMF), and C3F7COCl were charged in the same manner. The mixture was re-acted for 3 h at a designed temperature and cooled below 0 o C to stop the reaction temporarily. After an alkylating agent, (CH3)2SO4 or (C2H5)2SO4, was injected, the reactor was warmed up to a designed temperature again and fur-ther reaction was induced by additional stirring for 2 to 5 h. When C3F7COF (25 mmol, 5.40 g) was used instead of C3F7COCl, the first-step reaction last for 2.5 h at a design temperature. In the two-step reaction with dropping, C3F7COCl was added dropwise to the solution containing alkali metal fluoride during the first-step reaction, and an alkylating agent was also added dropwise during the sec-ond-step reaction. Small amount of liquid was sampled periodically, and the progress of reaction was checked by GC and GC-MS. After the reaction was completed, the products were discharged at 50 o C under vacuum and collected in a cold trap in liquid nitrogen. The collected solution was washed with ice-water, and the lower organ-ic layer was separated in a separatory funnel. A colorless solution was obtained by being dried with anhydrous MgSO4 and filtered.Characterization of ProductsProducts were identified by GC-MS, IR, 1H NMR, and 19F NMR. The major products were a group of C4F9OCH3, C3F7COOCH3, CH3Cl, and C3F7COF, or of C4F9OC2H5, C3F7COOC2H5, C2H5Cl, C2H5OOCH, and C3F7COF. Trace amount of CH3F or C2H5F was also found. Ac- cording to the clear boiling point difference, CH3Cl, C2H5Cl, C2H5OOCH, and C3F7COF were easily separable. The major products were characterized by analytical instru-ments as follows.C4F9OCH3 : MW 250.08; bp 60∼61 o C; d = 1.52; IR (gas, cm-1): 2975.5, 2878.09, 1461.0, 1352.4, 1313.5, 1248.7, 1147.0, 1118.4, 1064.9, 990.8, 936.4, 884.1, 740.3; 1H NMR (CDCl3): δ3.72 (3H, s); 19F NMR: δ- 81.68 (3F, t), -89.01 (2F), -126.86 (2F), -127.24 (2F); MS (EI) [m/e (species)] : 249 (C4F9OCH2+), 231 (C4F8OCH3+), 219 (C4F9+), 181 (CF2CF2CF2OCH3+), 169 (CF3CF2CF2+), 150 (CF3CF2CF+), 131 (CF2CF2OCH3+), 119 (CF3CF2+), 100 (CF2CF2+), 81 (CF2OCH3+), 69 (CF3+), 31 (OCH3+), 15 (CH3+).C3F7COOCH3 : MW 228.07; bp 80∼81 o C; d = 1.48; MS (EI) [m/e (species)]: 209 (C3F6COOCH3+), 197 (C3F7CO+), 181 (CF3CF2CF2C+), 169 (CF3CF2CF2+), 150 (CF3CF2CF+), 119 (CF3CF2+), 100 (CF2CF2+), 69 (CF3+), 59 (COOCH3+), 50 (CF2+), 31 (OCH3+), 15 (CH3+).C4F9OC2H5 : MW 264.11; bp 76∼78 o C; d = 1.43; MS (EI) [m/e (species)]: 263 (C4F9OCH2CH2+), 249 (C4F9 OCH2+), 219 (C4F9+), 169 (CF3CF2CF2+), 150 (CF3CF2 CF+), 131 (CF2CF2OCH3+), 119 (CF3CF2+), 100 (CF2 CF2+), 95 (CF2OC2H5+), 69 (CF3+), 29 (CH3CH2+), 15 (CH+3).C3F7COOC2H5 : MW 242.09; bp 95∼97 o C; d = 1.39; MS (EI) [m/e (species)]: 241 (C3F7COOCH2CH2+), 227 (C3F7COOCH2+), 213 (C3F7COO+), 197 (C3F7CO+), 169 (CF3CF2CF2+), 119 (CF3CF2+), 100 (CF2CF2+), 69 (CF3+), 29 (CH3CH2+).Results and DiscussionPreparation of C4F9OCH3 with C3F7COCl in one-pot reactionC4F9OCH3 seems producible from C3F7COCl in the presence of metal fluoride and an alkylating agent in one-pot reaction through one of following examples.C3F7C(=O)Cl + 2 MF + (CH3)2SO4→C4F9OCH3 + MCl + CH3SO4M(1)Sun-Hee Hwang, Ju-Ryun Kim, Sang Deuk Lee, Hyunjoo Lee, Hoon Sik Kim, and Honggon Kim540Figure 1. Product distribution change according to the reaction time in one-pot reaction (The area of CH 3Cl is compared with the total area of C 4 reactants and products).C 3F 7C(=O)Cl + 2 MF + CF 3SO 3CH 3 → C 4F 9OCH 3 +MCl + CF 3SO 3M (2)Liquid samples periodically collected during the reactionshowed that intermediates, such as C 3F 7COF and CH 3Cl, were formed. The reactant, C 3F 7COCl, was fluorinated and almost completely converted to C 3F 7COF within ini-tial 2 h, and the Cl - released by halogen exchange reacted with CH 3+ of an alkylating agent to form CH 3Cl. The main intermediate, C 3F 7COF, was produced in large amount during initial 2 h then remarkably reduced as be-ing converted to the final product, C 4F 9OCH 3. However, C 3F 7COF was not thoroughly converted in a batch re-action even after 20 h. In contrast to C 3F 7COF, the pro-duction of CH 3Cl proceeded for a while then gradually reduced even far after the production of C 4F 9OCH 3 ceased as shown in Figure 1. This proposed that the re-actant acyl chloride, C 3F 7COCl, firstly exchanges the halogen ion with alkali metal fluoride, MF, to form an acyl fluoride, C 3F 7COF. Then the acyl fluoride reacts with another MF to transform into an alkali metal non-afluorobutoxide (C 4F 9O -M +) in an aprotic polar solvent. The C 4F 9O -M + consecutively reacted in situ with the al-kylating agent to produce C 4F 9OCH 3 by electrophilic substitution of CH 3+ to M + on perfluorobutoxide (C 4F 9O -). Meanwhile, some free alkali metal ions seemed to rever-sibly react with an alkylating agent such as dimethyl sul-fate ((CH 3)2SO 4) or methyl triflate (CF 3SO 3CH 3) to form CH 3SO 4M or CF 3SO 3M and released a methyl group (CH 3+) which temporarily combined with free Cl - ex-pelled from the reactant to form CH 3Cl.There were several other by-products detected in trace amount: CH 3F in the effluent gas and methyl ether (CH 3OCH 3) and methyl formate (CH 3OOCH) in the solvent. When an alkylating agent such as dimethyl sul-fate is hydrolyzed, methanol is formed. And this reaction is known to be likely accelerated over 30 o C [10]. The methanol successively reacts with another dimethyl sul-fate or with DMF to produce methyl ether or methyl for-mate, respectively.(CH 3)2SO 4 + 2 H 2O → 2 CH 3OH + H 2SO 4 (3) (CH 3)2SO 4 + CH 3OH → CH 3OCH 3 + CH 3OSO 3H (4) (CH 3)2NC(=O)H + CH 3OH →CH 3OC(=O)H + (CH 3)2NH(5)Formation of the primary by-product, methyl hepta-fluorobutyrate (C 3F 7COOCH 3), could be attributed to the moisture in chemicals. When the reactant C 3F 7COCl con-tacted with moisture, it was likely to be converted to car-boxylic acid, C 3F 7COOH with release of HCl. Or, when the initial intermediate C 3F 7COF contacted with H 2O, it might be readily converted to C 3F 7C(OH)2F, then to C 3F 7COOH by releasing HF, too. The carboxylic acid wo- uld be consecutively converted to an ester C 3F 7COOCH 3 by in situ methylation or acid-catalyzed esterification. Following equations describe possible reactions for gen-erating the primary by-product.(CH 3)2SO 4C 3F 7COX (X=Cl, F) + H 2O → C 3F 7COOH → C 3F 7COOCH 3(6)C 3F 7COOH(CH 3O)2SO 4 + 2 H 2O → 2 CH 3OH + H 2SO 4 → C 3F 7COOCH 3(7) Therefore, in case of high moisture content, C 3F 7COOCH 3 would be remarkably produced, and the solution turned acidic by HCl, HF or H 2SO 4. In addition, HF or HCl pro-duced under high moisture content could hinder the pri-mary forward reaction to C 4F 9OCH 3 by reacting with C 4F 9O -M + to produce MF or MCl and unstable C 4F 9OH which readily turn backward to C 3F 7COF and HF [9]. This reaction was known to proceed well at high temper-ature so that more C 3F 7COOCH 3 was produced at an ele-vated reaction temperature while less C 4F 9OCH 3 was pro- duced. When a chemical-grade KF conventionally cal-cined was used as the alkali metal fluoride, C 3F 7COOCH 3 was primarily produced. Meanwhile, C 4F 9OCH 3 was the main product with spray-dried KF under the same re-action condition. This indicated that the spray-dried KF was less hygroscopic than the former, so that less mois-ture was involved in the reaction. In addition, the spray- dried KF was apt to effectively source nucleophilic fluo-ride because of its smaller particle size and higher sur-face area than the conventionally calcined KF. Effects of temperature, solvent, alkali metal fluoride, and alkylating agent on the one-pot reaction for C 4F 9OCH 3 were examined. On the basis of yield and selectivity of C 4F 9OCH 3 in the entries of 1 to 6 in Table 3, the yield of targeted C 4F 9OCH 3 decreased and the selectivity of by-product, C 3F 7COOCH 3, increased as the reaction tem-Preparation of Methoxy and Ethoxy Nonafluorobutanes541 Table 3. Effect of Reaction Conditions in One-pot Reactions of C4F9OCH3Entry Alkylating agent MF Solvent T (o C)Time (h)Product yield (%)Selectivity (%)C4F9OCH3C3F7COOCH31(CH3)2SO4KF DMF252060928 2(CH3)2SO4KF DMF5020346337 3(CH3)2SO4KF DMF1002095644 4(CH3)2SO4KF diglyme2520466832 5(CH3)2SO4KF diglyme5020244357 6(CH3)2SO4KF diglyme1002043862 7(CH3)2SO4CsF DMF252068937 8CF3SO3CH3KF DMF2520418911T (C)One-pot reaction : C3F7COCl + MF + (CH3)2SO4 C4F9OCH3 + MCl + (CH3)SO4MSolventProduct yield : calculated from the GC peak area percent of targeted C4F9OCH3 based on the reactant-related products and the weight of the separated organic layer.Selectivity : calculated from the GC peak area ratio of C4F9OCH3 and C3F7COOCH3.perature increased regardless of solvent type. Higheryield of C4F9OCH3 at lower reaction temperature was ob-tained possibly because the reaction between C3F7COCland KF proceeded in the liquid phase and moreC3F7COCl could be dissolved in the solvent when thetemperature was much lower than its boiling point, e.g.38∼39 o C at 1 atm. Since the boiling point of the inter-mediate C3F7COF is around 7 o C, it was also dissolvedmore in the liquid phase and produced more C4F9OCH3at lower temperature. This could be confirmed againfrom the cases run at 50 and 100 o C where the initial in-side pressures of the reactor were above 1 and about 2bars, respectively. The pressure gradually decreased asthe reaction proceeded. Higher selectivity of C3F7COOCH3at higher temperature was presumed because the side re-action between H2O and the intermediate, C4F9O-K+, wasaccelerated at higher temperature as mentioned before.It is known that an aprotic polar solvent, such as di-methyl formamide (DMF) or diglyme, can solvate K+ ionfrom the intermediate C4F9O-K+ to enhance the methyl-ation of C4F9O- [11]. For the effect of solvent type, theyield and the selectivity of C4F9OCH3 increased more inDMF than in diglyme (entries of 1 to 3 vs. entries of 4 to6). This emphasized the importance of the selection ofaprotic polar solvent capable of solvating K+ ion.In terms of alkali metal fluoride, the rate of consumingC3F7COCl and the yield of C4F9OCH3 were higher withCsF than with spray-dried KF (entries of 1 and 7). Thisindicated that the formation of intermediate, C4F9O-M+,and the yield of product, C4F9OCH3, seemed to be de-pendent on the degree of alkali metal fluoride dis-sociation in the following equilibrium reaction; R f COF(l)+ MF(s)⇔ R f CF2O-M+(s) (M = K, Rb, Cs; R f = CF3, C2F5,C3F7, etc.). As the metallic ion has higher positive charge density or smaller size, the polarity of the negative ion, R f CF2O-, increases and the salt, R f CF2O-M+, gets easily decomposed backwards to R f COF and MF at the same temperature [9]. In other words, metallic ions in large size tend to enhance the stability of metal perfluoalk-oxide and to induce stable methylation of the salt toward the product, R f CF2OCH3. Therefore, it can be rational-ized that CsF would be a more effective fluorination agent than KF.In terms of alkylating agent, methyl triflate (CF3SO3CH3), known as a representative strong methylating agent, pro-duced lower yield of C4F9OCH3 than (CH3)2SO4 as shown in the entries of 1 and 8. This could be explained by a relatively stronger interaction between methyl tri-flate and DMF, which might lessen the methylating abil-ity of methyl triflate [12,13].Preparation of C4F9OCH3 with C3F7COCl in two-step reactionIn two-step reaction, reactants were added stepwise but different temperatures were applied at each step in order to eliminate possible competitive reactions among re-actants which seemingly happened in the one-pot re-action and to increase the desired product. In other words, the reaction was conducted in two consecutive stages. C3F7COCl was introduced to a solvent containing alkali metal fluoride, KF, and fluorinated to form an in-termediate alkali metal perfluorobutoxide (C4F9O-K+) at a temperature during the first stage. Then an alkylating agent (dimethyl sulfate) was added and reacted with the C4F9O-K+ to produce C4F9OCH3 at another temperature during the second stage as shown in the following equation.① T (o C)② T (o C)C3F7COCl + KF C4F9O-K+C4F9OCH3DMF, stirring, 3 h (CH3)2SO4, stirring, 2 h(8)Sun-Hee Hwang, Ju-Ryun Kim, Sang Deuk Lee, Hyunjoo Lee, Hoon Sik Kim, and Honggon Kim542Table 4. Effect of Reaction Temperatures in Two-step Reactions of C 4F 9OCH 3Entry Alkylating agent MF Solvent T (o C) ①T (o C) ②Product yield (%)Selectivity (%)C 4F 9OCH 3C 3F 7COOCH 39(CH 3)2SO 4KF DMF 252560901010(CH 3)2SO 4KF DMF 255046891111(CH 3)2SO 4KF DMF 2510024623812(CH 3)2SO 4KF DMF 502542811913(CH 3)2SO 4KFDMF5050387723T ①T ②Two-step reaction : C 3F 7COCl + KF C 4F 9O-K4F 9OCH 3DMF, 3 h (CH 3)2SO 4, 2 hProduct yield : calculated from the GC peak area percent of targeted C 4F 9OCH 3 based on the reactant-related products and the weight of theseparated organic layer.Selectivity : calculated from the GC peak area ratio of C 4F 9OCH 3 and C 3F 7COOCH 3.Table 5. Effect of Reaction Temperatures in Two-step Reactions of C 4F 9OCH 3 with Dropwise Slow Addition of ReagentsEntry Alkylating agent MF Solvent T (o C) ①T (o C) ②Product yield (%)Selectivity (%)C 4F 9OCH 3C 3F 7COOCH 314(CH 3)2SO 4KF DMF 25255894615(CH 3)2SO 4KF DMF 255038851516(CH 3)2SO 4KFDMF5070267327C 3F 7COCl ①(CH 3)2SO 4 ② 4F 9O -K 4F 9OCH 3dropping, 0.5 h/mixing, 2.5 hdropping, 2 hAccording to Table 4, higher yield and selectivity of thetargeted product could be obtained at lower temperatures of both the first and the second stages as in the one-pot reactions. However, in comparing the entries of 2 and 13 running at the same temperature of 50 o C, the two-step reaction seemed to boost the overall reaction. In compar-ing the entries of 10, 12, and 13, lower temperature in ei-ther stage induced more production of the targeted product. Furthermore, the lower temperature in the first stage was more efficient in producing the targeted prod-uct and increasing its selectivity than that in the second stage. This indicated that a low temperature of the first stage enhanced the dissolution of C 3F 7COCl or C 3F 7COF in the liquid phase and converted it to more C 4F 9O -K +. However, the intermediate C 4F 9O -K + is known to be un-stable and easily decomposed backward to alkali metal fluoride and corresponding acyl fluoride R f COF to fol-low the equilibrium. In other words, the C 4F 9O -K + is rela-tively stable at below 50 o C but quickly decomposed to acyl fluoride at higher temperature like between 50 and 80 o C [14]. The same observation was confirmed in com-parison of the entries from 9 to 11 and from 12 to 13 where the production of C 4F 9OCH 3 was lowered at high-er temperatures. Even though low temperatures in both stages were preferred for high product yields, a low tem-perature in the first stage, where the intermediate C 4F 9O -K + was formed, was likely a more important keyfactor than that in the second stage, where the alkylation is proceeded.The two-step reaction was conducted in another manner where the reactants were slowly added and mixed drop by drop as described in equation (9).① T (o C)① T (o C)C 3F 74F 9O -K +dropping for 0.5 h2.5 h②T (℃)4F 9OCH 3dropping (CH 3)2SO 4 for 0.5 h, additional stirring for 1.5 h(9)It was expected that slow mixing of reactants in eachstep would induce the enhanced formation of C 4F 9O -K +and the effective methylation by avoiding temperature el-evation resulting from the exothermic reactions. How- ever, there was no remarkable improvement by the slow dropwise addition of reactants as in the comparison of entries 14 and 15 to the entries 9 and 10 in Table 5 at the same temperatures, respectively. Only the temperature effect was confirmed again.Preparation of C 4F 9OC 2H 5 with C 3F 7COClAs C 4F 9OCH 3, C 4F 9OC 2H 5 was produced in the two-step reaction with C 3F 7COCl, KF and diethyl sulfate. (equa- tion (10))Preparation of Methoxy and Ethoxy Nonafluorobutanes 543Table 6. Effect of Reaction Temperatures in Two-step Reactions of C 4F 9OC 2H 5Entry Alkylating agent MF Solvent T (o C) ①T (o C) ②Product yield(%)Selectivity (%)C 4F 9OC 2H 5C 3F 7COOC 2H 517(C 2H 5)2SO 4KF DMF 252569901018(C 2H 5)2SO 4KF DMF 255062861419(C 2H 5)2SO 4KFDMF5070427426T ①T ②Two-step reaction : C 3F 74F 9O-K +C 4F 9OC 2H 5DMF, 3 h(C 2H 5)2SO 4, 5 hTable 7. Effect of Reactant in Two-step Reactions with C 3F 7COFEntry Aalkylating agent MF Solvent T (o C) ①T (o C) ②time (h)Product yield(%)Selectivity (%)C 4F 9OR C 3F 7COOR20(CH 3)2SO 4KF DMF 2525 2.560891121(C 2H 5)2SO 4KFDMF25257648515T ①T ②Two-step reaction : C 3F 74F 9O -K +C 4F 9OCH 3DMF, 3 h(CH 3)2SO 4, 2 h① T (o C)② T (o C)C 3F 74F 9O -KC 4F 9OC 2H 5DMF, stirring, 3 h(C 2H 5)2SO 4, stirring, 5 h(10)Pressure during the reaction was higher than 1 bar and slowly decreased as the reactions for C 4F 9OCH 3, indicat-ing that the major factor causing the pressure might be the intermediates, C 3F 7COF and CH 3Cl. Likely as in the reactions for C 4F 9OCH 3, higher yield and selectivity of the targeted C 4F 9OC 2H 5 could be obtained at lower tem-peratures as shown in Table 6. More C 3F 7COOC 2H 5, the major by-product, was produced at higher reaction tem- perature. There was also trace amount of similar by-prod-ucts such as C 2H 5Cl, C 2H 5OOCH and C 2H 5OC 2H 5.Preparation of C 4F 9OR (R= CH 3, C 2H 5) with C 3F 7COF C 4F 9OCH 3 and C 4F 9OC 2H 5 were produced in the two- step reaction with an intermediate C 3F 7COF instead of C 3F 7COCl.T ① (oC)T ② (oC)C 3F 7 COF + KF C 4F 9O -K+C 4F 9OR3 hR 2SO 4, 2.5 h(11)C 3F 7COF was prepared from a reaction of C 3F 7COCl and KF in a separate reactor and transferred to the main reactor containing KF dispersed in a solvent at about 0o C. Once the temperature of the solvent was set to the temperature where the reaction was to start, the pressure initially higher than 1 bar was slowly reducing during thereaction. The completion of reaction was decided by no more change of C 3F 7COF. The product yields and se-lectivities of entries 20 and 21 in Table 7 were similar to those of entries 9 and 17, respectively. This indicated that the fluorination of C 3F 7COCl with KF to C 3F 7COF proceeded quickly, and the other reactions thereafter mi- ght be the rate controlling steps in producing C 4F 9OCH 3 and C 4F 9OC 2H 5. More by-products, C 3F 7COOCH 3 and C 3F 7COOC 2H 5, with C 3F 7COF than with C 3F 7COCl was possibly due to the moisture introduced during trans-ferring C 3F 7COF into the main reactor.ConclusionsMethoxy and ethoxy nonafluorobutanes, C 4F 9OCH 3 and C 4F 9OC 2H 5, could be prepared by reacting C 3F 7COCl or C 3F 7COF with alkali metal fluoride and alkylating agents having CH 3 and C 2H 5. A serial reaction mechanism is proposed; the formation of alkali metal perfluorobut-oxide, C 4F 9O -M +, is followed by the alkylation ofC 4F 9O -M + bydimethyl sulfate or diethyl sulfate. Two- step reactions showed noticeable improvement compar-ing to one-pot reactions. However, the reaction temper-ature was the main factor deciding the yield and the se-lectivity of the targeted alkoxynonafluorobutanes. Rev- iewing all results, we concluded that C 4F 9OCH 3 or C 4F 9OC 2H 5 was produced in better yield at lower tem- peratures. Even though reaction rates were likely to in-crease as the temperature increased as usual, high yields of the targeted products were obtained at a temperature as low as or lower than the boiling points of the re-。

NIBSC-WHO AFP标准物质

WHO International StandardAlphafoetoprotein, Human, 100,000 IUNIBSC code: AFPInstructions for use(Version 6.0, Dated 11/12/2012)1. INTENDED USEThis material was prepared from a pool of several hundred cord sera characterised by the Statens Serum Institut (SSI), Copenhagen, Denmark and it was established as the First WHO IS for AFP in 1975 (1). With effect from 1st July 1997, the National Institute for Biological Standards and Control (NIBSC), Potters Bar, UK became the custodian and distributor of this material.2. CAUTIONThis preparation is not for administration to humans.This preparation contains material of human origin. The ampouled material has been tested and found negative for HBsAg and anti-HIV. It gave a positive PCR test for HCV RNA. The preparation should be regarded as potentially hazardous to health. The container and its contents should be used and discarded according to your own laboratory procedures. Such procedures probably will include the wearing of protective gloves and avoiding the generation of aerosols. Care should be exercised in opening the container to avoid cuts3. UNITAGEEach ampoule of this preparation contains 100,000 International Units by definition (1,2,3).Uncertainty: the International unit of AFP is assigned without uncertainty. The uncertainty of the ampoule content of AFP may be considered to be the coefficient of variation, which was determined to be ± 0.42%.4. CONTENTSCountry of origin of biological material: DenmarkThe pool was distributed into ampoules in volumes of 2ml and freeze-dried. The ampoules were originally coded 72/225 and contain approx 70 mg of protein each.5. STORAGEUnopened ampoules should be stored, in the dark, at –20ºCPlease note: because of the inherent stability of lyophilized material, NIBSC may ship these materials at ambient temperature.6. DIRECTIONS FOR OPENINGTap the ampoule gently to collect the material at the bottom (labelled) end. Ensure ampoule is scored all round at the narrow part of the neck, with a diamond or tungsten carbide tipped glass knife file orother suitable implement before attemptingto open. Place theampoule in the ampoule opener, positioning the score at position 'A'; shown in the diagram below. Surround the ampoule with cloth or layers of tissue paper. Grip the ampoule and holder in the hand and squeeze at point 'B'. The ampoule will snap open. Take care to avoid cuts and projectile glass fragments that enter eyes. Take care that no material is lost from the ampoule and that no glass falls into the ampoule.Side view of ampoule opening device containing an ampoule positioned ready to open. 'A' is the score mark and 'B' the point of applied pressure.7. COLLABORATIVE STUDYThe standard preparation has been investigated in two collaborative studies (3,4) using several assay methods, such as radioimmunoassay, single radial immunodiffusion and immunoelectrophoresis. Six laboratories estimated that one IU approximately equals 1.21 (1.02 –1.43) nanograms of AFP (3).8. STABILITYThe total contents of an ampoule should be reconstituted in a suitable volume of the solvent to be used in the test. If 10ml of solvent is used, the resulting solution will have a concentration of 10,000 IU/ml. If not used shortly after reconstitution, the solution should be kept in a frozen state.It is the policy of WHO not to assign an expiry date to their international reference materials. They remain valid with the assigned potency and status until withdrawn or amended.Reference materials are held at NIBSC within assured, temperature-controlled storage facilities. Reference Materials should be stored on receipt as indicated on the label. For information specific to a particular biological standard, contact standards @.In addition, once reconstituted, diluted or aliquoted, users should determine the stability of the material according to their own method of preparation, storage and use.NIBSC follows the policy of WHO with respect to its reference materials.Users who have data supporting any deterioration in the characteristics of any reference preparation are encouraged to contact NIBSC.9. REFERENCES1. WHO Technical Report Series No.594,1976,142. Sizaret P, Anderson S G, The International Reference Preparation for alphafoetoprotein. J of Biol. Stand., 4, 149, 19763. Sizaret P et al, Equivalence between international units and mass units of alphafoetoprotein. Report of a collaborative study. Clinica Chimica Acta, 96, 59-65, 19794. Sizaret P, Breslow N, Anderson S G, and 12 other participants, Collaborative study of a preparation of human cord serum for its use as a reference in the assay of alphafoetoprotein, J Biol. Stand 3, 201-223, 197510. FURTHER INFORMATIONFurther information can be obtained as follows;This material: enquiries@WHO Biological Standards:http://www.who.int/biologicals/en/JCTLM Higher order reference materials:/en/committees/jc/jctlm/Derivation of International Units:/products/biological_reference_materials/frequently_ asked_questions/how_are_international_units.aspxOrdering standards from NIBSC:/products/ordering_information/frequently_asked_qu estions.aspxNIBSC Terms & Conditions:/terms_and_conditions.aspx11. CUSTOMER FEEDBACKCustomers are encouraged to provide feedback on the suitability or use of the material provided or other aspects of our service. Please send any comments to enquiries@12. CITATIONIn all publications, including data sheets, in which this material is referenced, it is important that the preparation's title, its status, the NIBSC code number, and the name and address of NIBSC are cited and cited correctly.14. LIABILITY AND LOSSIn the event that this document is translated into another language, the English language version shall prevail in the event of any inconsistencies between the documents.Unless expressly stated otherwise by NIBSC, NIBSC’s Standard Terms and Conditions for the Supply of Materials (available at /About_Us/Terms_and_Conditions.aspx or upon request by the Recipient) (“Conditions”) apply to the exclusion of all other terms and are hereby incorporated into this document by reference. The Recipient's attention is drawn in particular to the provisions of clause 11 of the Conditions.17. CERTIFICATE OF ANALYSISNIBSC does not provide a Certificate of Analysis for WHO Biological Reference Materials because they are internationally recognised primary reference materials fully described in the instructions for use. The reference materials are established according to the WHO Recommendations for the preparation, characterization and establishment of international and other biological reference standards http://www.who.int/bloodproducts/publications/TRS932Annex2_Inter_biol efstandardsrev2004.pdf (revised 2004). They are officially endorsed by the WHO Expert Committee on Biological Standardization (ECBS) based on the report of the international collaborative study which established their suitability for the intended use.。

Preparation and Characterization of a Novel

ORIGINAL ARTICLEPreparation and Characterization of a NovelExtracellular Polysaccharide with Antioxidant Activity,from the Mangrove-Associated Fungus Fusarium oxysporumYan-Li Chen &Wen-Jun Mao &Hong-Wen Tao &Wei-Ming Zhu &Meng-Xia Yan &Xue Liu &Tian-Tian Guo &Tao GuoReceived:1August 2013/Accepted:7January 2015/Published online:28January 2015#Springer Science+Business Media New York 2015Abstract Marine fungi are recognized as an abundant source of extracellular polysaccharides with novel structures.Mangrove fungi constitute the second largest ecological group of the marine fungi,and many of them are new or inadequate-ly described species and may produce extracellular polysac-charides with novel functions and structures that could be explored as a source of useful polymers.The mangrove-associated fungus Fusarium oxysporum produces an extracel-lular polysaccharide,Fw-1,when grown in potato dextrose-agar medium.The homogeneous Fw-1was isolated from the fermented broth by a combination of ethanol precipitation,ion-exchange,and gel filtration chromatography.Chemical and spectroscopic analyses,including one-and two-dimensional nuclear magnetic resonance spectroscopies showed that Fw-1consisted of galactose,glucose,and man-nose in a molar ratio of 1.33:1.33:1.00,and its molecular weight was about 61.2kDa.The structure of Fw-1contains a backbone of (1→6)-linked β-D -galactofuranose residues with multiple side chains.The branches consist of terminal α-D -glucopyranose residues,or short chains containing (1→2)-linked α-D -glucopyranose,(1→2)-linked β-D -mannopyranose,and terminal β-D -mannopyranose residues.The side chains are connected to C-2of galactofuranose res-idues of backbone.The antioxidant activity of Fw-1was eval-uated with the scavenging abilities on hydroxyl,superoxide,and 1,1-diphenyl-2-picrylhydrazyl radicals in vitro,and the results indicated that Fw-1possessed good antioxidant activ-ity,especially the scavenging ability on hydroxyl radicals.Theinvestigation demonstrated that Fw-1is a novel galactofuranose-containing polysaccharide with different structural characteristics from extracellular polysaccharides from other marine microorganisms and could be a potential source of antioxidant.Keywords Mangrove-associated fungus .Fusarium oxysporum .Extracellular polysaccharide .Preparation .Characterization .Antioxidant activityIntroductionMangroves grow in saline coastal sediment habitats in the tropics and subtropics harboring a great diversity of marine fungi (Shearer et al.2007).Mangrove fungi constitute the second largest ecological group of the marine fungi and may produce chemicals with novel functions and structures (Kobayashi and Tsuda 2004).Fungi often produce extracellu-lar polysaccharides that are secreted into the growth media or remain tightly attached to the cell surface (Seviour et al.1992).The research on extracellular polysaccharides from marine fungi is attempted for providing polysaccharide with novel functions and structures (Chen et al.2012;Sun et al.2011).The extracellular polysaccharides produced by marine fungi become an important research area in new drug discovery and show enormous development prospects (Kanekiyo et al.2005).Polysaccharides with hexofuranose units are of interest be-cause of their unique structures and specific properties (Leal et al.2010).The investigations showed that galactose is the most widespread hexose in furanose form in naturally occur-ring polysaccharides (Pedersen and Turco 2003;Peltier et al.2008).The galactofuranose-containing extracellularY .<L.Chen :W.<J.Mao (*):H.<W.Tao :W.<M.Zhu :M.<X.Yan :X.Liu :T.<T.Guo :T.GuoKey Laboratory of Marine Drugs,Ministry of Education,Institute of Marine Drugs and Foods,Ocean University of China,5Yushan Road,Qingdao 266003,People ’s Republic of China e-mail:wenjunmqd@Mar Biotechnol (2015)17:219–228DOI 10.1007/s10126-015-9611-6polysaccharides with novel structural characteristics have been isolated from the fermented broth or cell walls of some microorganisms(Gander et al.1974;Ikuta et al.1997;Latgéet al.1994;Unkefer and Gander1990).With today’s interest in new renewable sources of polymers,the galactofuranose-containing extracellular polysaccharides represent potential source to be explored.However,the galactofuranose-containing extracellular polysaccharides from marine fungi have not yet been fully studied.In the current study,a novel galactofuranose-containing extracellular polysaccharide was isolated from the fermented broth of the mangrove-associated fungus Fusarium oxysporum by a combination of ethanol precipitation,ion-exchange,and gel filtration chroma-tography,and its structural characterization was investigated using chemical and spectroscopic methods,including one-and two-dimensional nuclear magnetic resonance(1D and 2D NMR)spectroscopic analyses.The antioxidant activity of the extracellular polysaccharide was also evaluated by scavenging assays involving hydroxyl,superoxide,and1,1-diphenyl-2-picrylhydrazyl(DPPH)radicals.Materials and MethodsMaterialsMonosaccharides(D-glucose,L-rhamnose,D-xylose,L-arabi-nose,D-mannose,L-fucose,D-galactose,D-glucuronic acid,D-galacturonic acid,D-mannuronic acid,N-acetyl-β-D-glucos-amine),1,1-diphenyl-2-picrylhydrazyl,trifluoroacetic acid, thiobarbituric acid,trichloroacetic acid,and1-phenyl-3-meth-yl-5-pyrazolone were from Sigma-Aldrich(St.Louis,MO, USA).Pullulan standards(Mw=344,200,107,47.1,21.2, and9.6kDa)were from the Showa Denko K.K.(Tokyo, Japan).Q Sepharose Fast Flow and Sephacryl S-100were from GE healthcare(Piscataway,NJ,USA).Dialysis mem-branes(flat width,44mm;molecular weight cut-off,3500) were from Lvniao(Yantai,China).Microbial Strain and Culture ConditionsThe marine fungus F.oxysporum was isolated from fresh leaves of Ipomoea pes-caprae(Linn.)collected from South Sea,China.It was identified according to its morphological characteristics and18S rRNA sequences,and the accession number of Genbank was JN604549.Briefly,the fungus was cultivated in the liquid medium containing yeast extract(3g/ L),peptone(5g/L),glucose(20g/L),malt extract(3g/L),sea salt(24.4g/L),KH2PO4(0.5g/L),NH4Cl(0.5g/L),pH6.0–6.5,at25°C for40days,and50L of fermented broth was obtained.Preparation of the Extracellular PolysaccharideThe fermented broth was filtered through cheese cloth,the filtrate was concentrated to1/15of its original volume under reduced pressure at40°C,and a threefold of the volume of 95%(v/v)ethanol was added.The resulting precipitate was recovered by centrifugation at3600×g for10min,dialyzed in cellulose membrane tubing against distilled water for72h. The retained fraction was dried,and the protein in the fraction was removed as described by Matthaei et al.(1962).The crude polysaccharide was fractionated by anion-exchange chroma-tography using a Q Sepharose Fast Flow column(30×3cm) coupled to an AKTA FPLC system and elution with a step-wise gradient of0,0.2,and1.0M NaCl.The fractions were assayed for carbohydrate content by the phenol–sulfuric acid method.The fractions eluted with distilled water were pooled, dialyzed,and further purified on a Sephacryl S-100column (70×2cm)eluted with0.2M NH4HCO3at a flow rate of 0.3mL/min.The major polysaccharide fractions were pooled, freeze–dried,and designated as Fw-1.Determination of Purity and Molecular WeightPurity and molecular weight were determined by high-performance gel permeation chromatography(HPGPC)with a Shodex Ohpak SB804(7.8×300mm,Tokyo,Japan)column and a refractive index detector(Agilent RID-10A Series),and elution with0.1M Na2SO4at a flow rate of0.5mL/min(Li et al.2012).Of1%sample solutions in0.2M Na2SO4,20μL was injected.The molecular weight was estimated by refer-ence to a calibration curve made by pullulan standards.General AnalysisTotal sugar content was measured by the phenol–sulfuric acid method using galactose as the standard(Dubois et al.1956). Protein content was assayed according to the modified Lowry method(Bensadoun and Weinstein1976).Sulfate content was measured according to Silvestri et al.(1982).Uronic acid con-tent was determined by the carbazole–sulfuric acid method (Bitter and Muir1962).Analysis of Monosaccharide CompositionFive milligrams of polysaccharide was hydrolyzed with2M trifluoroacetic acid at100°C for6h.Excess acid was re-moved by co-distillation with methanol after the hydrolysis was completed.Sample was subjected to reversed-phase high-performance liquid chromatography(HPLC)after pre-column derivatization and UV detection(Li et al.2011). Sugar identification was done by comparison with reference sugars(D-glucose,L-rhamnose,D-xylose,L-arabinose,D-man-nose,L-fucose,D-galactose,D-glucuronic acid,D-galacturonicacid,D-mannuronic acid,N-acetyl-β-D-glucosamine). Calculation of the molar ratio of the monosaccharide was car-ried out on the basis of the peak area of the monosaccharide. Methylation AnalysisMethylation analysis was performed by the method of Hakomori(1964)with some modifications.In brief, 2mg of polysaccharide in dimethyl sulfoxide was meth-ylated using NaH and iodomethane,and the completion of methylation was confirmed by Fourier transform infrared (FTIR)spectroscopy by the disappearance of OH bands. After hydrolysis with2M trifluoroacetic acid at105°C for6h,the methylated sugar residues were converted to partially methylated alditol acetates by reduction with NaBH4,followed by acetylation with acetic anhydride. The derivatised sugar residues were extracted into dichlo-romethane and evaporated to dryness,and dissolved again in100μL of dichloromethane.The products were ana-lyzed by gas chromatography–mass spectrometry(GC-MS)on a DB225using a temperature gradient of100–220°C with heating at a rate of5°C/min and mainte-nance of a temperature at220°C for15min.GC-MS was performed on an HP6890II instrument.Identification of partially methylated alditol acetates was carried out on the basis of retention time and mass fragmentation patterns.IR Spectroscopy AnalysisFTIR spectra were measured on a Nicolet Nexus470spec-trometer.The polysaccharide was mixed with KBr powder, ground up,and then pressed into1-mm pellets for FTIR mea-surements in the frequency range of4000–500cm−1with a resolution of4.0cm−1and320scans co-addition.NMR Spectroscopy Analysis1H nuclear magnetic resonance(NMR)and13C NMR spectra were measured at23°C using a JEOL JNM-ECP600MHz spectrometer.60mg of polysaccharide was deuterium ex-changed by two successive freeze–drying steps in99%D2O and then dissolved in0.5mL of99.98%D2O.1H–1H corre-lated spectroscopy(COSY),1H–1H total correlation spectros-copy(TOCSY),1H–1H nuclear overhauser effect spectrosco-py(NOESY),1H–13C heteronuclear multiple quantum coher-ence spectroscopy(HMQC)and1H–13C heteronuclear multi-ple bond correlation spectroscopy(HMBC)experiments were also carried out.Chemical shifts are expressed in ppm using acetone as internal standard at2.225ppm for1H and 31.07ppm for13C.Analysis of Antioxidant ActivityScavenging ability of hydroxyl radicals was determined ac-cording to the method of Smirnoff and Cumbes(1989). Scavenging ability of superoxide radicals was assessed ac-cording to the method reported by Marklund and Marklund (1974).Scavenging ability of DPPH radicals was measured according to the method described by Shimada et al.(1992). The scavenging ability was calculated according to the equa-tion below:scavenging ability(%)=(1–A sample/A control)×100, where A control is the absorbance of control without the tested samples,and A sample is the absorbance in the presence of the tested samples.The EC50value(mg/mL)was the effective concentration at which the tested radicals were scavenged by 50%.Ascorbic acid was used as positive control in all anti-oxidant assays.All bioassay results were expressed as means ±standard deviation(SD).The experimental data were sub-jected to an analysis of variance for a completely random design,and three samples were prepared for assays of every antioxidant attribute.ResultsPreparation and Chemical Composition of the Extracellular PolysaccharideProcedures used for the preparation of the extracellular poly-saccharides from the fermented broth of the mangrove-associated fungus F.oxysporum were shown in Fig.1.Crude extracellular polysaccharide(0.59g/L)was obtained from the fermented broth,and fractionated using a Q Sepharose Fast Flow column(Fig.2a).The polysaccharide fraction,eluted with distilled water,was a major component of the crude polysaccharides.The fraction was further purified by a Sephacryl S-100column(Fig.2b),and a polysaccharide frac-tion Fw-1was obtained.The yield of Fw-1from crude polysaccharide was about 42.86%.Fw-1gave a single and symmetrical peak in the HPGPC chromatogram(Fig.2c),thus Fw-1could be a homo-geneous polysaccharide.The linear relationship between the logarithm of molecular weight of pullulan standards and re-tention time was obtained.The retention time in HPGPC chro-matogram of Fw-1was used to calculate its molecular weight by the obtained regression equation.Thus,the molecular weight of Fw-1was estimated to be about61.2kDa.Fw-1 contained91.3%total carbohydrate and minor amounts of protein(0.79%)and did not have any sulfate ester. Monosaccharide composition analysis by reversed-phase HPLC showed that Fw-1consisted of galactose,glucose, and mannose with a molar ratio of1.33:1.33:1.00.No acidic sugar and amino sugar were detected in Fw-1.Thepolysaccharide fraction Fs,eluted at 0.2M NaCl,was not further investigated due to the limit of sample amount.It is possible that fraction Fs contains an acidic polysaccharide,such as a polysaccharide with phosphate ester (Chen et al.2013).IR SpectroscopyFrom the FTIR spectrum of Fw-1,the broad and intense band at 3416cm −1was the result of valent vibrations OH groups.The signal at 2931cm −1was attributed to the stretch vibration of the C –H bond.The band at 1649cm −1was assigned to the bending vibrations of HOH,and the band at 1416cm −1originated from the bend-ing vibrations of O –H bond.The band at 1241cm −1was due to the stretch vibration of C –O –C linkages.The signal at 1032cm −1was assigned to the stretch vibration of C –O and change angle vibration of O –H.The characteristic ab-sorption bands at 876and 809cm −1suggested the pres-ences of furan ring and mannopyranose units,respectively (Ahrazem et al.2000;Mathlouthi and Koenig 1986).Methylation AnalysisIn order to determine the linkage pattern of the sugar residues,Fw-1was subjected to methylation analysis (Table 1).A large amount of 1,2,4,6-tetra-O -acetyl-3,5-di-O -methyl-galactitol,which originated from the (1→2,6)-linked galactofuranoseresidue,was detected in Fw-1,suggesting that Fw-1was a highly branched polysaccharide.1,5-di-O -acetyl-2,3,4,6-tet-ra-O -methyl-glucitol,1,2,5-tri-O -acetyl-3,4,6-tri-O -methyl-mannitol,and 1,2,5-tri-O -acetyl-3,4,6-tri-O -methyl-glucitol were also detected,indicating the presence of (1→)-linked glucopyranose,(1→2)-linked mannopyranose and (1→2)-linked glucopyranose residues.In addition,1,5-di-O -acetyl-2,3,4,6-tetra-O -methyl-mannitol,which originated from the (1→)-linked mannopyranose residue,was also found in Fw-1.The results suggested that the structure of Fw-1is com-posed of (1→2,6)-linked galactofuranose,(1→2)-linked glu-copyranose,(1→2)-linked mannopyranose,terminal gluco-pyranose,and mannopyranose residues.NMR SpectroscopyThe 1H NMR spectrum (Fig.3a )of Fw-1showed anomeric proton signals at 5.20,5.10,5.09,4.91,4.75,and 4.65ppm,which were labeled A –F from low to high field.The anomeric signals B and C almost overlapped.The anomeric proton sig-nals A –F had relative integrals of 1.0:0.5:0.5:0.25:0.25:0.25.A might be signal of β-galactofuranose residue.B and C were attributed to the signals of α-configuration pyranose units,and D –F were likely the signals of β-configuration pyranose units.The chemical shifts from 3.42to 4.26ppm were assigned to H2–H6of glycosidic ring.In the anomeric region of the 13C NMR spectrum (Fig.3b )of Fw-1,there were six main anomeric carbon signals that occurred at 107.8,102.4,101.8,101.3,99.6,and 99.5ppm.The anomeric carbon signal at 107.8ppm was due to signal of β-galactofuranose residue because of extremely low field shifts (Ahrazem et al.2006).As shown in the DEPT spectrum,the signal at 70.8ppm was assigned to the substituted C-6of β-galactofuranose units.The result confirmed the presence of the substituted C-6linkage patterns,which was in accordance to the methylation results.The 1H NMR spin systems chemical shifts of the polysac-charide were assigned by means of the 1H –1H COSY spec-trum (Fig.3c )and the 1H –1H TOCSY spectrum (Fig.3d ).Combined with the analysis of the 1H –13C HMQC spectrum of Fw-1(Fig.3e ),the observed 1H and 13C chemical shifts and the assignment of the sugar residues were given (Table 2).A was assigned to →2,6)-β-D -Gal f (1→because of the down-field chemical shifts of the C-2(88.1ppm)and C-6(70.8ppm).B and C were suggested to be Glc p because of the high field chemical shift of H-2(3.59and 3.69ppm).In the 1H –1H TOCSY spectrum,H-1of B and C showed the correlation peaks with H-2,H-3,H-4,and H-5,which con-firmed this speculation.The 1H –13C HMQC spectrum re-vealed the substitution of C at C-2due to the downfield chem-ical shift (77.0ppm)of C-2compared with the parent α-D -Glc p .Thus,B was attributed to α-D -Glc p (1→,and C was due to →2)-α-D -Glc p (1→.Combined with methylationanalysisFig.1Scheme for the preparation of the extracellular polysaccharide produced by the mangrove-associated fungus F .oxysporumand NMR spectra data (Takegawa et al.1997),E was assigned to →2)-β-D -Man p (1→because of C-2(78.0ppm)of E had a relative downfield chemical shifts.D and F were assigned to be β-D -Man p (1→,the different glycosidic bond and sugar rings,which linked with D and F,had different chemical en-vironments and chemical shifts.The sequence of glycosyl residues was determined from the 1H –1H NOESY spectrum,followed by confirmation with 1H –13C correlations obtained from the 1H –13C HMBC spec-trum.In the 1H –1H NOESY spectrum (Fig.3f )of Fw-1,A had a strong NOE contact of its H-1with the H-2of C,indicating C linked to the C-2position of A.B and C had a strongcontactFig.2Isolation and HPGPC chromatogram of the extracellular polysaccharide from the fermented broth of the mangrove-associated fun-gus F .oxysporum .a The crude polysaccharides were fractionated using a Q Sepharose Fast Flow column.The fraction eluted with distill water was pooled and named as Fw.b Fw was purified on a Sephacryl S-100column and eluted with 0.2M NH 4HCO 3.The peak fractions containing the polysaccharides were pooled and named as Fw-1.c HPGPC chro-matogram of Fw-1on a Shodex Ohpak SB-804column and the standard curve of molecular weightof its H-1with the H-2of A,suggesting B and C linked to theC-2position of A.D had a strong inter-residue contact be-tween its H-1and the H-2of E,indicating D linked to theC-2position of E.From the1H–13C HMBC spectrum ofFw-1(Fig.3g),the presence of strong cross peak H-1/C-4,C-6of A confirmed that A wasβ-galactofuranose configura-tion and→6)-β-D-Gal f(1→was the main pattern of linkage.The cross-peak H-1B,C/C-2A,and H-2A/C-1B,C indicatedthat B and C linked to the C-2of→6)-β-D-Gal f(1→.The 1H–13C HMBC spectrum of Fw-1also showed H-1F/C-2 C,H-1E/C-2C,H-1D/C-2E,H-2E/C-1D,B H-1/C-5crosspeaks,which further proved the existence ofβ-D-Man p(1→2)-β-D-Man p(1→2)-α-D-Glc p(1→andβ-D-Man p(1→2)-α-D-Glc p(1→.The results also revealed both the furanoid char-acter of A and the pyranoid structure of B–F.The NMR resultswere thus in agreement with methylation results.These anal-yses allowed the identification of most of the1H and13Csignals of the sugar residues.Thus,structure of Fw-1couldbe characterized to consist of the backbone of(1→6)-linked β-D-galactofuranose residues,with multiple branches at C-2 consisting of theα-D-Glc p(1→,β-D-Man p(1→2)-β-D-Man p(1→2)-α-D-Glc p(1→andβ-D-Man p(1→2)-α-D-Glc p(1→.The hypothetical structure of Fw-1was shown in Fig.4.Analysis of Antioxidant ActivityAs shown in Table3,the scavenging abilities of Fw-1on hydroxyl,DPPH,and superoxide radicals were in a concentration-dependent manner.Less scavenging of hydrox-yl radicals was observed with Fw-1at2mg/mL,but the scav-enging ability of Fw-1on hydroxyl radicals at10.0mg/mL was up to90.2%.Fw-1showed strong scavenging ability on hydroxyl radicals as evidenced by its low EC50value(1.1mg/ mL).The scavenging ability of Fw-1on superoxide radicals was50.2%at2.0mg/mL,and the scavenging ability of Fw-1 was up to89.2%at10.0mg/mL.The EC50value of scaveng-ing ability of Fw-1on superoxide radicals was2.0mg/mL. The scavenging ability of Fw-1on DPPH radicals was up to 88.2%at10.0mg/mL,and its EC50value was2.1mg/mL, indicating that Fw-1was also good effectiveness in the anti-oxidant attribute.The scavenging abilities of Fw-1on hydroxyl,superoxide and DPPH radicals were all relativelylower than that of ascorbic acid at the same concentrations. DiscussionA novel extracellular polysaccharide Fw-1is successfullyobtained from the mangrove-associated fungus F.oxysporum.Fw-1is an extracellular polysaccharidewith different structural characteristics from other extra-cellular polysaccharides produced by Fusarium sp.Thecell wall polysaccharides from F.oxysporum are com-posed of glucosamine and N-acetylglucosamine(Fukamizo et al.1992,1996),and the polysaccharidefrom Fusarium sp.M7-1consists of mannose,glucose,galactose,and glucuronic acid(Iwahara et al.1992).However,a small amount of→2)-β-D-Gal f(1→and→6)-α-D-Glc p(1→residues present in the cell wall polysac-charide of Fusarium sp.M7-1(Iwahara et al.1996).Somealkali-extractable and water-soluble extracellular polysac-charides from Fusarium species contain a backbone of β-(1→6)-linked galactofuranose residues almost fully branched at O-2by single residues of glucopyranose oracidic chains containing glucuronic acid and mannose.The extracellular polysaccharide from F.oxysporumY24-2is composed of→2)-β-D-Gal f(1→6)-α-D-Glc p(1→units(Guo et al.2013).The structure of Fw1also differs from othergalactofuranose-containing extracellular polysaccharides re-ported previously(Gómez-Miranda et al.2003;Leal et al.2010).The galactofuranans from Aspergillus niger, A.fumigatus,Trichophyton species and Penicillium charlesii,have been characterized as linear chains of(1→5)-linkedβ-D-galactofuranose units(Gander et al.1974;Latgéet al.1994; Unkefer and Gander1990;Ikuta et al.1997).For the extracel-lular polysaccharide from the deep-sea fungus P.griseofulvum,its galactofuranan chain consists of(1→5)-linkedβ-D-galactofuranose,with additional branches at C-6 consisting of(1→)-linkedβ-D-galactofuranose residues and phosphate esters(Chen et al.2013).Fw-1contains a backbone of(1→6)-linkedβ-D-galactofuranose residues with multipleTable1Results of methylation analysis of Fw-1Methylated sugar Primary mass fragments(m/z)Molar ratio Deduced linkage1,5-Di-O-acetyl-2,3,4,6-tetra-O-methyl-mannitol101,117,129,145,161,205 2.0Man p(→1,5-Di-O-acetyl-2,3,4,6-tetra-O-methyl-glucitol101,117,129,145,161,205 2.0Glc p(1→1,2,5-Tri-O-acetyl-3,4,6-tri-O-methyl-mannitol87,101,129,161,189 1.0→2)Man p(1→1,2,5-Tri-O-acetyl-3,4,6-tri-O-methyl-glucitol101,117,129,161,201,233,277 2.0→2)Glc p(1→1,2,4,6-Tetra-O-acetyl-3,5-di-O-methyl-galactitol87,101,117,129,173,189,201,233 4.0→2,6)Gal f(1→Fig.3NMR spectra of Fw-1.Spectra were performed at23°C on a JEOL ECP600MHz spectrometer Chemical shifts are expressed in ppm using acetone as internal standard at2.225ppm for1H and 31.07ppm for13C.a1H NMR spectrum.b13C NMR and DEPT spectra.c1H–1H COSY spectrum.d1H–1H TOCOSY spectrum.e 1H–13C HMQC spectrum.f1H–1H NOESY spectrum.g1H–13C HMBC spectrum.A→2,6)-β-D-Gal f(1→.Bα-D-Glc p(1→.C→2)-α-D-Glc p(1→.Dβ-D-Man p(1→,linked to→2)-β-D-Man p(l→.E→2)-β-D-Man p(l→.Fβ-D-Man p(1→,linked to→2)-α-D-Glc p(l→.Glcpglucopyranose,Manp mannopyranose,Galf galactofuranosebranches at C-2consisting of terminal α-glucopyranose resi-dues,or short chains containing (1→2)-linked α-D -glucopy-ranose,(1→2)-linked β-D -mannopyranose,and terminal β-D -mannopyranose residues.To the best of our knowledge,this is the first report of such kind of galactofuranose-containing mannoglucogalactan isolated from fermented broth of micro-organism.The present result suggested that mangrove-associated fungi could be a potential source of extracellular polysaccharides with unique structures to be worth being fur-ther studied.In order to investigate the antioxidant activity of Fw-1,the assays based on scavenging abilities of hydroxyl,superoxide,and DPPH radicals were carried out and compared with that of ascorbic acid,one standard antioxidant.Hydroxyl radical is considered to be a highly potent oxidant,which can react with most biomacromolecules functioning in living cells and in-duce severe damage to the adjacent biomolecules.In cellular oxidation reactions,superoxide radical is normally formed first,and its effects can be magnified because it produces hydrogen peroxide and hydroxyl radical through dismutationTable 21H and 13C chemical shifts for the extracellular polysaccharide Fw-1Sugar residuesChemical shifts (ppm)a H1/C1H2/C2H3/C3H4/C4H5/C5H6/C6A b 5.20/107.8 4.21/88.1 4.26/76.9 4.05/83.9 4.02/71.0 3.94,3.69/70.8B c 5.10/99.5 3.59/72.6 3.77/73.1 3.47/71.0 3.79/73.8 3.91,3.73/62.1C d 5.09/99.6 3.69/77.0 3.81/73.1 3.45/71.0 3.76/72.6 4.12,3.79/62.3D e 4.91/102.4 4.18/72.6 3.73/72.4 3.61/72.6 3.45/71.9 3.79,3.90/62.6E f 4.75/101.3 4.24/78.0 3.68/68.3 3.95/71.2 3.76/73.5 3.96,3.45/62.4F g4.65/101.84.02/71.93.73/72.43.96/71.13.80/73.63.47,3.86/62.3Glcp glucopyranose,Manp mannopyranose,Galf galactofuranoseaThe spectra were recorded using a JEOL JNM-ECP 600MHz spectrometer.Chemical shifts are referenced to internal acetone at 2.225ppm for 1H and 31.07ppm for 13C b →2,6)-β-D -Gal f (→c α-D -Glc p (1→d →2)-α-D -Glc p (1→e β-D -Man p (1→,linked to →2)-β-D -Man p (l →f →2)-β-D -Man p (l →gβ-D -Man p (1→,linked to →2)-α-D -Glc p (l→Fig.4One of the possible structures of Fw-1(Glcp gluco-pyranose,Manp ,mannopyranose,Galf ,galactofuranose,n ≈16)and other types of reaction and was the source of free radicals formed in vivo.DPPH is a useful reagent to evaluate the free radical scavenging ability of the hy-drogen donating antioxidant,which can transfer hydro-gen atoms or electrons to DPPH radicals.It was found that Fw-1had a more noticeable scavenging ability on hydroxyl radicals than the extracellular polysaccharide AVP produced by coral-associated fungus Aspergillus versicolor LCJ-5-4,and the EC50value of AVP was 4.0mg/mL(Chen et al.2012).Moreover,the scaveng-ing ability of Fw-1on superoxide radicals appears to be higher than that of the extracellular polysaccharide As1-1produced by marine fungi Aspergillus sp.Y16,and the EC50value of As1-1was 3.4mg/mL(Chen et al. 2011).Scavenging ability of Fw-1on DPPH radicals was similar to that of extracellular polysaccharide AVP produced by coral-associated fungus,A.versicolor LCJ-5-4,and its EC50value was2.05mg/mL(Chen et al. 2012).Fw-1had a higher scavenging ability on DPPH radicals than the extracellular polysaccharides PS2-1, PS1-2,and PS1-1isolated from marine fungus Penicillium sp.F23-2(EC50value 2.53–6.81mg/mL) (Sun et al.2009).The present result suggested that the extracellular polysaccharide Fw-1could be a potential antioxidant.The antioxidant activity of Fw-1may be attributed to the extracellular polysaccharide can connect with radicals,and terminate the radical chain reaction. However,the antioxidant mechanisms of polysaccha-rides are complex.Further study on antioxidant property of extracellular polysaccharides with different structural characterization will play an important role in the un-derstanding of the mechanism of antioxidant activity.In conclusion,the extracellular polysaccharide Fw-1pro-duced by the mangrove-associated fungus F.oxysporum is a galactofuranose-containing mannoglucogalactan differing from previously described extracellular polysaccharides.Fw-1exhibits good antioxidant activity in vitro.An in-depth investigation of the antioxidant activity of Fw-1will be re-quired to determine if the extracellular polysaccharide will be useful in the food and pharmaceutical industry. Acknowledgments This work was supported by the Science and Tech-nology Development Program of Shandong Province,China (2014GHY115015),NSFC-Shandong Joint Fund for Marine Science Re-search Centers(U1406402),and the National Oceanographic Center of Qingdao of China.ReferencesAhrazem O,Gómez-Miranda B,Prieto A,Barasoaín I,BernabéM,Leal JA(2000)An acidic water-soluble cell wall polysaccharide:a che-motaxonomic marker for Fusarium and Gibberella.Microbiol Res 104:603–610Ahrazem O,Prieto A,Giménez-Abián MI,Leal JA,Jiménez-Barberoa J, Bernabe M(2006)Structural elucidation of fungal polysaccharides isolated from the cell wall of Plectosphaerella cucumerina and Verticillium spp.Carbohydr Res341:246–252Bensadoun A,Weinstein D(1976)Assay of proteins in presence of in-terfering materials.Anal Chem70:241–256Bitter T,Muir HM(1962)A modified uronic acid carbazole reaction.Anal Biochem4:330–334Chen Y,Mao WJ,Tao HW,Zhu WM,Qi XH,Chen YL,Li HY,Zhao CQ, Yang YP,Hou YJ,Wang CY,Li N(2011)Structural characterization and antioxidant properties of an exopolysaccharide produced by the mangrove endophytic fungus Aspergillus sp.Y16.Bioresour Technol102:8179–8184Chen Y,Mao WJ,Yang YP,Teng XC,Zhu WM,Qi XH,Chen YL,Zhao CQ,Hou YJ,Wang CY,Li N(2012)Structure and antioxidant activity of an extracellular polysaccharide from coral-associated fun-gus,Aspergillus versicolor LCJ-5-4.Carbohydr Polym87:218–226 Chen Y,Mao WJ,Wang BF,Zhou LN,Gu QQ,Chen YL,Zhao CQ,Li N, Wang CY,Shan JM,Yan MX,Lin C(2013)Preparation and char-acterization of an extracellular polysaccharide produced by the deep-sea fungus Penicillium griseofulvum.Bioresour Technol132: 178–181Dubois C,Gilles KA,Hamilton JK,Rebers PA,Smith F(1956) Colorimetric method for determination of sugars and related sub-stances.Anal Chem28:350–356Table3Antioxidant activity of the extracellular polysaccharide Fw-1in vitroa The results were expressed as means±standard deviation(SD). The experimental data were subjected to an analysis of variance for a completely random design,and three samples were prepared for assays of every antioxidant attribute Sample Concentration(mg/mL)a0 2.0 4.0 6.08.010.0Scavenging ability on hydroxyl radicals(%)Fw-10.059.5±1.482.5±2.885.6±2.486.8±3.590.2±2.3 Ascorbic acid0.097.2±2.497.2±2.697.4±2.697.5±1.997.7±2.1 Scavenging ability on superoxide radicals(%)Fw-10.050.2±1.868.3±3.179.1±2.385.7±3.289.2±2.8 Ascorbic acid0.097.2±1.997.3±2.297.4±2.797.5±2.897.8±2.4 Scavenging ability on DPPH radicals(%)Fw-10.049.1±1.766.9±2.475.0±2.585.2±2.388.2±2.6 Ascorbic acid0.097.2±2.297.3±1.797.4±2.097.5±2.597.7±2.8。

Colloids and Surfaces B Biointerfaces

Colloids and Surfaces B:Biointerfaces 65(2008)239–246Contents lists available at ScienceDirectColloids and Surfaces B:Biointerfacesj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c o l s u r fbPhysico-chemical properties and cytotoxicity assessment of PEG-modiﬁed liposomes containing human hemoglobinVal ´erie Centis a ,Patrick Vermette a ,b ,∗aLaboratoire de Bioing´e nierie et de Biophysique de l’Universit´e de Sherbrooke,Department of Chemical Engineering,Universit´e de Sherbrooke,2500,boul.de l’Universit´e ,Sherbrooke (QC),Canada J1K 2R1bResearch Centre on Aging,Institut universitaire de g´e riatrie de Sherbrooke,1036,rue Belv´e d`e re Sud,Sherbrooke (QC),Canada J1H 4C4a r t i c l e i n f o Article history:Received 5December 2007Received in revised form 14April 2008Accepted 15April 2008Available online 24April 2008Keywords:PEGylated liposomes encapsulating hemoglobin Oxygen carriers Cytotoxicity HUVECCharacterizationTransmission electron microscopy Zeta potentialParticle size distributiona b s t r a c tPEGylated liposomes encapsulating human hemoglobin as oxygen carriers were prepared frompuriﬁed carbonylhemoglobin (HbCO)solution and a lipid mixture composed of 1,2-dipalmitoyl-sn -glycero-3-phosphatidylcholine (DPPC),cholesterol,1,2-dimyristoyl-sn -glycero-3-phosphoethanolamine-N -[poly(ethylene glycol)2000](DMPE-PEG 2000)and palmitic acid.Hemoglobin was extracted and puriﬁed from human blood samples.SDS-PAGE was used to assess its purity.Diameter of liposomes containing hemoglobin was controlled to approximately 200nm using extrusion as measured by dynamic light scattering and transmission electron microscopy.Liposome size distributions were shown to remain unimodal over 14days,even at different storage temperatures.Zeta potential measurements revealed that liposome containing hemoglobin have a net surface charge of −7.16±0.33mV.Also,hemoglobin encapsulated in liposomes was able to perform several cycles of oxygen loading and unloading using oxygen (O 2)and carbon monoxide (CO).The hemoglobin vesicle dispersion showed some toxicity as revealed by three in vitro assays in which endothelial cell (HUVECs)monolayers were exposed to these dispersions.Cytotoxicity was function of the liposome concentration in the culture medium.©2008Elsevier B.V.All rights reserved.1.IntroductionSince Rabiner et al.in 1967and Savitsky et al.in 1978[1,2]stud-ied stroma-free hemoglobin solution as a possible substitute for human blood,research on the development of universal,safe and effective oxygen carriers has been an ongoing subject.Such a solu-tion,despite its purpose to be used in blood replacement therapy,could also be used to supply oxygen in high-density cell culture sys-tems,where oxygen is often the limiting factor.This is particularly ﬂagrant in tissue engineering applications [3–7].Many perﬂuorocarbon emulsions and hemoglobin-based prepa-rations have been considered as oxygen carriers,with their loads of advantages and disadvantages.For a complete review on the dif-ferent types of oxygen carriers,their design and characterization,the reader is referred to Riess [8].Liposomes and biodegradable polymer capsules have been examined as conﬁnement vehicles for tetrameric hemoglobin [9–12].Liposomes are double-layered phospholipid vesicles able∗Corresponding author.Tel.:+18198218000x62826;fax:+18198217955.E-mail address:Patrick.Vermette@USherbrooke.ca (P.Vermette).to encapsulate different drugs or molecules [13].They have been thoroughly studied for the past 40years and the reader is directed to the work of Lasic and colleagues for more information [13–15].Surface modiﬁcation with PEG was reported to improve the cir-culation of liposomes in circulation [8,16].PEG is a biologically inert polymer extensively used in drug delivery to create,once conjugated,a steric barrier and/or a water structure around lipo-somes,with the aim to protect liposomes from plasma proteins in the body [17,18].The steric barrier created by PEG conjugation also prevents liposome fusion and aggregation and thus,stabilizes the dispersions during storage [17,18].It was shown by Sakai et al.[17]that PEGylated liposomes encapsulating hemoglobin stored in a deoxygenated state at 4◦C and 23◦C were stable for 1year,while dispersions stored at 40◦C were stable for 6months before liposome aggregation and hemoglobin leakage were observed [17].To be used in cell cultures,liposome dispersions should have limited cell toxicity and their formulations should be stable throughout the duration of the culture.Also,the liposome prepa-ration should be able to increase the oxygen concentration in the cultures and therefore,have a positive impact on the cell growth.Cytotoxicity of PEGylated liposomes encapsulating hemoglobin was assayed in different studies.For example,hemoglobin vesicles0927-7765/$–see front matter ©2008Elsevier B.V.All rights reserved.doi:10.1016/j.colsurfb.2008.04.009240V.Centis,P.Vermette/Colloids and Surfaces B:Biointerfaces65(2008)239–246were tested on human cord blood hematopoietic progenitor cells [19].Mild toxicity was observed on short-term exposures,while inhibition of proliferation was monitored at longer times[19].Also, the effect of liposome encapsulating hemoglobin(LEH)on human platelet function in plasma was studied.Wakamoto et al.observed neither aggregation,activation,nor adverse effects when human platelets were exposed to liposome dispersions[20].Similarly,the exposure of rat erythrocytes to PEG-LEH did not affect the struc-tural integrity of the blood structures[21].No signs of aggregation and deformation of the erythrocytes were observed,suggesting that liposomes exhibited low,if any toxicity[21].Another study examined the cytokine-induced adhesiveness of monocytic cells to HUVECs[22].The data suggested that,in this speciﬁc model,LEH did not induce leukocyte adhesion and could have a beneﬁcial effect in preventing leukocyte adhesion to vascular endothelium caused by inﬂammatory cytokines[22].To our knowledge,in vitro long-term cytotoxicity of LEH formulations towards human cells have not been fully addressed and such testing is aﬁrst and manda-tory step to investigate the applicability of these LEH in tissue engineering.Therefore,this work examines the preparation and characteri-zation of liposomes encapsulating human hemoglobin as potential oxygen carriers for tissue engineering purposes and presents a char-acterization of the LEH dispersions.To assess the feasibility of using LEH as oxygen transporters,size distribution,zeta potential,mor-phology and shape,and cytotoxicity of these LEH dispersions were examined.To our knowledge,no similar study has been reported.2.Materials and methods2.1.Materials1,2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine(DPPC,cat. #PCS-020)and1,2-dimyristoyl-sn-glycero-3-phosphoethanol-amine-N-[poly(ethylene glycol)2000](DMPE-PEG2000,cat.#PPE-010)were purchased from Northern Lipids(Vancouver,BC, Canada).Cholesterol(Chol,cat.#110525)was obtained from Avanti Polar Lipids(Alabaster,AL,USA).Palmitic acid(PA,cat.#P-0500),Medium199(M-199,cat.M-5017),Heparin(cat.#H-1027), Hank’s balances salt solution(HBSS,cat.#H-6648),fetal bovine serum(FBS,cat.#F-1051),endothelial cell growth supplement (ECGS,cat.#E-2759),pyridoxal5 -phosphate(PLP,cat.#P-3657) and dl-homocysteine(Hcy,cat.#H-4628)were purchased from Sigma–Aldrich(Oakville,ON,Canada).Chloroform,sodium dithionite,and dichloromethane used for hemoglobin puriﬁcation and liposome preparation were of analyt-ical grade and were obtained from Fisher Chemicals.All the water used in these experiments was taken from a Milli-Q Gradient(Milli-pore,Billerca,MA,USA)ultra-pure deionization water system with a resistivity of18.2M cm.CellTiter96®AQueous One Solution Cell Proliferation Assay Kit(cat.#G3580)and CytoTox96®Non-Radioactive Cytotoxicity Assay(Lactate deshydrogenase,cat.#G1780)were obtained from Promega(Madison,WI,USA).CyQuant®NF Cell Proliferation Assay Kit(cat.#C35006)was purchased from Invitrogen(Burlington,ON, Canada).2.2.Hemoglobin puriﬁcationHemoglobin was obtained from human whole blood of healthy volunteers.All procedures were approved by the Ethics Committee of the Research Centre on Aging(Sherbrooke,Qc).After the subjects were thoroughly informed about the nature and goal of the study, they provided written consent.The protein was puriﬁed accord-ing to an adapted method previously published[23].In summary: (1)hemoglobin was stabilized by CO complexion(HbCO)(60min at room temperature);(2)the removal of membranes and stroma was done by solvent treatment(20%(v/v)CH2Cl2,shaken for3min with subsequent centrifugation at1900×g for15min);(3)traces of dichloromethane were removed by slowly heating the sample with aﬂow of inert gas(Ar)(heating from40◦C to60◦C,120min);(4)virus inactivation was done by heating the hemoglobin solu-tion(60◦C,overnight,with magnetic stirring agitation);(5)dialysis (MW cutoff of25kDa,against ultra-pure water,for24h,with mag-netic stirring agitation)was used to remove small molecules;(6) ultra-ﬁltration(MW cutoff of30kDa,Millipore,USA)was used to ﬁlter out molecules with MW<30kDa.Solutions containing puri-ﬁed HbCO were aliquoted and preserved in liquid nitrogen with PLP(18mM)and Hcy(15mM).SDS-PAGE was used to assess the presence of hemoglobin.2.2.1.SDS-PAGEThe puriﬁcation of Hb was determined via SDS-PAGE using a FisherBiotech system(Fisher Scientiﬁc,cat.#FB200).All the sam-ples were prepared in a Laemmli buffer(Bio-rad,cat.#161–0158) with a4%acrylamide stacking gel and a15%acrylamide resolving gel(Bio-rad,cat.#161–0158).Human hemoglobin is known to have a MW of approximately 64kDa[8,24].The hemoglobin samples were run along with a pre-stained broad-range molecular weight marker(Bio-rad,cat. #161–0318),which consisted of proteins having molecular weights ranging between6.9and210kDa.10␮L of hemoglobin samples (2␮g/mL)and7␮L of the molecular weight marker(dilution ratio of1:75(v/v)i.e.,1part of the marker in75parts of a buffer)were deposited in each well.A puriﬁed commercial hemoglobin sam-ple(Sigma,cat.#H7379)was used as a control and was run at a concentration of0.1␮g/mL.All samples were run at115V until the migration front reached the bottom of the gel(±120min).The gels were then stained using Silver SNAP Stain kit II(Fisher Scientiﬁc, cat.#24612)to determine band intensities.2.3.Liposome preparationLiposomes were prepared in a slightly different way than the method previously reported[23].Brieﬂy,the mixture of phos-pholipids DPPC,Chol,PA and DMPE-PEG2000in a molar ratio of 5/5/1/(0.3%[mole/total moles of lipids])was dissolved in chloro-form.The organic solvent was then evaporated using a rotary evaporator(B¨uchi,Switzerland)under reduced pressure to remove all traces of solvent.The resulting thinﬁlm was then hydrated with a NaOH solution(0.9mM)overnight to allow theﬁlm to dissolve to yield a lipid concentration of50mg/mL.The solution was then diluted with pure water to obtain a concentration of25mg/mL of lipids.Then,ten cycles of freeze-thawing(−196◦C and40◦C)were performed.The solution was then frozen for10min in liquid nitro-gen and lyophilized for48h.After dispersing the lipid mixture into a hemoglobin solution(4g/mL)for2h,the resulting multilamel-lar vesicles were extruded with the LiposoFast-Basic(Avestin Inc, Ottawa,ON,Canada)using11passes at54◦C through polycarbon-ate membranes with pore sizes of200nm.The resulting solution was then ultra-centrifuged at50,000×g for30min(Optima TLX, Beckman Coulter,Mississauga,On)to recover and eliminate the top layer containing non-encapsulated Hb.2.4.Liposome characterization2.4.1.Particle size distributionThe intensity mean diameter of the liposomes and the polydis-persity index(PI)of the distribution were determined by dynamicV.Centis,P.Vermette/Colloids and Surfaces B:Biointerfaces65(2008)239–246241light scattering(DLS)using a Zetasizer Nanoseries(Malvern Instru-ments,UK).Samples of liposomes stored at4◦C,22◦C and37◦C were analyzed.The refractive index and viscosity of pure water were used as calculation parameters and each sample was mea-sured3times for25runs using the unimodal model for size distribution.All samples were diluted to an appropriate counting rate prior to analysis.2.4.2.Zeta potentialThe zeta potential was measured by electrophoresis at25◦C (Zetasizer Nanoseries,Malvern Instruments,UK).The surface charge of the particles was obtained by measuring the velocity of the dispersion in an electricalﬁeld.Samples were diluted and placed in an electrophoretic cell where a potential of±60mV was established.Actual values were calculated from the mean elec-trophoretic mobility using Smoluchowski’s equation.The viscosity and dielectric constant of pure water were used as calculation parameters and each sample was measured in quadruplicate.All samples were diluted to an appropriate counting rate prior to anal-ysis.2.4.3.Inﬂuence of pH and osmolaritySlight changes in pH and osmolarity can have deleterious effects on cells in culture.To ensure that seeded cells would not respond to a change in pH and osmolarity due to the addition of liposomes in culture media,these parameters were moni-tored over24h(6,12,and24h).HUVECs were seeded onto 24-well plates(6000cells/well)(Corning,cat.#3527)and let to adhere in normal incubator conditions.The next day,HUVECs were either exposed to:(1)LEH(2mg/mL),(2)“Empty”lipo-somes i.e.,liposomes with no hemoglobin(2mg/mL),or(3)M-199 media.After6,12and24h,250␮L were pipetted and used for osmolarity analyses using a pre-calibrated osmometer(Advanced Instruments,model3250,Norwood,MS,USA).The rest of the solution was used for pH monitoring(Orion Research,model 710A,Lausanne,Switzerland).Measurements were done in triplicate.2.5.Functionality of the LEH as oxygen carriersHemoglobin possesses characteristic bands throughout the vis-ible spectra.Q-band(around500–600nm)and Soret band(in the blue region,around400nm)are typical regions that provide infor-mation about the hemoglobin state.Therefore,functionality of hemoglobin entrapped in liposomes and its ability to shift from its oxygen-loaded position to unloaded position was assayed by spectrometry.LEH dispersions(2mg/mL,using HBSS buffer)were exposed to pure oxygen and halogen light(90min,400W)[25]and sub-sequently compared to carbon monoxide(CO)exposure(15min, in the dark).To obtain deoxygenated spectra of hemoglobin,solu-tions were equilibrated with inert gas(Ar)to remove most of the oxygen before treatment with dithionite[26].Wavelengths shifts of the Q-band and Soret band under these conditions were mea-sured using a micro-plate reader(Bio-Tek Instruments,Winooski, VT,USA).2.6.Transmission electron microscopy(TEM)analysesThe samples were diluted appropriately and were negatively stained with uranyl acetate and allowed to air-dry directly on TEM grids.Observations were made using an H-7500transmission elec-tron microscope(HITASHI,Pleasanton,CA,USA)at a voltage of 60kV.2.7.HUVEC cultureHuman Umbilical Vein Endothelial Cells(HUVEC,PromoCell, Heidelberg,Germany)were cultured in M-199supplemented with10%(v/v)de-complemented FBS.ECGS(20␮g/mL),heparin (90mg/L),and antibiotics(100U/mL penicillin G and100␮g/mL streptomycin)were also added.Culture media was replenished three times a week.All cells were maintained at37◦C in an incu-bator with a humidiﬁed atmosphere containing5%CO2.Cells of passages4and5were used throughout this study.2.8.Cytotoxicity of LEHFollowing cell exposure to LEH,cell proliferation assays were performed to evaluate the cytotoxicity of the LEH formulations.For this purpose,HUVECs were incubated with three concentrations of LEH dispersions(5,2and1mg/mL)for6,12,and24h.The M-199culture medium concentration was adjusted to compensate for the dilution caused by the addition of liposomes.Empty liposomes (5mg/mL)were used as controls.Three assays were performed to evaluate the cytotoxicity of the LEH dispersions.Different assays were used since it was reported that cellular metabolic processes vary greatly over time and that methodologies that rely only on measurements of ATP content can cause some problems in further cytotoxicity interpretation[27].LEH cytotoxicity wasﬁrst examined using the MTS assay,a colorimetric method optimized for adherent cells.Viable cells enzymatically reduce the colorless tetrazolium salt MTS to inten-sively colored MTS-formazan.Brieﬂy,4000cells/well were seeded inﬂat bottom96-well plates(Corning Incorporated,Corning,NY, USA)and incubated for24h.After removing the culture medium, 100␮L of LEH dispersions were applied,and the plates were incu-bated at37◦C for a period of4h.The absorbance was read at490nm on a micro-plate reader(Bio-Tek Instruments,Winooski,VT,USA). Values were corrected for background absorbance.Secondly,cell number was assessed using the CyQuant®Cell Proliferation Assay Kit,a highly sensitive,ﬂuorescence-based micro-plate assay[27].The CyQuant®assay measures the ability of CyQuant®dye to bind to the cellular nucleic acids of viable cells. Measurements of cellular proliferation provide a general measure of toxicity.Cell cultures were seeded at103cells/well in duplicate inﬂat bottom96-well plates.After exposure of the liposomes to adherent cells,cells were rinsed with HBSS buffer to remove dead cells no longer adhering to the plate,lysed,and the DNA was stained using the CyQuant®ﬂuorescent dye solution as recommended by the manufacturer.Plates were incubated at37◦C for60min.Flu-orescence was measured using aﬂuorescence micro-plate reader (Bio-Tek Instruments,Winooski,VT,USA).The excitation maximum was480nm and the emission maximum was530nm.Values were corrected for background absorbance.Finally,quantiﬁcation of the release of the cytoplasmic enzyme, lactate deshydrogenase(LDH)in culture media was evaluated.LDH liberation is correlated with the number of lysed cells.Brieﬂy,4000 cells/well were seeded inﬂat bottom96-well plates(Corning Incor-porated,Corning,NY,USA)and incubated for24h.After HUVEC exposure to the different concentrations of LEH,cells were lysed using a lysis buffer,as described by the manufacturer.LDH activ-ities in the culture media and in the corresponding cell lysates were measured at490nm on a micro-plate reader(Bio-Tek Instru-ments,Winooski,VT,USA).Values were corrected for background absorbance.2.8.1.Assessment of potential residual CH2Cl2cytotoxicityTo ensure that the dichloromethane used in the hemoglobin puriﬁcation process was not responsible for any cell death,a sample242V.Centis,P.Vermette/Colloids and Surfaces B:Biointerfaces65(2008)239–246 of M-199culture media was treated in the same way.In a con-trol experiment,M-199was exposed to CH2Cl2with subsequentcentrifugation and evaporation procedures(steps2–3,Section2.2).Then,10%(v/v)of de-complemented FBS was added to the media.This“CH2Cl2treated”M-199sample was then added inﬂat bot-tom96-well plates seeded with4000cells/well.Cells were thenincubated for24h with this M-199and a standard MTS assay wasperformed after the exposure.Normal M-199media was used ascomparison.The absorbance was read at490nm on a micro-platereader(Bio-Tek Instruments,Winooski,VT,USA).Values were onceagain corrected for background absorbance.Results were done intriplicate.2.9.Statistical analysisAll the data collected throughout the study were expressed asmeans±standard deviations(S.D.s).Analysis of variance(ANOVA)was used to determine if data were signiﬁcantly different usingp≤0.05.3.Results and discussion3.1.Hemoglobin puriﬁcation3.1.1.SDS-PAGEFig.1shows SDS-PAGE results for the Hb puriﬁcation proceduredescribed above.In this study,the SDS-PAGE technique was usedas a qualitative method to show the presence of hemoglobin in thesample.Proteins of interest were well deﬁned on the ne Bshows the molecular weight marker containing proteins rangingfrom6.9to210kDa while lanes A and C show puriﬁed samples ofhuman Hb and a commercial hemoglobin standard,respectively.It is possible to observe the tetramer,dimer and monomer com-posing hemoglobin at values of,respectively,64,32and16kDa(black arrows on left side,respectively noted1,2and3).The sub-units are visible since the sample was heated prior to its run,thusdenaturing the protein.When puriﬁed Hb was compared with thecommercial sample,it was possible to observe the same bandsexcept for one(under the last arrow),which was present only ontheFig.1.SDS-PAGE nes A and C show a puriﬁed human hemoglobin sample and a commercial hemoglobin ne B presents broad-range molecular weight markers.Black arrows on left side(1,2,and3)show,respectively,the64,32 and16kDa subunits of the protein.puriﬁed sample.It was published by Pearson et al.that this band is not apparent on freshly prepared specimens[28],as opposed to outdated blood,as in our case.Also,the observed streaking in the obtained SDS-PAGE analyses can be caused by the presence of other proteins.Therefore,the effect of the presence of these other proteins on the functionality of Hb has been tested(see below).3.2.Hemoglobin functionalitySpectra of hemoglobin encapsulated in liposomes are presented in Fig.2.Fig.2A clearly shows the difference between oxygen-loaded and unloaded states of the hemoglobin.This difference is even more evident in Fig.2B,showing the characteristic Q-bands and the shift from538and569nm to540and577nm.Panel C of Fig.2summarizes the shifts of hemoglobin from oxygen-loaded states to unloaded ones.Also,Fig.2C shows that hemoglobin can be oxygen-loaded and unloaded several times.The ability of the pro-tein to shift back and forth from one position to another(in thiscase,Fig.2.(A)Complete visible spectra of oxygen-loaded and unloaded puriﬁed human hemoglobin,(B)zoom on Q-bands showing the shift between oxygen-loaded and unloaded positions of puriﬁed human hemoglobin,and(C)variation of Q-band wavelengths after exposure to carbon monoxide and oxygen.V.Centis,P.Vermette /Colloids and Surfaces B:Biointerfaces 65(2008)239–246243Table 1Mean particle diameter of empty liposomes and liposomes encapsulating hemoglobin stored at 4◦C,22◦C and 37◦C over 2weeks4◦C22◦C37◦CEmpty liposomesLiposomes encapsulating hemoglobin Empty liposomes Liposomes encapsulating hemoglobin Empty liposomes Liposomes encapsulating hemoglobin Day 1220(39.6)226(48.5)240(45.6)269(47.2)229(48.1)239(53.3)Day 7229(41.8)237(47.7)245(46.2)252(45.1)249(44.2)255(42.7)Day 14240(40.7)239(57.6)249(45.9)247(47.8)248(45.6)254(50.1)Data in parentheses indicate the %Polydispersity (%Pd).from oxygen to carbon monoxide)demonstrates the functionality of hemoglobin even after its encapsulation into liposomes.3.3.Liposome characterization3.3.1.Particle size distributionResults of size distribution of liposomes encapsulating hemoglobin are presented in Table 1.It is evident from the data that the size distributions were unimodal and relatively narrow for all samples.%Polydispersity (%Pd)values of all LEH disper-sions were higher than 40%(Table 1).Therefore,LEH dispersions can be considered to as polydispersed.The term polydispersity is derived from the polydispersity index (PdI).PdI is a number cal-culated from a simple two-parameter ﬁt to the correlation data called a cumulants analysis.PdI is known as the relative variance,while %Pd is the coefﬁcient of variation or relative polydisper-sity and can be expressed as (PdI)1/2×100(Malvern Instruments,UK).As a rule of thumb,samples with %Pd ≤20%are considered monodisperse.The size of a particle is an important factor in determining its use as an oxygen carrier.Size is of great importance when one wishes to use these particle-systems in a body to supply increased (or opti-mized)oxygen concentration.Rapid extravasion from the body by the reticulo-endothelial system (RES)and immunogenic effects are only a few of the problems foreign particles encounter in the body [8,19,20,29–34].For tissue engineering purposes,particle size is also an important issue to consider.Liposomes added to a culture medium should be able,for example,to circulate through porous scaffolds often used in tissue engineering.Also,as for LEH,their small size (±200nm)should enable to increase the surface area between the liposomes and the dispersing medium phase allowing a better oxygen transfer.The results in Table 1show that LEH sizes are within the expected range of the 200-nm pores of the extrusion membrane used.These results are comparable and consistent with the ﬁndings of Ariﬁn and Palmer [35,36].Using asymmetric ﬁeld ﬂow fraction-ation coupled to a multi-angle static light scattering photometer,they found that liposomes extruded through membranes having 200-nm pore diameter exhibited a mean diameter that was close to that of the membrane.The dispersion observed was unimodal with distribution widths of 10–20nm.Mean LEH diameter does not vary signiﬁcantly with storage tem-perature (p ≤0.01).Still,LEH samples should be kept at 4◦C prior to their use in order to limit possible particle size enlargement.Stor-age time of 14days affected to some extent particle size growth in the samples (p ≥0.05).On the other hand,all the preparations exhibited a unimodal distribution,allowing to conclude,that over a short period of time (≤7days),the particles should not aggregate or fuse (p ≤0.05).PEG grafted onto the surface of the vesicles cer-tainly help to stabilize LEH by creating a steric hindrance around liposomes [18].The molar ratio of PEG used in our study is fairly small compared to those of other studies in which PEG was used.But this PEG concentration was sufﬁcient to stabilize the liposomes over 14days,a time corresponding to a culture period required to grow a network of vascular micro-vessels,for instance.The data presented in Table 1reveal that the encapsulation of hemoglobin molecules does not seem to affect the size distribution of the liposomes.Liposomes encapsulating hemoglobin exhibit the same size distribution behaviour as “empty”ones [37].3.3.2.Zeta potentialMeasurements of zeta potential can yield information about the colloidal stability of particle dispersions [36–39].On one hand,in a solution containing no proteins,no amino acids,and little to no multi-valent electrolytes,the larger the magnitude of the zeta potential the more stable the dispersion should be.Limited ﬂoccu-lation occurs between |5|and |15|mV [37].For “naked”liposomes (prepared in the same manner as LEH,but hydrated with M-199culture media instead of hemoglobin),it was shown that the zeta potential at the surface of liposomes was approximately −45mV [12,40].When PEG 2000was added to the liposome preparation,the zeta potential increased to reach a plateau around −5mV [12].ThisFig.3.TEM images revealing shape,structure,and sizes of liposomes encapsulating hemoglobin molecules.244V.Centis,P.Vermette/Colloids and Surfaces B:Biointerfaces65(2008)239–246increase of the surface charge was thought in part to be because of the drag caused by the presence of PEG chains on the liposome sur-face,decreasing the mobility of the liposomes,hence affecting the zeta potential[12,41].On the other hand,the presence of surface charge should be counter-balanced with a good steric hindrance in solutions containing proteins and amino acids(e.g.,culture media), as these often charged molecules can adsorb on the surface of the particles and enhance particle aggregation.For the PEGylated liposomes encapsulating Hb studied here,the mean zeta potential on the particle surface was−7.16±0.33mV. This indicates that limited aggregation of the particles should be observed and that stability over time can be,to some extent,con-trolled,as observed in Table1.In fact,over14days,the particle size did not vary for a given temperature and conﬁrmed the stability of the distribution.The values obtained can also be compared with results obtained by Sakai et al.[37].They reported LEH,without PEG and PA,with a surface charge of−21mV.PA was reported to decrease surface charge proportionally to its increase in concentra-tion in the lipid bilayer[37,42].3.3.3.pH and osmolarity of culture media with LEHpH and osmolarity of culture media containing either“empty”liposomes or LEH were monitored over24h.All the solutions had a constant pH of7.4.As for osmolarity measurements,for both“empty”liposomes and M-199controls,the values remained constant at approximately330±3mOsm.The osmolarity of LEH dispersions was319±2mOsm.From these data,it can be concluded that the addition of dispersions of“empty”liposomes and LEH does not affect the osmolarity of the culture medium used in this study.3.4.Transmission electron microscopy(TEM)analysesTEM images of LEH are presented in Fig.3.In the TEM images, the characteristic aspects of large unilamellar vesicles(LUV)can be observed[14].Large unilamellar vesicles are liposomal structures exhibiting a rather empty core that can be used to encapsulate different molecules,such as drugs or proteins[15].LUV’s can be prepared from large“onion-like”structures of multilamellar vesi-cles(MLV)by different methods such as extrusion.The TEM images clearly show that the extrusion method was convenient to produce LEHs.The distinctively recognizable double-layered membrane and the empty core are good indications of a LUV structure[13,14].TEM images revealed that LEH(over62individual liposomes)have a mean diameter of170±50nm.These results are in good agreement with the ones obtained from dynamic light scattering analyses. 3.5.Cytotoxicity of liposomes encapsulating hemoglobin(LEH)Fig.4shows the behaviour of HUVECs in contact with disper-sions made of different concentrations of LEH.Similar cytotoxicity trends were observed when the same experiments were conducted using humanﬁbroblasts from foreskin(data not shown).Fig.4A and B illustrate the percentage of live cells when HUVECs were exposed to LEH dispersions,while Fig.4C shows the percent-age of cell death after similar exposure.Statistical analyses of the results of these three cytotoxicity assays reveal that exposure time had a signiﬁcant effect on cell viability(p=0.05).The analyses also show that LEH concentration had an effect on the cell death rate.For example,a signiﬁcant difference(p≤0.05)was observed between 5and1mg/mL for all tested exposure times.From Fig.4,it can be seen that approximately50%cell loss occurred following24-h cell exposure to the LEH dispersions.The results obtained from the three assays are consistent with one another(p≤0.05).Unfor-tunately,these results cannot be directly correlated with thoseof Fig.4.Cytotoxicity assays of HUVECs exposed to PEGylated liposomes containing hemoglobin for different exposure times.(A)AQueous One assay,(B)CyQuant®assay,and(C)lactate deshydrogenase assay.All data are signiﬁcantly different with p≤0.05.other studies since very few long-term in vitro tests have been car-ried out.A recent study by Yamaguchi et al.[19]investigated the short and long-term effects of hemoglobin vesicles(HbVs)on the clono-genic and proliferative activity of human hematopoietic progenitor cells derived from umbilical cord blood.They found that,at a concentration of3%(v/v),continuous exposure of HbV signif-icantly decreased the number and size of mature-committed colonies[19].Moreover,HbVs also notably reduced the number of high-proliferative potential colony-forming cells and lead to the suppression of cellular proliferation and differentiation in liquid culture[19].On the other hand,the same study presents the effect of HbV exposure to cord blood for20h or3days.Yamaguchi et。

Preparation and Characterization of La0.8Sr0.2MnO3-δ Cathode for SOFCs Fabricated Using Azeotro

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materials characterization审稿意见

Materials Characterization审稿意见IntroductionMaterials characterization is an essential aspect of scientific research and industrial applications. In this article, we will discuss the importance of materials characterization and explore various techniques used in the field. Additionally, we will address the key considerations for reviewers when evaluating materials characterization studies.Importance of Materials CharacterizationMaterials characterization plays a crucial role in understanding the properties and behavior of various materials. It involves the analysis and evaluation of the structure, composition, and physical properties of materials. By characterizing materials, scientists and engineers can make informed decisions about their applications and optimize their performance.Techniques used in Materials Characterization1. Scanning Electron Microscopy (SEM)SEM is a widely used technique for characterizing materials at high resolution. It uses a focused beam of electrons to scan the surface of a sample, providing detailed information about its topography, composition, and elemental analysis. SEM is particularly useful for studying microstructures, surface morphology, and particle distribution.2. X-ray Diffraction (XRD)XRD is a technique that analyzes the crystal structure of materials. It works by shining X-rays onto a sample and measuring the diffraction pattern produced. This pattern contains information about the arrangement of atoms in the material, allowing researchers to determineits crystal structure, lattice parameters, and phase composition. XRD is commonly used to identify crystalline phases and study phase transformations in materials.3. Fourier Transform Infrared Spectroscopy (FTIR)FTIR is a spectroscopic technique used to identify functional groups and chemical bonds in organic and inorganic materials. It measures the absorption of infrared radiation by the sample, providing a unique fingerprint that can be used for identification. FTIR is widely used in materials characterization to determine the presence of specificchemical groups, analyze molecular structures, and investigate surface properties.4. Thermal AnalysisThermal analysis techniques, such as differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), are used to study thethermal behavior of materials. DSC measures the heat flow in a sample as a function of temperature, providing information about phase transitions, thermal stability, and thermal properties. TGA measures the weight loss of a sample as it is heated, allowing for the analysis of composition, decomposition, and moisture content.Key Considerations for ReviewersWhen evaluating materials characterization studies, reviewers should consider several key aspects to assess the quality and significance of the research. These considerations include:1.Sample Preparation: Reviewers should evaluate the adequacy of thesample preparation techniques used in the study. Proper samplepreparation is vital to obtain accurate and representative results.2.Characterization Techniques: Reviewers should assess thesuitability and reliability of the characterization techniquesemployed. The chosen techniques should be appropriate for theresearch objectives and should provide sufficient evidence tosupport the conclusions.3.Data Analysis: Thorough data analysis is essential for materialscharacterization studies. Reviewers should evaluate thestatistical methods, data interpretation, and conclusions drawnfrom the analysis. It is important to ensure that the conclusions are supported by the data presented.4.Reproducibility: Reviewers should consider the reproducibility ofthe results presented in the study. Materials characterizationstudies should provide sufficient information to allow otherresearchers to reproduce the experiments and obtain similarresults.5.Limitations and Future Directions: It is important for authors toacknowledge the limitations of their study and propose futuredirections for research. Reviewers should assess whether theseaspects are adequately addressed and if the study contributes tothe existing knowledge in the field.ConclusionMaterials characterization is an integral part of scientific research and technological advancements. By employing various characterization techniques, researchers gain insights into the properties and behavior of materials, leading to the development of new materials with enhanced functionalities. Reviewers play a crucial role in ensuring the quality and validity of materials characterization studies by thoroughly evaluating the sample preparation, characterization techniques, data analysis, reproducibility, and future directions of the research.。

2011年6月英语四级真题及答案(含解析)

2011年6月大学英语四级真题及答案解析作文部分Part I Writing (30 minutes) Directions:For this part, you are allowed 30 minutes to write a short essay on the topic of Online Shopping. You should write at least 120 words following the outline givenbelow:1．现在网上购物已成为一种时尚2．网上购物有很多好处，但也有不少问题3．我的建议Online Shopping注意：此部分试题在答题卡1上。

Part II Reading Comprehension (Skimming and Scanning) (15 minutes) Directions:In this part, you will have 15 minutes to go over the passage quickly and answer the questions on Answer Sheet 1. For questions 1-7, choose the best answer from thefour choices marked A), B), C) and D). For questions 8-10, complete the sentenceswith the information given in the passage.British Cuisine: the Best of Old and NewBritish cuisine (烹饪) has come of age in recent years as chefs (厨师) combine the best of old and new.Why does British food have a reputation for being so bad? Because it is bad! Those are not the most encouraging words to hear just before eating lunch at one of Hong Kong's smartest British restaurants, Alfie's by KEE, but head chef Neil Tomes has more to say."The past 15 years or so have been a noticeable period of improvement for food in England," the English chef says, citing the trend in British cuisine for better ingredients, preparation and cooking methods, and more appealing presentation. Chefs such as Delia Smith, Nigel Slater, Jamie Oliver and Gordon Ramsay made the public realise that cooking - and eating - didn't have to be a boring thing. And now, most of the British public is familiar even with the extremes of Heston BlumenthaPs molecular gastronomy, a form of cooking that employs scientific methods to create the perfect dish."It's no longer the case that the common man in England is embarrassed to show he knows about food," Tomes says.There was plenty of room for improvement. The problems with the nation's cuisine can be traced back to the Second World War. Before the War, much of Britain's food was imported and when German U-boats began attacking ships bringing food to the country, Britain went on rations (配给)."As rationing came to an end in the 1950s, technology picked up and was used to mass-produce food," Tomes says. "And by then people were just happy to have a decent quantity of food in their kitchens."They weren't looking for cured meats, organic produce or beautiful presentation; they were looking for whatever they could get their hands on, and this prioritisation of quantity over quality prevailed for decades, meaning a generation was brought up with food that couldn't compete with neighbouring France, Italy, Belgium or Spain.Before star chefs such as Oliver began making cooking fashionable, it was hard to find a restaurant in London that was open after 9pm. But in recent years the capital's culinary (烹饪的) scene has developed to the point that it is now confident of its ability to please the tastes of any international visitor.With the opening of Alfie's in April, and others such as The Pawn, two years ago, modern British food has made its way to Hong Kong. "With British food, I think that Hong Kong restaurants are keeping up," says David Tamlyn, the Welsh executive chef at The Pawn in Wan Chai. "Hong Kong diners are extremely responsive to new ideas or presentations, which is good news for new dishes."Chefs agree that diners in Hong Kong are embracing the modern British trend. Some restaurants are modifying the recipes (菜谱) of British dishes to breathe new life into the classics, while others are using better quality ingredients but remaining true to British traditions and tastes.Tamlyn is in the second camp. "We select our food very particularly. We use US beef, New Zealand lamb and for our custards (牛奶蛋糊)we use Bird's Custard Powder," Tamlyn says. "Some restaurants go for custard made fresh with eggs, sugar and cream, but British custard is different, and we stay true to that."Matthew Hill, senior manager at the two-year-old SoHo restaurant Yorkshire Pudding, also uses better ingredients as a means of improving dishes. "There are a lot of existing perceptions about British food and so we can't alter these too much. We're a traditional British restaurant so there are some staples (主菜) that will remain essentially unchanged."These traditional dishes include fish and chips, steak and kidney pie and large pieces of roasted meats. At Alfie's, the newest of the British restaurants in town and perhaps the most gentlemen's club-like in design, Neil Tomes explains his passion for provenance (原产地)."Britain has started to become really proud of the food it's producing. It has excellent organic farms, beautifully crafted cheeses, high-quality meats."However, the British don't have a history of exporting their foodstuffs, which makes it difficult for restaurants in Hong Kong to source authentic ingredients."We can get a lot of our ingredients once a week from the UK," Tamlyn explains. "But there is also pressure to buy local and save on food miles, which means we take our vegetables from the local markets, and there are a lot that work well with British staples."The Phoenix, in Mid-Levels, offers the widest interpretation of "British cuisine", while still trying to maintain its soul. The gastro-pub has existed in various locations in Hong Kong since 2002. Singaporean head chef Tommy Teh Kum Chai offers daily specials on a blackboard, rather than sticking to a menu. This enables him to reinterpret British cuisine depending on what is available in the local markets."We use a lot of ingredients that people wouldn't perhaps associate as British, but are pre-sented in a British way. Bell peppers stuffed with couscous, alongside ratatouille, is a very popular dish."Although the ingredients may not strike diners as being traditional, they can be found in dishes across Britain.Even the traditional chefs are aware of the need to adapt to local tastes and customs, while maintaining the Britishness of their cuisine.At Yorkshire Pudding, Hill says that his staff asks diners whether they would like to share their meals. Small dishes, shared meals and "mixing it up" is not something commonly done in Britain, but Yorkshire Pudding will bring full dishes to the table and offer individual plates for each diner. "That way, people still get the presentation of the dishes as they were designed, but can carve them up however they like," Hill says.This practice is also popular at The Pawn, although largely for rotisseries (烤肉馆),Tamlyn says. "Some tables will arrive on a Sunday, order a whole chicken and a shoulder of lamb or a baby pig, and just stay for hours enjoying everything we bring out for them."Some British traditions are too sacred (神圣的)to mess with, however, Tomes says. "I'd never change a full English breakfast."注意：此部分试题请在答题卡1上作答。

preparation and characterization

preparation and characterizationPreparation and CharacterizationPreparation and characterization are two important aspects of scientific research. Preparation refers to the process of obtaining and producing a material or sample,while characterization refers to the process of identifying and analyzing the properties of that material or sample. In this article, we will discuss the different steps involved in preparation and characterization.PreparationThe preparation step involves obtaining or producing the material or sample for study. Depending on the type ofmaterial or sample, different preparation methods may be used. For example, if the material is a chemical compound, it maybe synthesized in the laboratory using a specific reaction.On the other hand, if the material is a biological sample, it may need to be extracted from a tissue or fluid, and then purified.Once the material or sample has been prepared, it may need to be processed further for analysis. For example, ifthe material is a solid, it may need to be ground into fine particles to improve the surface area for further investigation. Alternatively, if the sample is a liquid or gas, it may need to be concentrated or diluted for proper analysis.CharacterizationOnce the sample or material has been prepared, it is ready for characterization. Characterization involvesidentifying the physical and chemical properties of the material or sample. This can be done using various analytical techniques such as microscopy, spectroscopy, and chromatography.Microscopy involves the use of a microscope to examine the physical structure of the material or sample at a microscopic level. This can provide information about the size, shape, and texture of the sample.Spectroscopy involves the use of various types of electromagnetic radiation to measure the energy levels and wavelengths of molecules in the sample. This can provide information about the chemical composition and molecular structure of the sample.Chromatography involves separating the various components of a sample based on their chemical properties using a chromatography column. This can provide information about the chemical composition and purity of the sample.ConclusionIn conclusion, preparation and characterization are important steps in scientific research that involve acquiring and analyzing a material or sample. Preparation involves the process of obtaining and producing the material or sample, while characterization involves identifying and analyzing the properties of that material or sample. Proper preparation and characterization are crucial for accurate scientific analysis and reliable results.。

Development and Characterization of a Cell Culture Manufacturing Process Using Quality

Development and Characterizationof a Cell Culture Manufacturing ProcessUsing Quality by Design (QbD)PrinciplesDaniel M.Marasco,Jinxin Gao,Kristi Grifﬁths,Christopher Froggatt,Tongtong Wang and Gan WeiAbstract The principles of quality by design (QbD)have been applied in cell culture manufacturing process development and characterization in the biotech industry.Here we share our approach and practice in developing and char-acterizing a cell culture manufacturing process using QbD principles for establishing a process control strategy.Process development and character-ization start with critical quality attribute identiﬁcation,followed by process parameter and incoming raw material risk assessment,design of experiment,and process parameter classiﬁcation,and conclude with a design space con-struction.Finally,a rational process control strategy is established and documented.Keywords Cell culture process characterization ÁCell culture process develop-ment ÁCell culture process scale-up ÁControl strategy ÁCritical quality attribute ÁDesign space ÁQuality by design ÁRisk assessment AbbreviationsQbDQuality by design QTPPQuality target product proﬁle CQACritical quality attributes DOEDesign of experiment CPMControl point matrix FMEA Failure modes and effects analysisD.M.Marasco (&)ÁJ.Gao ÁK.Grifﬁths ÁC.Froggatt ÁT.Wang ÁG.WeiBioproduct Research and Development,Lilly Research Laboratories,Eli Lilly and Company,Indianapolis,IN 46285,USAe-mail:marasco_daniel_m@Adv Biochem Eng Biotechnol (2014)139:93–121DOI:10.1007/10_2013_217ÓSpringer-Verlag Berlin Heidelberg 2013Published Online:5July 201394 D.M.Marasco et al. Contents1Introduction (94)2Development and Characterization of Cell Culture Manufacturing Process for Establishing a Process Control Strategy (96)2.1Construct CQA(s)Control Points Matrix (96)2.2Initial Process Parameter Risk Assessment (98)2.3Risk Mitigation/Initial Process Characterization Experiments (101)2.4Final Characterization Experiment (106)2.5FMEA Process Parameter Risk Assessment (106)2.6Classification of Process Parameters (108)2.7Process Excursion Studies (110)2.8Construction of the Design Space/Operating Space (110)2.9Cell Culture Process Control Strategy (110)3Case Study (111)3.1Construct CQA(s)Control Points Matrix (111)3.2Initial Process Parameter Risk Assessment (111)3.3Scale-Down Model (112)3.4Initial Process Characterization Experiments (112)3.5Final Process Characterization Experiment (115)3.6FMEA Process Parameter Risk Assessment (116)3.7Process Excursion Study (116)3.8Classification of Process Parameters (117)3.9Construction of Design Space (117)References (121)1IntroductionThe quality by design(QbD)concepts embodied in the International Conference on Harmonization(ICH)guidelines Q8(R2),Q9,Q10,and Q11have been applied to cell culture manufacturing process development and characterization[1–4].The January2011revised FDA Guidance for Industry,Process Validation:General Principles and Practices,integrates QbD principles into process validation prac-tices[5].These guidance documents outline the application of QbD principles in the lifecycle of a product from process design,process deﬁnition,and process characterization to process validation and continued process veriﬁcation.The expectation from regulatory agencies is that quality is designed or built into the product and its manufacturing process and quality cannot be adequately assured by testing[5].The beneﬁt of QbD is twofold:one is to provide a high level of assurance for product quality through lifecycle management of the product;the other is the potential forﬂexibility in the reporting responsibilities for movements within a registered design space[1].The implementation of QbD principles means product characteristics are designed and fully understood and their linkage to patient safety and clinical efﬁcacy is established,the interaction between critical product quality attributes and its manufacturing process are fully characterized,and control strategyDevelopment and Characterization of a Cell Culture Manufacturing Process95including design space is established to ensure that the manufacturing process is capable of consistently producing the product with the desired quality attributes [6,7].Figure1presents our approach in applying QbD principles to developing and characterizing a cell culture manufacturing process for establishing a process control strategy.Development of a cell culture manufacturing process control strategy starts from identifying drug substance critical quality attributes based on the quality target product proﬁle(QTTP).Critical quality attributes(CQAs)are identiﬁed through risk assessment that evaluates severity based on impact on patient safety and/or clinical efﬁcacy[8].The list of CQA(s)evolves during the development lifecycle.Then,a matrix is created to describe the interaction between critical quality attributes and process unit operations based on previous process development work,platform knowledge,literature information,andﬁrst principles.This control point matrix (CPM)visually indicates the origin,growth,reduction,or clearance of the quality attributes over the entire drug substance manufacturing process and demonstrates the process control points for each critical quality attribute.96 D.M.Marasco et al.Using the CPM as a guide,initial process parameter risk assessments are per-formed to evaluate the impact of process parameters and incoming raw materials systematically,within common cause variability,on critical product quality attributes.Process parameters are selected based on risk assessment for empirical evaluation using design of experiments(DOE)utilizing a qualiﬁed scale-down model.The purpose of the initial characterization study is to link process parameters to critical quality attributes.A resolution III or IV,fractional factional DOE is conducted depending on the number of parameters to be evaluated.Pro-cess parameters having statistically signiﬁcant impact on CQA(s)are selected for further study using response surface DOE.The functional relationships between these process parameters and CQA(s)are fully characterized.A secondary risk assessment,failure mode and effects analysis(FMEA),is performed during technology transfer to the commercial manufacturing site.Risks identiﬁed during the FMEA are further reduced or mitigated through process excursion and/or process challenge studies.Process parameters are classiﬁed as critical or noncritical postprocess charac-terization studies.The classiﬁcation is performed based on risk assessment and experimental results from process characterization studies.Based on risk assess-ments conducted throughout the development lifecycle,those process parameters assessed as not likely to affect CQAs are classiﬁed as noncritical.For process parameters evaluated in characterization studies,if a parameter is both statistically signiﬁcant and practically signiﬁcant in affecting CQA(s),it is classiﬁed as critical. Otherwise,it is classiﬁed as noncritical.A design space/operating space is constructed post parameter classiﬁcation.Per ICH Q8,design space is the multidimensional combination and interaction of input variables(e.g.,material attributes)and process parameters that have been dem-onstrated to provide assurance of quality.A cell culture process control strategy is established and documented based on information generated through risk assessments and process characterization studies during the development lifecycle.The establishment of analytical control strategy and microbiological control strategy is beyond the scope of this chapter.In the next sections,we describe our practices for process parameter risk assessments,CQA-driven process characterization by design of experiment,pro-cess parameter classiﬁcation,design space/operating space construction,and process control strategy establishment.2Development and Characterization of Cell Culture Manufacturing Process for Establishing a ProcessControl StrategyThe process development lifecycle consists of process design,process deﬁnition, process characterization,process validation,and continued process veriﬁcation.Development and Characterization of a Cell Culture Manufacturing Process97Table1Control points matrix describing the probable quality attribute control pointsCritical quality attribute Analytical method Unit operation inﬂuencing CQA(s)12345…N CQA#1OCQA#2OCQA#3O:XCQA#4O l X; CQA#5O X;O Origin of attribute at this unit operation:Growth of attribute at this unit operation;Reduction of attribute at this unit operationl Potential for growth or reduction of attribute at this unit operationX Signiﬁcant reduction/clearance of attribute at this unit operationAfter deﬁnition of an initial baseline process,characterization studies are initiated to understand fully the impact of process parameters and incoming raw material attributes,within common cause variability,on critical quality attributes.Process characterization starts with risk assessment.The intention of the initial risk assessment is systematically to evaluate the potential risk of process parameters and incoming raw material attributes from each unit operation,within common cause variability,on critical quality attributes.A cause and effect methodology is utilized in the initial risk assessment.2.1Construct CQA(s)Control Points MatrixPrior to initializing process characterization,sufﬁcient information should be available to describe,or reasonably estimate,the relationship between the unit operations and critical quality attributes.In order to facilitate the initial cause-and-effect risk assessment,a unit operation-based,control points matrix(CPM),is created to describe the probable control points(one or many)for each critical quality attribute.The matrix should include the most likely origin,growth, reduction,or clearance of the critical quality attributes across the entire drug substance manufacturing process.An example of a unit operation-based control point matrix is displayed in Table1.The control points matrix is used to guide the process parameter risk assessment by allowing unit operation characterization studies to focus only on the relevant critical quality attributes that are signiﬁcantly inﬂuenced by the purpose or design intent of the unit operation.The control points matrix is updated as additional information becomes available.2.2Initial Process Parameter Risk AssessmentInitial process parameter risk assessments are based on process knowledge,that is, a combination of practical experience and theoretical understanding.The process parameter risk assessment is performed iteratively throughout the development lifecycle to prioritize development efforts.Depending upon an organization’s experience and relative level of comfort conducting these risk assessments,they may be performed by a subject matter expert,or by a cross-functional team.Per ICH Q6,the degree of rigor and formality of quality risk management should reﬂect available knowledge and be commensurate with the complexity and/or criticality of the issue to be addressed.The initial process parameter risk assessment is performed in four basic steps: (1)identify output,(2)identify input process parameters,(3)evaluate the probablerisks,and(4)rank the process parameters by riskscore.The results from the risk assessment guide and prioritize the experimental program used to characterize each unit operation of the cell culture manufacturing process.2.2.1Identiﬁcation of OutputsCritical quality attributes are the main output analyzed in the initial process parameter risk assessment.Process performance indicators may also be considered.2.2.2Identiﬁcation of Input Process ParametersThe inputs,or process parameters,are identiﬁed based on the operational knowledge and mechanistic understanding of each unit operation in the manu-facturing process.A cause and effect diagram is a useful tool to organize and group process parameters systematically by function.The cause-and-effect diagram is constructed by placing the output(i.e.,product and process attributes of interest)at the right side of the diagram,with the potential design factors(i.e.,process parameters and incoming raw material attributes,e.g.,concentration accuracy)on a series of branches and subbranches extending from the output axis.The process parameters can be grouped by function or process step to ensure no process parameters are overlooked.98 D.M.Marasco et al.The level of branching can be moderated to facilitate efﬁcient communica-tion to ensure the level of detail is appropriate.An example cause-and-effect diagram describing a typical production bioreactor process is given in Fig.2[9].2.2.3Risk AnalysisAfter identifying the relevant process outputs (CQAs)and process inputs (process parameters)for each unit operation,the risks of common cause variability in the input parameters that may affect the output parameters are assessed.The risk analysis is based on ﬁrst principles,literature information,platform knowledge,manufacturing experience,scientiﬁc judgment of the subject matter experts,and molecule-speciﬁc empirical knowledge.The process parameters can be classiﬁed into two groups:those that have the potential to affect critical quality attributes and those that do not.Process parameters that do not have the potential to affect critical quality attributes may be assigned a low risk score.Typically,low-risk process parameters are not formally studied in laboratory models or designed experiments and are classiﬁedas Development and Characterization of a Cell Culture Manufacturing Process 99100 D.M.Marasco et al.noncritical with appropriate rationales.The remaining process parameters are classiﬁed as high risk,thus,they may have the potential to affect critical quality attributes and require additional evaluation to better understand,reduce,or miti-gate risks.The process parameter risk assessment follows the logic diagram pre-sented in Fig.3.The initial process parameter risk assessment is an integral part of the development of a control strategy;therefore,this assessment should be ade-quately documented.2.2.4Raw Material Risk AssessmentThe risks of variability inherent to the cell culture raw materials used to manu-facture drug substances on CQA(s)are evaluated in the development lifecycle.The raw material components are analyzed to assess the intrinsic risk(use of the correct raw materials)and the extrinsic risk(lot-to-lot variability)on CQA(s)and other quality attributes.The assessment includes the risks introduced from a quality,technical,and procurement perspective.The initial risk assessment occurs prior to the manufacture of pivotal clinical materials,and is reassessed as the process evolves.For example,technology transfer and/or changes in the process or supply chain may initiate a reassessment.The evaluation of raw material risk utilizes a series of weighted risk elements based on their criticality to the product or process,and the risk to the patient.Each raw material is assigned a three-tiered risk score(low=1,medium=3,or high=5)for each risk element using a combination of platform knowledge, manufacturing experience,opinions of the subject matter experts,and molecule-Development and Characterization of a Cell Culture Manufacturing Process101 speciﬁc empirical knowledge.The summation of the individual risk scores mul-tiplied by the risk element weight is calculated for each component.These values are used to rank the relative risks for each raw material component.As an example,the risk elements,and their respective weights,are described in table.Description of risk elementsWeight=5•Variability has the potential to affect the drug substance quality attributes•Ability of raw material to introduce bioburden,endotoxin,viral contaminates•Known issues with raw materialWeight=3•Molecular complexity•Potential to affect process performanceWeight=1•Experience with vendor•Manufactured for pharmaceutical industry2.3Risk Mitigation/Initial Process CharacterizationExperimentsFollowing the identiﬁcation of high-risk process parameters and raw materials,an experimental program is designed to characterize and mitigate the risks of iden-tiﬁed process parameters on critical quality attributes within common cause variability.2.3.1Experimental StrategyThe experimental program is designed to characterize the manufacturing process to ensure consistent robust manufacturing capability.The high-risk process parameters are studied in a series of designed experiments intended to understand and mitigate potential risks further.Scale-independent process parameters are explored using a laboratory scale-down model.Scale-dependent parameters may be studied using intermediate or at-scale bioreactors.The experimental program is typically initialized utilizing a highly leveraged design of experiments of a resolution sufﬁcient to identify the main effects and some quadratic effects.Depending upon the number of relevant process parameters identiﬁed in the risk assessment process,a single or a series of screening exper-iments can be planned.Multivariate fractional factorial design of experiments of resolution III or IV run using one or several blocks are common.Based on the output from the screening experiment,additional studies may be performed to102 D.M.Marasco et al. characterize parameters further that have a statistically and practically signiﬁcant effect on critical quality attributes.Prior to designing experiments,the high-risk process parameters should be examined while acknowledging that not all process parameters are independent of each other(i.e.,medium strength and medium osmolality).Potential correlations should be identiﬁed and taken into consideration.2.3.2Process Parameter Range of InterestDuring cell culture manufacturing process characterization studies,the target setpoints of process parameters are determined based on process design and def-inition experimentation;process parameter ranges selected are intended to eval-uate the impact of common cause variability in operations on critical quality mon cause variability is deﬁned as the expected level of variability experienced during normal unit operations in a manufacturing environment when executed according to the batch record instructions.The range of interest is determined from the current understanding of the at-scale control capability using a combination of operational variability,or the variance from target setpoints,and the measurement uncertainty of the device(s) that record the process measurement.Theoperationalvariabilityisameasureofperformancederived fromsampling unit operations in the clinical manufacturing or commercial manufacturing facilities.The range encompassing common cause variability is chosen so that the probability of the parameter values being within the range of the target setpoints±operational vari-ability is at least0.995(or99.5%).Generally,six times the operational variability is selectedtoensurethatthevaluesofagivenprocessparameterwillfallwithinthisrange irrespective of the underlying distribution[10].The measurement uncertainty characterizes the dispersion of the values that could be reasonably attributed to the measurement.The measurement uncertainty is designed to reduce the false acceptance rate and is selected to ensure95%of the recorded measurements fall within the desired range.The measurement uncer-tainty is derived from either the measurement system design speciﬁcation or historic calibration performance[11].The summation of operational variability(containing99.5%of the observed values)and measurement uncertainty(containing95%of the recorded measure-ments)deﬁnes the recommended minimum range of interest used to characterize the process,as displayed in Fig.4.2.3.3Laboratory Scale Models for Process CharacterizationIn most scenarios,performing process characterization studies at the manufac-turing scale is not practically feasible due to the cost of operation,and limited availability of large-scale bioreactors.Therefore,laboratory scale models are usedto perform process characterization experiments that deﬁne acceptable process ranges and establish predictive relationships between the scale-independent pro-cess parameters and critical product quality attributes.This approach is in align-ment with ICH guidance [4];small–scale models can be developed and used to support process development studies.The development of a model should account for scale effects and be representative of the proposed commercial process.A scientiﬁcally justiﬁed model can enable a prediction of product quality,and can be used to support the extrapolation of operating conditions across multiple scales and equipment.The cell culture manufacturing process includes a series of shake ﬂasks and conventional stirred-tank or disposable bioreactors to manufacture the unprocessed bulk drug substance.The culture expansion steps have a limited potential for impact on critical quality attributes due to negligible accumulation of product;therefore the focus of the scale-down model is typically on the production bio-reactor unit operation.The bioreactor conﬁguration has ﬁve primary control loops intended to measure and control culture temperature,dissolved oxygen,culture pH,agitation rate,and vessel pressure by manipulating caustic and acidic pH control loops,air,oxygen,and carbon dioxide gas ﬂow rates,vessel jacket heat exchanger,and the agitator drive.An example P&ID (piping and instrumentation diagram)is provided in Fig.5.The cell culture process parameters can be separated into two groups including scale-dependent and scale-independent parameters.The operating conditions for scale-independent parameters (i.e.,temperature,pH,dissolved oxygen concen-tration)are conserved across different scales.The scale-dependent parameters (i.e.,agitation rate,gas ﬂow rates,nutrient addition volume)are adjusted to conform to the scaling strategy employed.The scale-dependent parameters included in a bioreactor system are driven by gas–liquid and liquid–liquid mixing with the associated mass and heat transport phenomena.Mixing systems do not scale proportionally in all dimensions;therefore a basis for scaling up mixing unit operations must be chosen by bal-ancing the characteristics that are important to the process under consideration.Scaling strategies are typically based on a combination of geometric similarity,kinematic similarity,dynamic similarity,and/or power per unit volume input.TargetVariability6σr 2σmu2σmu 6σrTypically two of the four methods are selected,allowing the other characteristics to change.Bioreactor unit operations used for mammalian cell culture processes are usually scaled up by conserving the power per unit volume with geometrically similar vessels.When scaling up on the basis of geometric similarity and constant power per unit volume,the relative agitator tip speed and the bulk mixing time increase.Increasing the agitator tip speed may increase the risk of shear damage to the cells;however,prior experiments have demonstrated that the risk of damage is minimal over the normal operating range of interest.Increasing the bulk mixing time will result in an increased risk of vessel heterogeneity which could affect the product’s critical quality attributes and process performance.Equipment design and addi-tional experiments should be considered if there is a high risk of vessel hetero-geneity affecting culture performance or critical quality attributes.In cell culture processes the proper scaling of gas ﬂow rates to control dissolved carbon dioxide and dissolved oxygen levels is not trivial.As the process is scaled up,the mass transport of oxygen increases with vessel volume leading to a decreased volumetric ﬂow rate of oxygen necessary to meet the culture demand.The resulting decrease in volumetric ﬂow rate reduces the capability to remove carbon dioxide.An air balance is required in the sparger line to provide a sufﬁcient volumetric ﬂow for carbon dioxide removal.In addition,the medium chemistry and the proﬁle of metabolic by-products (i.e.,lactate concentration)may lead to a feedforward control strategy based on the interaction between dissolvedoxygen Fig.5Example bioreactor piping and instrumentation diagramand pH control loops.In our system,the gas sparger conﬁguration may be spec-iﬁed so that the amount of gasﬂow needed to maintain the dissolved oxygen control is the amount of gas needed for carbon dioxide removal.The carbon dioxide management in the at-scale and intermediate-scale bioreactors may be determined through process models that simultaneously solve the chemistry equilibrium and mass transfer equations through the course of the run assuming that the oxygen uptake rate and signiﬁcant metabolic by-products are deﬁned by the process conditions.The models are used to deﬁne a target airﬂow rate that allows for carbon dioxide off-gassing.The interaction between multiple scale-dependent control loops presents additional challenges when scaling down cell culture processes to the laboratory bench scale.The power per unit volume is difﬁcult to determine as the standard vessel geometry is modiﬁed to accommodate the reduced scale.In addition,the ratio between culture volume and surface area in contact with the head space increases,inﬂuencing the mass transfer rates for gases.As a result controlling the pCO2concentration at the laboratory scale is difﬁcult to model.Additional experiments may be performed to understand the risks better that elevated carbon dioxide levels have on culture performance and/or product critical quality attributes.The capabilities of the laboratory scale models are monitored throughout the development lifecycle and the risk,whether the scale-down models are repre-sentative of at-scale processes,is analyzed as sufﬁcient large-scale information becomes available.The laboratory-scale models are analyzed by comparing results between the scale-down and at-scale processes for outcomes including critical quality attributes,other product quality attributes,and process performance indicators.The scale comparison data for quality attributes are explored using statistical methods.The data from bioreactors run at process targets in the scale-down model (from process characterization and process design and deﬁnition studies)are compared to the data generated from at-scale clinical material manufacturing campaigns.An equivalence test(two-one-sided t test,TOST)with a predeﬁned practical difference is used to test for equivalency between critical and other product quality attributes[12].A practical difference threshold should be sufﬁcient to support the claims,or intended use of the scale-down model.Based on these criteria,the suitability of the scale-down model relative to the at-scale process can be assessed.The process performance indicators are also explored qualitatively by exam-ining the process trends over parisons are made relative to the direc-tionality and closeness of the time-series data.If the performance of the scale-down model is not equivalent,additional analysis should be performed to determine if the process characterization results are sufﬁcient to construct an adequate control strategy.If not,additional work should be performed to develop a better model,or generate additional data to mitigate risks.。

Preparation and Characterization of Component Materials for Intermediate Temperature Solid Oxide

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Novel Strategies for Preparation and Characterization of Functional Polymer-metal Nanocomposites

A r ticle ID:042727104(2007)0520630202R 22B y D N M ,,2D M @N ovel Strategies for Preparation and Character izationof Functional Polymer 2metal N anocomposites f orElectr ochemical ApplicationsD.N.Muravie v(Depa rt ment of Chemist ry ,Autonomous U niv ersity of Ba rcelona ,08193Bellater ra ,Ba rcelona ,Spain)K eyw o r ds :n ov el strategies;polymer 2metal ;nan ocompositesCLC number :O 636.9 1　IntroductionThe synt hesi s and charact erization of Metal Nano Part icles (MN Ps)has at tracted great i nterest of scien 2ti st s and technologist s wit hi n t he last years due t heir unique physical and chemical properties ,which substan 2tially differ f rom t hose of bot h bul k material and si ngle atoms.These properties provide various pract ical appli 2cations of MNPs i ncluding cat alysi s 2and elect rocatalysi s 2based processes ,which occur in ,for example ,f uel cells of different types or in various sensing devices (e.g.amperomet ric sensors and biosensors ).The main draw 2back ,which still li mit s t heir wide applicat ions ,is i nsufficient stabilit y of MN Ps deali ng wit h t hei r high t rend to aggregate.Coalescence of MNPs result s in t he loss of t heir nanomet ric size and s pecial properties.St abilization of MNPs i n polymeric mat rices of different t ypes has been proven to be one of t he most promi sing strategies to prevent t hei r aggregat ion and to save t hei r properties [1].The Polymer 2Stabilized MNPs (PSMN Ps )and t he polymer 2metal nanocomposite materials on t heir base start to fi nd wide applications in various fields of science and technology.2　ResultsIn t his com munication we demonst rat e t hat Metal 2Polymer Nanocomposite Membranes (MPNCMs )con 2t aining MN Ps can be easily prepared in a Sulfonat ed PolyEt herEt her K etone (SP EE K )polymeric mat ri x by using t he polymeric membranes as nanoreactors to bot h synthesize and to characterize t he composition and ar 2chi tect ure of t he formed MN Ps [2-3].Metal ions (or met al ion c omplexes )are fi rst i nc orporated in the poly 2meric matrix where t hey undergo a reduct ion reaction t hat leads to t he formation of corresponding MPNCMs.Since t his t echnique allows for carrying out successive met al loading 2reduction cycles ,it permit s to synt hesize bot h monometallic and bi metallic (e.g.,core 2shell )MNPs.The proposed approach i s illust rated by t he resul ts obt ained by t he synt hesi s and characterization of MPNCMs containing Pt ,Pd (monometallic )and Pt @Cu and Pd @Cu (core 2shell )PSMN Ps along wit h t hei r application i n electrochemical sensor and biosens or const ruc 2tions.第46卷　第5期2007年10月复旦学报(自然科学版)Journal of Fudan U niversity (Natural Science )Vol.46No.5Oct.2007eceived da te :20070704iogra ph :..uraviev Corres ponde nce author E mail :imit ri.uraviev ua b.es.References:[1]　A D Pomogailo,G I Dzhardimalieva,A S Rozenberg,et al.K inetics a nd mechanism of in situ simultaneous forma2tion of metal nan oparticles in stabilizing polymer Matrix[J].J Na nopar ticle Res,2003,5:4972519.[2]　D N Muraviev.Intermatrix synthesis of polymer2stabilized metal nano2par ticles for sens or applications[J].Con2tributions to Science,2005,3(1),17230.[3]　D N Muraviev,J Maca nás,M Farre,et al.Novel routes for inter2matrix synt hesis and c haracterization of polymerstabilized metal nano2particles for molecular recognition devices[J].Sensors&Act uators,2006,118(122):4082 417.(C ont inued from page629)References:[1]　Valeeva I L,Lachinov A N.Synth Met,1993,57(1):4115.[2]　Z hereb ov A Yu,Lachin ov A N.Synth Metals,1991,44:99.[3]　Lachinov A N,Z hereb ov A Yu,K or nilov V M.Pisma v J ET F,1990,52(2):742.[4]　Vorobieva N V,Lac hinov A N,Loginov B A.Surf ace,2006,5:22.[5]　K ornilov V M,Lachin ov A N.J ET F,1997,111(4):1513.[6]　Lachinov A N,Z hereb ov A Yu,et al.Synt h Metals,1993,59:377.[7]　K ornilov V M,Lachin ov A N.Pisma v J E T F,1995,61(6):504.[8]　Lachinov A N,K ornilov V M,et a l.J SID,2004,12(2):149.[9]　Ionov I N,Lachinov A N,Rench R.Pisma v J TF,2002,28(14):69.[10]　K ornilov V M,Lac hinov A N.Mic ro system Technique,2003,3:78.[11]　K ornilov V M,Lac hinov A N.Phy sics of Low2Dimen sional Str ucture s,2004,1/2:145.[12]　Lachinov A N,K ornilov V M,et al.J ET P,2006,102(4):640.1 36　第5期 D.N.Muraviev:Novel Strategies for Prepa ration and Characterization of Functional… 。

英文文献翻译

Preparation and characterization of Ag-TiO2 hybrid clusters powders[1]（Ag-TiO2混合团簇粉末的制备和表征）Abstract：液相电弧放电法被用于制备纳米Ag-TiO2复合超细粉末。

XRD和TEM图表明颗粒呈葫芦状形态，分布狭窄。

我们讨论了实验条件对产品的影响，比较了这种方法制备的粉末和其他γ射线辐照法制备的粉末。

Introduction：材料合成技术，提高了研究特定电子和光学特性的能力。

这也导致了设备和不同效应的快速发展，如集成光学型偏振器[1]和量子霍耳效应。

所需的长度尺度对于这些结构的控制是在纳米级别的[ 2 ]。

科学家面临的一个新的挑战是半导体量子点的生长，它具有新的光学响应，引起了对其基础物理方面和三阶非线性光致发光的应用等的研究兴趣。

这方面的一个例子是Ag-TiO2复合材料通过胶体方法合成[ 3 ]或由γ射线辐照法合成[ 4 ]。

对比其他制备超细金属颗粒的方法，γ射线辐照法能在室温的环境压力下产生粉末。

在这封信中，我们开发了一种新的方法，即液相电弧放电法，用以制备纳米复合材料，当它经水热处理可以得到纳米级别的超细粉。

Preparation and photocatalytic activity of immobilized composite photocatalyst (titania nanoparticle/activated carbon)[2]固定化复合光催化剂（TiO2纳米颗粒/活性炭）的制备和光催化活性研究Abstract：制备了一种固定化复合光催化剂——TiO2纳米颗粒/活性炭（AC），并研究了它在降解纺织染料的光催化活性。

AC通过油菜籽壳制备。

碱性红18（BR18）和碱性红46（BR46）被用来作为模型染料。

并采用了傅里叶变换红外（FTIR），波长色散X射线光谱（WDX），扫描电子显微镜（SEM），紫外可见分光光度法，化学需氧量（COD）和离子色谱（IC）分析。

文献检索第三章CA

Benzoic acid 苯甲酸 Benzylacetate 乙酸苄酯 Benzylalcohol 苯甲醇 Bromo-2ˊ-deoxyuridine 5-Br-2’-脱氧尿苷 Bromocinnamic acid 2-溴代肉桂酸 Bromocinnamic acid 3-溴代肉桂酸 Bromocinnamic acid 4-溴代肉桂酸 Bromomethyl-7-methoxycoumain 4-溴甲基-7-甲氧基香豆素 Butyl-3-methylpyrazine 2-丁基-3-甲基吡嗪
。
CA报道内容几乎涉及化学家和药物学家感兴趣的所有领域，其中除包括无机化学、有机化学、分析化学、物理化学、高分子化学外，还包括冶金学、地球化学、辐射化学、药物学、毒物学、环境化学、生物学以及物理学等很多学科领域。根据统计，CA摘录了世界化学文献的 98%。CA以其历史悠久，收录内容广泛，索引齐备，被世界公认为最完美的化学化工文献检索工具和最有权威的文摘刊物之一。
美国化学文摘 Chemical Abstracts
•常见的动词
Preparate(制备） Synthesis（合成）
Manufacture(制造）
Analysis（分析） Assay（测定） determine （测定） Stir（搅拌） Crystallize（结晶）
Recrystallize（重结晶） Evapo萃取) Separate(分离) purify (提纯）
Farnesol 法呢醇/金合欢醇 Fenchyl alcohol 葑醇 Ferulic acid ethylester 阿魏酸乙酯 Ferulic acid methylester 阿魏酸甲酯 Flavanone diacetyl hydrazone 黄烷酮二乙酰腙 Flavanone hydrazone 黄烷酮腙 Flavanone 黄烷酮 Flavone 黄酮

Preparation and Characterization of PVC Based Form Stable Phase Change Materials

Preparation and characterization of PVC-based form-stable phasechange materialsXiaoming Jin a,1,Jianli Li b,2,Ping Xue a,n,Mingyin Jia a,na Beijing University of Chemical Technology,Beijing100029,Chinab Beijing Institute of Petrochemical Technology,Beijing102617,Chinaa r t i c l e i n f oArticle history:Received25March2014Received in revised form2July2014Accepted10July2014Keywords:Form-stable phase change materialsMicrocapsulesPVCHDPEWoodﬂoura b s t r a c tSeveral different form-stable phase change materials(FSPCMs),which comprise microencapsulatedphase change materials(MEPCMs)as latent heat storage medium and poly(vinyl chloride)/woodﬂourcompound as supporting material,were prepared by blending and compression molding method for theapplication of potential latent heat thermal energy storage(LHTES).SEM images reveal that preparedFSPCMs have homogeneous constitution and most MEPCM particles in them are undamaged.Both theshell of MEPCM and the matrix prevent the molten parafﬁn from leakage.DSC results show that meltingand freezing temperatures as well as latent heats of the prepared FSPCMs are suitable for potentialLHTES applications.Results of thermal performance tests show that the surface temperatureﬂuctuationof the FSPCMs is obviously less than that of plate without MEPCM.Results of mechanical property testsindicate that bending strength,and bending modulus,as well as tensile and impact strength of theFSPCMs with10wt%MEPCM are26.7MPa,3.48GPa,9.8MPa and10kJ mÀ2respectively.The mechan-ical properties of FSPCMs with poly(vinyl chloride)/woodﬂour compound as supporting material aresuperior to those of HDPE-based FSPCMs.&2014Elsevier B.V.All rights reserved.1.IntroductionThe building and industrial sectors are the leading energyconsumers around the world taking up28%of the overall energyconsumption[1].Forecast made by the EIA suggests that energyuse in the building environment will grow by34%in the next20years[2].Thermal energy storage acts as a useful tool for improv-ing energy efﬁciency and energy savings.It bridges time gapbetween energy requirement and energy supply[3].Latent heatstorage is the most attractive thermal energy storage techniquebecause of high storage density and small temperature changefrom storage to retrieval.In latent heat thermal energy storage(LHTES)systems,energy is stored during melting and recoveredduring freezing of a phase change material(PCM)[4,5].A great variety of inorganic and organic solid–liquid PCMs andtheir mixtures have been studied as latent heat storage materials[2,6–10].Among different solid–liquid PCMs,parafﬁn has manymerits such as large latent heat,little or no super-cooling,lowvapor pressure,good thermal and chemical stability and self-nucleating behavior[11,12].So,parafﬁn is found to be widely usedfor LHTES applications.The major difﬁculty for solid–liquid PCMs'wide applications is the leakage of the molten PCMs.Becauseform-stable phase change materials(FSPCMs)can be directly usedwithout encapsulation,they are more feasible than solid–liquidPCMs for practical applications[11].The FSPCMs can be preparedby impregnating PCMs into polymer-based materials through meltblending or vacuum impregnation.Many scholars have prepareddifferent FSPCMs with various structure supporting componentssuch as high-density polyethylene(HDPE)[13–16],polypropylene(PP)[17,18],styrene–butadiene–styrene(SBS)triblock copolymer[19,20],acrylic resins[21,22],poly(methyl methacrylate)(PMMA)[23,24]and polyurethane block copolymer[8,25,26].However,justa few researchers employ poly(vinyl chloride)(PVC)as supportingmaterial to prepare FSPCMs.Feldman et al.[27]researched thepossibility of developing FSPCMs on the basis of PVC and fattyacids.Sarıand Kaygusuz[28]studied the compositions of differentfatty acids as PCMs and PVC as supporting material.Though polymers or porous materials have been employed assupporting material to package solid–liquid PCMs,the leakageproblem of liquid PCMs is not well solved.Zhang et al.[29]pointedout that parafﬁn in polymer-based FSPCMs tends to diffuse ontosurface and is lost little by little,which results in some problemssuch as leakage,frosting and quick deterioration of thermalproperties.Recently,microencapsulated phase change materials(MEPCMs)have been added into different supporting materials toovercome the leakage problem of liquid PCMs.Schossig et al.[30]Contents lists available at ScienceDirectjournal homepage:/locate/solmatSolar Energy Materials&Solar Cells/10.1016/j.solmat.2014.07.0130927-0248/&2014Elsevier B.V.All rightsreserved.n Corresponding authors.Tel.:þ861064436016.E-mail addresses:buct_nme@(X.Jin),lijianli1979@(J.Li),xueping@(P.Xue),jiamy@(M.Jia).1Tel.:þ861064426911.2Tel.:þ861081292115.Solar Energy Materials&Solar Cells130(2014)435–441integrated MEPCM into plaster and performed building simula-tions and material testing to identify reasonable applications of PCM/plaster compound.Mehling et al.[31]also introduced MEPCM into gypsum for lightweight buildings.Considering leak-age and lipophilicity of parafﬁn,Liu et al.[32]encapsulated FSPCMs with80wt%parafﬁn and20wt%HDPE into silica gel polymer by in situ polymerization.In our previous studies[33]a series of FSPCMs,which comprised MEPCM as the latent heat storage medium and HDPE/woodﬂour composite as the matrix, were prepared by blending and compression molding method.The shell of MEPCM prevents parafﬁn from dispersion in the support-ing material,reduces evaporation and reaction of parafﬁn with the outside environment,provides an increased heat-transfer area and a constant volume,and allows an easy application without affect-ing properties of parent material.Like HDPE/woodﬂour compo-sites,PVC/woodﬂour compound is also a popular wood–plastic composite.However,adding MEPCM into PVC/woodﬂour com-pound to prepare FSPCMs has not been investigated.In this study,PVC-based FSPCMs with MEPCM as latent heat storage medium and PVC/woodﬂour composite as the matrix were prepared by the compression molding method.The micro-structures and thermal properties of the prepared FSPCMs were studied with SEM and DSC techniques respectively.The phase change behaviors of the FSPCMs under the condition which was different from that of the DSC method were studied by melting and freezing performance test.Furthermore,the mechanical properties of the FSPCMs were measured to clarify of whether they have desirable mechanical properties for practical applications.2.Preparation of polymer-based FSPCMs2.1.MaterialsMEPCM(average particle size:7.29μm,as shown in Fig.2;bulk density:900kg mÀ3)was supplied by Beijing Julongbofang Science and Technology Research Institute.PVC(DG-1000K)was provided by Tianjin Dagu Chemical Co.,Ltd.HDPE(melting index: 0.20–0.40g(10min)À1,bulk density:942–950kg mÀ3)was pro-vided by No.2Assistant Reagent Factory of Beijing Oriental Petroleum and Chemical Industry Co.,Ltd.Lead composite stabi-lizer,used as the thermal stabilizer for PVC,was provided by Beijing Daxing Guangming Chemical Factory.Woodﬂour(average particle size:350μm)was obtained from Hebei Xinglong Wood Flour Factory.Silane coupling agent(KH-560)and titanate ester were used to improve interface afﬁnity between woodﬂour and resin.Chlorinated polyethylene(CPE)was used to enhanceﬂame retardancy,improve the mechanical properties and reduce costs. Calcium carbonate was used as aﬁller to improve shape stability of the composite and reduce costs.Acrylic copolymer(ACR)is used as the impact modiﬁer to improve the anti-impact property of PVC.2.2.Pretreatment of woodﬂourFirst,weighed woodﬂour was dried for1h in a drying oven (SFG-02.400,China)at1051C,and was mixed for2min at a low speed.Then titanate ester was sprayed into the blend system and stirred for5min at a high speed.2.3.Preparation of PVC-based FSPCMsFirst,premix A was obtained by modifying calcium carbonate with1%KH-560and premix B was obtained by adding6%Lead composite stabilizer into PVC,both of which were mixed in a high-speed mixer(SHR-50A model,China).Then the PVC mixture was obtained by mixing2.6wt%premix A,69.7wt%premix B,19.7wt% dried woodﬂour,5.9wt%CPE and2.0wt%ACR in the high-speed mixer for3min at alow speed(50r minÀ1).Theﬁnal material used to prepare the PVC-based FSPCMs are obtained by mixing PVC mixture and MEPCM in the high-speed mixer for10min at a high speed(200r minÀ1)according to the formulation in Table1.Finally,a certain number of materials were put into the mold (200mmÂ200mmÂ35mm)and the product was shaped via a hot-press machine(M3-500Â500model,China)at a temperature ranging from1701C to1801C and pressure ranging from5MPa to 7MPa.2.4.Preparation of HDPE-based FSPCMsTheﬁnal material for preparing the HDPE-based FSPCM(No. 7in Fig.1)was obtained by mixing25wt.%dried woodﬂour,50wt %HDPE and25wt%MEPCM for5min at high speed(200r minÀ1) in the high-speed mixer.Then some content ofﬁnal materials (about176g)was put into the mold andﬁnally the HDPE-based FSPCM with the thickness of$4mm was shaped via a hot-press machine at a temperature ranging from1501C to1701C with pressure ranging from8MPa to10MPa.Photos of the prepared PVC-based and HDPE-based FSPCMs are shown in Fig.1.2.5.Characterization of polymer-based FSPCMsSEM(Hitach S-4700,Hitach Co.,Japan)was used to analyze the microstructures of MEPCM and FSPCMs.DSC(NETZSCH DSC204 F1,Germany)was employed to measure the phase transition temperature and latent heat.A universal material testing machine (INSTRON1185,USA)was employed to test bending strength and bending modulus as well as elongation at break of the FSPCMs according to China national standards of GB1042-79.Thermal performance test was performed by using the drying oven and a refrigerator as heating and cooling devices respectively.The set temperatures of the drying oven and the refrigerator were301C andÀ101C,respectively.3.Results and discussion3.1.Microcosmic imagesSEM images of MEPCM and polymer-based FSPCMs are shown in Fig.2.It can be seen from Fig.2(a)that the MEPCM has smooth surface and uniform particle size distribution.Fig.2(b)and(c) shows that MEPCM particles are well dispersed in the prepared PVC-based and HDPE-based FSPCMs.From Fig.2,it can also be seen that most of MEPCM particles are intact though some of them are distorted or even ruptured.Table1Components of PVC-based FSPCMs.No.PVC mixture(wt%)MEPCM(wt%)Thickness of FSPCM plates(mm)11000.0 4.0295 5.0 4.039010.0 4.048515.0 4.058020.0 4.067525.0 4.0X.Jin et al./Solar Energy Materials&Solar Cells130(2014)435–441 4363.2.Phase-transition temperature and enthalpiesThe DSC results of MEPCM and FSPCMs are shown in Fig.3and Table 2.According to Fig.3,the difference of melting peak temperature between MEPCM and FSPCMs is less than 0.51C,and that of solidi ﬁcation peak temperature is 1.51C.It can be safely said the DSC curves of MEPCM and FSPCMs are very similar,which indicates that the shell materials of microcapsule provide good protection to the core materials and keep physical and chemical properties of MEPCM.The measured melting enthalpy value (15.92kJ kg À1)of the PVC-based FSPCM with 15wt%MEPCM is slightly lower than the calculated value,17.07kJ kg À1,which equals the sum of product of the latent heats of MEPCM and the mass fraction of MEPCM in FSPCMs.Similarly,the measured solidi ﬁcation enthalpy value is lower than the calculated value.The measured melting and solidi ﬁcation enthalpy values of the HDPE-based FSPCM with 25wt%MEPCM are 8.42%and 12.11%lower than corresponding calculated values,respectively.These differences stem from the rupture of a small part of MEPCM particles,which leads to the leakage of phase change materials.In a word,the prepared FSPCMs store thermal energy when the ambient temperature is greater than $101C and release latent heat of lower than $131C.According to estimates,the novel PVC-based FSPCM with 15wt%MEPCM can store more than 40kJ kg À1thermal energy at the temperature ranging between 5and151C.Consequently,the novel FSPCMs have potential for low-temperature thermal energy storage and self-regulation for environment temperature.For example,they can be used as energy storage module in mechanical ventilation system to store low-temperature heat at night for reducing air conditioning energy consumption during the daytime.3.3.Thermal performance testHeating and cooling curves of the PVC-based composite without MEPCM and FSPCM with 15wt%MEPCM are shown respectively in Figs.4and 5.In Figs.4and 5,the curve of surface temperature ﬂuctuation every 15s is used to characterize the temperature control effect of the FSPCMs.According to Figs.4and 5,the melting and freezing processes of PVC-based FSPCM occur respectively within the temperature range of 8–18.81C and 0.7–13.11C,which is in concordance with the DSC results.In Fig.4temperature remaining almost constant for $25min can be identi ﬁed on the heating curve of the PVC-based FSPCM sample with 15wt%MEPCM,and the value of relevant average temperature ﬂuctuation equals 0.1081C 15s À1while that of PVC-based composite with-out MEPCM is 0.2371C 15s À1.Similar situations can be found in Fig.5;that is temperature remaining almost constant for 24min can be determined on the cooling curve of the PVC-based FSPCM sample with 15wt%MEPCM,and the value of relevant average temperature ﬂuctuation equals 0.1271C 15s À1while that of PVC-based composite without MEPCM is 0.2231C 15s À1.The average temperature ﬂuctuation of the FSPCM with 15wt%MEPCM in the corresponding temperature range of 8–18.81C and 0.7–13.11C are respectively 54.4%and 43%lower than that of PVC-basedcompositeFig.1.Photos of FSPCMs.(1)PVC-based composite without MEPCM.(2)PVC-based FSPCM with 5wt%MEPCM.(3)PVC-based FSPCM with 10wt%MEPCM.(4)PVC-based FSPCM with 15wt%MEPCM.(5)PVC-based FSPCM with 20wt%MEPCM.(6)PVC-based FSPCM with 25wt%MEPCM.(7)HDPE-based FSPCM with 25wt%MEPCM.X.Jin et al./Solar Energy Materials &Solar Cells 130(2014)435–441437without MEPCM.Since the novel FSPCMs can be used to reduce the amplitude and frequency of temperature ﬂuctuation within the range of phase transition temperature,they have potential applica-tions in ﬁelds such as building air conditioning,greenhouse,storageand transportation of temperature-sensitive goods for temperature smoothness and regulation.3.4.Mechanical properties3.4.1.DensityThe relationship between the density of PVC-based FSPCMs and the mass fraction of MEPCM is shown in Fig.6.It can be seen that the density of PVC-based FSPCMs gradually decreases with increasing mass fraction of MEPCM as the density of MEPCM is lower than that of PVC/wood ﬂour composite.In contrast,the density of PVC-based FSPCMs is greater than that of HDPE-based FSPCMs (996.1kg m À3[33]).In China,common interior decoration plates such as shaving boards,solid wood panels,plasterboard and wood –plastic composite boards have very different densities.The density of shaving board is in the range of 750–1000kg m À3,that of solid wood panels changes from o 350kg m À3to 41130kg m À3,that of plasterboard is about 1000kg m À3and that of wood –plastic composite boards is 600–1400kg m À3.So the density of PVC-based FSPCMs is comparatively moderate during the practical application.3.4.2.Tensile strength,bending strength and modulusLarge test samples with the dimension of 120mm (length)Â15mm (width)Â9mm (thickness)were made for themeasurementFig.2.SEM images of MEPCM and Polymer-based FSPCM plates.(a)MEPCM Â800.(b)PVC-based FSPCM plates with 15wt%MEPCM Â2000.(c)HDPE-based FSPCM plates with 15wt%MEPCM Â4000.Fig. 3.DSC curves of MEPCM and FSPCM plates.(1)MEPCM.(2)HDPE-based FSPCM with 25wt%MEPCM.(3)PVC-based FSPCM with 15wt%MEPCM.X.Jin et al./Solar Energy Materials &Solar Cells 130(2014)435–441438of mechanical properties at 231Cand 70%relative humidity.The bearing width and loading speed for the test of bending strength and bending modulus at break of FSPCM samples were 100mm and 2mm min À1respectively.The test results are shown in Fig.7.It can be seen that (1)the mass fraction of MEPCM notably in ﬂuences the bending strength and modulus;(2)greater mass fraction of MEPCM leads to poorer bending strength and there is an almost constant region in the bending strength curve of PVC-based FSPCMs in the interval of 5–15wt%.In this region,bending strength changes between 25.7and27.8MPa and it is smaller than that of PVC-based composite without MEPCM (35.3MPa)by 21.7–27.0%.Compared with the bend-ing strength of HDPE-based FSPCMs with 25wt%MEPCM (14.38MPa [33]),the bending strength of PVC-based FSPCMs increases by 78.0%–93.3%.Thus,PVC is used as a base material to improve the bending strength dramatically;(3)the bending modulus of FSPCMs increases when the mass fraction of MEPCM increases from 0%to 10wt%whileit decreases at the other MEPCM fractions.When the mass fraction of MEPCM is 10wt%,the bending modulus is pared with the base materials'bending modulus (3.06GPa),the increase in amplitude is about 13.7%.The reason is that a fraction of MEPCM in PVC-based materials is dispersed homogenously,and MEPCM particles toughen the materials.The bending modulus of the plates decreases when the mass fraction of MEPCM exceeds 10wt%.The reason might be that small MEPCM particles coalesce with each other due to their large surface energy and the bending modulus is pared with the bending modulus of HDPE-based FSPCMs with 25wt%MEPCM (757.86MPa [33]),the bending modulus of PVC-based FSPCMs increases more than 3times;(4)the tensile strength of FSPCM is decreased with increasing mass fraction of MEPCM.Some reduction of MEPCM should be considered during the practical applicationTable 2DSC results of MEPCM and PVC-base FSPCMs.SampleMass (mg)Rates of heating/cooling (1C min À1)Melting process Solidifying processT s (1C)T p (1C)λ(kJ kg À1)T s /1C T p /1C λ(kJ kg À1)MEPCM5.211011.3815.14113.813.17 4.85114.1PVC-based FSPCM a 7.61011.3114.8115.92(17.07)c 12.32 6.2616.66(17.12)HDPE-based FSPCM b 5.61010.7014.6527.31(29.82)11.104.7226.27(29.89)Notes :a The mass fraction of MEPCM in the PVC-based FSPCMs is 15%.b The mass fraction of MEPCM in the HDPE-based FSPCMs is 25%.cThe numeral value in brackets equals the product of the latent heats of MEPCM and the mass fraction of MEPCM inFSPCMs.Fig.4.Heating curves of PVC-based composite without MEPCM and FSPCM with 15wt%MEPCM.Fig.5.Cooling curves of PVC-based composite without MEPCM and FSPCM with 15wt%MEPCM.Fig.6.Effect of MEPCM on the density of PVC-basedFSPCMs.Fig.7.Effect of MEPCM on the mechanical properties of PVC-based FSPCMs.X.Jin et al./Solar Energy Materials &Solar Cells 130(2014)435–441439where larger tensile strength is required.When the mass fraction of MEPCM increases from 0to 25wt%,the tensile strength of FSPCMs decreases by 52.1%.Furthermore,many other factors such as the fraction and particle size of wood ﬂour,the properties of coupling agent and processing parameter greatly in ﬂuence the mechanical properties of FSPCMs.3.4.3.Impact strengthThe effect of MEPCM on the impact strength of PVC-based FSPCMs is shown in Fig.8.When the mass fraction of MEPCM is 5wt%,the impact strength reaches 10.93kJ m Àpared with the impact strength of base materials (8.71kJ m À2),the relative increment is 25.5%.With more MEPCM,the impact strength decreases.When the mass fraction of MEPCM is 25wt%,it is down to 7.81kJ m À2and $10.3%less than that of the base materials.The reason which can be explained is that appropriate amount of MEPCM in PVC-based FSPCMs is dispersed homoge-nously and it toughens the materials.MEPCM particles begin to aggregate with the increase of MEPCM because of its large surface energy,which leads to the decline of the impact strength.Besides,it should be noted that the impact strength of rigid PVC ranges from 22to 108kJ m À2.Therefore,adding MEPCM to PVC/wood ﬂour composite will decrease the impact strength of PVC-based FSPCMs.4.ConclusionPreparation and properties (thermal and mechanical)charac-terization of ﬁve novel PVC-based FSPCMs are presented in this paper.Micrographs taken on SEM reveal that most MEPCM particles in the PVC-based FSPCMs are undamaged.Thus both the shell of MEPCM and the matrix prevent molten paraf ﬁn from leakage.DSC results show that the melting and freezing tempera-tures as well as latent heats of the novel FSPCMs are suitable for potential LHTES applications.Temperature remaining almost con-stant for $25min can be identi ﬁed in the heating and cooling curves of the PVC-based FSPCM sample with 15wt%MEPCM.And according to these curves,the average surface temperature ﬂuc-tuations every 15s of the FSPCM with 15wt%MEPCM in the corresponding temperature range of 8–18.81C and 0.7–13.11C are respectively 54.4%and 43%lower than that of PVC-based composite without MEPCM.So the novel FSPCMs can be used to reduce the amplitude and frequency of temperature ﬂuctuation within the range of phase transition temperature.The results of mechanical properties test indicate that the mechanical properties of PVC-basedFSPCM are better than that of HDPE-based FSPCM.The PVC-based FSPCM with 10wt%MEPCM has the best mechanical properties.Based on their proper phase change temperatures,outstanding leak tightness of molten paraf ﬁn,good temperature control feature and mechanical properties,the novel PVC-based FSPCMs have the potential for LHTES applications such as building air conditioning,greenhouse,storage and transportation of temperature-sensitive goods for temperature smoothness and regulation.AcknowledgmentsThe authors would like to acknowledge Beijing Excellent Talents Program (NO.2013D009016000002)for support.References[1]S.A.Memon,T.Y.Lo,S.Barbhuiya,W.Xu,Development of form-stablecomposite phase change material by incorporation of dodecyl alcohol into ground granulated blast furnace slag,Energy Build.62(2013)360–367.[2]Energy Information Administration,International Energy Outlook 2006,U.S.Department of Energy,2006.[3]B.Tang,M.Qiu,S.Zhang,Thermal conductivity enhancement of PEG/SiO 2composite PCM by in situ Cu doping,Sol.Energty Mater.Sol.Cells 105(2012)242–248.[4]A Sharma,V Tyagi,CR Chen,D.,Buddhi,Review on thermal energy storagewith phase change materials and applications,Renew.Sustain.Energy Rev.13(2)(2009)318–345.[5]MK Rathod,J,Banerjee,Thermal stability of phase change materials used inlatent heat energy storage systems:a review,Renew.Sustain.Energy Rev.18(2013)246–258.[6]B.Tang,C.Wu,M.Qiu,X.Zhang,S.Zhang,PEG/SiO 2–Al 2O 3hybrid form-stablephase change materials with enhanced thermal conductivity,Mater.Chem.Phys.(2014).[7]M.Silakhori,H.S.C.Metselaar,T.M.I.Mahlia,H.Fauzi,S.Baradaran,M.S.Naghavi,Palmitic acid/polypyrrole composites as form-stable phase change materials for thermal energy storage,Energy Convers.Manage.80(2014)491–497.[8]K.Chen,X.Yu,C.Tian,J.Wang,Preparation and characterization of form-stable paraf ﬁn/polyurethane composites as phase change materials for ther-mal energy storage,Energy Convers.Manage.77(2014)13–21.[9]J.Zeng,F.Zhu,S.Yu,Z.Xiao,W.Yan,S.Zheng,L.Zhang,L.Sun,Z.Cao,Myristicacid/polyaniline composites as form stable phase change materials for thermal energy storage,Sol.Energy Mater.Sol.Cells 114(2013)136–140.[10]M.Kenisarin,K.Mahkamov,Solar energy storage using phase changematerials,Renew.Sustain.Energy Rev.11(2007)1913–1965.[11]F.Chen,M.P.Wolcott,Miscibility studies of paraf ﬁn/polyethylene blends asform-stable phase change materials,Eur.Polym.J.(2013).[12]Y.Cai,Q.Wei,F.Huang,S.Lin,F.Chen,W.Gao,Thermal stability,latent heatand ﬂame retardant properties of the thermal energy storage phase change materials based on paraf ﬁn/high density polyethylene composites,Renew.Energy 34(2009)2117–2123.[13]W.Cheng,R.Zhang,K.Xie,N.Liu,J.Wang,Heat conduction enhanced shape-stabilized paraf ﬁn/HDPE composite PCMs by graphite addition:preparation and thermal properties,Sol.Energy Mater.Sol.Cells 94(2010)1636–1642.[14]K.Kaygusuz,A.Sari,High density polyethylene/paraf ﬁn composites as form-stable phase change material for thermal energy storage,Energy Sources Part A 29(2007)261–270.[15]Y.Hong,G.Xin-shi,Preparation of polyethylene –paraf ﬁn compound as a form-stable solid-liquid phase change material,Sol.Energy Mater.Sol.Cells 64(2000)37–44.[16]H.Inaba,P.Tu,Evaluation of thermophysical characteristics on shape-stabilized paraf ﬁn as a solid –liquid phase change material,Heat Mass Transfer 32(1997)307–312.[17]C.Alkan,K.Kaya, A.Sar ı,Preparation,thermal properties and thermalreliability of form-stable paraf ﬁn/polypropylene composite for thermal energy storage,J.Polym.Environ.17(2009)254–258.[18]I.Krupa,G.Miková, A.Luyt,Polypropylene as a potential matrix for thecreation of shape stabilized phase change materials,Eur.Polym.J.43(2007)895–907.[19]M.Xiao,B.Feng,K.Gong,Preparation and performance of shape stabilizedphase change thermal storage materials with high thermal conductivity,Energy Convers.Manage.43(2002)103–108.[20]M.Xiao,B.Feng,K.Gong,Thermal performance of a high conductive shape-stabilized thermal storage material,Sol.Energy Mater.Sol.Cells 69(2001)293–296.[21]K.Kaygusuz,C.Alkan,A.Sari,O.Uzun,Encapsulated fatty acids in an acrylicresin as shape-stabilized phase change materials for latent heat thermal energy storage,Energy Sources Part A 30(2008)1050–1059.[22]A.Sari, C.Alkan,U.Kolemen,O.Uzun,Eudragit S (methyl methacrylatemethacrylic acid copolymer)/fatty acid blends as form ‐stable phasechangeFig.8.Effect of MEPCM on the impact strength of PVC-based FSPCMs.X.Jin et al./Solar Energy Materials &Solar Cells 130(2014)435–441440material for latent heat thermal energy storage,J.Appl.Polym.Sci.101(2006) 1402–1406.[23]Y.Wang,T.D.Xia,H.X.Feng,H.Zhang,Stearic acid/polymethylmethacrylatecomposite as form-stable phase change materials for latent heat thermal energy storage,Renew.Energy36(2011)1814–1820.[24]A.Sarı,C.Alkan,A.Karaipekli,O.Uzun,Poly(ethylene glycol)/poly(methylmethacrylate)blends as novel form‐stable phase‐change materials for ther-mal energy storage,J.Appl.Polym.Sci.116(2010)929–933.[25]J.Su,P.Liu,A novel solid–solid phase change heat storage material withpolyurethane block copolymer structure,Energy Convers.Manage.47(2006) 3185–3191.[26]Q.Cao,P.Liu,Hyperbranched polyurethane as novel solid–solid phase changematerial for thermal energy storage,Eur.Polym.J.42(2006)2931–2939. [27]D.Feldman,M.Shapiro,P.Fazio,A heat storage module with a polymerstructural matrix,Polym.Eng.Sci.25(1985)406–411.[28]A.Sari,K.Kaygusuz,Studies on poly(vinyl chloride)/fatty acid blends as shape-stabilized phase change material for latent heat thermal energy storage, Indian J.Eng.Mater.Sci.13(2006)253.[29]Y.Zhang,K.Lin,R.Yang,H.Di,Y.Jiang,Preparation,thermal performance andapplication of shape-stabilized PCM in energy efﬁcient buildings,Energy Build.38(2006)1262–1269.[30]P.Schossig,H.-M.Henning,S.Gschwander,T.Haussmann,Micro-encapsulatedphase-change materials integrated into construction materials,Sol.Energy Mater.Sol.Cells89(2005)297–306.[31]H.Mehling,R.Krippner, A.Hauer,Research project on PCM in wood–lightweight-concrete,in:Proceedings of the2nd Workshop of IEA ECES IA, Annex17,2002.[32]X.Liu,H.Liu,S.Wang,L.Zhang,H.Cheng,Preparation and thermal propertiesof form stable parafﬁn phase change material encapsulation,Energy Convers.Manage.47(2006)2515–2522.[33]J.Li,P.Xue,W.Ding,J.Han,G.Sun,Micro-encapsulated parafﬁn/high-densitypolyethylene/woodﬂour composite as form-stable phase change material for thermal energy storage,Sol.Energy Mater.Sol.Cells93(2009)1761–1767.X.Jin et al./Solar Energy Materials&Solar Cells130(2014)435–441441。

普通化学实验课程体系思政建设

Univ. Chem. 2023, 38 (5), 61–66 61收稿：2022-07-04；录用：2022-11-01；网络发表：2022-12-01*通讯作者，Email:****************.cn基金资助：2021年度拔尖计划2.0研究课题(20212048)；浙江省课程思政示范基层教学组织；浙江大学一流本科专业综合改革项目；浙江大学2021年度第一批校级课程思政建设项目•教学研究与改革• doi: 10.3866/PKU.DXHX202207020 普通化学实验课程体系思政建设徐孝菲，李宁*，陈晨，赵玲丽，蒋银土，张嘉捷，林旭锋浙江大学化学系，杭州 310058摘要：普通化学实验课程体系以化学实验为载体，构建了涵盖家国情怀、文化自信、科学精神、科学伦理、社会责任、化学与生活等六大方面的思政元素的课程思政案例库，从中国特色、浙大特色、学科特色和课程特色四个方面，将思想价值引领巧妙生动地融合到知识传授过程中，践行了立德树人的根本任务，学生反馈良好。

关键词：普通化学实验；课程思政；中国特色；浙大特色；学科特色；课程特色中图分类号：G64；O6The Ideological and Political Education in the Courses of General Chemistry LaboratoryXiaofei Xu, Ning Li *, Chen Chen, Lingli Zhao, Yintu Jiang, Jiajie Zhang, Xufeng LinDepartment of Chemistry, Zhejiang University, Hangzhou 310058, China.Abstract: Taking chemistry experiments as the carrier, the courses of General Chemical Laboratory have built a curricular case library covering six ideological and political elements including patriotism, cultural confidence, scientific spirit, scientific ethics, social responsibility, chemistry and life. They have lively incorporated ideological and moral education into the teaching process from four aspects: Chinese characteristics, Zhejiang University characteristics, disciplinary characteristics and curricular characteristics. During the process, a university’s fundamental task of moral education and talent fostering has been performed and good feedback has been received from students.Key Words: General chemistry laboratory; Curricular ideological and political education;Chinese characteristics; Zhejiang University characteristics; Disciplinary characteristics;Curricular characteristics为全面贯彻党的十九大和全国高校思想政治工作会议精神，全面执行教育部《高等学校课程思政建设指导纲要》，浙江大学化学实验教学中心积极开展化学实验类课程思政建设，努力构建具有浙江大学特色的科教融合式化学实验类课程体系。

CA申请文书解析丨可以有很多发挥空间

CA申请文书解析丨可以有很多发挥空间有意愿申请美国大学的同学们，一定听说过Common Application（通用申请系统），全世界有将近900所高等院校使用这个系统来进行招生工作，它是美国大学本科申请中最大的一站式网站。

根据Common Application官宣，2020-2021年度CA的主文书题目与上个申请季保持一致，申请者需要从CA提供的七个文书题目中，选择一个最能体现自身性格特点的题目来撰写文章，字数要求在250~650个英文单词。

但是，5月份的时候，CA宣布给2021的申请者新增文书题目，阐述对于COVID-19对学生的影响。

这道题optional，是“可选的”，申请者可根据自已的意愿来提交文书。

现在，我们说回CA主文书，这可就不是optional啦，一定要选择一个题目来写。

在详细探讨CA申请文书之前，需要同学们先了解，大学招生官想要从你的Application Essay中“看到”什么？1.你是谁？2.你能为我们的校园做出有价值的贡献吗？3.你的写作能力？这几个问题应该贯穿头脑风暴和写作的全过程，清楚地了解自己的特点和价值并表达出来。

好了，下面奉上CA文书题目的详细解析，希望可以助童鞋们一臂之力，也愿你们文思泉涌，斩获好录取！1Some students have a background, identity, interest, or talent so meaningful they believe their application would be incomplete without it. If this sounds like you, please share your story.你是否来自一种背景，一个身份，或者你有一个兴趣，一项特长，它对于你非常有意义（重要），对于别人认识你也不可或缺。

飛渡君：无论是写背景，身份，还是兴趣、特长，要通过具体叙述写出它对自己的重要性（不可或缺），即题目中讲的“meaningful”-有意义，它如何帮助你重新认识了自己？借此向读者呈现“我是个什么样的人”，即我作为一个独特的个体，最大的特点是什么？A：一种背景/一个身份：1.可介绍你来自的一种成长环境（或家庭，或学校，或某个团体等），一种身份（族群-如客家人/潮汕人等，种族- 如黑人，白人等，个人品质/特质-独立的，喜欢安静的，有创造力的，甚至肥胖的等），聚焦于一到两个2.他们如何帮助你形成了一种对自己的认知，更了解自己？3.为此你如何坚信自己与众不同？B：一个兴趣/特长/天赋：1.你的兴趣/特长/天赋是什么？2.通过不同时间点，或具体的事件，说明它如何塑造了不一样的你？3.为此你如何坚信自己与众不同？2The lessons we take from obstacles we encounter can be fundamental to later success. Recount a time when you faced a challenge, setback, or failure. How did it affect you, and what did you learn from the experience?我们从遇到的困境/阻碍获得的经验教训是我们之后成功的基础。

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Materials Science and Engineering B132(2006)307–310Preparation and characterization of carbonﬁbers coated byFe3O4nanoparticlesJing Xu a,Haibin Yang a,b,∗,Wuyou Fu a,Yongming Sui a,Hongyang Zhu a,Minghui Li a,Guangtian Zou aa National Laboratory of Superhard Materials,Jilin University,Changchun130012,PR Chinab Institute of Materials Science&Engineering,Henan Polytechnic University,Jiaozuo454003,PR ChinaReceived4March2006;received in revised form24April2006;accepted29April2006AbstractCarbonﬁbers have been successfully coated with Fe3O4nanoparticles coating layers via sol–gel method combined with annealing under vacuum. The phase structures and morphologies of the composite have been characterized by XRD and FESEM.The results show that a complete and uniform Fe3O4nanoparticles coating on carbonﬁbers can be obtained in the temperature range of300–550◦C.The coating layers with thickness ca.800nm are composed by Fe3O4nanoparticles of mean sizes ca.30nm.The surface properties of Fe3O4nanoparticles detached from carbon ﬁbers/Fe3O4composite have been analyzed by XPS and FTIR.©2006Elsevier B.V.All rights reserved.Keywords:Fe3O4nanoparticles;Carbonﬁbers;Sol–gel1.IntroductionWith the fast development of information and communication technology in recent years,microwave absorptive materials draw more and more attention because of their widespread applica-tions for electromagnetic compatibility(EMC)[1–3].They are important parts of stealthy defense system for aircraft,sea or land vehicles,and essential parts of absorbing and shielding the environmental pollution from microwave irradiation.Magnetic absorption materials continue to play a leading role in the inves-tigation and application of microwave absorption materials[4]. However,the conventional absorption materials,such as ferrite and metal,are quite heavy.The problem restricts their useful-ness in many applications[5,6].One of the ways to overcome the problem is to couple ferrite and metal materials with low den-sity materials.Carbonﬁbers(CFs)are dominant in low density materials because of their low density,high strength,and per-fect electrical property[7,8].CFs coated by magnetic materials can decrease the weight of the microwave absorptive compos-ite,and improve the conductivity and strength of the composite. It has been demonstrated that iron-coated CFs can be obtained ∗Corresponding author.Tel.:+864315168763;fax:+864315168258.E-mail address:yanghb@(H.Yang).by electrochemical method and used as electromagnetic radia-tion absorptive composite[9,10].Huang et al.reported nickel and copper/nickel can be loaded on CFs for EMI shielding by electroless method[11].However,very little work has been done for combining ferrite with CFs.Huang et al.prepared CFs coated with barium ferrite by sol–gel method and analyzed its electromagnetic properties theoretically[12].Even though the composite is an absorbing microwave absorbing materials,it still has some limitations, such as high density and high-temperature treat.Magnetite (Fe3O4)has attracted long-standing interest in nanostructure synthesis because of its extensive applications in ferroﬂuid,and ultrahigh density magnetic storage media[13,14].Magnetite (Fe3O4)as a type of relatively low density ferrite can be obtained in a relatively low temperature(compared with barium ferrite). However,little work has been done to couple CFs with Fe3O4 nanoparticles so far.In this paper,Fe3O4nanoparticles have been coated on CFs by sol–gel method based on the reaction of ferric nitrate and ethylene glycol under vacuum in the temperature range of300–550◦C.The Fe3O4coating layers with thickness ca. 800nm are composed by Fe3O4nanoparticles of mean sizes ca.30nm.The Fe3O4coating on CFs surfaces can be further functionalized with a large variety of other nanostructure mate-0921-5107/$–see front matter©2006Elsevier B.V.All rights reserved. doi:10.1016/j.mseb.2006.04.038308J.Xu et al./Materials Science and Engineering B 132(2006)307–310Fig.1.The scheme of the procedure used to synthesize the CFs/Fe 3O 4composite.rials by utilized the chemistry of Fe 3O 4nanoparticles to create novel multilayer coating on CFs.And the magnetic,thermal,and microwave absorption properties of CFs/Fe 3O 4would be considered in near future.2.Experimental 2.1.CFs and chemicalsPAN-based CFs with 6–8␮m in diameter and 3mm in length were supplied by Jillin Carbon Group Co.,Ltd.Chemicals,ferric nitrate (Fe(NO 3)3·9H 2O),ethylene glycol (C 2H 6O 2),nitric acid (HNO 3,65%)and acetone (C 3H 6O),used in the experiment are of analytical grade and used without further puriﬁcation.2.2.Preparation of CFs/Fe 3O 4compositeThe synthesis procedure for the CFs/Fe 3O 4composite is illustrated in Fig.1and exhaustively described as follows:before deposition,CFs were immersed in acetone for 2h to clean the CFs surfaces,then CFs were treated with HNO 3(65%)for 24h at room temperature to introduce various functional groups (such as COOH,OH,and CO)on CFs surfaces.These functional groups can act as nucleation sites for Fe 3O 4nanoparticles on CFs.A 0.2mol ferric nitrate was ﬁrstly dissolved in 100ml ethy-lene glycol with vigorously stirring for 2h at 40◦C,to be usedasFig.2.TG-DTA curves of CFs/precursor in N 2.coating solution.Then 2g of acid-treated CFs were immersed in the above sol.The system was ultrasonically dispersed for 2h at room temperature,and then the excess sol was ﬁltered from CFs with the precursor of Fe 3O 4(CFs/precursor).After being dried at 120◦C,CFs/precursor was further annealed at different temperatures under vacuum for 2h.After repeating the process several times,CFs would be coated by a uniform Fe 3O 4nanoparticles layer.2.3.CharacterizationThe morphologies,particle size,and thickness of Fe 3O 4nanoparticles coating on CFs were investigated by a ﬁeld emis-sion scanning electron microscopy (FESEM,JSM-6700F).The phase structures of CFs/precursor annealed were character-ized by X-ray powder diffraction (XRD,D/Max-rA)with Cu K ␣radiation (λ=0.15418nm).Thermal analysis (TG-DTA)of the CFs/precursor was conducted in N 2on a thermal ana-lyzer (TA,SDT-2960)at a heating rate of 10◦C/min from room temperature to 900◦C.The residual organic materials on the surface of Fe 3O 4nanoparticles were examined by X-ray diffraction photoelectron spectroscopy (XPS,ESCALAB Mark II)and Fourier transform infrared spectrophotometer (FTIR,UV-3101).Fig.3.XRD patterns of CFs/precursor annealed at different temperatures under vacuum for 2h:(a)250◦C,(b)300◦C,(c)550◦C and (d)700◦C.J.Xu et al./Materials Science and Engineering B132(2006)307–3103093.Results and discussion3.1.Characterization of CFs/Fe3O4compositeA typical TG-DTA curve for CFs/precursor is shown in Fig.2. The TGA curve exhibits three distinct weight loss steps and the DTA curve shows three exothermic peaks.Theﬁrst obvi-ous weight loss step in the temperature range of150–250◦C is accompanied with an exothermic peak around200◦C in the DTA curve.We believe this result is due to the combustion of organic residue in the CFs/precursor.The second weight loss step in the temperature range of250–320◦C and the broad exothermic peak around290◦C in the DTA curve are considered as a result of crystallization of Fe3O4phase.Almost no weight loss was observed from300to530◦C,implying the unique presence of CFs/Fe3O4in this temperature range.The exothermic peak at 620◦C in the DTA curve and the weight loss process continued until about680◦C are presumed to be associated with the crys-tallization of␣-Fe phase.The XRD analysis was carried out to probe the composition at different temperatures(Fig.3),which conﬁrmed the results of TG-DTA.Fig.3shows the XRD patterns of CFs/precursor annealed at different temperatures under vacuum for2h.At250◦C(Fig.3a), no other diffraction peaks except the(002)peak of graphite appears,indicating that the Fe3O4coating layer on CFs has not been formed.Fig.3b and c illustrate that CFs/Fe3O4composite can be obtained in the temperature range from300to550◦C. The diffraction peaks at2θ=35.48◦,62.62◦,30.12◦,57.02◦, and43.12◦can be assigned to(311),(440),(220),(511) and(400)planes of Fe3O4(JCPDS88-0866),respectively.No other diffraction peaks can be observed but those of graphite and Fe3O4,indicating that the coating layer is only consisted of Fe3O4nanoparticles.When CFs/precursor were annealed at 700◦C(Fig.3d),the phase of␣-Fe appears and the(002)peakof Fig.4.FESEM micrographs of(a)bare CFs,(b)multiple CFs/Fe3O4composite,(c)single CFs/Fe3O4composite,and(d)the magniﬁed view of Fe3O4nanoparticles coating layer.310J.Xu et al./Materials Science and Engineering B132(2006)307–310Fig.5.XPS spectrum of Fe3O4nanoparticles separated from CFs/Fe3O4com-posite of O1s.graphite still remains,showing that the CFs/precursor changed to CFs/␣-Fe at high-temperature under vacuum.No obvious peaks corresponding to ferrite nitrite or other iron oxide,such as␣-Fe2O3and␥-Fe2O3,are detected(Fig.3).Fig.4shows the FESEM micrographs of bare CFs, CFs/Fe3O4composite(CFs/precursor annealed at300◦C).The diameters of bare CFs with smooth surfaces are about6–8␮m (Fig.4a).The direct evidence of the formation of Fe3O4nanopar-ticles coating on CFs is given by FESEM in Fig.4b.It is apparent that almost all CFs have been fully coated with uniform Fe3O4 nanoparticles without separated Fe3O4nanoparticles conglom-erations next to the coating layers.The ca.800nm thickness of coating layer is observed distinctly in Fig.4c,and Fig.4d is the magniﬁed micrograph of Fe3O4nanoparticles coating, which reveals that the perfect coating are composed of Fe3O4 nanoparticles with sizes of about30nm.3.2.The surface properties of Fe3O4nanoparticles detached from CFs/Fe3O4compositeTo analyze the surface properties of Fe3O4nanoparticles, we separated Fe3O4nanoparticles from CFs/Fe3O4composite. XPS analysis(Fig.5)was performed to characterize the surface properties of Fe3O4nanoparticles detached from CFs/Fe3O4 composite.The oxygen1s peak is deconvoluted into three spec-tral bands at530.6,532.0,and533.4eV.The most intense peak at530.6eV is attributed to the lattice oxygen in the metal oxide. The532.0eV of binding energy is due to the carbonyl(C O) and the relatively small peak at533.4eV represents C–O.The results indicate that some organic residual materials exist with the Fe3O4nanoparticles.To further conﬁrm the results,FTIR analysis was carried out to probe the surface properties of Fe3O4 nanoparticles separated from CFs/Fe3O4composite(Fig.6). Vibrational features at3400,1634and1400cm−1are assigned to the O–H,C O and C–O stretching motion.The FTIR studies correctly agree with the results of XPS.Thus,we concludethat Fig.6.FTIR spectrum of Fe3O4nanoparticles detached from CFs/Fe3O4com-posite.some organic materials have been absorbed on Fe3O4nanopar-ticles.4.ConclusionsFe3O4nanoparticles have been successfully coated on CFs surfaces by sol–gel method.The CFs/Fe3O4composite is present at300–550◦C,and the Fe3O4coating layers with thick-ness ca.800nm are composed by Fe3O4nanoparticles of mean sizes ca.30nm.The surface properties of Fe3O4nanoparti-cles separated from CFs/Fe3O4composite have been studied by XPS and FTIR analysis.The results indicate that some organic residue exist with Fe3O4nanocrystalline.CFs/Fe3O4compos-ite is potential microwave absorbing materials,and the magnetic properties and microwave absorption properties of CFs/Fe3O4 will be considered in our future work.References[1]A.N.Yusoffa,M.H.Abdullah,J.Magn.Magn.Mater.269(2004)271.[2]P.-H.Martha,J.Magn.Magn.Mater.215–216(2000)171.[3]T.Giannakopoulou,L.Kompotiatis,A.Kontogeorgakos,G.Kordas,J.Magn.Magn.Mater.246(2002)360.[4]H.How,C.Vittoria,J.Appl.Phys.69(1991)5183.[5]K.Hatakeyama,T.Inui,IEEE Trans.Magn.20(1984)1261.[6]M.Matsumoto,Y.Miyata,IEEE Trans.Magn.33(1994)4459.[7]G.Dorey,J.Phys.D:Appl.Phys.20(1987)245.[8]J.B.Donnet,R.C.Bansal,M.J.Wang,CFs,Marcel Dekker,New York,1990.[9]Y.Yang,B.S.Zhang,W.D.Xu,Y.B.Shi,N.S.Zhou,H.X.Lu,J.AlloyCompd.365(2004)300.[10]Y.Yang,B.S.Zhang,W.D.Xu,Y.B.Shi,Z.S.Jiang,N.S.Zhou,B.X.Gu,H.X.Lu,J.Magn.Magn.Mater.256(2003)129.[11]C.Y.Huang,W.W.Mo,M.L.Roan,Surf.Coat.Tech.184(2004)163.[12]X.Z.Huang,X.D.Li,C.X.Feng,P.Peng,J.Funct.Mater.4(2000)446.[13]K.Raj,R.Moskowitz,J.Magn.Magn.Mater.85(1990)223.[14]H.Zeng,J.Li,J.P.Liu,Z.L.Wang,S.H.Sun,Nature420(2002)395.。