Biological and Chemical Materials
Choline chloride and xylitol were purchased from Aladdin Chemistry Co., Ltd (Shanghai, China). All other reagents of analytical grade were directly used without any further purification. ESO and ELO were purchased from a domestic market.
Free Lipase G50 (Penicillium camemberti) was purchased from Amano Enzyme Products Company Japan (Japan). The wild-type Lipase SMG1 and Lipase SMG1-F278N were produced in our lab according to the method described previously [21]. Lipase SMG1, Lipase SMG1-F278N and Lipase G50 were immobilized onto epoxy resins of ECR 8285 following the procedures of Li et al [18]. The immobilized Lipase SMG1, Lipase SMG1-F278N and Lipase G50 were obtained by filtration and then dried in vacuum at 20 °C for 12 h. The recovered enzymes were stored at 4 °C until use. The esterification activity of immobilized Lipase SMG1, Lipase SMG1-F278N and Lipase G50 were 258, 328 and 423 U/g, respectively.
Extraction of SPO
SPO was extracted by petroleum ether using a Soxhlet extractor. After reflux at 30 oC for 8 h, the solvent was distilled off in a rotary vacuum evaporator (0.09 MPa). Then the obtained silkworm pupae extracts were dried with anhydrous sodium sulfate for 24 h [21].
Preparation of DESs
The DESs consisting of Choline chloride (ChCl, recrystallized from ethanol, filtered, and dried in vacuum) and xylitol (1:1, mol/mol) were mixed and heated at 80 °C in a rotary evaporator for 1 h. The formed colorless, homogeneous liquids were transferred into a tightly sealed bottle, then placed in a desiccator for further use.
General Procedures for Epoxidation of SPO
Firstly, SPO (4.3 g) and DESs (4.6 g) were added to a conical flask. The mixture was supplemented with 4.6 g of hydrogen peroxide (H2O2 : double bonds = 2:1, mol/mol). The epoxidation reaction was initiated by addition of enzymes (20 U/g oil). The reaction medium was mixed for 12 h at 400 rpm. The products were collected at a fixed interval (2 h) and centrifuged for 3 min at 10000 rpm. The excess H2O2 was removed by ddH2O. The immobilized enzymes were recycled after being rinsed with excess ddH2O and dried in vacuum at 20 °C.
A Scale-up Reaction
A scale-up reaction (~100-fold) was carried out under the optimized conditions. 108 g of SPO, 115 g of DESs and 115 g of hydrogen peroxide (H2O2 : double bonds = 2:1, mol/mol) were added to the reaction vessel. After the addition of Lipase SMG1-F278N (20 U/g oil), the epoxidation reaction was catalyzed at 30 °C for 8 h.
Analysis of Fatty Acid Composition by Gas Chromatography
The fatty acid composition was determined as fatty acid methyl esters (FAMEs) according to the standard method described in ISO 5509:2000 (E). The FAMEs were analyzed by gas chromatography-flame ionization detector (GC-FID) equipped with a CP-Sil 88 capillary column (60 m × 0.25 mm, 0.2 µm film thickness; Dikma Technologies, Beijing, China) [22].
Determination of Physicochemical Indices
The iodine value, oxirane value, acid value, density and moisture of the products were determined by titration according to the American Oil Chemists’ Society (AOCS) Official Methods Tg 1a-64, Cd 9-57, Cd 3d-63, Cc 10c-95 and Ac 2-41, respectively.
Characteristics of ESPO
The formation of oxirane ring in ESPO was confirmed by fourier transform infrared spectrometer (FT-IR) and carbon-13 nuclear magnetic resonance (13C NMR) spectra. The FT-IR spectra of the products were recorded on an FT-IR (Thermo Scientific, model Nicolet 6700, Waltham, MA, USA) with a resolution of 4 cm−1. Data were acquired using the FT-IR software (Thermo Scientific, OMNIC series suite, Waltham). The 1H and 13C NMR spectra of SPO and the derivatives (10~15 mg/mL in CDCl3) were recorded by a 600 MHz Brucker NMR spectrophotometer.
The thermal degradation of the obtained polymers was determined by thermogravimetric analysis (TGA) using Mettler-Toledo TGA/SDTA 851e. TG analysis was performed using a thermal weightlessness analyzer (TG209-F3, NETZSCH Co., Germany) in the temperature range of 25-700 °C with a rate of 10 °C/min under N2 (20 mL/min).
The curing behaviors of epoxidized oils were characterized by a differential scanning calorimeter (DSC) (Netzsch Geratebau, model 204 C, Germany), in the temperature range of -60-80 °C with a rate of 10 °C/min under N2.
The Mechanical Properties of PVC Samples
In the study, the tensile strength and elongation at break of PVC films made of ESPO were investigated to evaluate the comprehensive performance of various epoxy greases according to the American Society for Testing and Materials (ASTM) standards (ASTM D638).
The Thermal Stability and Migration Tests of The PVC Samples
The plastisol was prepared by mixing 50, 60, 70, 80, 90, and 100 phr of plasticizer with 100 phr PVC for 15 min, respectively. Subsequently, the mixture was compounded by double-roller blending rolls (SK-160B, Shanghai Rubber Machinery Factory) at 165 °C for 8 min. The sample was pressed on a flat plate vulcanizing machine (HY-100TA, Shanghai Hengyu Instrument Co., Ltd.) at 170 °C for 6 min to make film with a thickness of 5 mm. The final film was obtained after the temperature of the flat-plate vulcanizing machine dropped to 50 ℃.
The thermal stability of PVC samples was tested according to the Congo red method in GB/T 2917.1-2002. PVC samples were cut into 2 mm × 2 mm and placed in test tubes. Then the test tubes were incubated in an oil bath at 170 ℃. The time required for color changes of the Congo red test paper represented the thermal stability.
The migration tests were performed according to the following procedures. Firstly, all samples were immersed in n-hexane at 50 ℃ for 2 h. The weight loss was measured gravimetrically. It is considered that the weight of residue extracts present in n-hexane is equal to the plasticizer extracted [8].
Secondly, the mass loss of a plastic piece placed between two absorbent films was measured according to ISO 177. Samples were placed on two plates (3 mm thick) of low density polyethylene (LDPE) at 70 °C for 24 h with a circular cylinder (1 mm thick) of PVC plasticized with ESPO in between. The amount of plasticizer migrated was determined according to the weight differences before and after tests [8].
The third migration test was conducted using activated carbon as per ASTM D 1203-94 test method B. A wire cage was used to prevent from direct contact between the plasticized PVC and carbon. The migrated volatile components were measured after the circular cylinder was placed at 100 °C for 24 h.
Statistical Analysis
All experiments were performed in triplicate. The data were presented as mean values ± standard deviations.