2.1 Reagents and instruments
Reagents: chitosan(BR)was obtained from Sinopharm. β-cyclodextrin (BR), absolute ethyl alcohol were obtained from Shanghai huishi biochemical reagent Ltd. Glutaraldehyde (AR), glycerol were obtained from Tianjin Damao Chemical Reagent Factory. Cos Brilliant Blue G250 was obtained from Beijing Dingguo Changsheng Biotechnology Lt. Citric acid (AR) was obtained from Macklin; Polyethylene Glycol 400(AR), sodium dehydration phosphate (AR), glacial acetic acid (AR), 85% phosphoric acid (AR) were obtained from Beijing Chemical Works.
Instrument: DF-101S heat-collecting constant-temperature heating magnetic stirrer (GongyiCity Yuhua Instrument Ltd.). Electric heating drying oven (Tianjin Laiboterui Instrument Equipment Ltd.); PX124ZH/E Electronic Balance (Ohaus Instrument Ltd.); 3K15 Centrifuge (Xima Centrifuge Ltd.); NTS-4000 Constant Temperature Oscillating Water Tank (Tokyo Physical and Chemical EYELA); Nicolis5ft-IR Fourier infrared spectrometer (Thermo Fisher Scientific Shier Technology); IMark microplate reader (bio-rad, USA); SL200KS optical contact angle meter (Kono Industrial Co., Ltd., USA); QJ210-50N Universal Mechanical Testing Equipment.
2.2 Methods
2.2.1 Preparation of β-CDP
Citric aci(2g), polyethylene glycol 400(1g), sodium phosphate monobasic (0.25g), β-CD (10g) were added to 80 mL ultrapure water, dissolve to transparency stored at 145℃ for 4.5 h. Take out the powder for grinding, add a small amount of water to dissolve it, and then make alcohol precipitation with 95% ethanol. Take the precipitate for filtration and drying, and grind the dried solid powder.
2.2.2 Preparation of CS/β-CDP composite membrane
Chitosan solution is mixed with an aqueous solution of β-CDP polymer in Fig. 1. Then, glutaraldehyde was added and the mixture was stirred at a constant temperature to obtain a homogeneous solution. The CS/β-CDP composite membrane was prepared by the flow method.
2.2.3 The absorption of BSA.
The adsorption effect of CS/β-CDP composite membrane of BSA was analyzed. 1g of CS/β-CDP composite membrane was placed in 10 mL 0.5 mg/mL BSA solution, and the solution was shaken at 100 r/min 1 h to determine the BSA content before and after shaking. The adsorption capacity of BSA was calculated using Eq. 1.
Where, q is adsorption capacity (mg/g); C0 is BSA initial concentration (mg/mL); C is adsorbed BSA concentration (mg/mL); V is BSA solution volume (mL); M is composite film quality (g).
2.2.4 Optimization of preparation conditions of CS/β-CDP composite membrane
2.2.4.1 Single factor experiment
Adsorbed BSA was carried out to examine the adsorption capacity of CS/β-CDP composite membrane with different mass ratio (1:1,1:1.5,1:2,1:2.5,1:3), temperature (20,30,40,50,60℃) and glutaraldehyde addition (0.5,1,1.5,2,2.5,3mL).
2.2.4.2 Response surface methodology experiment
Response surface optimization design: According to the results of single factor investigation, the BSA adsorption capacity was as the response value, the Box-Behnken design was used to conduct the 3-factor 3-level experiment.
2.2.5 Effects of adsorption temperature and pH on adsorption capacity of BSA
The optimum adsorption condition of BSA was determined by changing the temperature and pH, respectively. At the optimal membrane preparation conditions, the adsorption capacity of CS/β-CDP composite membrane for BSA was investigated at different temperature (15,25,35,45,55℃) and pH (3,4,5,6,7).
2.2.6 Mechanical performance test
The mechanical properties of the membrane were controlled tested by an electronic universal testing machine. The membranes were cut into strips with a size of 10 mm × 7 mm, and then were tested in conditions using a Universal Mechanical Testing Equipment. A crosshead speed of 500mm/min was applied until the specimen ruptured. Ten specimens were tested per condition to calculate the mean and standard deviation values of tensile strength, elongation at the break, and Young’s modulus.
2.2.7 swelling degree
The swelling was determined as describing by Bagher[24] with some modifications. The sample membrane was accurately weighed (m0). They were immersed in distilled water at room temperature for 24 h. The membrane was taken out of the water, its surface moisture was absorbed by filter paper, and the membrane was weighed (m1). The swelling degree of the membrane was calculated using Eq. 2.
Swelling=\(\frac{{m}_{1}-{m}_{0}}{{m}_{0}}\times 100\%\) (1)
Where m0 is the initial mass of the sample film (g) and m1 is the mass of the sample membrane (g).
2.2.8 Contact angle measurements
The water contact angle of chitosan membrane surface was determined with optical contact angle meter. Adding 5 µL droplets to the middle of chitosan membrane and CS/β-CDP composite membrane, respectively, then the contact angle was measured after 30 s. Three parallel measurements were conducted and the average value was determined.
2.2.9 Scanning electron microscopy (SEM)
SEM analysis was performed to visualize the surface morphology of prepared before and after adsorption of BSA by CS/β-CDP composite membrane using scanning electron microscope. The membranes were cut into strips with a size of 0.3 mm × 0.3 mm and were gold platted for better conductivity, and then glued on aluminum stub using double adhesive carbon tape. The surface morphology was observed.
2.2.10 FT-IR analysis
FT-IR analysis was carried out to monitor the functional group of CS/β-CDP composite membrane before and after absorption BSA. Samples and the controls were tested with a resolution of 2 cm− 1 under the scan range of 4000− 1 to 600 cm− 1.
2.2.11 X-ray diffraction (XRD) analysis
XRD analysis was carried out to monitor the crystalline structure of CS/β-CDP composite membrane before and after absorption BSA. An angular step of 0.02° and a scanning speed of 2°/min was selected, and analyses were performed in a copper tube with a current of voltage of 40 kV. The datum was collected in the range of 10–60° at 2Ɵ.
2.2.12 Batch adsorption studies
2.2.12.1 Adsorption isotherm studies
The concentrations of the BSA solution were determined at knowing concentration intervals. 0.1g CS/β-CDP composite membrane was added to adsorb BSA. The concentration of the BSA from 0.2 mg/mL to 1.4 mg/mL. The mixture was shaken for 1 h at room temperature. To apprehend the adsorption mechanism, Langmuir, Freundlich and Temkin models were applied in Table 1.
Table 1
Adsorption isotherm equation
Isothermal equation
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Langmuir
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Freundlich
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Temkin
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Original equation
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\(q=\frac{abC}{1+bC}\)
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\(q=K{C}^{\frac{1}{n}}\)
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\(q=b\text{ln}\left(AC\right)\)
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linear equation
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\(\frac{1}{q}=\frac{1}{abC}+\frac{1}{a}\)
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\(\text{ln}q=\text{ln}K+\frac{1}{n}\text{ln}C\)
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\(q=b\text{ln}A+b\text{ln}C\)
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Where q is adsorption capacity (mg/g); C is Adsorbed BSA concentration (mg/mL) ; a、b、K、n are constants
2.2.12.2 Kinetic studies
The concentrations of the BSA solution were determined by knowing time intervals. 0.1g CS/β-CDP composite membrane was added to adsorb BSA. The time of absorption BSA from 10 min to 150 min. To apprehend the adsorption mechanism, pseudo-first-order, pseudo-second-order and Intraparticle diffusion kinetic models were applied in Table 2.
Table 2
dynamical equation
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pseudo-first-order
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pseudo-second-order
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Intraparticle diffusion
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Original equation
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\(\text{ln}\left({q}_{e}-q\right)=\text{ln}{q}_{e}-kt\)
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\(\frac{1}{q}=\frac{a}{t}+b\)
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\(q=k{t}^{\frac{1}{2}}+C\)
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Where qe is the maximum adsorption quantity (mg/g); q is the adsorption quantity at time t (mg/g); a、b、k、C are constants
2.2.12.3 Thermodynamic of adsorption
The concentrations of the BSA solution were determined at knowing temperature intervals. 0.1g CS/β-CDP composite membrane was added to adsorb BSA. The temperature of absorption BSA from 15℃ to 55℃. The mixture was shaken for 1 h at room temperature. To apprehend the adsorption mechanism, thermodynamic parameters, including enthalpy changes, entropy changes, and Gibbs free energy changes of BSA adsorption on CS/β-CDP composite membrane were applied to Table 3. LnKC vs. of 1/T and obtaining ΔH and ΔS.
Table 3
Thermodynamic parameters
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ΔH
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ΔG
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\(\text{lnKc}\)
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Original equation
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\(\varDelta {\rm H}=\varDelta G+T\varDelta S\)
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\(\varDelta G=-RT\text{ln}{K}_{c}\)
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\(\text{lnKc}=\frac{\varDelta S}{R}-\frac{\varDelta H}{RT}\)
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Where ΔH is enthalpy changes (kJ/mol); ΔG is Gibbs free energy changes (kJ/mol); ΔS is entropy changes (J/mol·K);T is temperature (K); R is gas constant(8.314 kJ/mol·K); Kc is thermodynamic equilibrium constant.