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Determination of microstructural characteristics of advanced biocompatible nanofibrous membranes
Microporous and Mesoporous Materials xxx (xxxx) xxxx
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Determination of microstructural characteristics of advanced biocompatible nanofibrous membranes Karel Soukup∗, Vladimír Hejtmánek, Olga Šolcová Institute of Chemical Process Fundamentals of the CAS, v. v. i, Rozvojová 135, CZ-165 02, Prague 6, Czech Republic
ARTICLE INFO
ABSTRACT
Keywords: Electrospinning Electrospun biocompatible nanofibrous membrane Mass transfer Wicke-Kallenbach cell Effective transport parameters
Effective transport properties of two biocompatible nanofibrous membranes—gelatin and chitosan—were evaluated using the gas transport measurement. The assessments involve the counter-current diffusion carried out both in Graham's and Wicke-Kallenbach cells under isothermal steady-state conditions. Additionally, the isothermal quasi-stationary gas permeation was also performed in modified Wicke-Kallenbach cell. It was found that the obtained transport characteristics reflect the gas transport mechanism which takes place predominantly in the continuum regime due to the prevailing macroporosity of the electrospun nanofibrous membranes. The gas permeation transport characteristics were evaluated from permeation cell measurements carried out at low pressures. The actual transport mechanism corresponded to the Knudsen flow dominating over continuous flow. The accuracy of the transport characteristics was estimated as the 95% confidence regions. It was confirmed that the confidence region shape of the optimized transport characteristics was intimately connected with the prevailing mass transport mechanism.
1. Introduction Biocompatible nanofibrous membranes have attracted much attention in the last decade owing to their potential use in a broad range of medical applications since the nanoscale is the typical scale related directly to human body. Therefore, advanced biocompatible and biodegradable nanofibrous materials can be utilized as wound dressing, bone tissue engineering, artificial organ, tissue templates, prostheses or drug delivery [1–3]. Biocompatible nanofibers can be generally prepared using a number of different techniques including phase separation, templating, drawing, or vapor phase polymerization based on physical, chemical, thermal as well as electrostatic phenomena [1]. However, the electrospinning method represents currently both the simplest and the cheapest fabrication route of the biocompatible nanofibrous system. The most general electrospinning setup consists of the two major parts—a high voltage power supply and two opposite charged electrodes [2]. During electrospinning process, the polymer solution flows through a hollow electrode which is usually represented by the thin nozzle or syringe and receives some quantum of the charge from this electrode. Between the electrode and the grounded collector, a strong electrostatic field is applied (usually tens of kV). The electric field draws solution droplet into a microscopic spatial object commonly known as
∗
the Taylor cone [3]. As soon as the electric forces overcome the surface tension of the polymer solution or melt, the polymer is attracted to the collector and a polymer jet is created. As the jet dries out in flight, it is elongated by a whipping process caused by the electrostatic repulsion initiated at small bends in the fiber until it is finally deposited on the surface of grounded collector (most using collectors are in the form of plate or drum). Produced electrospun membranes reveal outstanding properties such as high macroporosity, high surface to volume ratio, low density of layers, and high length to diameter ratio or tunable surface morphologies. The diameter of fibers prepared by electrospinning are usually in the range from microns down to tens of nanometers depending on the polymer solution parameters (its viscosity, concentration, conductivity, dielectric constant, surface tension), processing conditions (electric potential, flow rate, distance between spinning electrodes and their geometric shape) and ambient parameters (temperature, pressure, air humidity) [1]. Gelatin is the natural biopolymer that can be obtained from native collagen by means of the thermal denaturation treating or by chemical and physical degradation. Gelatin is the most abundant structural proteins found in the animal body of skin, tendon, cartilage and bone [4]. Owing to a wealth of merits such as biological origin, non-immunogenicity, biodegradability, biocompatibility, and commercial
Corresponding author. E-mail address: [email protected] (K. Soukup).
https://doi.org/10.1016/j.micromeso.2019.02.015 Received 16 June 2018; Received in revised form 18 January 2019; Accepted 11 February 2019 1387-1811/ © 2019 Published by Elsevier Inc.
Please cite this article as: Karel Soukup, Vladimír Hejtmánek and Olga Šolcová, Microporous and Mesoporous Materials, https://doi.org/10.1016/j.micromeso.2019.02.015
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availability at relatively low cost, pure gelatin or mixed with another (synthetic) biopolymer [5] has been widely used in the pharmaceutical and medical fields in a variety of applications. There are included tissue engineering [5], wound dressing, drug delivery and gene therapy [7] as sealants for vascular prostheses, carrier for drug delivery as well as dressings for wound healing [8]. It is dissolvable in water; however, it was shown [9] that the aqueous solution of gelatin cannot be easily processed by electrospinning under laboratory conditions. The main reason for this behavior is the formation of a three-dimensional interconnected network below the sol-gel transition temperature (approx. 30 °C). In early works [5,6,10] polar solvents based on fluorinated alcohols have been firstly utilized. Recently, detailed study [11,12] dealing with non-toxic solvents utilization based on acetic acid, aqueous solution of acetic acid or ethyl acetate have been appeared. Chitosan is a linear semi-crystalline polysaccharide produced by deacetylation of chitin that typically reveals biocompatibility, biodegradability and non-toxicity. Besides that, chitosan shows the anti-microbial activity and hydrating effects [13], analgesic [14] and hemostatic [15] properties. All these unique features make chitosan the appropriate candidate for medical [16], biological, and even for many industrial applications including the waste water treatment (chitosan is able to absorb and chelate many cations [17]). Ohkawa et al. [18] studied in detail the influence of the both solvent of acidic character together with chitosan concentration on the morphology of the prepared nanofibrous mats. They found that the increase of the chitosan concentration caused changes in the nanoparticle shape from the spherical beads into the interconnected nanofibrous network. In the follow-up study [19] authors found an optimum viscosity of the chitosan solution with respect to the requested fiber diameter. Chitosan can also form the structures with other bio-macromolecules such as collagen [20], poly(vinyl alcohol) [21], agarose [22] or zein [23] to produce blended biocompatible nanofibrous systems exhibiting unique properties. The majority of the published articles devoted to the biocompatible electrospun membranes has been typically focused on the process conditions. However, there is a lack of the original papers concerning the detailed study of microstructure properties assessed the texture analysis and the gas transport measurements. The main benefit of textural analysis appears from the wealth of the well-established experimental methods and evaluation procedures. Physical adsorption of inert gases or high-pressure mercury porosimetry govern the multilayer physical adsorption and condensation or the liquid metal intrusion into pores. Nevertheless, these methods do not provide any information related directly to the effective transport properties of porous materials. It is evident that due to preparation of the biocompatible electrospun materials with desired microstructure properties suitable for the given medical applications (wound dressing), it is important the knowledge on their pore network permeability. In principle, the classical experimental methods based on the direct measurement of the mass transport in porous solids (such as gas diffusion, gas permeation, or combined gas
diffusion-permeation) primary developed for heterogeneous catalysts or ceramic adsorbents [24–27] can be after some modification applied for determination of the permeability of biocompatible nanofibrous systems. The main objective of the present study is to evaluate the effective transport properties of different biocompatible electrospun membranes based on gelatin and chitosan representing a new class of biomedical nanomaterials. The relevant transport characteristics were estimated from the gas transport measurements under isothermal steady-state regime including counter-current diffusion (carried out both in Graham's and Wicke-Kallenbach cells) and isothermal quasi-stationary permeation (performed in the modified Wicke-Kallenbach cell). Detailed information of these material constants related to the microstructure characterization of nanofibers could be used for a tailored design of other biocompatible nanofibrous materials in terms of their medical applications in the future. 2. Experimental 2.1. Preparation of biocompatible membranes At the beginning, polymer solutions with the optimal electro-physical properties (the selection of appropriate solvent or mixture of solvents, optimal polymer solution concentration, its viscosity and conductivity) were prepared with respect to the electrospinning process. Gelatin (gelatin from porcine skin, gel strength 300, Sigma-Aldrich, powder) was dissolved in a mixture of acetic acid (p.a. grade, Penta Chemikalie, Czech Republic) and deionized water (specific electric conductivity below 2 μS/cm) with volume ratio of 1:1. Mixture of chitosan (low molecular weight, Sigma-Aldrich, powder) and poly(vinyl alcohol) (Mowiol 10–98, Kuraray, powder) in ratio of 1:10 (w/w) were dissolved in the mixture of acetic acid (p.a. grade, Penta Chemikalie, Czech Republic) and deionized water with the volume ratio of 1:1. The weight concentration of polymers was adjusted to 14.5% (w/v) and the specific electric conductivity to 1100 μS/cm by adding sodium chloride to the prepared polymeric solution, which was found as optimum for the electrospinning process. Nanofibrous membranes were prepared immediately from the fresh solutions by the electrospinning technique. The electrospinning equipment (SPUR, Czech Republic) depicted schematically in Fig. 1 involves the steel multi-jet spinning electrode and the steel plate as the collecting electrode. The process parameters of the multi-jet electrospinning were adjusted: the voltage +80 kV DC, electrode distance of 180 mm, temperature 21 ± 2 °C, relative humidity 25 ± 2.5% and solution flow rate 0.4 mL/min. 2.2. Determination of texture characteristics and morphology The standard methods of texture analysis including the physical adsorption of nitrogen, the high-pressure mercury porosimetry and the
Fig. 1. Electrospinning setup. 2
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helium pycnometry were utilized for determination of the basic texture characteristics of the tested electrospun samples. The BET surface area SBET, the mesopore surface area Smeso and the micropore volume Vmicro were evaluated from the nitrogen physical adsorption-desorption isotherms measured at 77 K obtained with the ASAP 2020 physisorption analyzer (Micromeritics, USA). The high-pressure mercury porosimetry performed on AUTOPORE IV 9520 instrument (Micromeritics, USA) were used for determination of the intrusion volume Vintr, the apparent density ρHg and the pore-size distribution curves. Skeletal density, ρHe, was determined using the AccuPyc II 1340 pycnometer (Micromeritics, USA) and porosity ε of all studied samples was determined according to ε = 1 − (ρHg/ρHe). To guarantee the precision of the obtained data, purity of nitrogen or helium (Linde Gas, Czech Republic) was grade of 99.999%. Before analysis, the samples were dried at 50 °C for 24 h in high vacuum (< 0.1 Pa). Morphology description of the produced electrospun mats was performed with the scanning electron microscope Vega II LSH (Tescan, Czech Republic). The image processing and image analysis were accomplished with aid of the public domain software ImageJ 1.44 (National Institutes of Health, USA).
stands for the Knudsen coefficient. According to the MTPM approach [28], r is the mean transport-pore radius and ψ includes porosity, εp, and tortuosity, qt of the transport pores related as ψ = εp/qt. In the case of the binary diffusion (gases A and B), the ratio of molar diffusion fluxes meets the conditions of Graham's law [26], the pair of constitutive equation (1) can be replaced by a single constitutive equation (2) according to
The Graham's diffusion cell [25,26,31] was operated at ambient laboratory conditions under steady-state regime. The cylindrical cell was divided into the two flow-through chambers of equal volume of 150 mL, separated by an impermeable disc holding the electrospun mat fixed by a sealing ring. During these experiments, the combination of pure gases (binary diffusion) or their mixtures (ternary diffusion) flowing steadily through the individual chambers was utilized (the total flow rate corresponding to 150 mL/min). Thereafter, until the steadystate was established, the gas flow through the lower chamber was stopped and the net diffusion volumetric flux was measured by the digital bubble flowmeter (Optiflow HFM 570, Agilent Technologies, USA). The cell was operated to achieve an ideally mixed regime inside the both chambers. Then, the effect of an external diffusion on the net diffusion flux was fully eliminated. In this described cell arrangement, the gas diffusion is considered like one dimensional from the macroscopic point of view. Therefore, the constitutive Maxwell-Stefan equation can be applied in terms of the Mean Transport Pore Model (MTPM) [28] as follows
j=1 j i
yj Nid
yi N jd Dijm
,
MA /MB ) m DAB
1
c
dyA dx
(2)
Counter-current gas diffusion was observed in the common WickeKallenbach cell operated at a constant total pressure [29,30,36,37]. Each side of the electrospun mat carefully tighten in an impermeable metal disc, was exposed to a different gas stream. The streams of pure gases were fed from the pressure cylinders through the mass flow controllers (Brooks, the Netherlands). High inlet space velocities were adjusted to significantly reduce the external diffusion resistance. Isobaric conditions were achieved by manually adjusting two fine needle valves mounted in the outlet tubes and by merging the two outlet gas streams in front of a vent. Consequently, the pressure difference between the cell chambers, measured by a sensitive differential pressure transducer PX654–0.05BD5V (Omega Engineering Inc., USA), was less than 0.2 Pa. Therefore, the forced flow, induced by a tiny total pressure gradient, did not affect the diffusion flow. Downstream from the cell, small amount of gaseous mixtures was removed by means of a six-way valve and analyzed online by a thermal conductivity detector (Raczek, Germany). The mass transport of gases through porous nanofiber sample was considered to be macroscopically one-dimensional. The constitutive equation commonly applied for the reduction of isobaric experimental data involving the diffusion of the gas mixture in the continuum, transition region or Knudsen regions reads as equation (2). To be readily integrated equation (2), the experiment under stationary conditions must be accomplished, i.e. to achieve the constant fluxes of the both gases through porous sample. A set of non-linear equations given by the mass balance of the cell and the integrated form of equation (2) represented the mathematical model. These equations were solved for unknown mole fractions of the outlet streams. The maximum likelihood estimate of the model parameters, ψ and r was obtained by minimizing the quantity according to the objective function (equation (3))
2.4. Graham's diffusion cell
n
KA
yA (1
2.5. Wicke-Kallenbach diffusion cell
The samples had a circle shape with diameter of 10 mm and they were prepared from the parent electrospun sheet (100 × 100 mm) by using a die cutting tool. All sample manipulation had to be performed very carefully with respect to the soft and easily deformable nanofibrous structure. Four non-adsorbing gases including hydrogen, helium, nitrogen and argon (grade of 99.99%, Linde Gas, Czech Republic) were used for all mass transport measurements in the diffusion and permeation cells. The appropriate difference in their molar weight and viscosity enable good observation and measurement of studied electrospun mats. Consequently, the inertness of selected gases prevents the unwanted appearance of surface diffusion.
dyi Nid = + dx r Ki
r
+
where M stands for the molecular weight. This equation can be integrated analytically between x = 0 and x = L (bottom/upper part of the nanofibrous sample) where the mole fraction of component A are, yAU and yAL , respectively.
2.3. Transport phenomenon measurements and evaluation of effective transport properties
c
1
NA =
n 2
=
yAi
y( , r
i=1
i
, ai)
2
(3)
where ai stands for the vector of known parameters that specify each run (particularly inlet concentrations, inlet flow rates and total pressure), n is the number of measurements, yAi is the experimental mole fraction of A and σi is its standard deviation. The minimization of χ2 was achieved using the Levenberg-Marquardt method [33] under Matlab R2016b, The Mathworks, Inc., USA. 2.6. Permeation cell
i = 1, ...,n
The design of the permeation cell operating in the quasi-steady state regime was similar to Graham's and Wicke-Kallenbach cell. It consists of two chambers separated by a metallic holder with a thin sheet of the nanofibrous membrane. In principle, time dependences of the small pressure differences changing during the inert gas permeation through the porous sample at the appropriate pressure level (1–25 kPa) are
(1)
where yi stands for the mole fraction of gas i, x denotes the length coordinate in the diffusion direction and Nid is the molar diffusion flux density of the gas i per unit total cross-section of the porous solid. Dijm represents the binary bulk diffusion coefficient of the gas pair i–j and Ki 3
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monitored. At the beginning of every run, slight pressure difference was created in the space of the input chamber (500–900 Pa) by the admission of little amount of the inert gas. The time dependent attenuation of the pressure difference between both chambers was sampled from the differential pressure transducer (Baratron, MKS Instruments GmbH., Germany, accuracy 0.1%) and logged in a PC. The length of this response spans was ranging from 2 to 10 min. Usually, 15–20 runs for all inert gases per sample of the electrospun membrane at ambient temperature were collected. Gas permeation transport through the nanofibrous membrane can be described by the subsequent constitutive equation:
Ni p =
r
Ki +
r2
Pi µi
1 dPi R g T dx
nanofibers have diameter in the range from 25 to 475 nm with the maximum at 175 nm. On the other hand, the chitosan membrane revealed nanofibers with diameters ranged from 200 nm up to 700 nm with the broader maximum at 250–300 nm. Textural properties including the BET surface area (SBET), the total intrusion volume (Vintr), the true (ρHe) and the apparent (ρHg) densities and the porosity (ε) of the tested nanofibrous membranes are summarized in Table 1. It can be seen that both biocompatible electrospun membranes possess rather low BET surface area, 13.0 and 14.5 m2/g for gelatin and chitosan, respectively. On the contrary, membranes reveal high porosity—more than 80% for both samples. Porosity of electrospun membranes is formed by the voids (gaps) between individual fibers, since the own polymeric fibers are practically compact without inner pore network structure. On the basis of our recent studies [34,35], rather macropores structure of the prepared membranes was considered. Therefore, mercury intrusion measurement was utilized as an appropriate method to determine the pore size distribution function. Obtained pore size distribution curves for the both membranes are depicted in Fig. 4. It is evident that membranes show monodispersed pore structure which corresponds to the free-space between fibers with the similar values of an average fiber diameter. Fig. 4 indicates that gelatin reveals prevailing pore diameter at 130 nm and chitosan at 140 nm.
(4)
where Ni is the permeation molar flux through the cross-sectional area of the nanofibrous membrane, Ki is Knudsen coefficient, Rg is the gas constant and x is the spatial coordinate. The least-square non-linear fitting of the pressure difference responses (Matlab, Mathworks R2016b, USA) including the assumption of errors in both pressure differences and time was used for evaluation of the transport parameters. p
3. Results and discussion 3.1. Morphology and texture properties
3.2. Determination of the transport parameters
The aim of our study was focused on preparation of the beads-free nanofibrous membranes as well as a system isotropy in terms of the microstructural properties. It appears from the previous studies [16–19] that both the final morphology and transport properties of nanofibrous membranes mostly depend on the process conditions during an electrospinning process. In accordance with this intention, the viscosity, which can be controlled directly by the concentration of polymer, and the electric conductivity of the polymer solution were recognized as the most important parameters related to formation of the beads on a fiber [32]. From the SEM micrographs given in Fig. 2, it can be clearly seen that the both kinds of membranes showed fairly good uniformity with predominantly bead-free, uniform and smooth fibers. For the statistical calculation of the fiber diameter distribution, approximately 100 randomly distributed fibers over the SEM micrographs were taken into account. Calculated histograms related to gelatin or chitosan nanofibrous membranes are presented in Fig. 3, respectively. Both tested membranes consist of nanometer diameter-size fibers. Gelatin
The Mean Transport Pore Model approach [28] was used to evaluate the transport parameters such as follows: ψ, r , and r 2 . These characteristics enable the comparison of the effective permeability of various porous materials. They represent the material constants independent of the experimental conditions (temperature and pressure) and composition of the gases used. The measurements carried out in Graham's and Wicke-Kallenbach diffusion cell provide the transport parameter pair r and ψ, only, while the pair r and r 2 is accessible from the permeation measurement. Graham's cell was employed for determination of the transport characteristics of electrospun materials by means of the gas diffusion measurement. The binary and ternary gas diffusion were used to obtain sufficiently large set of primary data (the net diffusion fluxes versus binary/ternary composition). Then, accuracy of the evaluated transport parameters and their confidence limits were substantially improved by this way. The isothermal binary diffusion measurement accomplished in Wicke-Kallenbach cell under steady-state conditions were sequentially
Fig. 2. SEM micrographs of gelatin (a) and chitosan (b) electrospun nanofibrous membrane. 4
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Fig. 3. Distribution of nanofiber diameters for gelatin (a) and chitosan (b) electrospun nanofibrous membrane. Table 1 Textural properties. Sample
Gelatin Chitosan
Table 2 Optimized transport parameters from diffusion measurements.
SBET
Vintr
ρHe
ρHg
ε
[m2/g]
[cm3/g]
[g/cm3]
[g/cm3]
[−]
13.0 14.5
4.22 3.61
1.16 1.19
0.20 0.22
0.83 0.81
Sample
Wicke-Kallenbach cell
r
Gelatin Chitosan
carried out at three levels of the total pressure (14, 26, 50 kPa). This operating pressure range brings an advantage to obtain the transport parameters of improved precision, because the mass transport mechanism is altered (Knudsen region). Consequently, a mean free path of gas molecules at lower pressure are more lengthened and its magnitude are comparable with pore size. This fact is positive especially for macroporous material [36]. Similarly, the permeation experiments under quasi steady-state conditions were made at pressure level in the range 2–13 kPa, to obtain information-rich data set enabling reliable estimation of transport characteristics. Optimized transport parameters evaluated from diffusion and permeation measurements are summarized in Tables 2 and 3, respectively. The transport characteristic ψ obtained from the both diffusional measurements independently is of comparable magnitude each together for gelatin and chitosan nanofibers. Value of ψ has a connection to the microstructural properties such as porosity and tortuosity. Because of gelatin and chitosan have approximately the same porosity (∼0.8) and gelatin has lower ψ value in relative to chitosan, the inert gas molecule must “travel” longer distance in gelatin nanofibrous material. Therefore, gelatin is less permeable for inert gases than chitosan. This difference in permeability of both materials can be visually presumed
Graham's cell
r
ψ
ψ
[nm]
[−]
[nm]
[−]
100963 123127
0.117 0.128
988 61.5
0.119 0.130
Table 3 95% Confidence intervals of the optimized transport parameters from permeation measurements. Sample
Gelatin Chitosan
r
[nm]
r2
[nm2]
Lower limit
Mean
Upper limit
Lower limit
Mean
Upper limit
167.2 254.2
167.5 254.5
167.9 254.7
27397 45124
27968 45598
28539 46072
from Fig. 2. The nanofibrous network of gelatin appears as denser than chitosan one. However, this fact is not so evident from classical texture measurements (see Table 1). The parameter r reflects the size of transport pore in the MTPM model was estimated, either from the diffusion or permeation measurements. Obtained parameters reveal a huge dispersion of values. The reason for that consists in the macroporous character of nanofibers. Despite of the mass transport measurement in the range of lower pressure (14–50 kPa) the Knudsen number has excessively low
Fig. 4. Pore-size distribution functions for gelatin (a) and chitosan (b) electrospun nanofibrous membrane from mercury porosimetry. 5
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magnitude, thus the continuum flow regime dominates over Knudsen one (see more in the Chapter 3.3). Therefore, the parameter r moves in the four orders and can not be relevantly determined. The pressure range theoretically calculated on the MTPM predict diffusional measurements within Knudsen regime at 10 Pa for both nanofiber membranes.
obtained from Wicke-Kallenbach cell (see Fig. 6). These results imply the higher relevance of the characteristic ψ than r . It can be concluded on the basis of the confidence region shapes that the continuous flow regime is typical for the both tested electrospun membranes. These findings are fully in agreement with the texture analysis indicating the high portion of pores above 150 nm of both nanofibrous membranes (see Fig. 4). The 95% confidence intervals of the optimized transport parameters r and r 2 for gelatin and chitosan samples are summarized in Table 3. It appears that during the gas permeation in nanofibrous mats (owing to the measurements under lower pressure which is inversely proportional to the mean free path of the gas molecules) the Knudsen transport mechanism dominates under these circumstances. This fact can be reflected through the narrow confidence interval of parameter r for both nanofibrous membranes. The uncertainty of the estimated parameter r from diffusion measurement is higher. The permeation cell working at low pressure and monitoring the pressure difference enables to get the more relevant estimation of parameter r . The relevance of the parameter r 2 reflected the viscous flow mechanism is then lower.
3.3. Influence of the gas transport mechanism on optimized transport parameters Generally, the mass transport of gases through the pore network takes place by Knudsen or viscous flow (molecular or bulk flow) mechanism [28]. The actual flow regime in pores is controlled by the Knudsen number, Kn, which is defined as the ratio of molecular meanfree path to the characteristic pore size. In the Knudsen regime, Kn→ ∞, the molecule-wall collisions are prevailed and the gas flow is governed by the Knudsen mechanism. Contrary, in the continuum regime, Kn→ 0, the mutual collisions of type molecule-molecule dominate over the molecule-wall collisions. In addition, there is transition flow region, where the both kinds of collisions take place with similar statistical significance. Within of our previous studies [27,36], the generally valid relation between transport parameters and the prevailing gas flow regime was confirmed. In accordance with equation (1), parameter r has a connection to the Knudsen flow regime, while the transport parameter ψ is directly connected with the continuum flow regime. The setup of Wicke-Kallenbach cell enables to perform the isothermal gas diffusion measurements under conditions of the different total pressure levels. This is the advantage of the Wicke-Kallenbach cell since the diffusion measurement can be adapted to the broad range of the microstructural properties of various porous materials. Especially, for nanofibrous material with macroporous network structure is this possibility promised. The low-pressure level could be adjusted to gain relevant experimental data for confidential evaluation of the transport parameters [36]. Contrary, Graham's diffusion cell, which is during measurements opened to the atmosphere via flowmeter, may operate under ambient pressure only. The statistical reliability of the obtained transport parameters was assessed by means of their confidence regions evaluated at the significance level of 5%. Corresponding 95% confidence regions based on the relative critical sum of squared deviations for gelatin and chitosan membranes are depicted in Fig. 5 (Graham's cell) and Fig. 6 (WickeKallenbach cell). The optimum parameter pairs are highlighted as the full circles. The shape of the confidence regions are situated in the way that the parameter ψ has lower uncertainty than r and the regions are significantly elongated along the r axis. This tendency is even more evident for the confidence regions of transport parameters
4. Conclusions Diffusion and permeation of selected inert gases (H2, He, N2, Ar) were newly used for characterization of biocompatible nanofibrous membranes. The separation materials from gelatin or chitosan were prepared by the electrospinning method. Their permeable properties were tested in Graham's and Wicke-Kallenbach diffusion cells as well as in the permeation cell. The transport characteristics ψ, r , or r 2 were estimated from the experimental data of three independent apparatuses. Based on confidence regions, their relevance was thoroughly discussed. The chitosan nanofibrous membrane revealed a higher density (and lower permeability) than gelatin one. These results were confirmed by the SEM images and by the classical methods of textural analysis. Consequently, the macroporosity of membranes has considerable effect on a flow regime of inert gases through diffusion or permeation measurements and thus, it has an impact on a transport parameter relevance. Due to the prevailing macroporosity of tested nanofibers, the evaluated transport characteristics reflect the gas transport mechanism in considerable extend. The flow regime reversion can be achieved at the lower pressure levels as a result of Knudsen number change. Therefore, the transport parameter r was obtained with higher relevance by experiments in permeation cell. This study has also shown the potential meaning of the effective transport parameters like the material constants of nanofibrous membranes determined with aid of mass transport experiments. Especially,
Fig. 5. The 95% confidence region of optimized transport parameters (full circle) for gelatin (a) and chitosan (b) from diffusion measurements performed in Graham's diffusion cell. 6
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Fig. 6. The 95% confidence region of optimized transport parameters (full circle) for gelatin (a) and chitosan (b) from diffusion measurements performed in WickeKallenbach cell.
for the assessment of suitability of nanofibrous mats utilization in various medical applications based on the quantitative prediction of their permeability. The mapping of effective transport properties of the biocompatible electrospun membranes can also contribute to their final design.
142–150. [14] M. Aranaz, M. Mengibar, R. Harris, I. Panos, B. Miralles, N. Acosta, G. Galed, A. Heras, Curr. Chem. Biol. 3 (2009) 203–230. [15] J. Yang, F. Tian, Z. Wang, Q. Wang, Y.J. Zeng, S. Q, J. Biomed. Mater. Res. Part B 85B (2007) 131–137. [16] Y. Kamari, M. Ghiaci, Microporous Mesoporous Mater. 234 (2016) 361–369. [17] M.Z. Elabee, H.F. Naguib, R.E. Morsi, Mat. Sci Mater. Sci. Eng., C 32 (2012) 1711–1726. [18] K. Ohkawa, D. Cha, H. Kim, A. Nishida, H. Yamamoto, Macromol. Rapid Commun. 25 (2004) 1600–1605. [19] K. Ohkawa, K.I. Minato, G. Kumagai, S. Hayashi, H. Yamamoto, Biomacromolecules 7 (2006) 3291–3294. [20] Z. Chen, X. Mo, F. Qing, Mater. Lett. 61 (2007) 3490–3494. [21] M. Ignatova, K. Starbova, N. Markova, N. Manolova, I. Rashkov, Carbohydr. Res. 341 (2006) 2098–2107. [22] Matsuda, G. Kagata, R. Kino, J. Tanaka, J. Nanosci. Nanotechnol. 7 (2007) 852–855. [23] G.W. Selling, A. Biswas, A. Patel, D.J. Walls, C. Dunlap, Y. Wei, Macromol. Chem. Phys. 208 (2007) 1002–1010. [24] V. Hejtmánek, P. Čapek, O. Šolcová, P. Schneider, Chem. Eng. J. 70 (1998) 189–195. [25] J. Valuš, P. Schneider, Appl. Catal. 1 (1981) 355–366. [26] K. Soukup, O. Šolcová, P. Schneider, Chem. Eng. Sci. 63 (2008) 1003–1011. [27] K. Soukup, O. Šolcová, P. Schneider, Wicke-Kallenbach and Graham's diffusion cell: limits of application for low surface area porous solids, Chem. Eng. Sci. 63 (2008) 4490–4493. [28] P. Schneider, Chem. Eng. Sci. 33 (1978) 1311–1319. [29] P. Čapek, V. Hejtmánek, L. Brabec, A. Zikánová, M. Kočiřík, Chem. Eng. Res. Des. 86 (2008) 713–722. [30] K. Hou, M. Fowles, R. Hughes, Chem. Eng. Res. Des. 77 (1999) 55–61. [31] K. Soukup, O. Šolcová, P. Schneider, Chem. Eng. Sci. 63 (2008) 4490–4493. [32] H. Fong, I. Chun, D.H. Reneker, Polymer 40 (1999) 4585–4592. [33] W.H. Press, S.A. Teukolsky, W.T. Vetterling, B.P. Flannery, Numerical Recipes in C: the Art of Scientific Computing, second ed., Cambridge University Press, Cambridge, 1992. [34] K. Soukup, D. Petráš, P. Topka, P. Slobodian, O. Šolcová, Catal. Today 193 (2012) 165–171. [35] K. Soukup, V. Hejtmánek, D. Petráš, O. Šolcová, Colloids Surf., A 437 (2013) 133–140. [36] P. Čapek, M. Veselý, V. Hejtmánek, Chem. Eng. Sci. 118 (2014) 192–207. [37] P. Čapek, A. Seidel-Morgenstern, Appl. Catal. Gen. 211 (2001) 227–237.
Acknowledgement The financial support of the Technology Agency of the Czech Republic (The Competence Centres, project TE01020080 and the National Centres of Competence, project TN0100048/5) is gratefully acknowledged. References [1] D. Pisignano, Polymer Nanofibers Building Blocks for Nanotechnology, The Royal Society of Chemistry, Dorchester, 2013. [2] J.-H. He, Y. Liu, L.-F. Mo, Y.-Q. Wan, L. Xu, Electrospun Nanofibres and Their Applications, iSmithers, Shawbury, 2008. [3] S. Ramakrishna, K. Fujihara, W.-E. Teo, T.-C. Lim, Z. Ma, An Introduction to Electrospinning and Nanofibers, World Scientific Publishing, Singapore, 2005. [4] A.G. Ward, A. Courts, The Science and Technology of Gelatin, Academic Press, London, 1977. [5] R.K. Ravichandran, D. Sundaramurthi, S. Gandhi, S. Sethuraman, U.M. Krishnan, Microporous Mesoporous Mater. 187 (2014) 53–62. [6] Z.M. Huang, Y.Z. Zhang, S. Ramakrishna, C.T. Lim, Polymer 45 (2004) 5361–5368. [7] J.F. Mano, G.A. Silva, H.S. Azevedo, P.B. Malafaya, R.A. Sousa, S.S. Silva, L.F. Boesel, J.M. Oliveira, T.C. Santos, A.P. Marques, N.M. Neves, R.L. Reis, J. R. Soc. Interface 4 (2007) 999–1030. [8] Y.Z. Zhang, J. Venugopal, Z.M. Huang, C.T. Lim, S. Ramakrishna, Polymer 47 (2006) 2911–2917. [9] C. Tonda-Turo, E. Cirpiani, S. Gnavi, V. Chiono, C. Mattu, P. Gentile, I. Perroteau, M. Zanetti, G. Ciardelli, Mater. Sci. Eng. C 33 (2013) 2723–2735. [10] M. Li, M.J. Mondrinos, M.R. Gandhi, F.K. Ko, A.S. Weiss, P.I. Lelkes, Biomaterials 26 (2005) 5999–6008. [11] J.H. Song, H.E. Kim, H.W. Kim, J. Mater. Sci. Mater. Med. 19 (2008) 95–102. [12] S.Y. Gu, Z.M. Wang, J. Ren, C.Y. Zhang, Mater. Sci. Eng. C 29 (2009) 1822–1828. [13] R. Jayakumar, M. Prabaharan, S.V. Nair, H. Tamura, Biotechnol. Adv. 28 (2010)
7
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Microporous and Mesoporous Materials journal homepage: www.elsevier.com/locate/micromeso
Determination of microstructural characteristics of advanced biocompatible nanofibrous membranes Karel Soukup∗, Vladimír Hejtmánek, Olga Šolcová Institute of Chemical Process Fundamentals of the CAS, v. v. i, Rozvojová 135, CZ-165 02, Prague 6, Czech Republic
ARTICLE INFO
ABSTRACT
Keywords: Electrospinning Electrospun biocompatible nanofibrous membrane Mass transfer Wicke-Kallenbach cell Effective transport parameters
Effective transport properties of two biocompatible nanofibrous membranes—gelatin and chitosan—were evaluated using the gas transport measurement. The assessments involve the counter-current diffusion carried out both in Graham's and Wicke-Kallenbach cells under isothermal steady-state conditions. Additionally, the isothermal quasi-stationary gas permeation was also performed in modified Wicke-Kallenbach cell. It was found that the obtained transport characteristics reflect the gas transport mechanism which takes place predominantly in the continuum regime due to the prevailing macroporosity of the electrospun nanofibrous membranes. The gas permeation transport characteristics were evaluated from permeation cell measurements carried out at low pressures. The actual transport mechanism corresponded to the Knudsen flow dominating over continuous flow. The accuracy of the transport characteristics was estimated as the 95% confidence regions. It was confirmed that the confidence region shape of the optimized transport characteristics was intimately connected with the prevailing mass transport mechanism.
1. Introduction Biocompatible nanofibrous membranes have attracted much attention in the last decade owing to their potential use in a broad range of medical applications since the nanoscale is the typical scale related directly to human body. Therefore, advanced biocompatible and biodegradable nanofibrous materials can be utilized as wound dressing, bone tissue engineering, artificial organ, tissue templates, prostheses or drug delivery [1–3]. Biocompatible nanofibers can be generally prepared using a number of different techniques including phase separation, templating, drawing, or vapor phase polymerization based on physical, chemical, thermal as well as electrostatic phenomena [1]. However, the electrospinning method represents currently both the simplest and the cheapest fabrication route of the biocompatible nanofibrous system. The most general electrospinning setup consists of the two major parts—a high voltage power supply and two opposite charged electrodes [2]. During electrospinning process, the polymer solution flows through a hollow electrode which is usually represented by the thin nozzle or syringe and receives some quantum of the charge from this electrode. Between the electrode and the grounded collector, a strong electrostatic field is applied (usually tens of kV). The electric field draws solution droplet into a microscopic spatial object commonly known as
∗
the Taylor cone [3]. As soon as the electric forces overcome the surface tension of the polymer solution or melt, the polymer is attracted to the collector and a polymer jet is created. As the jet dries out in flight, it is elongated by a whipping process caused by the electrostatic repulsion initiated at small bends in the fiber until it is finally deposited on the surface of grounded collector (most using collectors are in the form of plate or drum). Produced electrospun membranes reveal outstanding properties such as high macroporosity, high surface to volume ratio, low density of layers, and high length to diameter ratio or tunable surface morphologies. The diameter of fibers prepared by electrospinning are usually in the range from microns down to tens of nanometers depending on the polymer solution parameters (its viscosity, concentration, conductivity, dielectric constant, surface tension), processing conditions (electric potential, flow rate, distance between spinning electrodes and their geometric shape) and ambient parameters (temperature, pressure, air humidity) [1]. Gelatin is the natural biopolymer that can be obtained from native collagen by means of the thermal denaturation treating or by chemical and physical degradation. Gelatin is the most abundant structural proteins found in the animal body of skin, tendon, cartilage and bone [4]. Owing to a wealth of merits such as biological origin, non-immunogenicity, biodegradability, biocompatibility, and commercial
Corresponding author. E-mail address: [email protected] (K. Soukup).
https://doi.org/10.1016/j.micromeso.2019.02.015 Received 16 June 2018; Received in revised form 18 January 2019; Accepted 11 February 2019 1387-1811/ © 2019 Published by Elsevier Inc.
Please cite this article as: Karel Soukup, Vladimír Hejtmánek and Olga Šolcová, Microporous and Mesoporous Materials, https://doi.org/10.1016/j.micromeso.2019.02.015
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availability at relatively low cost, pure gelatin or mixed with another (synthetic) biopolymer [5] has been widely used in the pharmaceutical and medical fields in a variety of applications. There are included tissue engineering [5], wound dressing, drug delivery and gene therapy [7] as sealants for vascular prostheses, carrier for drug delivery as well as dressings for wound healing [8]. It is dissolvable in water; however, it was shown [9] that the aqueous solution of gelatin cannot be easily processed by electrospinning under laboratory conditions. The main reason for this behavior is the formation of a three-dimensional interconnected network below the sol-gel transition temperature (approx. 30 °C). In early works [5,6,10] polar solvents based on fluorinated alcohols have been firstly utilized. Recently, detailed study [11,12] dealing with non-toxic solvents utilization based on acetic acid, aqueous solution of acetic acid or ethyl acetate have been appeared. Chitosan is a linear semi-crystalline polysaccharide produced by deacetylation of chitin that typically reveals biocompatibility, biodegradability and non-toxicity. Besides that, chitosan shows the anti-microbial activity and hydrating effects [13], analgesic [14] and hemostatic [15] properties. All these unique features make chitosan the appropriate candidate for medical [16], biological, and even for many industrial applications including the waste water treatment (chitosan is able to absorb and chelate many cations [17]). Ohkawa et al. [18] studied in detail the influence of the both solvent of acidic character together with chitosan concentration on the morphology of the prepared nanofibrous mats. They found that the increase of the chitosan concentration caused changes in the nanoparticle shape from the spherical beads into the interconnected nanofibrous network. In the follow-up study [19] authors found an optimum viscosity of the chitosan solution with respect to the requested fiber diameter. Chitosan can also form the structures with other bio-macromolecules such as collagen [20], poly(vinyl alcohol) [21], agarose [22] or zein [23] to produce blended biocompatible nanofibrous systems exhibiting unique properties. The majority of the published articles devoted to the biocompatible electrospun membranes has been typically focused on the process conditions. However, there is a lack of the original papers concerning the detailed study of microstructure properties assessed the texture analysis and the gas transport measurements. The main benefit of textural analysis appears from the wealth of the well-established experimental methods and evaluation procedures. Physical adsorption of inert gases or high-pressure mercury porosimetry govern the multilayer physical adsorption and condensation or the liquid metal intrusion into pores. Nevertheless, these methods do not provide any information related directly to the effective transport properties of porous materials. It is evident that due to preparation of the biocompatible electrospun materials with desired microstructure properties suitable for the given medical applications (wound dressing), it is important the knowledge on their pore network permeability. In principle, the classical experimental methods based on the direct measurement of the mass transport in porous solids (such as gas diffusion, gas permeation, or combined gas
diffusion-permeation) primary developed for heterogeneous catalysts or ceramic adsorbents [24–27] can be after some modification applied for determination of the permeability of biocompatible nanofibrous systems. The main objective of the present study is to evaluate the effective transport properties of different biocompatible electrospun membranes based on gelatin and chitosan representing a new class of biomedical nanomaterials. The relevant transport characteristics were estimated from the gas transport measurements under isothermal steady-state regime including counter-current diffusion (carried out both in Graham's and Wicke-Kallenbach cells) and isothermal quasi-stationary permeation (performed in the modified Wicke-Kallenbach cell). Detailed information of these material constants related to the microstructure characterization of nanofibers could be used for a tailored design of other biocompatible nanofibrous materials in terms of their medical applications in the future. 2. Experimental 2.1. Preparation of biocompatible membranes At the beginning, polymer solutions with the optimal electro-physical properties (the selection of appropriate solvent or mixture of solvents, optimal polymer solution concentration, its viscosity and conductivity) were prepared with respect to the electrospinning process. Gelatin (gelatin from porcine skin, gel strength 300, Sigma-Aldrich, powder) was dissolved in a mixture of acetic acid (p.a. grade, Penta Chemikalie, Czech Republic) and deionized water (specific electric conductivity below 2 μS/cm) with volume ratio of 1:1. Mixture of chitosan (low molecular weight, Sigma-Aldrich, powder) and poly(vinyl alcohol) (Mowiol 10–98, Kuraray, powder) in ratio of 1:10 (w/w) were dissolved in the mixture of acetic acid (p.a. grade, Penta Chemikalie, Czech Republic) and deionized water with the volume ratio of 1:1. The weight concentration of polymers was adjusted to 14.5% (w/v) and the specific electric conductivity to 1100 μS/cm by adding sodium chloride to the prepared polymeric solution, which was found as optimum for the electrospinning process. Nanofibrous membranes were prepared immediately from the fresh solutions by the electrospinning technique. The electrospinning equipment (SPUR, Czech Republic) depicted schematically in Fig. 1 involves the steel multi-jet spinning electrode and the steel plate as the collecting electrode. The process parameters of the multi-jet electrospinning were adjusted: the voltage +80 kV DC, electrode distance of 180 mm, temperature 21 ± 2 °C, relative humidity 25 ± 2.5% and solution flow rate 0.4 mL/min. 2.2. Determination of texture characteristics and morphology The standard methods of texture analysis including the physical adsorption of nitrogen, the high-pressure mercury porosimetry and the
Fig. 1. Electrospinning setup. 2
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helium pycnometry were utilized for determination of the basic texture characteristics of the tested electrospun samples. The BET surface area SBET, the mesopore surface area Smeso and the micropore volume Vmicro were evaluated from the nitrogen physical adsorption-desorption isotherms measured at 77 K obtained with the ASAP 2020 physisorption analyzer (Micromeritics, USA). The high-pressure mercury porosimetry performed on AUTOPORE IV 9520 instrument (Micromeritics, USA) were used for determination of the intrusion volume Vintr, the apparent density ρHg and the pore-size distribution curves. Skeletal density, ρHe, was determined using the AccuPyc II 1340 pycnometer (Micromeritics, USA) and porosity ε of all studied samples was determined according to ε = 1 − (ρHg/ρHe). To guarantee the precision of the obtained data, purity of nitrogen or helium (Linde Gas, Czech Republic) was grade of 99.999%. Before analysis, the samples were dried at 50 °C for 24 h in high vacuum (< 0.1 Pa). Morphology description of the produced electrospun mats was performed with the scanning electron microscope Vega II LSH (Tescan, Czech Republic). The image processing and image analysis were accomplished with aid of the public domain software ImageJ 1.44 (National Institutes of Health, USA).
stands for the Knudsen coefficient. According to the MTPM approach [28], r is the mean transport-pore radius and ψ includes porosity, εp, and tortuosity, qt of the transport pores related as ψ = εp/qt. In the case of the binary diffusion (gases A and B), the ratio of molar diffusion fluxes meets the conditions of Graham's law [26], the pair of constitutive equation (1) can be replaced by a single constitutive equation (2) according to
The Graham's diffusion cell [25,26,31] was operated at ambient laboratory conditions under steady-state regime. The cylindrical cell was divided into the two flow-through chambers of equal volume of 150 mL, separated by an impermeable disc holding the electrospun mat fixed by a sealing ring. During these experiments, the combination of pure gases (binary diffusion) or their mixtures (ternary diffusion) flowing steadily through the individual chambers was utilized (the total flow rate corresponding to 150 mL/min). Thereafter, until the steadystate was established, the gas flow through the lower chamber was stopped and the net diffusion volumetric flux was measured by the digital bubble flowmeter (Optiflow HFM 570, Agilent Technologies, USA). The cell was operated to achieve an ideally mixed regime inside the both chambers. Then, the effect of an external diffusion on the net diffusion flux was fully eliminated. In this described cell arrangement, the gas diffusion is considered like one dimensional from the macroscopic point of view. Therefore, the constitutive Maxwell-Stefan equation can be applied in terms of the Mean Transport Pore Model (MTPM) [28] as follows
j=1 j i
yj Nid
yi N jd Dijm
,
MA /MB ) m DAB
1
c
dyA dx
(2)
Counter-current gas diffusion was observed in the common WickeKallenbach cell operated at a constant total pressure [29,30,36,37]. Each side of the electrospun mat carefully tighten in an impermeable metal disc, was exposed to a different gas stream. The streams of pure gases were fed from the pressure cylinders through the mass flow controllers (Brooks, the Netherlands). High inlet space velocities were adjusted to significantly reduce the external diffusion resistance. Isobaric conditions were achieved by manually adjusting two fine needle valves mounted in the outlet tubes and by merging the two outlet gas streams in front of a vent. Consequently, the pressure difference between the cell chambers, measured by a sensitive differential pressure transducer PX654–0.05BD5V (Omega Engineering Inc., USA), was less than 0.2 Pa. Therefore, the forced flow, induced by a tiny total pressure gradient, did not affect the diffusion flow. Downstream from the cell, small amount of gaseous mixtures was removed by means of a six-way valve and analyzed online by a thermal conductivity detector (Raczek, Germany). The mass transport of gases through porous nanofiber sample was considered to be macroscopically one-dimensional. The constitutive equation commonly applied for the reduction of isobaric experimental data involving the diffusion of the gas mixture in the continuum, transition region or Knudsen regions reads as equation (2). To be readily integrated equation (2), the experiment under stationary conditions must be accomplished, i.e. to achieve the constant fluxes of the both gases through porous sample. A set of non-linear equations given by the mass balance of the cell and the integrated form of equation (2) represented the mathematical model. These equations were solved for unknown mole fractions of the outlet streams. The maximum likelihood estimate of the model parameters, ψ and r was obtained by minimizing the quantity according to the objective function (equation (3))
2.4. Graham's diffusion cell
n
KA
yA (1
2.5. Wicke-Kallenbach diffusion cell
The samples had a circle shape with diameter of 10 mm and they were prepared from the parent electrospun sheet (100 × 100 mm) by using a die cutting tool. All sample manipulation had to be performed very carefully with respect to the soft and easily deformable nanofibrous structure. Four non-adsorbing gases including hydrogen, helium, nitrogen and argon (grade of 99.99%, Linde Gas, Czech Republic) were used for all mass transport measurements in the diffusion and permeation cells. The appropriate difference in their molar weight and viscosity enable good observation and measurement of studied electrospun mats. Consequently, the inertness of selected gases prevents the unwanted appearance of surface diffusion.
dyi Nid = + dx r Ki
r
+
where M stands for the molecular weight. This equation can be integrated analytically between x = 0 and x = L (bottom/upper part of the nanofibrous sample) where the mole fraction of component A are, yAU and yAL , respectively.
2.3. Transport phenomenon measurements and evaluation of effective transport properties
c
1
NA =
n 2
=
yAi
y( , r
i=1
i
, ai)
2
(3)
where ai stands for the vector of known parameters that specify each run (particularly inlet concentrations, inlet flow rates and total pressure), n is the number of measurements, yAi is the experimental mole fraction of A and σi is its standard deviation. The minimization of χ2 was achieved using the Levenberg-Marquardt method [33] under Matlab R2016b, The Mathworks, Inc., USA. 2.6. Permeation cell
i = 1, ...,n
The design of the permeation cell operating in the quasi-steady state regime was similar to Graham's and Wicke-Kallenbach cell. It consists of two chambers separated by a metallic holder with a thin sheet of the nanofibrous membrane. In principle, time dependences of the small pressure differences changing during the inert gas permeation through the porous sample at the appropriate pressure level (1–25 kPa) are
(1)
where yi stands for the mole fraction of gas i, x denotes the length coordinate in the diffusion direction and Nid is the molar diffusion flux density of the gas i per unit total cross-section of the porous solid. Dijm represents the binary bulk diffusion coefficient of the gas pair i–j and Ki 3
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monitored. At the beginning of every run, slight pressure difference was created in the space of the input chamber (500–900 Pa) by the admission of little amount of the inert gas. The time dependent attenuation of the pressure difference between both chambers was sampled from the differential pressure transducer (Baratron, MKS Instruments GmbH., Germany, accuracy 0.1%) and logged in a PC. The length of this response spans was ranging from 2 to 10 min. Usually, 15–20 runs for all inert gases per sample of the electrospun membrane at ambient temperature were collected. Gas permeation transport through the nanofibrous membrane can be described by the subsequent constitutive equation:
Ni p =
r
Ki +
r2
Pi µi
1 dPi R g T dx
nanofibers have diameter in the range from 25 to 475 nm with the maximum at 175 nm. On the other hand, the chitosan membrane revealed nanofibers with diameters ranged from 200 nm up to 700 nm with the broader maximum at 250–300 nm. Textural properties including the BET surface area (SBET), the total intrusion volume (Vintr), the true (ρHe) and the apparent (ρHg) densities and the porosity (ε) of the tested nanofibrous membranes are summarized in Table 1. It can be seen that both biocompatible electrospun membranes possess rather low BET surface area, 13.0 and 14.5 m2/g for gelatin and chitosan, respectively. On the contrary, membranes reveal high porosity—more than 80% for both samples. Porosity of electrospun membranes is formed by the voids (gaps) between individual fibers, since the own polymeric fibers are practically compact without inner pore network structure. On the basis of our recent studies [34,35], rather macropores structure of the prepared membranes was considered. Therefore, mercury intrusion measurement was utilized as an appropriate method to determine the pore size distribution function. Obtained pore size distribution curves for the both membranes are depicted in Fig. 4. It is evident that membranes show monodispersed pore structure which corresponds to the free-space between fibers with the similar values of an average fiber diameter. Fig. 4 indicates that gelatin reveals prevailing pore diameter at 130 nm and chitosan at 140 nm.
(4)
where Ni is the permeation molar flux through the cross-sectional area of the nanofibrous membrane, Ki is Knudsen coefficient, Rg is the gas constant and x is the spatial coordinate. The least-square non-linear fitting of the pressure difference responses (Matlab, Mathworks R2016b, USA) including the assumption of errors in both pressure differences and time was used for evaluation of the transport parameters. p
3. Results and discussion 3.1. Morphology and texture properties
3.2. Determination of the transport parameters
The aim of our study was focused on preparation of the beads-free nanofibrous membranes as well as a system isotropy in terms of the microstructural properties. It appears from the previous studies [16–19] that both the final morphology and transport properties of nanofibrous membranes mostly depend on the process conditions during an electrospinning process. In accordance with this intention, the viscosity, which can be controlled directly by the concentration of polymer, and the electric conductivity of the polymer solution were recognized as the most important parameters related to formation of the beads on a fiber [32]. From the SEM micrographs given in Fig. 2, it can be clearly seen that the both kinds of membranes showed fairly good uniformity with predominantly bead-free, uniform and smooth fibers. For the statistical calculation of the fiber diameter distribution, approximately 100 randomly distributed fibers over the SEM micrographs were taken into account. Calculated histograms related to gelatin or chitosan nanofibrous membranes are presented in Fig. 3, respectively. Both tested membranes consist of nanometer diameter-size fibers. Gelatin
The Mean Transport Pore Model approach [28] was used to evaluate the transport parameters such as follows: ψ, r , and r 2 . These characteristics enable the comparison of the effective permeability of various porous materials. They represent the material constants independent of the experimental conditions (temperature and pressure) and composition of the gases used. The measurements carried out in Graham's and Wicke-Kallenbach diffusion cell provide the transport parameter pair r and ψ, only, while the pair r and r 2 is accessible from the permeation measurement. Graham's cell was employed for determination of the transport characteristics of electrospun materials by means of the gas diffusion measurement. The binary and ternary gas diffusion were used to obtain sufficiently large set of primary data (the net diffusion fluxes versus binary/ternary composition). Then, accuracy of the evaluated transport parameters and their confidence limits were substantially improved by this way. The isothermal binary diffusion measurement accomplished in Wicke-Kallenbach cell under steady-state conditions were sequentially
Fig. 2. SEM micrographs of gelatin (a) and chitosan (b) electrospun nanofibrous membrane. 4
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Fig. 3. Distribution of nanofiber diameters for gelatin (a) and chitosan (b) electrospun nanofibrous membrane. Table 1 Textural properties. Sample
Gelatin Chitosan
Table 2 Optimized transport parameters from diffusion measurements.
SBET
Vintr
ρHe
ρHg
ε
[m2/g]
[cm3/g]
[g/cm3]
[g/cm3]
[−]
13.0 14.5
4.22 3.61
1.16 1.19
0.20 0.22
0.83 0.81
Sample
Wicke-Kallenbach cell
r
Gelatin Chitosan
carried out at three levels of the total pressure (14, 26, 50 kPa). This operating pressure range brings an advantage to obtain the transport parameters of improved precision, because the mass transport mechanism is altered (Knudsen region). Consequently, a mean free path of gas molecules at lower pressure are more lengthened and its magnitude are comparable with pore size. This fact is positive especially for macroporous material [36]. Similarly, the permeation experiments under quasi steady-state conditions were made at pressure level in the range 2–13 kPa, to obtain information-rich data set enabling reliable estimation of transport characteristics. Optimized transport parameters evaluated from diffusion and permeation measurements are summarized in Tables 2 and 3, respectively. The transport characteristic ψ obtained from the both diffusional measurements independently is of comparable magnitude each together for gelatin and chitosan nanofibers. Value of ψ has a connection to the microstructural properties such as porosity and tortuosity. Because of gelatin and chitosan have approximately the same porosity (∼0.8) and gelatin has lower ψ value in relative to chitosan, the inert gas molecule must “travel” longer distance in gelatin nanofibrous material. Therefore, gelatin is less permeable for inert gases than chitosan. This difference in permeability of both materials can be visually presumed
Graham's cell
r
ψ
ψ
[nm]
[−]
[nm]
[−]
100963 123127
0.117 0.128
988 61.5
0.119 0.130
Table 3 95% Confidence intervals of the optimized transport parameters from permeation measurements. Sample
Gelatin Chitosan
r
[nm]
r2
[nm2]
Lower limit
Mean
Upper limit
Lower limit
Mean
Upper limit
167.2 254.2
167.5 254.5
167.9 254.7
27397 45124
27968 45598
28539 46072
from Fig. 2. The nanofibrous network of gelatin appears as denser than chitosan one. However, this fact is not so evident from classical texture measurements (see Table 1). The parameter r reflects the size of transport pore in the MTPM model was estimated, either from the diffusion or permeation measurements. Obtained parameters reveal a huge dispersion of values. The reason for that consists in the macroporous character of nanofibers. Despite of the mass transport measurement in the range of lower pressure (14–50 kPa) the Knudsen number has excessively low
Fig. 4. Pore-size distribution functions for gelatin (a) and chitosan (b) electrospun nanofibrous membrane from mercury porosimetry. 5
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magnitude, thus the continuum flow regime dominates over Knudsen one (see more in the Chapter 3.3). Therefore, the parameter r moves in the four orders and can not be relevantly determined. The pressure range theoretically calculated on the MTPM predict diffusional measurements within Knudsen regime at 10 Pa for both nanofiber membranes.
obtained from Wicke-Kallenbach cell (see Fig. 6). These results imply the higher relevance of the characteristic ψ than r . It can be concluded on the basis of the confidence region shapes that the continuous flow regime is typical for the both tested electrospun membranes. These findings are fully in agreement with the texture analysis indicating the high portion of pores above 150 nm of both nanofibrous membranes (see Fig. 4). The 95% confidence intervals of the optimized transport parameters r and r 2 for gelatin and chitosan samples are summarized in Table 3. It appears that during the gas permeation in nanofibrous mats (owing to the measurements under lower pressure which is inversely proportional to the mean free path of the gas molecules) the Knudsen transport mechanism dominates under these circumstances. This fact can be reflected through the narrow confidence interval of parameter r for both nanofibrous membranes. The uncertainty of the estimated parameter r from diffusion measurement is higher. The permeation cell working at low pressure and monitoring the pressure difference enables to get the more relevant estimation of parameter r . The relevance of the parameter r 2 reflected the viscous flow mechanism is then lower.
3.3. Influence of the gas transport mechanism on optimized transport parameters Generally, the mass transport of gases through the pore network takes place by Knudsen or viscous flow (molecular or bulk flow) mechanism [28]. The actual flow regime in pores is controlled by the Knudsen number, Kn, which is defined as the ratio of molecular meanfree path to the characteristic pore size. In the Knudsen regime, Kn→ ∞, the molecule-wall collisions are prevailed and the gas flow is governed by the Knudsen mechanism. Contrary, in the continuum regime, Kn→ 0, the mutual collisions of type molecule-molecule dominate over the molecule-wall collisions. In addition, there is transition flow region, where the both kinds of collisions take place with similar statistical significance. Within of our previous studies [27,36], the generally valid relation between transport parameters and the prevailing gas flow regime was confirmed. In accordance with equation (1), parameter r has a connection to the Knudsen flow regime, while the transport parameter ψ is directly connected with the continuum flow regime. The setup of Wicke-Kallenbach cell enables to perform the isothermal gas diffusion measurements under conditions of the different total pressure levels. This is the advantage of the Wicke-Kallenbach cell since the diffusion measurement can be adapted to the broad range of the microstructural properties of various porous materials. Especially, for nanofibrous material with macroporous network structure is this possibility promised. The low-pressure level could be adjusted to gain relevant experimental data for confidential evaluation of the transport parameters [36]. Contrary, Graham's diffusion cell, which is during measurements opened to the atmosphere via flowmeter, may operate under ambient pressure only. The statistical reliability of the obtained transport parameters was assessed by means of their confidence regions evaluated at the significance level of 5%. Corresponding 95% confidence regions based on the relative critical sum of squared deviations for gelatin and chitosan membranes are depicted in Fig. 5 (Graham's cell) and Fig. 6 (WickeKallenbach cell). The optimum parameter pairs are highlighted as the full circles. The shape of the confidence regions are situated in the way that the parameter ψ has lower uncertainty than r and the regions are significantly elongated along the r axis. This tendency is even more evident for the confidence regions of transport parameters
4. Conclusions Diffusion and permeation of selected inert gases (H2, He, N2, Ar) were newly used for characterization of biocompatible nanofibrous membranes. The separation materials from gelatin or chitosan were prepared by the electrospinning method. Their permeable properties were tested in Graham's and Wicke-Kallenbach diffusion cells as well as in the permeation cell. The transport characteristics ψ, r , or r 2 were estimated from the experimental data of three independent apparatuses. Based on confidence regions, their relevance was thoroughly discussed. The chitosan nanofibrous membrane revealed a higher density (and lower permeability) than gelatin one. These results were confirmed by the SEM images and by the classical methods of textural analysis. Consequently, the macroporosity of membranes has considerable effect on a flow regime of inert gases through diffusion or permeation measurements and thus, it has an impact on a transport parameter relevance. Due to the prevailing macroporosity of tested nanofibers, the evaluated transport characteristics reflect the gas transport mechanism in considerable extend. The flow regime reversion can be achieved at the lower pressure levels as a result of Knudsen number change. Therefore, the transport parameter r was obtained with higher relevance by experiments in permeation cell. This study has also shown the potential meaning of the effective transport parameters like the material constants of nanofibrous membranes determined with aid of mass transport experiments. Especially,
Fig. 5. The 95% confidence region of optimized transport parameters (full circle) for gelatin (a) and chitosan (b) from diffusion measurements performed in Graham's diffusion cell. 6
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Fig. 6. The 95% confidence region of optimized transport parameters (full circle) for gelatin (a) and chitosan (b) from diffusion measurements performed in WickeKallenbach cell.
for the assessment of suitability of nanofibrous mats utilization in various medical applications based on the quantitative prediction of their permeability. The mapping of effective transport properties of the biocompatible electrospun membranes can also contribute to their final design.
142–150. [14] M. Aranaz, M. Mengibar, R. Harris, I. Panos, B. Miralles, N. Acosta, G. Galed, A. Heras, Curr. Chem. Biol. 3 (2009) 203–230. [15] J. Yang, F. Tian, Z. Wang, Q. Wang, Y.J. Zeng, S. Q, J. Biomed. Mater. Res. Part B 85B (2007) 131–137. [16] Y. Kamari, M. Ghiaci, Microporous Mesoporous Mater. 234 (2016) 361–369. [17] M.Z. Elabee, H.F. Naguib, R.E. Morsi, Mat. Sci Mater. Sci. Eng., C 32 (2012) 1711–1726. [18] K. Ohkawa, D. Cha, H. Kim, A. Nishida, H. Yamamoto, Macromol. Rapid Commun. 25 (2004) 1600–1605. [19] K. Ohkawa, K.I. Minato, G. Kumagai, S. Hayashi, H. Yamamoto, Biomacromolecules 7 (2006) 3291–3294. [20] Z. Chen, X. Mo, F. Qing, Mater. Lett. 61 (2007) 3490–3494. [21] M. Ignatova, K. Starbova, N. Markova, N. Manolova, I. Rashkov, Carbohydr. Res. 341 (2006) 2098–2107. [22] Matsuda, G. Kagata, R. Kino, J. Tanaka, J. Nanosci. Nanotechnol. 7 (2007) 852–855. [23] G.W. Selling, A. Biswas, A. Patel, D.J. Walls, C. Dunlap, Y. Wei, Macromol. Chem. Phys. 208 (2007) 1002–1010. [24] V. Hejtmánek, P. Čapek, O. Šolcová, P. Schneider, Chem. Eng. J. 70 (1998) 189–195. [25] J. Valuš, P. Schneider, Appl. Catal. 1 (1981) 355–366. [26] K. Soukup, O. Šolcová, P. Schneider, Chem. Eng. Sci. 63 (2008) 1003–1011. [27] K. Soukup, O. Šolcová, P. Schneider, Wicke-Kallenbach and Graham's diffusion cell: limits of application for low surface area porous solids, Chem. Eng. Sci. 63 (2008) 4490–4493. [28] P. Schneider, Chem. Eng. Sci. 33 (1978) 1311–1319. [29] P. Čapek, V. Hejtmánek, L. Brabec, A. Zikánová, M. Kočiřík, Chem. Eng. Res. Des. 86 (2008) 713–722. [30] K. Hou, M. Fowles, R. Hughes, Chem. Eng. Res. Des. 77 (1999) 55–61. [31] K. Soukup, O. Šolcová, P. Schneider, Chem. Eng. Sci. 63 (2008) 4490–4493. [32] H. Fong, I. Chun, D.H. Reneker, Polymer 40 (1999) 4585–4592. [33] W.H. Press, S.A. Teukolsky, W.T. Vetterling, B.P. Flannery, Numerical Recipes in C: the Art of Scientific Computing, second ed., Cambridge University Press, Cambridge, 1992. [34] K. Soukup, D. Petráš, P. Topka, P. Slobodian, O. Šolcová, Catal. Today 193 (2012) 165–171. [35] K. Soukup, V. Hejtmánek, D. Petráš, O. Šolcová, Colloids Surf., A 437 (2013) 133–140. [36] P. Čapek, M. Veselý, V. Hejtmánek, Chem. Eng. Sci. 118 (2014) 192–207. [37] P. Čapek, A. Seidel-Morgenstern, Appl. Catal. Gen. 211 (2001) 227–237.
Acknowledgement The financial support of the Technology Agency of the Czech Republic (The Competence Centres, project TE01020080 and the National Centres of Competence, project TN0100048/5) is gratefully acknowledged. References [1] D. Pisignano, Polymer Nanofibers Building Blocks for Nanotechnology, The Royal Society of Chemistry, Dorchester, 2013. [2] J.-H. He, Y. Liu, L.-F. Mo, Y.-Q. Wan, L. Xu, Electrospun Nanofibres and Their Applications, iSmithers, Shawbury, 2008. [3] S. Ramakrishna, K. Fujihara, W.-E. Teo, T.-C. Lim, Z. Ma, An Introduction to Electrospinning and Nanofibers, World Scientific Publishing, Singapore, 2005. [4] A.G. Ward, A. Courts, The Science and Technology of Gelatin, Academic Press, London, 1977. [5] R.K. Ravichandran, D. Sundaramurthi, S. Gandhi, S. Sethuraman, U.M. Krishnan, Microporous Mesoporous Mater. 187 (2014) 53–62. [6] Z.M. Huang, Y.Z. Zhang, S. Ramakrishna, C.T. Lim, Polymer 45 (2004) 5361–5368. [7] J.F. Mano, G.A. Silva, H.S. Azevedo, P.B. Malafaya, R.A. Sousa, S.S. Silva, L.F. Boesel, J.M. Oliveira, T.C. Santos, A.P. Marques, N.M. Neves, R.L. Reis, J. R. Soc. Interface 4 (2007) 999–1030. [8] Y.Z. Zhang, J. Venugopal, Z.M. Huang, C.T. Lim, S. Ramakrishna, Polymer 47 (2006) 2911–2917. [9] C. Tonda-Turo, E. Cirpiani, S. Gnavi, V. Chiono, C. Mattu, P. Gentile, I. Perroteau, M. Zanetti, G. Ciardelli, Mater. Sci. Eng. C 33 (2013) 2723–2735. [10] M. Li, M.J. Mondrinos, M.R. Gandhi, F.K. Ko, A.S. Weiss, P.I. Lelkes, Biomaterials 26 (2005) 5999–6008. [11] J.H. Song, H.E. Kim, H.W. Kim, J. Mater. Sci. Mater. Med. 19 (2008) 95–102. [12] S.Y. Gu, Z.M. Wang, J. Ren, C.Y. Zhang, Mater. Sci. Eng. C 29 (2009) 1822–1828. [13] R. Jayakumar, M. Prabaharan, S.V. Nair, H. Tamura, Biotechnol. Adv. 28 (2010)
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