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Membrane formation at interfaces examined by analytical ultracentrifugation techniques
Colloids and Surfaces A: Physicochemical and Engineering Aspects 180 (2001) 141 – 153 www.elsevier.nl/locate/colsurfa
Membrane formation at interfaces examined by analytical ultracentrifugation techniques Christine Wandrey *, Artur Bartkowiak 1 Laboratory of Polyelectrolytes and BioMacromolecules, Department of Chemistry, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland Received 10 April 2000; accepted 18 September 2000
Abstract For the first time, the formation of polyelectrolyte complex membranes has been examined by applying analytical ultracentrifugation’s synthetic boundary technique coupled with the twin scanning UV/vis and Rayleigh interference optics. Systematic basic studies demonstrate the efficiency of the new method, as well as its advantages and limitations. In particular, various phases of the membrane formation process have been identified. These include initial membrane formation, membrane growth, membrane equilibration, and, in some cases, phase separation or dissolution. The technique presented herein offers new and extended possibilities to study complex and/or structure formation by two components at interfaces dependent on polymer concentration, pH, ionic strength, and the component ratio. Temperature control, between 4 and 40°C, allows precise studies to be carried out under physiological conditions. Moreover, the membrane of defined thickness and structure and having an area of approximately 300 mm2, can be recovered from the cell for additional investigations. The polyanion sodium alginate and the polycation chitosan were employed as a model system in this investigation. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Polyelectrolyte complex; Hydrogel membrane; Analytical ultracentrifugation; Sodium alginate; Chitosan
1. Introduction Membrane formation, through the electrostatic interaction of oppositely charged polymers, has recently attracted increasingly scientific and commercial attention [1,2]. This is primarily due to the * Corresponding author. Tel.: +41-21-6933672; fax: + 4121-6935690. E-mail address: [email protected] (C. Wandrey). 1 Present address: Polymer Institute, Technical University of Szczecin, Pulaskiego 10, 70-322 Szczecin, Poland.
broad potential applications of the resulting membranes. Such materials may be employed in a variety of geometries including as components of flat devices or as self-contained hollow fibers, macrocapsules, or microcapsules [1–4]. Numerous combinations of anionic and cationic polylectrolytes have been examined in regards to their ability to form stable membranes having well defined characteristics. However, only a relatively few polymer pairs have been identified for this purpose [1,5–21]. Generally, the spontaneously
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created polyelectrolyte complex precipitates in aqueous solution without any stable macroscopic structure formation [22]. The ultimate properties of the polyelectrolyte complex depend on several parameters. These include both molecular characteristics of the polyelectrolyte molecules and conditions of the medium. The most influential molecular parameters are the charge density, the molar mass/chain length, the chemical structures of the charged groups and the polymer backbones, as well as the polydispersities in charge density and molar mass [1,21–25]. On the other hand, the concentration of the components, the pH of the solution, the ionic strength, and the temperature during the membrane formation can also strongly influence the key membrane properties including mechanical stability and permeability [26]. In particular, these two key parameters vary with the thickness and the structure of the membrane. It should also be noted that polyelectrolyte complex membranes are not static materials, and all of their properties are equilibrium characteristics, influenced by the environment’s electrochemical characteristics. Various analytical methods have been employed to study polyelectrolyte complex formation [1,22]. The complex stoichiometry, the membrane structure and surface characteristics have been investigated extensively by scattering methods, microscopic techniques, and surface scanning methods [1,22,27,28]. Analytical ultracentrifugation (AUC) has been employed for the characterization of polyelectrolyte complexes though not in terms of membrane formation [29]. In order to investigate the membrane formation process, through the electrostatic interaction of oppositely charged polyelectrolytes, we have adopted AUC-based techniques [30]. This paper presents the experimental principle on the basis of the synthetic boundary technique. Inside the analytical ultracentrifuge membrane formation, growth kinetics, and equilibration can be detected on-line. Basic investigations illustrate the efficiency of the method, its advantages and limitations. The polyanion sodium alginate and the polycation oligochitosan were used as a model system. Separation properties for membranes of this specific polyelectrolyte combination have
been presented recently [26]. Subsequent publications will report the influence of a broad variety of experimental influences on the membrane formation and other specific polyelectrolyte combinations.
2. Experimental
2.1. Equipment An Optima XL-I analytical ultracentrifuge (Beckman, Palo Alto, CA) was used for all the experiments. This instrument is designed to detect, measure, record, and analyze the movement of molecules in solution under a defined centrifugal field. It included two integrated detection systems, scanning UV/vis and Rayleigh interference optics. The scanning UV/vis detection operated in wavelength or radial scanning mode within a wavelength range 190–800 nm or a minimum radial increment of 0.001 cm, respectively. One absorbance reading could be performed approximately every 2 ×10 − 2 s. At the applied speed, in the range 3000–8000 rpm, absorbance readings could be performed every 60–120 s for a radial increment of 0.001 cm and a radial range 2–2.5 mm. The interference optics had a 30 mW laser (670 nm) as light source. The resolution of the detecting CCD camera was 2048× 96 pixels. Analytical ultracentrifugation offers numerous techniques to investigate the physico-chemical properties of macromolecules and colloids. Modern aspects using this equipment have been recently described [31].
2.2. Materials Sodium alginate (Keltone HV, lot 54650A, Kelco/NutraSweet, San Diego, CA) and degraded samples of chitosan (radical degradation [32] of raw material from E-055, Hutchinson/McNeil International, Philadelphia, PA) were been used as components for the membrane formation. The controlled radical degradation process, and the characterization of the resulting oligomers, have been described previously [26].
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2.3. Description of the experiments One of the principal techniques of the AUC is the synthetic boundary experiment. Special centerpieces for the analytical cells (each cylindrical analytical cell consists of a centerpiece, two window assemblies, and one cell housing) are available which allow solvent to be layered over a sample of a solution while the cell is spinning at moderately low speed (typically below 10 000 rpm). For this purpose the two sectors of the cylindrical double-sector centerpiece are connected on the upper surface with two capillary channels, one for the liquid and the second one for the back flow of air (see Fig. 1). These centerpieces are useful for preparing an artificial sharp boundary for measuring boundary spreading in measurements of diffusion coefficients. They can also be applied to examine sedimentation of small molecules (molar mass below 12 000 g mol − 1) for which the rate of sedimentation is insufficient to produce a sharp boundary that clears the meniscus [30]. In addition, the synthetic boundary technique has been used to monitor products from enzyme reactions [33], to detect crystallization in situ at the reaction boundary [34], to measure
Fig. 1. Principle of the membrane formation experiments in a synthetic boundary cell. Component positions and absorption scans (a): before layering, (b): during layering and (c): after layering is finished, though, chitosan is not yet completely complexed. , Air; a, chitosan I; b chitosan II; , membrane; b, alginate, …, capillary. Ranges 1–5 correspond to Table 1. The trapeziform cross-section of the two sectors is not shown.
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differential sedimentation coefficients [35], and to estimate extinction coefficients [36]. The centerpieces selected to study the membrane formation from oppositely charged polyelectrolytes were 12 mm high. This height represents the thickness of the liquid column that the light of the two optics will pass through. The standard sector length was 14 mm in radial direction, from sector top to sector bottom, with the capillary 6 mm from the bottom (compare the schematic in Fig. 1(a, b, c)). The maximum sector volume was 450 ml, however, for synthetic boundary experiments a solution volume of 150 ml is recommended for the solution sector [37]. The less viscous polycation solution possessing a lower density was layered over the higher viscous polyanion solution. The horizontal projection (view from above) of a synthetic boundary centerpiece and the principle of the experiment are illustrated in Fig. 1. Additionally, the appropriate absorbance plots are schematically presented in this figure. Each absorbance signal results from the difference of the absorbance measured in the solution sector (polyanion solution) and the one measured in the solvent sector (polycation solution). In principle five such differences are possible. However, various of these radial ranges will be detected dependent on the progress of the experiment. Table 1 summarizes the detection ranges during the experiment. The three radial ranges 1, 2, and 5 are expected before layering (Fig. 1(a)) whereas all five signal differences are visible during the layering (Fig. 1(b)). Finally, when the layering is finished the four ranges 1, 3, 4, and 5 exist (Fig. 1(c)). Since the absorbance of sodium alginate and air is negligible in comparison to the absorbance of oligochitosan, above 350 nm, a negative signal having the same value is detected for the total radius range of oligochitosan before the layering takes place (Fig. 1(a)). As soon as the polycation solution passes the capillary and is layered on the surface of the polyanion solution a spontaneous structure formation begins in the boundary zone. A chitosan concentration higher than that in the chitosan solution is detected by an absorbance
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Table 1 Absorbance signals during the experimentsa Range
Absorbance signal
Before layering
During layering
Finished layering
Long time experiment
1 2 3 4 5
AairII−AairI = 0 AairII−AchIB0 AchII−AchI50 Amem−AchI\0 Aalg−AchIB0
Yes Yes No No Yes
Yes Yes Yes Yes Yes
Yes No Yes Yes Yes
Yes No Yes Yes Yes/no
a AairI, absorbance of air in the solvent sector; AairII, absorbance of air in the solution sector; AchI, absorbance of the chitosan solution in the solvent sector; AchII, absorbance of the chitosan solution in the solution sector; Aalg, absorbance of the alginate solution; and Amem, absorbance of the membrane.
signal\0 (range 4) at the position of the boundary indicating the membrane formation (Fig. 1(b)). To answer the question to which extent the turbidity of the membrane contributes to the absorbance signal requires further detailed studies. The layering is completed when the solution meniscus in both sectors has reached the same radial position (Fig. 1(c)). The decrease of the absorbance in the chitosan I/chitosan II range reflects the consumption of chitosan (range 3 in Fig. 1(c)). In some cases, dependent on the reaction conditions, range 5 can partly or completely disappear at the end of a long-time experiment (compare Fig. 9). The simultaneous detection by the interference optics is not specific to one of the components. The scans monitor refractive index differences from the two cell sectors. For our system the refractive index signal was composed from the contributions of the sodium alginate, chitosan, water, and sodium chloride which was already added to the polymer solutions or results from the reaction of the oppositely charged polymers corresponding to: (sodium–polyanion) + (polycation – chloride) (polyanion – polycation) + sodium chloride. Though the interference optics is not specific to one of the components, it will be shown below that the information therefrom can support the evaluation of the absorption scans and contribute to the general understanding of the membrane formation and kinetics.
3. Results and discussion Membrane formation is influenced by various factors, some of which are experimentally defined. These include the type of the polymers, their structural and macromolecular characteristics, their concentration, the ratio polyanion to polycation, the pH, and the ionic strength. Others, such as the total polymer solution volume, the wavelength, the run velocity, the temperature, or scan resolution are limited in their variability by equipment specifications. Since membrane formation using an analytical ultracentrifuge has, to our knowledge, not been reported before, this paper develops, and optimizes, the method.
3.1. Methological aspects 3.1.1. Polyanion to polycation ratio The polyanion to polycation ratio determines the number of possible network links in the polyelectrolyte complex, whereas the network structure (swollen or collapsed) primarily depends on the local charge density. The latter varies with the chemical structure of the polymer, the solution concentration and the pH for non-permanently charged polyelectrolytes [1,22,23]. The membrane thickness and its structure have been regulated in our experiment by the molecular characteristics and the concentration of the polyanion, sodium alginate, as well as the polycation, chitosan. In addition, the added total amount of chitosan plays an important role. By varying the volume of the chitosan solution, as well as its concentration, the complex formation
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reaction time can be varied between 5 and 60 min. Less chitosan should, in general, result in thinner membranes under otherwise constant conditions. However, the geometry of the synthetic boundary centerpiece reduces the possible range of manipulation of the polyanion to polycation ratio. Both the solvent and the solution volume can regulate this ratio. Since the sedimentation force depends on the radial position, the filling height was kept constant for the polyanion solution during the method development in order to minimize the number of variables. The volume limit of the polycation solution results from the total volume of the sectors. For example, for a 100 ml polyanion solution the maximum volume for layering is 170 ml (before layering 440 ml polycation solution in the solvent sector and 100 ml polyanion solution in the solution sector, after layering: 270 ml in both sectors). If the polyanion solution is increased to 150 ml then the maximum for layering will be 145 ml.
3.1.2. Surface meniscus and run 6elocity Absorption scans can have a meniscus peak. This would influence the detection of the membrane growth at the meniscus interface. Therefore, the occurrence of such a meniscus peak dependent on the solution concentration, the wavelength and the spin velocity was studied for the components sodium alginate and chitosan. For this purpose, three standard double sector cells were filled on the solution side with three alginate solutions having different concentrations. The cells were placed into a 4-hole rotor for simultaneous measurements of the three concentrations. All solvent sectors were filled with the same chitosan solution. Absorption scans, resulting from the difference Aalg −Ach, are summarized in Fig. 2(a–f) for various run conditions. The differences of the radial positions for the three concentrations result from slightly different volumes in the solution sectors. The meniscus peak decreases in size with increasing velocity (Fig. 2(a – c)) and decreasing wavelength, eventually disappearing. However, the signal becomes noisier at lower wavelengths (Fig. 2(d–e)). Therefore, an optimum with respect to the signal-to-noise rate had to be defined.
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Additionally, a sedimentation force should be applied where the polycation solution passes the capillary between the two cell sectors sufficiently fast, while avoiding the sedimentation of any component. Table 2 summarizes the peak evaluation dependent on velocity and wavelength. For the system investigated an optimum is assigned to a velocity range 3000–8000 rpm and a wavelength range 360–380 nm. Interestingly, in all cases when a stable membrane was formed, it did not sediment even at run velocities of 20 000 rpm (Fig. 3). Fig. 3 was taken after approximately 2 h of reaction time at a run velocity 20 000 rpm. Even the deceleration, removal of the cell from the rotor and cell handling for the photograph did not change the vertical position of the flat membrane in the centerpiece sector implying that analytical centrifugation can be used to examine growth and diffusion kinetics. The membrane was also stable over days permitting further off-line characterization.
3.2. Membrane formation process Once the experiment had begun, the membrane formation process could be divided into three phases: 1. Layering and initial membrane formation.
Fig. 2. Solution meniscus detection as a function of sodium alginate concentration, run velocity and wavelength. Sodium alginate concentration: — , 2%;-- -, 1.5%; ···, 1.0%; (a) 3000 rpm, 380 nm; (b) 5000 rpm, 380 nm; (c) 12 000 rpm, 380 nm; (d) 3000 rpm, 360 nm; (e) 5000 rpm, 360 nm; (f) 10 000 rpm, 360 nm.
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Table 2 Evaluation of the meniscus peak of the absorption scans for various run velocities and wavelengths (sodium alginate 1.5% in 0.9% NaCl)a Velocity (rpm)
Wavelength (nm) 380
3000 5000 8000 10 000 20 000 a
360
350
h
w
h
w
0.76 0.42 0.25 :0.05 Noise B0.05 Noise
100 70 60
:0.1 Noise :0.15 Noise No experiment :0.1 Noise −0.3 80
h
340 w
No experiment No experiment No experiment No experiment −0.6 60
h
w
:0.4 Noise :0.5 Noise No experiment No experiment No experiment
h, High of the meniscus peak (absorbance units); w, width of the meniscus peak (mm); noise is provided in absorbance units.
2. Consumption of both polyions (membrane growth) until one component is completely bound in the complex, immobilized inside the membrane, or can no longer come in contact with the second polyion. 3. Membrane equilibration by diffusion of one or both components. Under certain conditions a destabilization of the membrane occurs with time. Figs. 4 –7 illustrate the steps involved in membrane formation, as observed by AUC. Fig. 4 provides the absorbance and interference scans taken immediately after increasing the velocity to 5000 rpm (2 min). The interference scan is very fast (seconds) and is always detected at the onset. However, since minutes are required to scan the total radius range of the centerpiece sector (radial position 5.8 – 7.2 cm) by the absorption optics with a step width of 10 mm, the absorbance appears with a time delay (ca. 6 min). During this time, the meniscus in the solvent (polycation) sector moved from a radial position 5.989 to 6.047 cm. Therefore, it is recommended to scan only a range close to the meniscus of the alginate solution if rapid replication is desired or is necessary, in order to follow membrane growth. To record a radius range of 2.5 mm requires approximately 1 min. For systematic studies of the membrane formation process such a limited range has been monitored. Fig. 4 serves as a demonstration of the experimental principle and in order to provide, in addition, the five ranges
corresponding to Fig. 1(b) for both the absorption and interference measurements. Fig. 5 demonstrates the progress of the membrane formation process during the 21 min following the polycation–polyanion contact. The polycation consumption at the membrane surface (on the left side of the membrane peak) is very fast during a period where the initial membrane is formed spontaneously. After 12 min the membrane growth slows, probably regulated by the diffusion of the components through the initial membrane.
Fig. 3. Membrane location in the right (here upper) centerpiece sector of a synthetic boundary cell after completing the experiment.
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Fig. 4. Absorption (—, A) and interference (···, I) scan after starting layering at 5000 rpm, 370 nm and 20°C. The scans were taken with a time shift of approximately 6 min caused by the slowness of the UV/vis scanning. During this time the meniscus of the citosan solution moved from the radial position 5.989 cm (I scan) to the radial position 6.047 cm (UV/vis scan). The scan ranges 1–5 correspond to Fig. 1(b).
As is shown in Fig. 6 the membrane grows as long as chitosan is available. For the selected experimental conditions the membrane widens (membrane thickness equals the width of the scan peak at a given time) in both the top (left) and bottom (right) directions. Furthermore, the absorbance increases at the maximum and decreases in range 3. It has to be mentioned that the main growth direction was found to be dependent on experimental conditions. When all chitosan is reacted the membrane starts to equilibrate, extending in one preferred
Fig. 5. Initial membrane growth at 5000 rpm, 370 nm and 20°C.
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Fig. 6. Membrane growth during the progress of layering and consumption of the polycation at 5000 rpm, 370 nm and 20°C.
direction (Fig. 7). This can be in the direction of the top (left) or the bottom (right) of the cell and is dependent on the molecular characteristics of the reacting polyelectrolytes and the particular polyanion and polycation concentration. At higher run velocities the sedimentation force may influence this direction. Under certain conditions this equilibration can result in structures consisting of two phases, one of which is very compact whereas the second one is like a week gel. Fig. 8 shows the microscopic photograph of such a two-phase complex structure taken from another experiment than that shown in Figs. 4–7 after more than 22 h. The photo demonstrates that the scans of Fig. 9 corre-
Fig. 7. Membrane equilibration after the complete polycation consumption at 5000 rpm, 370 nm and 20°C.
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Fig. 8. Photomicrograph after 22 h 49 min reaction time of 1% sodium alginate and 1% chitosan in 0.9% NaCl solution at 5000 rpm, 20°C, and approximately 30 min storage in 0.9% NaCl.
spond to real polyelectrolyte complex structures of different optical density and absorbance, respectively. The appropriate absorption and interference scans are summarized in Fig. 9. Both plots represent ranges 3 (absorbance B0) and 4 (absorbance \ 0) of the schematic in Fig. 1(c). Range 3, from the origin to 6.878 cm (UV/vis scan) corresponds to the layered chitosan solution. For both scans, UV/vis and interference, the signal is parallel to the x-axis indicating no change in chitosan concentration (UV) and refractive index (interference). Only close to the mem-
brane surface (6.878 cm) does the interference signal slightly increase, probably due to minor differences in the salt concentration. The steep increase of the absorbance at 6.882 cm corresponds to the well-defined membrane surface in Fig. 8. After the maximum at 6.940 cm the absorbance decreases and levels off to the bottom, however, always detecting some chitosan/turbidity. This intercept with the signal still at \0 corresponds to the less compact membrane part in Fig. 8. In this case the extension occurred into the bottom direction of the cell sector. Range 5 not longer exists. Additionally, both plots show their maxima at the same radial position. This can be correlated to the most dense structure but needs, however, evidence by complementary methods. The interference plot, which is less specific for this multi-component system, can be used in the presented case for data interpretation only in combination with the UV/vis plot. Nevertheless, it confirms the principal ranges of the last-mentioned plot. Figs. 8 and 9 are presented to demonstrate the agreement between scans and microscopic visualization for an extreme case. It has to be emphasized that Figs. 4–9 are selected as examples for the various phases of the membrane formation process and do not correspond to a desired optimal membrane. The occurrence and the extent of the specific phases strongly depend on the experimental conditions. This holds in particular for the growth direction, the membrane thickness, its equilibration, and the phase separation. While Figs. 4–6 correspond to the common membrane formation process, the results shown in Figs. 7–9 are more rare cases. The latter are, in general, undesirable for practical membrane applications. The membrane production process has to be stopped, for example, by washing procedures.
3.3. E6aluation of the scans
Fig. 9. Absorption (—, A) and interference (···, I) scan of the reaction product, which is shown in Fig. 8. Ranges (a) 3 and 4 correspond to Table 1. Range 4 is extended to the cell bottom.
Various levels of information can be concluded from the absorption (A) and interference (I) scans. These include, in principle, the velocity of layering (I), the chitosan concentration (A), the membrane and solution positions (A, I), the membrane thickness (A, I), the membrane growth
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Fig. 10. Interference scans for the calculation of the layering velocity, scan time in min. Inset: time dependent change of the radial position of the menisci in both cell sectors. , Solvent sector; , solution sector.
direction (A, I), the membrane growth velocity (A, I), and the membrane structure (A, I). Italic letters denote scans whose information are only qualitatively or not completely clear and needs more experimental data as well as the comparison with data from other polyanion/polycation systems. It is expected that the combination of the information delivered by the two optics will give quantitative results when more data are available. An example of the detection of the layering velocity is presented in Fig. 10. The movement of the signal jumps at the position of the meniscus of the chitosan solutions in the solvent and the solution sectors can be taken as measures for the layering velocity. After starting the layering the first-mentioned meniscus moves in the direction of the bottom from 6.095 to 6.270 cm within 12 min, the second one in the opposite direction from 6.773 to 6.608 cm. The meniscus movements are provided in the insert of Fig. 10. The meniscus air/chitosan in the solvent sector (lower curve in the inset of Fig. 10) is recommended for velocity evaluation because of its more precise detection. Furthermore, no influence of mixing or complex formation occurs in this sector. Because of the trapeziform cross-section of the sectors the meniscus movement is not expected to be linear. It does decrease slightly with increasing radius. However, from the known
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sector geometry (2.5° sector angle) the volume can be calculated [38]. For the experiment shown in Fig. 10 (sodium alginate/chitosan) a layering velocity of 4.9 ml min − 1 results. In general the layering velocity depends on the run velocity, the capillary diameter, and the solution properties of the solution to be layered. For the membrane formation experiments a variation of the capillary diameter is recommended in order to realise similar velocities for varying solution properties. Information about the layering velocity can also be got from the absorption scans. Since absorption scans, which record a broad radius range, require more time fast repetitions are limited. From the layering velocity the amount of available polycation at a given time can be calculated. Together with the detection of the non-reacted chitosan conclusions about the total amount of complexed chitosan seem to be possible. A variety of factors such as concentration or porosity of the initial membrane influences the chitosan consumption. In particular since the diameter of the capillaries of the synthetic boundary centerpieces varies, the determination of the layering velocity and therefrom the available chitosan is recommended for each experiment. The membrane, as well as solution positions, are readable from the signal jumps of both scan types (compare, for example, Fig. 4). The membrane dimensions become more precisely visible from the absorption plot rather than from the interference signal to which contributes, in particular in the fast initial formation phase, the low molar mass salt. Initial membrane thickness has been estimated in the range 50–300 mm. It is strongly influenced by the experimental conditions. The growth velocity of the membrane may be evaluated by various means, separately for the two possible growth directions or as the total growth. The first seems to be more informative. Such an evaluation is shown in Fig. 11 for the data taken from Figs. 5–7. Additionally, the growth above a certain absorbance value is useful to evaluate the compact membrane part separately from a more gel-like structure. Therefore, the movements of the scan fronts at one ab-
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sorbance unit are also plotted in Fig. 11. It should be noted that the determination of the total membrane thickness, immediately after the contact of the polycation and the polyanion solution, is accompanied by some difficulties. It seems that initially the formation of a more loose membrane structure in the direction into the chitosan solution occurs (compare the scan after 9 min in Fig. 5) followed by a contraction which results in a better defined membrane peak (after 12 min in Fig. 5). This observation requires more detailed studies. For the investigated growth period later then 12 min, the curvatures of the total membrane growth and the growth at absorbance one are similar for both growth directions, thought the growth is asymmetrical. After a total period of approximately 90 min the membrane starts to extend further in the top direction, whereas in the opposite direction the growth velocity decreases. In particular the increase of the compact membrane part seems to be finished on the bottom side. The 90 min correspond to the time when all chitosan has reacted (compare Fig. 7). The preferred growth direction is clearly visible. The small difference between the total peak broadening and the central part indicates that there is probably no, or only minor, gel formation on the membrane surface during the 150 min studied. The quantitative evaluation of the membrane structure is more complicated. The absorbance
signal results from the absorption of chitosan and that of the chitosan containing hydrogel polyelectrolyte complex membrane. Additionally, the polydispersity of the chitosan leads to weak molar mass dependent absorbance. At present only the qualitative comparison of membrane structures which have been produced under different experimental conditions seems reasonable. Fig. 12 presents such a comparison. Under otherwise constant conditions the polyanion concentration has been changed from 1.0 to 1.5% in 0.9% NaCl solution. The scans taken after 20 and 45 min clearly show the more asymmetric plots for the higher alginate concentration but, however, similar maximum intensities. For a quantitative conclusion comparison with structure information from other methods, for example microscopy, also have to be considered. Additional experimental data will likely improve the scan evaluation regarding the membrane structure.
3.4. Reproducibility and sensiti6ity Repeated runs, under equivalent experimental conditions, have been performed in order to test the reproducibility and the sensitivity of the membrane experiments. Fig. 13 provides an example of the reproducibility after different reaction times. After 25 min reaction time (Fig. 13(a)) membrane thickness is well reproducible. The small differences in the
Fig. 11. Evaluation of the membrane growth. Membrane growth as a function of the reaction time. , Total growth into the chitosan solution; , growth at one absorbance unit into the chitosan solution; , total growth into the sodium alginate solution;
, growth at one absorbance unit into the sodium alginate solution.
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Fig. 12. Evaluation of the membrane structure for the reaction of 1.0% chitosan in 0.9% NaCl solution with different sodium alginate concentrations in 0.9% NaCl solution at 20 000 rpm, 380 nm and 20°C. (a) 1.0% Sodium alginate: — , 20 min;-- -, 45 min. (b) 1.5% Sodium alginate: —, 20 min; ···, 45 min.
chitosan concentration on the left side of the membrane peak (radius B6.85 cm) result probably from fluctuations during the layering. These differences disappear with chitosan consumption (Fig. 13(b)). The experiments gave good reproducibility concerning the membrane thickness and its symmetry if the variation of the layering velocity was less than 20%. The smoothness of the curves is limited by the stepwidth of the detection, which has its minimum at 10 mm. That the experimental result is very sensitive to any change is demonstrated by the third plot in Fig. 13. For this experiment another polycation solution was applied having slightly different macromolecular characteristics (Mn = 3000 g mol − 1 and Mn/Mw =1.8 instead of Mn =2800 g mol − 1 and Mn/Mw =1.7). This resulted in changes of the membrane thickness and symmetry demonstrating the sensitivity of the method to variations of the polymer characteristics. Therefore, AUC can be used to optimize polymer selection for certain applications such as cell encapsulation or drug delivery systems.
4. Summary It was shown that the synthetic boundary technique of analytical ultracentrifugation could be
employed for the study of membrane formation from oppositely charged water-soluble polyelectrolytes. To the authors’ knowledge there is no other technique which can visualize on-line membrane growth and properties such as membrane thickness, symmetry, or stability in this way. The membrane formation occurs at the well-defined
Fig. 13. Reproducibility and sensitivity of the membrane formation experiments. 1.5% Sodium alginate and 1.0% chitosan in 0.9% NaCl solution at 5000 rpm and 20°C. — And ···, chitosan Mn =2800 g mol − 1 and Mw/Mn =1.7;-- -, chitosan Mn 3000 g mol − 1 and Mw/Mn =1.8. Reaction time (a) 25 min, (b) 70 min. For a better comparison the curves from the three independent experiments have been shifted in order to compensate the different radial positions of the menisci.
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flat interface of the polyanion solution while it comes in contact with the layered polycation solution. Since the membrane properties are dependent on the macromolecule characteristics, which can be distinguished by UV/vis and Rayleigh interference optics, AUC provides the means to optimize the polymer characteristics for a desired membrane. Time dependent experiments allow the investigation of various phases including membrane formation, equilibration, and aging. The duration of the several phases can be significantly influenced by the variation of the polyanion to the polycation ratio. The new method is, in particular, applicable to more concentrated systems which are of practical interest, for example, in biotechnology. This paper also demonstrates the applicability of the method under extreme conditions. Overall, AUC can be used to rapidly distinguish between polymers, according to properties such as molar mass and molar mass distribution, in terms of ideal membrane properties. This advance is likely to be of fundamental importance as well as accelerating development time of products such as those in drug delivery and tissue engineering. The results showing the influence of the polymer concentrations, the pH, the ionic strength, and the temperature on the membrane formation of sodium alginate and chitosan will be discussed in a subsequent paper.
Acknowledgements We thank D. Hunkeler, Laboratory of Polyelectrolytes and BioMacromolecules, Swiss Federal Institute of Technology, Lausanne, for critical and helpful discussions. A. Rehor is acknowledged for the photomicrographs. Furthermore, the authors gratefully acknowledge financial support by the Swiss Federal Institute of Technology.
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[2] D. Hunkeler, Trends Polym. Sci. 5 (1997) 286. [3] D.E. Awrey, M. Tse, G. Hortelano, P.L. Chang, Biotechnol. Bioeng. 52 (1996) 472. [4] P.L. Chang, Transfus. Sci. 17 (1996) 35. [5] Y. Kikuchi, N. Kubota, K. Maruo, Y. Goto, Makromol. Chem. 188 (1987) 2631. [6] S.D. Mundkur, J.C. Watters, Sep. Sci. Technol. 28 (1992) 1157. [7] H.H. Schwarz, K. Richau, D. Paul, Polym. Bull. 25 (1991) 95. [8] I. Schroeter, H. Roedicker, N. Radke, Chem. Technol. 44 (1992) 172. [9] H. Dautzenberg, F. Loth, W. Wagenknecht, B. Philipp, Das Papier 39 (1985) 601. [10] Y. Kikuchi, N. Kubuta, J. Appl. Polym. Sci 30 (1985) 2565. [11] Y. Kikuchi, N. Kubota, Makromol. Chem. Rapid Commun. 9 (1988) 727. [12] F. Lim, A.M. Sun, Science 210 (1980) 908. [13] C.J. Kim, P.I. Lee, Pharm. Res. 9 (1992) 1268. [14] M. Goetze, H. Torner, P. Kauffold, I. Ke´nig, H. Dautzenberg, F. Loth, Pharmazie 41 (1986) 250. [15] M.M. Daly, D. Knorr, Biotechnol. Prog. 4 (1988) 76. [16] J.E. Loefroth, L. Johansson, A.C. Norman, K. Wettstro¨m, Prog. Colloid Polym. Sci. 84 (1991) 78. [17] Y. Kawashima, T. Handa, A. Kasai, H. Takenaka, S. Lin, Y. Ando, J. Pharm. Sci. 74 (1985) 264. [18] C.J. Davison, K.E. Smith, L.E.F. Hutchinson, J. O’Mullane, J. Bioact. Compat. Polym. 5 (1990) 267. [19] K. Kono, F. Tabata, T. Takagishi, J. Membr. Sci. 76 (1993) 233. [20] A.B. Scranton, B. Rangarajan, J. Klier, Adv. Polym. Sci. 122 (1995) 1. [21] A. Prokop, D. Hunkeler, A.C. Powers, R.R. Whitesell, T.G. Wang, Adv. Polym. Sci. 136 (1998) 53. [22] B. Philipp, H. Dautzenberg, K.-J. Linow, J. Ko¨tz, W. Dawydoff, Prog. Polym. Sci. 14 (1989) 91. [23] J. Mikulik, Z. Vinkla´rek, M. Vondruska, Collect. Czech. Chem. Commun. 58 (1993) 713. [24] R. Daniels, E.M. Mittermaier, J. Microencapsul. 12 (1995) 591. [25] Y. Kikuchi, N. Kubota, K. Maruo, Y. Goto, Macromol. Chem. 188 (1987) 2631. [26] A. Bartkowiak, D. Hunkeler, Chem. Mater. 12 (2000) 206. [27] H. Arabi, S.A. Hashemi, M. Fooladi, J. Microencapsul. 13 (1996) 527. [28] J. Lukas, K. Richau, H.-H. Schwarz, D. Paul, J. Membr. Sci. 131 (1997) 39. [29] N. Karibyants, H. Dautzenberg, H. Co¨lfen, Macromolecules 30 (1997) 7803. [30] G. Ralston, Introduction to Analytical Ultracentrifugation, Beckman Instruments, Palo Alto, CA, 1993. [31] H. Co¨lfen, Polym. News 23 (1998) 152. [32] I.R. Mullagaliev, Pokl. Akad. Nauk 345 (1995) 199. [33] D.L. Kemper, J. Evers, in: C.H.W. Hirs, S.N. Timasheff
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Membrane formation at interfaces examined by analytical ultracentrifugation techniques Christine Wandrey *, Artur Bartkowiak 1 Laboratory of Polyelectrolytes and BioMacromolecules, Department of Chemistry, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland Received 10 April 2000; accepted 18 September 2000
Abstract For the first time, the formation of polyelectrolyte complex membranes has been examined by applying analytical ultracentrifugation’s synthetic boundary technique coupled with the twin scanning UV/vis and Rayleigh interference optics. Systematic basic studies demonstrate the efficiency of the new method, as well as its advantages and limitations. In particular, various phases of the membrane formation process have been identified. These include initial membrane formation, membrane growth, membrane equilibration, and, in some cases, phase separation or dissolution. The technique presented herein offers new and extended possibilities to study complex and/or structure formation by two components at interfaces dependent on polymer concentration, pH, ionic strength, and the component ratio. Temperature control, between 4 and 40°C, allows precise studies to be carried out under physiological conditions. Moreover, the membrane of defined thickness and structure and having an area of approximately 300 mm2, can be recovered from the cell for additional investigations. The polyanion sodium alginate and the polycation chitosan were employed as a model system in this investigation. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Polyelectrolyte complex; Hydrogel membrane; Analytical ultracentrifugation; Sodium alginate; Chitosan
1. Introduction Membrane formation, through the electrostatic interaction of oppositely charged polymers, has recently attracted increasingly scientific and commercial attention [1,2]. This is primarily due to the * Corresponding author. Tel.: +41-21-6933672; fax: + 4121-6935690. E-mail address: [email protected] (C. Wandrey). 1 Present address: Polymer Institute, Technical University of Szczecin, Pulaskiego 10, 70-322 Szczecin, Poland.
broad potential applications of the resulting membranes. Such materials may be employed in a variety of geometries including as components of flat devices or as self-contained hollow fibers, macrocapsules, or microcapsules [1–4]. Numerous combinations of anionic and cationic polylectrolytes have been examined in regards to their ability to form stable membranes having well defined characteristics. However, only a relatively few polymer pairs have been identified for this purpose [1,5–21]. Generally, the spontaneously
0927-7757/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 5 7 ( 0 0 ) 0 0 7 6 7 - 6
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created polyelectrolyte complex precipitates in aqueous solution without any stable macroscopic structure formation [22]. The ultimate properties of the polyelectrolyte complex depend on several parameters. These include both molecular characteristics of the polyelectrolyte molecules and conditions of the medium. The most influential molecular parameters are the charge density, the molar mass/chain length, the chemical structures of the charged groups and the polymer backbones, as well as the polydispersities in charge density and molar mass [1,21–25]. On the other hand, the concentration of the components, the pH of the solution, the ionic strength, and the temperature during the membrane formation can also strongly influence the key membrane properties including mechanical stability and permeability [26]. In particular, these two key parameters vary with the thickness and the structure of the membrane. It should also be noted that polyelectrolyte complex membranes are not static materials, and all of their properties are equilibrium characteristics, influenced by the environment’s electrochemical characteristics. Various analytical methods have been employed to study polyelectrolyte complex formation [1,22]. The complex stoichiometry, the membrane structure and surface characteristics have been investigated extensively by scattering methods, microscopic techniques, and surface scanning methods [1,22,27,28]. Analytical ultracentrifugation (AUC) has been employed for the characterization of polyelectrolyte complexes though not in terms of membrane formation [29]. In order to investigate the membrane formation process, through the electrostatic interaction of oppositely charged polyelectrolytes, we have adopted AUC-based techniques [30]. This paper presents the experimental principle on the basis of the synthetic boundary technique. Inside the analytical ultracentrifuge membrane formation, growth kinetics, and equilibration can be detected on-line. Basic investigations illustrate the efficiency of the method, its advantages and limitations. The polyanion sodium alginate and the polycation oligochitosan were used as a model system. Separation properties for membranes of this specific polyelectrolyte combination have
been presented recently [26]. Subsequent publications will report the influence of a broad variety of experimental influences on the membrane formation and other specific polyelectrolyte combinations.
2. Experimental
2.1. Equipment An Optima XL-I analytical ultracentrifuge (Beckman, Palo Alto, CA) was used for all the experiments. This instrument is designed to detect, measure, record, and analyze the movement of molecules in solution under a defined centrifugal field. It included two integrated detection systems, scanning UV/vis and Rayleigh interference optics. The scanning UV/vis detection operated in wavelength or radial scanning mode within a wavelength range 190–800 nm or a minimum radial increment of 0.001 cm, respectively. One absorbance reading could be performed approximately every 2 ×10 − 2 s. At the applied speed, in the range 3000–8000 rpm, absorbance readings could be performed every 60–120 s for a radial increment of 0.001 cm and a radial range 2–2.5 mm. The interference optics had a 30 mW laser (670 nm) as light source. The resolution of the detecting CCD camera was 2048× 96 pixels. Analytical ultracentrifugation offers numerous techniques to investigate the physico-chemical properties of macromolecules and colloids. Modern aspects using this equipment have been recently described [31].
2.2. Materials Sodium alginate (Keltone HV, lot 54650A, Kelco/NutraSweet, San Diego, CA) and degraded samples of chitosan (radical degradation [32] of raw material from E-055, Hutchinson/McNeil International, Philadelphia, PA) were been used as components for the membrane formation. The controlled radical degradation process, and the characterization of the resulting oligomers, have been described previously [26].
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2.3. Description of the experiments One of the principal techniques of the AUC is the synthetic boundary experiment. Special centerpieces for the analytical cells (each cylindrical analytical cell consists of a centerpiece, two window assemblies, and one cell housing) are available which allow solvent to be layered over a sample of a solution while the cell is spinning at moderately low speed (typically below 10 000 rpm). For this purpose the two sectors of the cylindrical double-sector centerpiece are connected on the upper surface with two capillary channels, one for the liquid and the second one for the back flow of air (see Fig. 1). These centerpieces are useful for preparing an artificial sharp boundary for measuring boundary spreading in measurements of diffusion coefficients. They can also be applied to examine sedimentation of small molecules (molar mass below 12 000 g mol − 1) for which the rate of sedimentation is insufficient to produce a sharp boundary that clears the meniscus [30]. In addition, the synthetic boundary technique has been used to monitor products from enzyme reactions [33], to detect crystallization in situ at the reaction boundary [34], to measure
Fig. 1. Principle of the membrane formation experiments in a synthetic boundary cell. Component positions and absorption scans (a): before layering, (b): during layering and (c): after layering is finished, though, chitosan is not yet completely complexed. , Air; a, chitosan I; b chitosan II; , membrane; b, alginate, …, capillary. Ranges 1–5 correspond to Table 1. The trapeziform cross-section of the two sectors is not shown.
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differential sedimentation coefficients [35], and to estimate extinction coefficients [36]. The centerpieces selected to study the membrane formation from oppositely charged polyelectrolytes were 12 mm high. This height represents the thickness of the liquid column that the light of the two optics will pass through. The standard sector length was 14 mm in radial direction, from sector top to sector bottom, with the capillary 6 mm from the bottom (compare the schematic in Fig. 1(a, b, c)). The maximum sector volume was 450 ml, however, for synthetic boundary experiments a solution volume of 150 ml is recommended for the solution sector [37]. The less viscous polycation solution possessing a lower density was layered over the higher viscous polyanion solution. The horizontal projection (view from above) of a synthetic boundary centerpiece and the principle of the experiment are illustrated in Fig. 1. Additionally, the appropriate absorbance plots are schematically presented in this figure. Each absorbance signal results from the difference of the absorbance measured in the solution sector (polyanion solution) and the one measured in the solvent sector (polycation solution). In principle five such differences are possible. However, various of these radial ranges will be detected dependent on the progress of the experiment. Table 1 summarizes the detection ranges during the experiment. The three radial ranges 1, 2, and 5 are expected before layering (Fig. 1(a)) whereas all five signal differences are visible during the layering (Fig. 1(b)). Finally, when the layering is finished the four ranges 1, 3, 4, and 5 exist (Fig. 1(c)). Since the absorbance of sodium alginate and air is negligible in comparison to the absorbance of oligochitosan, above 350 nm, a negative signal having the same value is detected for the total radius range of oligochitosan before the layering takes place (Fig. 1(a)). As soon as the polycation solution passes the capillary and is layered on the surface of the polyanion solution a spontaneous structure formation begins in the boundary zone. A chitosan concentration higher than that in the chitosan solution is detected by an absorbance
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Table 1 Absorbance signals during the experimentsa Range
Absorbance signal
Before layering
During layering
Finished layering
Long time experiment
1 2 3 4 5
AairII−AairI = 0 AairII−AchIB0 AchII−AchI50 Amem−AchI\0 Aalg−AchIB0
Yes Yes No No Yes
Yes Yes Yes Yes Yes
Yes No Yes Yes Yes
Yes No Yes Yes Yes/no
a AairI, absorbance of air in the solvent sector; AairII, absorbance of air in the solution sector; AchI, absorbance of the chitosan solution in the solvent sector; AchII, absorbance of the chitosan solution in the solution sector; Aalg, absorbance of the alginate solution; and Amem, absorbance of the membrane.
signal\0 (range 4) at the position of the boundary indicating the membrane formation (Fig. 1(b)). To answer the question to which extent the turbidity of the membrane contributes to the absorbance signal requires further detailed studies. The layering is completed when the solution meniscus in both sectors has reached the same radial position (Fig. 1(c)). The decrease of the absorbance in the chitosan I/chitosan II range reflects the consumption of chitosan (range 3 in Fig. 1(c)). In some cases, dependent on the reaction conditions, range 5 can partly or completely disappear at the end of a long-time experiment (compare Fig. 9). The simultaneous detection by the interference optics is not specific to one of the components. The scans monitor refractive index differences from the two cell sectors. For our system the refractive index signal was composed from the contributions of the sodium alginate, chitosan, water, and sodium chloride which was already added to the polymer solutions or results from the reaction of the oppositely charged polymers corresponding to: (sodium–polyanion) + (polycation – chloride) (polyanion – polycation) + sodium chloride. Though the interference optics is not specific to one of the components, it will be shown below that the information therefrom can support the evaluation of the absorption scans and contribute to the general understanding of the membrane formation and kinetics.
3. Results and discussion Membrane formation is influenced by various factors, some of which are experimentally defined. These include the type of the polymers, their structural and macromolecular characteristics, their concentration, the ratio polyanion to polycation, the pH, and the ionic strength. Others, such as the total polymer solution volume, the wavelength, the run velocity, the temperature, or scan resolution are limited in their variability by equipment specifications. Since membrane formation using an analytical ultracentrifuge has, to our knowledge, not been reported before, this paper develops, and optimizes, the method.
3.1. Methological aspects 3.1.1. Polyanion to polycation ratio The polyanion to polycation ratio determines the number of possible network links in the polyelectrolyte complex, whereas the network structure (swollen or collapsed) primarily depends on the local charge density. The latter varies with the chemical structure of the polymer, the solution concentration and the pH for non-permanently charged polyelectrolytes [1,22,23]. The membrane thickness and its structure have been regulated in our experiment by the molecular characteristics and the concentration of the polyanion, sodium alginate, as well as the polycation, chitosan. In addition, the added total amount of chitosan plays an important role. By varying the volume of the chitosan solution, as well as its concentration, the complex formation
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reaction time can be varied between 5 and 60 min. Less chitosan should, in general, result in thinner membranes under otherwise constant conditions. However, the geometry of the synthetic boundary centerpiece reduces the possible range of manipulation of the polyanion to polycation ratio. Both the solvent and the solution volume can regulate this ratio. Since the sedimentation force depends on the radial position, the filling height was kept constant for the polyanion solution during the method development in order to minimize the number of variables. The volume limit of the polycation solution results from the total volume of the sectors. For example, for a 100 ml polyanion solution the maximum volume for layering is 170 ml (before layering 440 ml polycation solution in the solvent sector and 100 ml polyanion solution in the solution sector, after layering: 270 ml in both sectors). If the polyanion solution is increased to 150 ml then the maximum for layering will be 145 ml.
3.1.2. Surface meniscus and run 6elocity Absorption scans can have a meniscus peak. This would influence the detection of the membrane growth at the meniscus interface. Therefore, the occurrence of such a meniscus peak dependent on the solution concentration, the wavelength and the spin velocity was studied for the components sodium alginate and chitosan. For this purpose, three standard double sector cells were filled on the solution side with three alginate solutions having different concentrations. The cells were placed into a 4-hole rotor for simultaneous measurements of the three concentrations. All solvent sectors were filled with the same chitosan solution. Absorption scans, resulting from the difference Aalg −Ach, are summarized in Fig. 2(a–f) for various run conditions. The differences of the radial positions for the three concentrations result from slightly different volumes in the solution sectors. The meniscus peak decreases in size with increasing velocity (Fig. 2(a – c)) and decreasing wavelength, eventually disappearing. However, the signal becomes noisier at lower wavelengths (Fig. 2(d–e)). Therefore, an optimum with respect to the signal-to-noise rate had to be defined.
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Additionally, a sedimentation force should be applied where the polycation solution passes the capillary between the two cell sectors sufficiently fast, while avoiding the sedimentation of any component. Table 2 summarizes the peak evaluation dependent on velocity and wavelength. For the system investigated an optimum is assigned to a velocity range 3000–8000 rpm and a wavelength range 360–380 nm. Interestingly, in all cases when a stable membrane was formed, it did not sediment even at run velocities of 20 000 rpm (Fig. 3). Fig. 3 was taken after approximately 2 h of reaction time at a run velocity 20 000 rpm. Even the deceleration, removal of the cell from the rotor and cell handling for the photograph did not change the vertical position of the flat membrane in the centerpiece sector implying that analytical centrifugation can be used to examine growth and diffusion kinetics. The membrane was also stable over days permitting further off-line characterization.
3.2. Membrane formation process Once the experiment had begun, the membrane formation process could be divided into three phases: 1. Layering and initial membrane formation.
Fig. 2. Solution meniscus detection as a function of sodium alginate concentration, run velocity and wavelength. Sodium alginate concentration: — , 2%;-- -, 1.5%; ···, 1.0%; (a) 3000 rpm, 380 nm; (b) 5000 rpm, 380 nm; (c) 12 000 rpm, 380 nm; (d) 3000 rpm, 360 nm; (e) 5000 rpm, 360 nm; (f) 10 000 rpm, 360 nm.
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Table 2 Evaluation of the meniscus peak of the absorption scans for various run velocities and wavelengths (sodium alginate 1.5% in 0.9% NaCl)a Velocity (rpm)
Wavelength (nm) 380
3000 5000 8000 10 000 20 000 a
360
350
h
w
h
w
0.76 0.42 0.25 :0.05 Noise B0.05 Noise
100 70 60
:0.1 Noise :0.15 Noise No experiment :0.1 Noise −0.3 80
h
340 w
No experiment No experiment No experiment No experiment −0.6 60
h
w
:0.4 Noise :0.5 Noise No experiment No experiment No experiment
h, High of the meniscus peak (absorbance units); w, width of the meniscus peak (mm); noise is provided in absorbance units.
2. Consumption of both polyions (membrane growth) until one component is completely bound in the complex, immobilized inside the membrane, or can no longer come in contact with the second polyion. 3. Membrane equilibration by diffusion of one or both components. Under certain conditions a destabilization of the membrane occurs with time. Figs. 4 –7 illustrate the steps involved in membrane formation, as observed by AUC. Fig. 4 provides the absorbance and interference scans taken immediately after increasing the velocity to 5000 rpm (2 min). The interference scan is very fast (seconds) and is always detected at the onset. However, since minutes are required to scan the total radius range of the centerpiece sector (radial position 5.8 – 7.2 cm) by the absorption optics with a step width of 10 mm, the absorbance appears with a time delay (ca. 6 min). During this time, the meniscus in the solvent (polycation) sector moved from a radial position 5.989 to 6.047 cm. Therefore, it is recommended to scan only a range close to the meniscus of the alginate solution if rapid replication is desired or is necessary, in order to follow membrane growth. To record a radius range of 2.5 mm requires approximately 1 min. For systematic studies of the membrane formation process such a limited range has been monitored. Fig. 4 serves as a demonstration of the experimental principle and in order to provide, in addition, the five ranges
corresponding to Fig. 1(b) for both the absorption and interference measurements. Fig. 5 demonstrates the progress of the membrane formation process during the 21 min following the polycation–polyanion contact. The polycation consumption at the membrane surface (on the left side of the membrane peak) is very fast during a period where the initial membrane is formed spontaneously. After 12 min the membrane growth slows, probably regulated by the diffusion of the components through the initial membrane.
Fig. 3. Membrane location in the right (here upper) centerpiece sector of a synthetic boundary cell after completing the experiment.
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Fig. 4. Absorption (—, A) and interference (···, I) scan after starting layering at 5000 rpm, 370 nm and 20°C. The scans were taken with a time shift of approximately 6 min caused by the slowness of the UV/vis scanning. During this time the meniscus of the citosan solution moved from the radial position 5.989 cm (I scan) to the radial position 6.047 cm (UV/vis scan). The scan ranges 1–5 correspond to Fig. 1(b).
As is shown in Fig. 6 the membrane grows as long as chitosan is available. For the selected experimental conditions the membrane widens (membrane thickness equals the width of the scan peak at a given time) in both the top (left) and bottom (right) directions. Furthermore, the absorbance increases at the maximum and decreases in range 3. It has to be mentioned that the main growth direction was found to be dependent on experimental conditions. When all chitosan is reacted the membrane starts to equilibrate, extending in one preferred
Fig. 5. Initial membrane growth at 5000 rpm, 370 nm and 20°C.
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Fig. 6. Membrane growth during the progress of layering and consumption of the polycation at 5000 rpm, 370 nm and 20°C.
direction (Fig. 7). This can be in the direction of the top (left) or the bottom (right) of the cell and is dependent on the molecular characteristics of the reacting polyelectrolytes and the particular polyanion and polycation concentration. At higher run velocities the sedimentation force may influence this direction. Under certain conditions this equilibration can result in structures consisting of two phases, one of which is very compact whereas the second one is like a week gel. Fig. 8 shows the microscopic photograph of such a two-phase complex structure taken from another experiment than that shown in Figs. 4–7 after more than 22 h. The photo demonstrates that the scans of Fig. 9 corre-
Fig. 7. Membrane equilibration after the complete polycation consumption at 5000 rpm, 370 nm and 20°C.
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Fig. 8. Photomicrograph after 22 h 49 min reaction time of 1% sodium alginate and 1% chitosan in 0.9% NaCl solution at 5000 rpm, 20°C, and approximately 30 min storage in 0.9% NaCl.
spond to real polyelectrolyte complex structures of different optical density and absorbance, respectively. The appropriate absorption and interference scans are summarized in Fig. 9. Both plots represent ranges 3 (absorbance B0) and 4 (absorbance \ 0) of the schematic in Fig. 1(c). Range 3, from the origin to 6.878 cm (UV/vis scan) corresponds to the layered chitosan solution. For both scans, UV/vis and interference, the signal is parallel to the x-axis indicating no change in chitosan concentration (UV) and refractive index (interference). Only close to the mem-
brane surface (6.878 cm) does the interference signal slightly increase, probably due to minor differences in the salt concentration. The steep increase of the absorbance at 6.882 cm corresponds to the well-defined membrane surface in Fig. 8. After the maximum at 6.940 cm the absorbance decreases and levels off to the bottom, however, always detecting some chitosan/turbidity. This intercept with the signal still at \0 corresponds to the less compact membrane part in Fig. 8. In this case the extension occurred into the bottom direction of the cell sector. Range 5 not longer exists. Additionally, both plots show their maxima at the same radial position. This can be correlated to the most dense structure but needs, however, evidence by complementary methods. The interference plot, which is less specific for this multi-component system, can be used in the presented case for data interpretation only in combination with the UV/vis plot. Nevertheless, it confirms the principal ranges of the last-mentioned plot. Figs. 8 and 9 are presented to demonstrate the agreement between scans and microscopic visualization for an extreme case. It has to be emphasized that Figs. 4–9 are selected as examples for the various phases of the membrane formation process and do not correspond to a desired optimal membrane. The occurrence and the extent of the specific phases strongly depend on the experimental conditions. This holds in particular for the growth direction, the membrane thickness, its equilibration, and the phase separation. While Figs. 4–6 correspond to the common membrane formation process, the results shown in Figs. 7–9 are more rare cases. The latter are, in general, undesirable for practical membrane applications. The membrane production process has to be stopped, for example, by washing procedures.
3.3. E6aluation of the scans
Fig. 9. Absorption (—, A) and interference (···, I) scan of the reaction product, which is shown in Fig. 8. Ranges (a) 3 and 4 correspond to Table 1. Range 4 is extended to the cell bottom.
Various levels of information can be concluded from the absorption (A) and interference (I) scans. These include, in principle, the velocity of layering (I), the chitosan concentration (A), the membrane and solution positions (A, I), the membrane thickness (A, I), the membrane growth
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Fig. 10. Interference scans for the calculation of the layering velocity, scan time in min. Inset: time dependent change of the radial position of the menisci in both cell sectors. , Solvent sector; , solution sector.
direction (A, I), the membrane growth velocity (A, I), and the membrane structure (A, I). Italic letters denote scans whose information are only qualitatively or not completely clear and needs more experimental data as well as the comparison with data from other polyanion/polycation systems. It is expected that the combination of the information delivered by the two optics will give quantitative results when more data are available. An example of the detection of the layering velocity is presented in Fig. 10. The movement of the signal jumps at the position of the meniscus of the chitosan solutions in the solvent and the solution sectors can be taken as measures for the layering velocity. After starting the layering the first-mentioned meniscus moves in the direction of the bottom from 6.095 to 6.270 cm within 12 min, the second one in the opposite direction from 6.773 to 6.608 cm. The meniscus movements are provided in the insert of Fig. 10. The meniscus air/chitosan in the solvent sector (lower curve in the inset of Fig. 10) is recommended for velocity evaluation because of its more precise detection. Furthermore, no influence of mixing or complex formation occurs in this sector. Because of the trapeziform cross-section of the sectors the meniscus movement is not expected to be linear. It does decrease slightly with increasing radius. However, from the known
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sector geometry (2.5° sector angle) the volume can be calculated [38]. For the experiment shown in Fig. 10 (sodium alginate/chitosan) a layering velocity of 4.9 ml min − 1 results. In general the layering velocity depends on the run velocity, the capillary diameter, and the solution properties of the solution to be layered. For the membrane formation experiments a variation of the capillary diameter is recommended in order to realise similar velocities for varying solution properties. Information about the layering velocity can also be got from the absorption scans. Since absorption scans, which record a broad radius range, require more time fast repetitions are limited. From the layering velocity the amount of available polycation at a given time can be calculated. Together with the detection of the non-reacted chitosan conclusions about the total amount of complexed chitosan seem to be possible. A variety of factors such as concentration or porosity of the initial membrane influences the chitosan consumption. In particular since the diameter of the capillaries of the synthetic boundary centerpieces varies, the determination of the layering velocity and therefrom the available chitosan is recommended for each experiment. The membrane, as well as solution positions, are readable from the signal jumps of both scan types (compare, for example, Fig. 4). The membrane dimensions become more precisely visible from the absorption plot rather than from the interference signal to which contributes, in particular in the fast initial formation phase, the low molar mass salt. Initial membrane thickness has been estimated in the range 50–300 mm. It is strongly influenced by the experimental conditions. The growth velocity of the membrane may be evaluated by various means, separately for the two possible growth directions or as the total growth. The first seems to be more informative. Such an evaluation is shown in Fig. 11 for the data taken from Figs. 5–7. Additionally, the growth above a certain absorbance value is useful to evaluate the compact membrane part separately from a more gel-like structure. Therefore, the movements of the scan fronts at one ab-
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sorbance unit are also plotted in Fig. 11. It should be noted that the determination of the total membrane thickness, immediately after the contact of the polycation and the polyanion solution, is accompanied by some difficulties. It seems that initially the formation of a more loose membrane structure in the direction into the chitosan solution occurs (compare the scan after 9 min in Fig. 5) followed by a contraction which results in a better defined membrane peak (after 12 min in Fig. 5). This observation requires more detailed studies. For the investigated growth period later then 12 min, the curvatures of the total membrane growth and the growth at absorbance one are similar for both growth directions, thought the growth is asymmetrical. After a total period of approximately 90 min the membrane starts to extend further in the top direction, whereas in the opposite direction the growth velocity decreases. In particular the increase of the compact membrane part seems to be finished on the bottom side. The 90 min correspond to the time when all chitosan has reacted (compare Fig. 7). The preferred growth direction is clearly visible. The small difference between the total peak broadening and the central part indicates that there is probably no, or only minor, gel formation on the membrane surface during the 150 min studied. The quantitative evaluation of the membrane structure is more complicated. The absorbance
signal results from the absorption of chitosan and that of the chitosan containing hydrogel polyelectrolyte complex membrane. Additionally, the polydispersity of the chitosan leads to weak molar mass dependent absorbance. At present only the qualitative comparison of membrane structures which have been produced under different experimental conditions seems reasonable. Fig. 12 presents such a comparison. Under otherwise constant conditions the polyanion concentration has been changed from 1.0 to 1.5% in 0.9% NaCl solution. The scans taken after 20 and 45 min clearly show the more asymmetric plots for the higher alginate concentration but, however, similar maximum intensities. For a quantitative conclusion comparison with structure information from other methods, for example microscopy, also have to be considered. Additional experimental data will likely improve the scan evaluation regarding the membrane structure.
3.4. Reproducibility and sensiti6ity Repeated runs, under equivalent experimental conditions, have been performed in order to test the reproducibility and the sensitivity of the membrane experiments. Fig. 13 provides an example of the reproducibility after different reaction times. After 25 min reaction time (Fig. 13(a)) membrane thickness is well reproducible. The small differences in the
Fig. 11. Evaluation of the membrane growth. Membrane growth as a function of the reaction time. , Total growth into the chitosan solution; , growth at one absorbance unit into the chitosan solution; , total growth into the sodium alginate solution;
, growth at one absorbance unit into the sodium alginate solution.
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Fig. 12. Evaluation of the membrane structure for the reaction of 1.0% chitosan in 0.9% NaCl solution with different sodium alginate concentrations in 0.9% NaCl solution at 20 000 rpm, 380 nm and 20°C. (a) 1.0% Sodium alginate: — , 20 min;-- -, 45 min. (b) 1.5% Sodium alginate: —, 20 min; ···, 45 min.
chitosan concentration on the left side of the membrane peak (radius B6.85 cm) result probably from fluctuations during the layering. These differences disappear with chitosan consumption (Fig. 13(b)). The experiments gave good reproducibility concerning the membrane thickness and its symmetry if the variation of the layering velocity was less than 20%. The smoothness of the curves is limited by the stepwidth of the detection, which has its minimum at 10 mm. That the experimental result is very sensitive to any change is demonstrated by the third plot in Fig. 13. For this experiment another polycation solution was applied having slightly different macromolecular characteristics (Mn = 3000 g mol − 1 and Mn/Mw =1.8 instead of Mn =2800 g mol − 1 and Mn/Mw =1.7). This resulted in changes of the membrane thickness and symmetry demonstrating the sensitivity of the method to variations of the polymer characteristics. Therefore, AUC can be used to optimize polymer selection for certain applications such as cell encapsulation or drug delivery systems.
4. Summary It was shown that the synthetic boundary technique of analytical ultracentrifugation could be
employed for the study of membrane formation from oppositely charged water-soluble polyelectrolytes. To the authors’ knowledge there is no other technique which can visualize on-line membrane growth and properties such as membrane thickness, symmetry, or stability in this way. The membrane formation occurs at the well-defined
Fig. 13. Reproducibility and sensitivity of the membrane formation experiments. 1.5% Sodium alginate and 1.0% chitosan in 0.9% NaCl solution at 5000 rpm and 20°C. — And ···, chitosan Mn =2800 g mol − 1 and Mw/Mn =1.7;-- -, chitosan Mn 3000 g mol − 1 and Mw/Mn =1.8. Reaction time (a) 25 min, (b) 70 min. For a better comparison the curves from the three independent experiments have been shifted in order to compensate the different radial positions of the menisci.
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flat interface of the polyanion solution while it comes in contact with the layered polycation solution. Since the membrane properties are dependent on the macromolecule characteristics, which can be distinguished by UV/vis and Rayleigh interference optics, AUC provides the means to optimize the polymer characteristics for a desired membrane. Time dependent experiments allow the investigation of various phases including membrane formation, equilibration, and aging. The duration of the several phases can be significantly influenced by the variation of the polyanion to the polycation ratio. The new method is, in particular, applicable to more concentrated systems which are of practical interest, for example, in biotechnology. This paper also demonstrates the applicability of the method under extreme conditions. Overall, AUC can be used to rapidly distinguish between polymers, according to properties such as molar mass and molar mass distribution, in terms of ideal membrane properties. This advance is likely to be of fundamental importance as well as accelerating development time of products such as those in drug delivery and tissue engineering. The results showing the influence of the polymer concentrations, the pH, the ionic strength, and the temperature on the membrane formation of sodium alginate and chitosan will be discussed in a subsequent paper.
Acknowledgements We thank D. Hunkeler, Laboratory of Polyelectrolytes and BioMacromolecules, Swiss Federal Institute of Technology, Lausanne, for critical and helpful discussions. A. Rehor is acknowledged for the photomicrographs. Furthermore, the authors gratefully acknowledge financial support by the Swiss Federal Institute of Technology.
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