Analytica Chimica Acta 819 (2014) 116–121
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Asymmetrical flow field-flow fractionation analysis of water suspensions of polymer nanofibers synthesized via RAFT-mediated emulsion polymerization Julien Gigault c , Wenjing Zhang b , Gaetane Lespes a , Bernadette Charleux b , Bruno Grassl a,∗ a
Laboratoire de Chimie Analytique BioInorganique et Environnement, UMR IPREM 5254 UPPA/CNRS-Technopole Hélioparc, Université de Pau et des Pays de l’Adour (UPPA), Av. du Président Angot, 64053 Pau cedex, France b C2P2 (Chemistry, Catalysis, Polymers & Processes), CNRS, UMR 5265, CPE Lyon, Université Lyon 1, Université de Lyon, Team LCPP Bat 308 F, 43 Bd du 11 Novembre 1918, 69616 Villeurbanne, France c National Institute of Standards and Technology, Material Measurement Science Division, 100 Bureau Drive, Gaithersburg, 20899 MD, USA
h i g h l i g h t s • Flow-field-flow-fractionation to characterize self-assembled nanofibers (NFs). • Accurate NFs length and aggregation number per unit length determination. • Characterization of new self-assembled polymers with various architectures.
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Article history: Received 2 December 2013 Received in revised form 31 January 2014 Accepted 9 February 2014 Available online 12 February 2014 Keywords: Controlled/living radical polymerization Asymmetric flow field-flow fractionation Nanofiber Length Aggregation
a b s t r a c t The aim of this work is to present a method based on asymmetric flow-field-flow-fractionation coupled on-line to a static light scattering (AF4-UV-SLS) detector to characterize self-assembled nanofibers (NFs). The method developed herein allows the determination of both the length distribution of the NFs as well as the distribution in terms of aggregation number per unit length (Agg ). Given the remaining synthetic challenges of better controlling the structural homogeneity and particle dimensions, the NF length and aggregation number per unit length are becoming essential for the improvement and control of their chemical processes and a better understanding of their properties. The results obtained with this AF4-UV-SLS method indicate that a well-resolved NF length distribution characterization and Agg determination were attained. These results provide critical information concerning the physical properties of the investigated NFs and open the door to the characterization of new self-assembled polymers with various asymmetrical architectures. Published by Elsevier B.V.
1. Introduction With the development of polymerization-induced micellization or polymerization-induced self-assembly (PISA) based on controlled/living radical polymerization (CRP), the field of polymer architectures has expanded considerably in the last few years [1–4]. Recently, various morphologies, such as crew-cut micelles, vesicles, elongated micelles, and nanofibers (NFs), were shown to be easily synthesized in the aqueous phase via these emulsion and dispersion processes [5].
∗ Corresponding author. E-mail address: [email protected] (B. Grassl). http://dx.doi.org/10.1016/j.aca.2014.02.011 0003-2670/Published by Elsevier B.V.
Self-assembled amphiphilic block copolymer nanofibers can be synthesized directly in aqueous suspensions at high concentrations (>20 wt%) via PISA. As illustrated in Fig. 1, the use of water-soluble macromolecular reversible addition–fragmentation chain transfer (RAFT) agent in the emulsion polymerization of styrene enables fast polymerizations and high final conversions with the formation of well-defined amphiphilic block copolymers without residual hydrophilic reactive chains [6–9]. The final product is an in situ created suspension of self-assembled amphiphilic nano-objects, the morphologies of which can be tuned from micelles to nanofibers through variation of certain parameters, such as the pH and the molar mass of the hydrophilic and hydrophobic blocks. Because of the complexity and chemical heterogeneity of these new nano-objects based on polymers, one of the major challenges
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in analytical chemistry is their size characterization, especially in the case of samples with the NF morphology. The ability to obtain the length (L) distribution and the distribution of the aggregation number per unit length (Agg ) of NFs is becoming essential to improve and control their chemical processes and to better understand their properties. Aqueous suspensions of polymer nanofibers could be used for their viscoelastic properties [9–11], whereas dried nanofibers may find direct applications in materials science or biotechnology, including organ repair and the treatment of osteoporosis and various other diseases [12]. In general, self-assembled nanostructures of amphiphilic and double hydrophilic block copolymers have been increasingly utilized as potent polymeric nanocarriers of therapeutic drugs, genes, bioactive molecules, and imaging/contrast agents because of their improved water solubility, bioavailability, and extended blood circulation duration [13]. The remaining synthetic challenges are related to the better control of the structural homogeneity and of the particle dimensions, especially their length and Agg . The application of the process to a broader range of polymers, thereby allowing access to new properties, also appears to be extremely important [5,14]. Currently, the lengths of one-dimensional micelles are normally characterized using imaging techniques [15], such as transmission electron microscopy (TEM), [16] fluorescence microscopy [17], or atomic force microscopy (AFM) [18]. However, more information can be obtained from scattering techniques. Static light scattering (SLS) was used to determine the length of elongated structures, whether the structures are rigid or flexible, and the mass per unit length of the structures [19,20]. Small-angle X-ray scattering (SAXS) [21,22] and neutron scattering (SANS) [23] can also provide information about the mass per unit length as well as information about the size of the micellar cross-section. Finally, dynamic light scattering (DLS) has been widely used to evaluate the hydrodynamic cross-section of natural fibrils [24–29] and elongated micelles [29,30]. Even if both static and dynamic light scattering techniques are widely used in batch-mode, the polydispersity of the size and shape of the NFs leads to biased measurements and inaccurate results related to these physical parameters. One interesting analytical strategy consists of using hyphenated techniques. The coupling of a separation technique on-line to a static light scattering (SLS) technique allows for the fractionation of nanofibers according to one or several physical properties, followed by characterization. Size-exclusion chromatography (SEC) coupled to SLS can be used to characterize NFs such as carbon nanotubes [31–34]. Some studies have shown that SEC can lead to a reasonable size resolution; however, the exclusion limit of the SEC column, which is controlled by the pore size, restricts the NFs that can be separated to those shorter than 1 m. The asymmetric flow field-flow fractionation
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(AF4) technique may be a feasible alternative. The results obtained for NFs such as carbon nanotubes show that AF4 is a promising technique because of its versatility and size-resolution potential [35–43]. By coupling AF4 with static light scattering detectors, carbon nanotubes can be fractionated on the basis of their length and their dispersion states (i.e., individually dispersed CNT and aggregates) can be determined. However, to our knowledge, the fractionation of NFs by methods such as those proposed in this work has not been reported. From the perspective of recent development of PISA of amphiphilic block copolymers based on controlled/living free radical polymerization in water, we demonstrate in this work the highly promising capabilities of asymmetric flow field-flow fractionation for the accurate measurement of the distributions of the length and Agg of self-assembled NFs from their crude aqueous suspensions. The novel determination of the Agg demonstrated in this work could provide critical information on the physical properties of the investigated NFs. 2. Experimental 2.1. Chemical Ammonium nitrate (NH4 NO3 , 99.5%) was purchased from Sigma Aldrich. Milli-Q 18 M cm water (Millipore System) was used in this study. The mobile phase used in AF4 was filtered through Millipore Durapore 0.1 m filters. The polystyrene standard reference materials (PSL) (mass fraction of 1%) were manufactured by Thermo Scientific and were traceable in size according to the National Institute of Standards and Technology (NIST) in the United States. The identification of any commercial product or trade name does not imply endorsement or recommendation by NIST. 2.2. Samples Four crude aqueous suspensions of the NF were prepared and used via the RAFT-mediated emulsion polymerization of styrene (samples A, B1, and B2) or methyl methacrylate (sample C) in water (see Fig. 1), which was performed in the presence of a P(MAA-co-PEOMA) macroRAFT agent bearing a trithiocarbonate reactive group. The experimental conditions were based on the one-pot, two-step strategy reported elsewhere [6,7,9]. The characteristics of the nanofibers were tuned by changing the pH of the emulsion and the molar mass and composition of the macroRAFT agent and the initial concentration ratio of the macroRAFT agent to styrene or MMA. The molecular parameters of the amphiphilic block copolymers constitutive of the NFs and the structure of the latter confirmed by transmission electron microscopy (TEM)
Fig. 1. Schematic representation of the one-pot NF synthesis: chemical composition (P(MAA-co-PEOMA) stands for poly(methacrylic acid-co-poly(ethylene oxide) methyl ether methacrylate), PS for polystyrene and PMMA for poly(methyl methacrylate)) and length (L) and aggregation number (Agg ) characteristics analyzed by AF4.
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analysis (see supplemental information, Fig. S2), were described in a previous work [9] with the same experiment numbers. 2.3. Instrumentation An Eclipse 3 asymmetrical flow field-flow fractionation system (Wyatt Technology, Dernbach, Germany) was used in this study. The trapezoidal channel was 26.5 cm in length and 0.6 and 2.1 cm in width. Flow rates were controlled using an Agilent Technologies 1100 series isocratic pump equipped with a micro-vacuum degasser. All injections were performed with an autosampler (Agilent Technologies 1100 series). The detection chain consisted of a variable-wavelength UV–vis spectrophotometer (Agilent Technologies 1100 series from Agilent, Tokyo, Japan) that was tuned to 254 nm with a SLS detector (DAWN HELEOS, Wyatt Technology, Santa Barbara, USA). Data from the SLS detector were collected and processed using the Astra 6.0.3 software (Wyatt Technology). Data were saved in the native ASTRA format and then exported as text files for analysis, as described below. Delay volume and normalization were determined using a polystyrene standard latex sample (mean hydrodynamic size of 20 nm), and the RI calibration constant was determined using a monodisperse poly(ethylene glycol) sample (Mw = 2 × 104 g mol−1 , Mw /Mn = 1.05, dn/dc = 0.135 mL g−1 ). The refractive index increments, dn/dc, were determined according to a method described elsewhere by our group [44]. The AF4 fractionation parameters used in this study are summarized in Table 1. The AF4 fractionation parameters were optimized for NF length characterization according to our previous detailed analytical setup used for carbon nanotube aqueous dispersion studies [37–40]. The run sequence began with two short and consecutive steps of elution and focusing without injection to reach equilibrium. During these steps, all of the residual impurities present in the diluted crude sample were also removed via cross-flow through a membrane. At the end of the fractionation process, a rinse step without cross-flow was applied. Each run was replicated 3 to 6 times to determine the experimental uncertainties. 3. Results and discussion The PISA method developed in previous work (illustrated in Fig. 1) relies on the use of a hydrophilic living polymer precursor prepared via CRP that is extended with a hydrophobic second block in an aqueous environment. This process leads to amphiphilic block copolymers that self-assemble in situ into self-stabilized nanoobjects in the frame of an emulsion or dispersion polymerization process. Depending on the nature and structure of the so-formed copolymer, aside from spherical particles, NF morphologies are also formed that can be found in the phase diagram of an amphiphilic block copolymer in a selective solvent.
Table 1 Optimal AF4 conditions for NF fractionation. Parameter Mobile phase Membrane Injection step
Focus step Elution step
Optimal condition Nature Concentration Nature Molecular weight cut-off Time Flow Volume Time Cross(focus)-flow Time Cross-flow Detector flow
Ammonium nitrate (NH4 NO3 ) 1 × 10−5 mol L−1 Regenerated cellulose 10 kDa 5 min 0.2 mL min−1 100 L 6 min 3.0 mL min−1 17 min 0.5 mL min−1 1.0 mL min−1
3.1. Length and length distribution of nanofibers As illustrated in the supplemental information and previously published works [9], the microscopy analyses indicate the wormlike nature of the NFs. However, the lack of standard reference materials made the characterization of the asymmetrical shape by AF4 coupled to UV–vis and SLS challenging. The nature of the analytes has recently been suggested to influence their retention behavior in the AF4 channel [45]. In the case of carbon-based analytes, the densities are close to that of water, and no significant difference appears. For fiber-shaped nanomaterials, such as carbon nanotubes, a strategy was recently proposed that consisted of using polystyrene standard reference materials (PSL) to calibrate the AF4 according to the equivalent sphere radius (Rs ) [41]. The advantages of this approach are the convenient light-scattering characterization that can be achieved for PSL analytes and the well-known hydrodynamic behavior of this spherical shape for carbon-based particles. Indeed, under optimal fractionation conditions, AF4 enables the separation of analytes with a retention ratio R (i.e., linked to the retention times as t0 /tR ) that correlates linearly to the coefficient of diffusion according to the equation (see supplemental material for more details on the AF4 theory): R =
V0 D 6Vc ω2
(1)
where V0 is the void volume (mL), Vc is the cross-flow rate (mL s−1 ), ω is the channel thickness and D is the analyte diffusion coefficient (m2 s−1 ). By taking into account the AF4 void time and by considering hard-sphere analytes, we can write the linear relationship between tR and the hydrodynamic radius (RH ) (through the Stokes–Einstein relation, see supplemental materials for more details) when the normal elution mode occurs (R = 6 < 0.17) and the cross-flow and main flow rates are kept constant [46–48]: tR =
Vc ω2 ln(1 + ) RH Vout kB T
(2)
where is the viscosity of the mobile phase (kg m−1 s−1 ), T is the temperature of the medium (K), Vout is the main channel flow (mL min−1 ) and kB is the Boltzmann constant. By using static light scattering coupled on-line to the AF4, the radius of gyration, Rg , can be determined. The Rg parameter was recently shown to be more accurate for the determination of the size parameter for asymmetrical shapes because it takes into account the mass distribution due to the shape variation that can occur during the elution process through the AF4 channel [40,41]. Moreover, without considering the shape of the object characterized, Rg can be directly assimilated to the hydrodynamic radius by using the shape factor () [49]: =
Rg Rh
(3)
As is constant, the same linear relationship between Rh and tR (Eq. (2)) can be made between Rg and tR . The first step was to analyze PSL analytes by AF4 under the fractionation conditions optimized for NFs (see AF4 fractionation parameters in Section 2). In the case of fibrous nanomaterials (nanofibers), the radius of gyration (Rg ) can be directly linked to a spherical particle in terms of apparent length (L). We used the geometric relationship developed for a rod that relates Rg to the cross-sectional rod radius (Rcs ) and to length L by the equation [50] Rg2 =
L2 R2 L2 + cs = , with L Rcs 12 2 12
(4)
With this approach, Rg is transformed into L using an appropriate shape model. Fig. 2a shows the superposition of UV fractograms of four NFs (samples B1, B2, C, and A) and the size calibration curve
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(L = f(t)). The void time was identified at t0 = 0.5 min. Some populations appear at retention times close to the void time (tR ranging from t0 and tr = 3.8 min) that can be attributed to some residual species, such as spherical micelles and vesicles [7], with more significant diffusing properties according to previous studies (i.e., a smaller size, estimated to range from 20 to 80 nm) [6]. By integration of the UV signal, the relative quantity of residual micelles and/or vesicles in the crude NF solutions was determined by a method previously published [51] and was estimated to be less than 4 wt%. Concerning the NF signal (with a corresponding retention time longer than 3.8 min), the number-average particle length (Ln ), the weight-average particle length (Lw ), and the particle-length dispersity (IP) are calculated from the UV signal and size calibration according to Eqs. (5)–(7); the corresponding length distribution curves are presented in Fig. 2b: Ln =
wi wi /Li
(5)
Lw =
wi Li wi
(6)
IP =
Lw Ln
(7)
where wi is the weight fraction of the NF determined from the UV signal under the assumption that the NFs have the same chemical composition. The results are reported in Table 2, and the average lengths are compared to those recently obtained by rheological studies (L ) from the measured viscoelastic parameters [9]. The rheology of the same self-stabilized polymeric NFs in water suspensions has been previously investigated [9]. The critical concentration, ˚0 , between the dilute and the semidilute regimes was determined from the crossover of the scaling law on the zero shear viscosity (0 ∝ ˚−1 and 0 ∝ ˚−3 for dilute and semidilute regimes, respectively). From a quantitative point of view, two sets of suspensions can be differentiated depending on their aspect ratio (L/d ≈70 for A, and L/d ≈54 for B1, B2, and C). The average length, noted as L , of these NFs ranges from 2.4 to 3.8 m (Table 2). The experimental ratio Lw /L is constant and equal to 2.0 ± 0.1. The model used in either the AF4 or the rheological study can explain the systematic observed difference in the length. In the AF4 study, Lw is the apparent length based on the equivalent diffusive properties of the spherical polystyrene latex standards in the channel. In rheological measurements, the aspect ratio of the NFs, and subsequently the length L , are calculated from the critical concentration ˚0 according to: ˚0 = (
2 ˇ L )d L or = ( ) 4 d 4˚0
0.5
(8)
where = ˇ/L3 is the number of fibers per unit volume of the suspension, and ˇ is a dimensionless constant found to be approximately 30 according to studies based on carbon nanotubes in polydimethylsiloxane suspensions [52]. With low ˇ values (i.e., ˇ < 30), even if the lengths of two NFs are the same, a lower value of can be simply explained by considering that the chemical structure between macromolecular NFs and carbon nanotubes differs substantially. 3.2. Aggregation number per unit length from static light scattering (SLS) In light-scattering measurements, the wavevector q is defined as 4n/sin( /2), where n is the refractive index of the solvent, is the wavelength of light used, and is the scattering angle. When qRg < 1, the Rg and the weight-average molar mass, Mw , of the self-assembled amphiphilic nano-objects can be determined,
Fig. 2. (a) UV fractograms of NFs (samples B1, B2, C, and A, left to right) and the size calibration curve (L = f(t)). The void time is identified at 0.5 min. (b) Length distribution curves (B1, B2, C, and A samples, left to right).
from which the aggregation number can be calculated. For wormlike nanofibers, qRg > 1 that does not leads the determination of Mw . Thus, only the mass per unit length can be measured by SLS from the Holtzer [18,19] plot, as illustrated in Fig. 3a, which shows R q/Kc as a function of q (where R is the Rayleigh ratio, K = 42 n2 /4 Nav (dn/dc)2 is the optical constant, Nav is Avogadro’s number, c is the mass concentration of the object in solution, and dn/dc the refractive index increment). The dn/dc of the NFs (A, B1, and B2) in water was measured as 0.19 ± 0.02 mL g−1 . The plateau value in Fig. 3a corresponds to the molar mass per unit length. The Agg is obtained by dividing the plateau value by the molar mass of the diblock copolymer (see Table 1 in previous work [9]).
Table 2 NF length and associated IP determined by AF4 and rheology measurements [9]. The aggregation numbers according to the different samples of nanofibers are listed in the last column. Sample
Lw (nm)
IP
L (nm)
Lw /L
A B1 B2 C
1870 1260 1380 1620
1.04 1.03 1.03 1.05
3800 2400 2800 3500
2.04 1.90 2.02 2.16
Agg (nm-1 ) 49 ± 5 73 ± 7 69 ± 6
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length of self-assembled NFs from their crude aqueous suspensions. With this method, the Rg obtained for the fractionated NFs is transformed into their length using an appropriate shape model. From the AF4-UV signal and the AF4 size calibration, the number-average particle length (Ln ), the weight-averaged particle length (Lw ) and the particle-length dispersity (IP) were calculated and compared to those reported in previous rheological studies on these polymeric NFs. The method appears to be highly relevant and accurate for the determination of the length and length distribution of these new types of NFs. To our knowledge, the unprecedented determination of Agg resulted in a constant value with a standard deviation of ±15%. The novel determination of the aggregation number per unit length demonstrated in this work provides critical information on the physical properties of the NFs investigated herein, such as a decreasing value of the NF Agg with the length of the polystyrene hydrophobic block. Compared to the other techniques generally used to characterize NFs (e.g., microscopy or rheology), the AF4 methodology developed herein allows for a more representative NF characterization to be obtained in a reasonable acquisition time through the use of a simple template of data analysis. Acknowledgment The authors thank Gerald Clisson for his technical assistance during the AF4 experiments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.aca.2013.12.001http://dx.doi.org/10.1016/j.aca.2013.12.001. References Fig. 3. (a) A representative Holtzer plot for samples A (triangles), B1 (squares), and B2 (circles), corresponding to the peak maxima. (b) The value of the plateau results in an aggregation number for the whole peak.
Fig. 3b illustrates the fractograms obtained for NFs (samples A, B1, and B2) with the corresponding Agg determined. The Agg values determined are constant, with a standard deviation of ±15%. The error limit of 15% on Agg is based on the average and standard deviation from multiple injections. The mean values of Agg are reported in Table 1. Given this standard deviation value, the NFs can be considered as homogeneous in structure along their entire size distribution. The Agg of the NFs decreases with the length of the polystyrene hydrophobic block increases (see Table 2 in previous work [9], where the number-average degree of polymerization of the polystyrene block is respectively 400, 350 and 350 for samples A, B1 and B2). The explanation is not obvious and this result could be explained by the formation of NFs during the synthesis. Previous work has shown that NFs resulted from the aggregation and coalescence of initially formed diblock copolymer spherical micelles swollen with non converted monomers [8]. This process occurred rather rapidly after approximately 50% styrene conversion and led to wormlike micelles that became thermodynamically frozen upon increased monomer conversion. The Agg is most likely fixed, and the mass of unit length increases with monomer conversion. 4. Conclusions In this work, we developed a method based on asymmetric flow field-flow fractionation coupled to SLS for the accurate measurement of the distributions in length and aggregation number per unit
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Asymmetrical flow field-flow fractionation analysis of water suspensions of polymer nanofibers synthesized via RAFT-mediated emulsion polymerization Julien Gigault c , Wenjing Zhang b , Gaetane Lespes a , Bernadette Charleux b , Bruno Grassl a,∗ a
Laboratoire de Chimie Analytique BioInorganique et Environnement, UMR IPREM 5254 UPPA/CNRS-Technopole Hélioparc, Université de Pau et des Pays de l’Adour (UPPA), Av. du Président Angot, 64053 Pau cedex, France b C2P2 (Chemistry, Catalysis, Polymers & Processes), CNRS, UMR 5265, CPE Lyon, Université Lyon 1, Université de Lyon, Team LCPP Bat 308 F, 43 Bd du 11 Novembre 1918, 69616 Villeurbanne, France c National Institute of Standards and Technology, Material Measurement Science Division, 100 Bureau Drive, Gaithersburg, 20899 MD, USA
h i g h l i g h t s • Flow-field-flow-fractionation to characterize self-assembled nanofibers (NFs). • Accurate NFs length and aggregation number per unit length determination. • Characterization of new self-assembled polymers with various architectures.
a r t i c l e
i n f o
Article history: Received 2 December 2013 Received in revised form 31 January 2014 Accepted 9 February 2014 Available online 12 February 2014 Keywords: Controlled/living radical polymerization Asymmetric flow field-flow fractionation Nanofiber Length Aggregation
a b s t r a c t The aim of this work is to present a method based on asymmetric flow-field-flow-fractionation coupled on-line to a static light scattering (AF4-UV-SLS) detector to characterize self-assembled nanofibers (NFs). The method developed herein allows the determination of both the length distribution of the NFs as well as the distribution in terms of aggregation number per unit length (Agg ). Given the remaining synthetic challenges of better controlling the structural homogeneity and particle dimensions, the NF length and aggregation number per unit length are becoming essential for the improvement and control of their chemical processes and a better understanding of their properties. The results obtained with this AF4-UV-SLS method indicate that a well-resolved NF length distribution characterization and Agg determination were attained. These results provide critical information concerning the physical properties of the investigated NFs and open the door to the characterization of new self-assembled polymers with various asymmetrical architectures. Published by Elsevier B.V.
1. Introduction With the development of polymerization-induced micellization or polymerization-induced self-assembly (PISA) based on controlled/living radical polymerization (CRP), the field of polymer architectures has expanded considerably in the last few years [1–4]. Recently, various morphologies, such as crew-cut micelles, vesicles, elongated micelles, and nanofibers (NFs), were shown to be easily synthesized in the aqueous phase via these emulsion and dispersion processes [5].
∗ Corresponding author. E-mail address: [email protected] (B. Grassl). http://dx.doi.org/10.1016/j.aca.2014.02.011 0003-2670/Published by Elsevier B.V.
Self-assembled amphiphilic block copolymer nanofibers can be synthesized directly in aqueous suspensions at high concentrations (>20 wt%) via PISA. As illustrated in Fig. 1, the use of water-soluble macromolecular reversible addition–fragmentation chain transfer (RAFT) agent in the emulsion polymerization of styrene enables fast polymerizations and high final conversions with the formation of well-defined amphiphilic block copolymers without residual hydrophilic reactive chains [6–9]. The final product is an in situ created suspension of self-assembled amphiphilic nano-objects, the morphologies of which can be tuned from micelles to nanofibers through variation of certain parameters, such as the pH and the molar mass of the hydrophilic and hydrophobic blocks. Because of the complexity and chemical heterogeneity of these new nano-objects based on polymers, one of the major challenges
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in analytical chemistry is their size characterization, especially in the case of samples with the NF morphology. The ability to obtain the length (L) distribution and the distribution of the aggregation number per unit length (Agg ) of NFs is becoming essential to improve and control their chemical processes and to better understand their properties. Aqueous suspensions of polymer nanofibers could be used for their viscoelastic properties [9–11], whereas dried nanofibers may find direct applications in materials science or biotechnology, including organ repair and the treatment of osteoporosis and various other diseases [12]. In general, self-assembled nanostructures of amphiphilic and double hydrophilic block copolymers have been increasingly utilized as potent polymeric nanocarriers of therapeutic drugs, genes, bioactive molecules, and imaging/contrast agents because of their improved water solubility, bioavailability, and extended blood circulation duration [13]. The remaining synthetic challenges are related to the better control of the structural homogeneity and of the particle dimensions, especially their length and Agg . The application of the process to a broader range of polymers, thereby allowing access to new properties, also appears to be extremely important [5,14]. Currently, the lengths of one-dimensional micelles are normally characterized using imaging techniques [15], such as transmission electron microscopy (TEM), [16] fluorescence microscopy [17], or atomic force microscopy (AFM) [18]. However, more information can be obtained from scattering techniques. Static light scattering (SLS) was used to determine the length of elongated structures, whether the structures are rigid or flexible, and the mass per unit length of the structures [19,20]. Small-angle X-ray scattering (SAXS) [21,22] and neutron scattering (SANS) [23] can also provide information about the mass per unit length as well as information about the size of the micellar cross-section. Finally, dynamic light scattering (DLS) has been widely used to evaluate the hydrodynamic cross-section of natural fibrils [24–29] and elongated micelles [29,30]. Even if both static and dynamic light scattering techniques are widely used in batch-mode, the polydispersity of the size and shape of the NFs leads to biased measurements and inaccurate results related to these physical parameters. One interesting analytical strategy consists of using hyphenated techniques. The coupling of a separation technique on-line to a static light scattering (SLS) technique allows for the fractionation of nanofibers according to one or several physical properties, followed by characterization. Size-exclusion chromatography (SEC) coupled to SLS can be used to characterize NFs such as carbon nanotubes [31–34]. Some studies have shown that SEC can lead to a reasonable size resolution; however, the exclusion limit of the SEC column, which is controlled by the pore size, restricts the NFs that can be separated to those shorter than 1 m. The asymmetric flow field-flow fractionation
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(AF4) technique may be a feasible alternative. The results obtained for NFs such as carbon nanotubes show that AF4 is a promising technique because of its versatility and size-resolution potential [35–43]. By coupling AF4 with static light scattering detectors, carbon nanotubes can be fractionated on the basis of their length and their dispersion states (i.e., individually dispersed CNT and aggregates) can be determined. However, to our knowledge, the fractionation of NFs by methods such as those proposed in this work has not been reported. From the perspective of recent development of PISA of amphiphilic block copolymers based on controlled/living free radical polymerization in water, we demonstrate in this work the highly promising capabilities of asymmetric flow field-flow fractionation for the accurate measurement of the distributions of the length and Agg of self-assembled NFs from their crude aqueous suspensions. The novel determination of the Agg demonstrated in this work could provide critical information on the physical properties of the investigated NFs. 2. Experimental 2.1. Chemical Ammonium nitrate (NH4 NO3 , 99.5%) was purchased from Sigma Aldrich. Milli-Q 18 M cm water (Millipore System) was used in this study. The mobile phase used in AF4 was filtered through Millipore Durapore 0.1 m filters. The polystyrene standard reference materials (PSL) (mass fraction of 1%) were manufactured by Thermo Scientific and were traceable in size according to the National Institute of Standards and Technology (NIST) in the United States. The identification of any commercial product or trade name does not imply endorsement or recommendation by NIST. 2.2. Samples Four crude aqueous suspensions of the NF were prepared and used via the RAFT-mediated emulsion polymerization of styrene (samples A, B1, and B2) or methyl methacrylate (sample C) in water (see Fig. 1), which was performed in the presence of a P(MAA-co-PEOMA) macroRAFT agent bearing a trithiocarbonate reactive group. The experimental conditions were based on the one-pot, two-step strategy reported elsewhere [6,7,9]. The characteristics of the nanofibers were tuned by changing the pH of the emulsion and the molar mass and composition of the macroRAFT agent and the initial concentration ratio of the macroRAFT agent to styrene or MMA. The molecular parameters of the amphiphilic block copolymers constitutive of the NFs and the structure of the latter confirmed by transmission electron microscopy (TEM)
Fig. 1. Schematic representation of the one-pot NF synthesis: chemical composition (P(MAA-co-PEOMA) stands for poly(methacrylic acid-co-poly(ethylene oxide) methyl ether methacrylate), PS for polystyrene and PMMA for poly(methyl methacrylate)) and length (L) and aggregation number (Agg ) characteristics analyzed by AF4.
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analysis (see supplemental information, Fig. S2), were described in a previous work [9] with the same experiment numbers. 2.3. Instrumentation An Eclipse 3 asymmetrical flow field-flow fractionation system (Wyatt Technology, Dernbach, Germany) was used in this study. The trapezoidal channel was 26.5 cm in length and 0.6 and 2.1 cm in width. Flow rates were controlled using an Agilent Technologies 1100 series isocratic pump equipped with a micro-vacuum degasser. All injections were performed with an autosampler (Agilent Technologies 1100 series). The detection chain consisted of a variable-wavelength UV–vis spectrophotometer (Agilent Technologies 1100 series from Agilent, Tokyo, Japan) that was tuned to 254 nm with a SLS detector (DAWN HELEOS, Wyatt Technology, Santa Barbara, USA). Data from the SLS detector were collected and processed using the Astra 6.0.3 software (Wyatt Technology). Data were saved in the native ASTRA format and then exported as text files for analysis, as described below. Delay volume and normalization were determined using a polystyrene standard latex sample (mean hydrodynamic size of 20 nm), and the RI calibration constant was determined using a monodisperse poly(ethylene glycol) sample (Mw = 2 × 104 g mol−1 , Mw /Mn = 1.05, dn/dc = 0.135 mL g−1 ). The refractive index increments, dn/dc, were determined according to a method described elsewhere by our group [44]. The AF4 fractionation parameters used in this study are summarized in Table 1. The AF4 fractionation parameters were optimized for NF length characterization according to our previous detailed analytical setup used for carbon nanotube aqueous dispersion studies [37–40]. The run sequence began with two short and consecutive steps of elution and focusing without injection to reach equilibrium. During these steps, all of the residual impurities present in the diluted crude sample were also removed via cross-flow through a membrane. At the end of the fractionation process, a rinse step without cross-flow was applied. Each run was replicated 3 to 6 times to determine the experimental uncertainties. 3. Results and discussion The PISA method developed in previous work (illustrated in Fig. 1) relies on the use of a hydrophilic living polymer precursor prepared via CRP that is extended with a hydrophobic second block in an aqueous environment. This process leads to amphiphilic block copolymers that self-assemble in situ into self-stabilized nanoobjects in the frame of an emulsion or dispersion polymerization process. Depending on the nature and structure of the so-formed copolymer, aside from spherical particles, NF morphologies are also formed that can be found in the phase diagram of an amphiphilic block copolymer in a selective solvent.
Table 1 Optimal AF4 conditions for NF fractionation. Parameter Mobile phase Membrane Injection step
Focus step Elution step
Optimal condition Nature Concentration Nature Molecular weight cut-off Time Flow Volume Time Cross(focus)-flow Time Cross-flow Detector flow
Ammonium nitrate (NH4 NO3 ) 1 × 10−5 mol L−1 Regenerated cellulose 10 kDa 5 min 0.2 mL min−1 100 L 6 min 3.0 mL min−1 17 min 0.5 mL min−1 1.0 mL min−1
3.1. Length and length distribution of nanofibers As illustrated in the supplemental information and previously published works [9], the microscopy analyses indicate the wormlike nature of the NFs. However, the lack of standard reference materials made the characterization of the asymmetrical shape by AF4 coupled to UV–vis and SLS challenging. The nature of the analytes has recently been suggested to influence their retention behavior in the AF4 channel [45]. In the case of carbon-based analytes, the densities are close to that of water, and no significant difference appears. For fiber-shaped nanomaterials, such as carbon nanotubes, a strategy was recently proposed that consisted of using polystyrene standard reference materials (PSL) to calibrate the AF4 according to the equivalent sphere radius (Rs ) [41]. The advantages of this approach are the convenient light-scattering characterization that can be achieved for PSL analytes and the well-known hydrodynamic behavior of this spherical shape for carbon-based particles. Indeed, under optimal fractionation conditions, AF4 enables the separation of analytes with a retention ratio R (i.e., linked to the retention times as t0 /tR ) that correlates linearly to the coefficient of diffusion according to the equation (see supplemental material for more details on the AF4 theory): R =
V0 D 6Vc ω2
(1)
where V0 is the void volume (mL), Vc is the cross-flow rate (mL s−1 ), ω is the channel thickness and D is the analyte diffusion coefficient (m2 s−1 ). By taking into account the AF4 void time and by considering hard-sphere analytes, we can write the linear relationship between tR and the hydrodynamic radius (RH ) (through the Stokes–Einstein relation, see supplemental materials for more details) when the normal elution mode occurs (R = 6 < 0.17) and the cross-flow and main flow rates are kept constant [46–48]: tR =
Vc ω2 ln(1 + ) RH Vout kB T
(2)
where is the viscosity of the mobile phase (kg m−1 s−1 ), T is the temperature of the medium (K), Vout is the main channel flow (mL min−1 ) and kB is the Boltzmann constant. By using static light scattering coupled on-line to the AF4, the radius of gyration, Rg , can be determined. The Rg parameter was recently shown to be more accurate for the determination of the size parameter for asymmetrical shapes because it takes into account the mass distribution due to the shape variation that can occur during the elution process through the AF4 channel [40,41]. Moreover, without considering the shape of the object characterized, Rg can be directly assimilated to the hydrodynamic radius by using the shape factor () [49]: =
Rg Rh
(3)
As is constant, the same linear relationship between Rh and tR (Eq. (2)) can be made between Rg and tR . The first step was to analyze PSL analytes by AF4 under the fractionation conditions optimized for NFs (see AF4 fractionation parameters in Section 2). In the case of fibrous nanomaterials (nanofibers), the radius of gyration (Rg ) can be directly linked to a spherical particle in terms of apparent length (L). We used the geometric relationship developed for a rod that relates Rg to the cross-sectional rod radius (Rcs ) and to length L by the equation [50] Rg2 =
L2 R2 L2 + cs = , with L Rcs 12 2 12
(4)
With this approach, Rg is transformed into L using an appropriate shape model. Fig. 2a shows the superposition of UV fractograms of four NFs (samples B1, B2, C, and A) and the size calibration curve
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(L = f(t)). The void time was identified at t0 = 0.5 min. Some populations appear at retention times close to the void time (tR ranging from t0 and tr = 3.8 min) that can be attributed to some residual species, such as spherical micelles and vesicles [7], with more significant diffusing properties according to previous studies (i.e., a smaller size, estimated to range from 20 to 80 nm) [6]. By integration of the UV signal, the relative quantity of residual micelles and/or vesicles in the crude NF solutions was determined by a method previously published [51] and was estimated to be less than 4 wt%. Concerning the NF signal (with a corresponding retention time longer than 3.8 min), the number-average particle length (Ln ), the weight-average particle length (Lw ), and the particle-length dispersity (IP) are calculated from the UV signal and size calibration according to Eqs. (5)–(7); the corresponding length distribution curves are presented in Fig. 2b: Ln =
wi wi /Li
(5)
Lw =
wi Li wi
(6)
IP =
Lw Ln
(7)
where wi is the weight fraction of the NF determined from the UV signal under the assumption that the NFs have the same chemical composition. The results are reported in Table 2, and the average lengths are compared to those recently obtained by rheological studies (L ) from the measured viscoelastic parameters [9]. The rheology of the same self-stabilized polymeric NFs in water suspensions has been previously investigated [9]. The critical concentration, ˚0 , between the dilute and the semidilute regimes was determined from the crossover of the scaling law on the zero shear viscosity (0 ∝ ˚−1 and 0 ∝ ˚−3 for dilute and semidilute regimes, respectively). From a quantitative point of view, two sets of suspensions can be differentiated depending on their aspect ratio (L/d ≈70 for A, and L/d ≈54 for B1, B2, and C). The average length, noted as L , of these NFs ranges from 2.4 to 3.8 m (Table 2). The experimental ratio Lw /L is constant and equal to 2.0 ± 0.1. The model used in either the AF4 or the rheological study can explain the systematic observed difference in the length. In the AF4 study, Lw is the apparent length based on the equivalent diffusive properties of the spherical polystyrene latex standards in the channel. In rheological measurements, the aspect ratio of the NFs, and subsequently the length L , are calculated from the critical concentration ˚0 according to: ˚0 = (
2 ˇ L )d L or = ( ) 4 d 4˚0
0.5
(8)
where = ˇ/L3 is the number of fibers per unit volume of the suspension, and ˇ is a dimensionless constant found to be approximately 30 according to studies based on carbon nanotubes in polydimethylsiloxane suspensions [52]. With low ˇ values (i.e., ˇ < 30), even if the lengths of two NFs are the same, a lower value of can be simply explained by considering that the chemical structure between macromolecular NFs and carbon nanotubes differs substantially. 3.2. Aggregation number per unit length from static light scattering (SLS) In light-scattering measurements, the wavevector q is defined as 4n/sin( /2), where n is the refractive index of the solvent, is the wavelength of light used, and is the scattering angle. When qRg < 1, the Rg and the weight-average molar mass, Mw , of the self-assembled amphiphilic nano-objects can be determined,
Fig. 2. (a) UV fractograms of NFs (samples B1, B2, C, and A, left to right) and the size calibration curve (L = f(t)). The void time is identified at 0.5 min. (b) Length distribution curves (B1, B2, C, and A samples, left to right).
from which the aggregation number can be calculated. For wormlike nanofibers, qRg > 1 that does not leads the determination of Mw . Thus, only the mass per unit length can be measured by SLS from the Holtzer [18,19] plot, as illustrated in Fig. 3a, which shows R q/Kc as a function of q (where R is the Rayleigh ratio, K = 42 n2 /4 Nav (dn/dc)2 is the optical constant, Nav is Avogadro’s number, c is the mass concentration of the object in solution, and dn/dc the refractive index increment). The dn/dc of the NFs (A, B1, and B2) in water was measured as 0.19 ± 0.02 mL g−1 . The plateau value in Fig. 3a corresponds to the molar mass per unit length. The Agg is obtained by dividing the plateau value by the molar mass of the diblock copolymer (see Table 1 in previous work [9]).
Table 2 NF length and associated IP determined by AF4 and rheology measurements [9]. The aggregation numbers according to the different samples of nanofibers are listed in the last column. Sample
Lw (nm)
IP
L (nm)
Lw /L
A B1 B2 C
1870 1260 1380 1620
1.04 1.03 1.03 1.05
3800 2400 2800 3500
2.04 1.90 2.02 2.16
Agg (nm-1 ) 49 ± 5 73 ± 7 69 ± 6
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length of self-assembled NFs from their crude aqueous suspensions. With this method, the Rg obtained for the fractionated NFs is transformed into their length using an appropriate shape model. From the AF4-UV signal and the AF4 size calibration, the number-average particle length (Ln ), the weight-averaged particle length (Lw ) and the particle-length dispersity (IP) were calculated and compared to those reported in previous rheological studies on these polymeric NFs. The method appears to be highly relevant and accurate for the determination of the length and length distribution of these new types of NFs. To our knowledge, the unprecedented determination of Agg resulted in a constant value with a standard deviation of ±15%. The novel determination of the aggregation number per unit length demonstrated in this work provides critical information on the physical properties of the NFs investigated herein, such as a decreasing value of the NF Agg with the length of the polystyrene hydrophobic block. Compared to the other techniques generally used to characterize NFs (e.g., microscopy or rheology), the AF4 methodology developed herein allows for a more representative NF characterization to be obtained in a reasonable acquisition time through the use of a simple template of data analysis. Acknowledgment The authors thank Gerald Clisson for his technical assistance during the AF4 experiments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.aca.2013.12.001http://dx.doi.org/10.1016/j.aca.2013.12.001. References Fig. 3. (a) A representative Holtzer plot for samples A (triangles), B1 (squares), and B2 (circles), corresponding to the peak maxima. (b) The value of the plateau results in an aggregation number for the whole peak.
Fig. 3b illustrates the fractograms obtained for NFs (samples A, B1, and B2) with the corresponding Agg determined. The Agg values determined are constant, with a standard deviation of ±15%. The error limit of 15% on Agg is based on the average and standard deviation from multiple injections. The mean values of Agg are reported in Table 1. Given this standard deviation value, the NFs can be considered as homogeneous in structure along their entire size distribution. The Agg of the NFs decreases with the length of the polystyrene hydrophobic block increases (see Table 2 in previous work [9], where the number-average degree of polymerization of the polystyrene block is respectively 400, 350 and 350 for samples A, B1 and B2). The explanation is not obvious and this result could be explained by the formation of NFs during the synthesis. Previous work has shown that NFs resulted from the aggregation and coalescence of initially formed diblock copolymer spherical micelles swollen with non converted monomers [8]. This process occurred rather rapidly after approximately 50% styrene conversion and led to wormlike micelles that became thermodynamically frozen upon increased monomer conversion. The Agg is most likely fixed, and the mass of unit length increases with monomer conversion. 4. Conclusions In this work, we developed a method based on asymmetric flow field-flow fractionation coupled to SLS for the accurate measurement of the distributions in length and aggregation number per unit
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