Field-Flow Fractionation of Cationic Cellulose Derivatives
The asymmetric flow field-flow fractionation (AF4) method was developed for cationic cellulose derivatives. AF4 is the method of choice especially for high-molar mass samples, which are challenging to characterize with conventional chromatographic techniques such as size-exclusion chromatography (SEC). The cationic charge of macromolecules also complicates the size-based separations where no interaction between the analytes and the column stationary phase (SEC) or membrane (AF4) should occur. However, many column matrices and membranes carry negative charge and thus preventing interactions between cationic analytes and negatively charged separation support should be taken into consideration when doing method development. In this study, two eluent compositions, neutral and acidic, were tested for AF4 separation of cationic hydroxyethyl celluloses with varying charge densities. The eluent composition with a pH below the isoelectric point of regenerated cellulose membrane, which was used in this AF4 study, enabled the size-based separation with close to 100% analysis recovery. Macromolecular parameters (molar mass and radius of gyration) and conformation were investigated by coupling a multi-angle light scattering detector and differential refractometer to the AF4 system.
KeywordsField-flow fractionation Separation Cationic polysaccharides Charge density
Cationic polymers are used in various applications such as carriers of genetic material (gene delivery), constituents in cosmetics and hair care solutions, and as flocculants in waste-water purification, to name a few . Currently, there is a growing demand for replacement of synthetic petroleum-based polymers with sustainable, biobased polymers. The properties and functionality of biopolymers can be altered by chemical, physical, or enzymatic modification. Adding charged groups to the polysaccharide backbone is one good example from structural tailoring. Reports on the cationization can be found for several polysaccharides, but most of the work on the cationization of polysaccharides has been conducted on starch and cellulose [1, 2, 3]. Both starch and cellulose have a high molar mass, which is required for certain applications. For example, only high-molar mass cationic polymers act as efficient flocculants in waste-water treatments . Thus, determination of macromolecular characteristics, including molar mass, is a prerequisite for evaluation of the functionality of the modified biopolymers.
Macromolecular separation and characterization of cationic (bio)polymers is a challenging task. The most commonly used technique for separation and molar mass determination of polymers is size-exclusion chromatography (SEC). Even though SEC has been widely used for characterization of cationic polymers, the technique has some limitations. In SEC, no enthalpic interactions between the analytes and column packing material should exist. Many commonly used SEC stationary phases, however, carry a negative charge, which might contribute to the unwanted interactions between positively charged analytes and negatively charged column material. Another limitation of SEC is the incapability of the technique to characterize high-molar mass polymers and polysaccharides accurately [5, 6]. SEC has been, however, successfully used for characterization of commercial cationic hydroxyethyl cellulose derivatives .
Another technique for the separation of polymers is field-flow fractionation (FFF). FFF has different variants depending on the external field (flow, sedimentation, thermal, electrical), which is used to enhance the separation. Flow FFF and especially asymmetric flow FFF (AF4) is most commonly used for the separation of (bio)polymers . In AF4, the analytes are injected into a thin, open channel where they separate in a parabolic flow based on their diffusion coefficients. The external flow, namely cross-flow, which is perpendicular to the main parabolic flow, enables the separation. Since the smaller molecules diffuse more quickly towards the center of the channel where the flow streams are faster, they elute from the channel earlier than the larger molecules with a lower diffusion coefficient. The AF4 channel consists of a solid top plate and porous bottom plate, which is covered by an ultrafiltration membrane with defined porosity. More information about the AF4 instrumentation and the theory can be found in the following references [9, 10]. Even though both SEC and AF4 can be used for characterization of many (bio)macromolecules, AF4 has some advantages over SEC especially for high-molar mass, charged analytes. First, AF4 is a gentler technique than SEC because separation takes place in an open channel. Second, the surface area in AF4 is around a few tens of square centimeters whereas in SEC the surface area of the porous stationary phase is in the order of the 107 cm2 . Thus, the risk of interactions between charged analytes and the SEC stationary phase is greater in SEC than the risk of having interactions between the analytes and membrane in AF4.
In this study, the AF4 method was developed for cationic hydroxyethyl celluloses (HEC) with varying degrees of cationization. Two different aqueous eluents (NaNO3 solution and acidic NaCl solution) were tested and analysis recovery was monitored to see if the interactions between the charged analytes and membrane occurred. A multi-angle light scattering (MALS) detector allowed the determination of molar mass and radius of gyration (RG) for cationic HEC samples. Conformation information was obtained from the relationship between molar mass and RG across the separated molecular species.
Materials and Methods
Materials and Reagents
The cationic HEC derivatives (quaternary ammonium salt of hydroxyethylcelluloses) with different degrees of cationization were obtained from The Dow Chemical Company (Amerchol Corp., Philadelphia, USA). The trade names for the samples used here are Polymer JR-400 (N% 1.5–2.2), Polymer LR-400 (N% 0.8–1.1), and Polymer LK (N% 0.4–0.6). The nitrogen content indicates the charge density (i.e., cationic group per repeating unit) of the polymers. According to the manufacturer, all samples have similar viscosities, which indicates similarity in molar mass (however, as can be seen from the results of this study; molar mass differences could be detected due to differences in the lengths of the branches). Sodium nitrate and sodium chloride were purchased from Merck (Darmstadt, Germany).
Determination of Refractive Index Increment (∂n/∂c)
Molar mass averages (Mw, Mn), dispersities (Đ), z average radius of gyration (RGz), AF4 analysis recoveries, and refractive index increments (∂n/∂c) for cationic hydroxyethyl celluloses
Mw (kg mol−1)
Mn (kg mol−1)
∂n/∂c (mL g−1)
HEC N% 0.4–0.6
HEC N% 0.8–1.1
HEC N% 1.5–2.2
Results and Discussion
Comparison of Two Eluents for AF4 Separation of Cationic HEC Samples
Eluent composition plays an important role in the separation of cationic macromolecules. The SEC column stationary phases and AF4 membrane materials commonly carry a weak negative charge. To mask the anionic sites at the column packing material or at the surface of the AF4 membrane, mobile phases with relatively high salt content are used to reduce the possible ionic interactions between the analytes and the stationary phase/membrane. Thus, we decided to test 0.8 M NaNO3, which has been successfully used for SEC separation of cationic celluloses . The other eluent tested was acidic salt solution (0.135 M NaCl in 0.012 M HNO3, pH ~ 2). In acidic conditions (below pH of 3.4 which is the isoelectric point for the RC membrane), the RC membrane is weakly positively charged [12, 13]. The repulsion between the positively charged membrane and cationic analytes likely prevents unwanted interactions between the membrane and the cationic HEC molecules.
The overlay of fractograms (light scattering signal at 90 °C) obtained with two eluent conditions for cationic HEC with nitrogen content of 1.5–2.2% is presented in Fig. 2. As can be seen from both fractograms, elution within 20 min could be achieved using the exponentially decaying cross-flow gradient. Molar masses across the peaks are also presented in Fig. 2. In the case of acidic eluent, the molecules elute according to the AF4 principles (e.g., smaller molecules with higher diffusion coefficient elutes before the larger molecules with lower diffusion coefficients). The elution of sample in 0.8 M NaNO3, however, seemed to be biased. The elution order of molecules for most of the peak was opposite to that observed in acidic eluent (and opposite to what is suggested by the AF4 theory). In addition, molar mass across the peak was higher in 0.8 M NaNO3 than in acidic eluent, indicating that the cationic HEC sample was not dissolved as the level of individual molecules but existed in the form of aggregates. The intensity difference of light scattering signals shown in Fig. 2 also indicates the difference in the molar masses of the sample analyzed in two eluent conditions. The AF4 analysis recovery was determined based on the refractive index signals and measured ∂n/∂c values. The recovery in the 0.8 M NaNO3 eluent was low at 17%, whereas recoveries for all the samples in acidic eluent were close to 100% (Table 1). Based on these AF4 trials on the two eluents, the acidic eluent was proven to be superior for the characterization of cationic HEC samples.
Molar Mass and Size of Cationic HEC with Different Charge Densities
To obtain reliable molar mass information, ∂n/∂c values for all samples dissolved in acidic eluent (0.135 M NaCl in 0.012 M HNO3) were measured by off-line refractometry. As can be seen in Fig. 1 and Table 1, the ∂n/∂c values for samples varied from 0.132 to 0.141 mL g−1. This range is typical for polysaccharides in aqueous solution [14, 15]. No clear correlation between the degree of cationization and ∂n/∂c was observed. For comparison, ∂n/∂c of the HEC sample with a nitrogen content of 1.5–2.2% dissolved in 0.8 M NaNO3 was measured but eluent composition had no effect on the measured ∂n/∂c values (similar ∂n/∂c values of 0.141 for both eluents).
Conformation of Cationic HEC
AF4 is a separation technique especially for high-molar mass macromolecules which are challenging to separate using conventional chromatographic techniques. In this study, the AF4 method was developed for cationic hydroxyethyl cellulose (quaternary ammonium salt of hydroxyethyl celluloses) samples with varying degrees of cationization. The eluent compositions tested were: 0.8 M NaNO3 and 0.135 M NaCl in 0.012 M HNO3. The separation in the neutral eluent resulted in poor sample recovery, which was likely due to the unwanted interactions between the cationic analytes and membrane carrying a negative charge. In addition, the separation behavior clearly deviated from what was expected based on the AF4 retention theory. In acidic eluent (pH below 3.4), the regenerated cellulose membrane carries a weak positive charge, which seemed to prevent the interactions between the membrane and cationic molecules. The analysis recovery in acidic eluent was near 100% for all the samples and molecules eluted in the order suggested by AF4 theory (molecules with higher translational diffusion coefficients elutes before the molecules with lower diffusion coefficients). MALS/DRI detection allowed determination of molar mass, size, and conformation of the cationic hydroxyethyl celluloses. Interestingly, the conformation plots suggested that the sample with the highest nitrogen content contains elongated side chains in contrast to the samples with lower nitrogen content. The methodology presented here can be used for separation and characterization of other cationic polysaccharides, which might have functions in several applications such as acting as flocculating agents in waste-water purification.
Open access funding provided by Aalto University. The authors would like to express their gratitude to Vladimir Aseyev for guidance with refractive index increment analyses. Magnus Ehrnroot Foundation is also acknowledged for funding.
Compliance with Ethical Standards
Conflict of interest
The authors declare no conflicts of interest.
Human and animal rights statement
This article does not contain any studies with human participants or animals performed by any of the authors.
- 4.Levine S, Friesen WI (1987) In: Attia YA (ed) Flocculation in biotechnology and separation science. Elsevier, AmsterdamGoogle Scholar
- 5.Striegel AM, Yau WW, Kirkland JJ, Bly DD (2009) Modern size-exclusion liquid chromatography. Practice of gel permeation and gel filtration chromatography. Wiley, New YorkGoogle Scholar
- 14.Theisen A, Johann C, Deacon MP, Harding SE (2000) Refractive increment data-book. Nottingham University Press, NottinghamGoogle Scholar
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