Biomacromolecules 2003, 4, 232-239
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Polyelectrolyte Complexes: Interactions between Lignosulfonate and Chitosan Guro E. Fredheim† Østfold College, P.O. Box 1192, NO-1705 Sarpsborg, Norway
Bjørn E. Christensen* Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology, Norwegian University of Science and Technology (NTNU), NO-7491 Trondheim, Norway Received August 2, 2002; Revised Manuscript Received December 2, 2002
The interactions between high molecular weight chitosans (fraction of acetylated units (FA) ) 0.10 or 0.50) and lignosulfonates of varying molecular weights (5000-400000 g/mol) and degrees of sulfonation (0.390.64) were studied. Lignosulfonates and chitosans form primarily insoluble polyelectrolyte complexes when mixed at pH 4.5, where the polymers are oppositely charged. In contrast, no complex formation occurred at pH 8, as shown by using a chitosan with FA ) 0.50, which is soluble at this pH. Thus, a positively charged chitosan is a prerequisite for interactions leading to insoluble complexes with lignosulfonates. It is therefore unlikely that complex formation involves the formation of covalent sulfonylamide linkages as proposed in the literature. The composition of the complexes varied to some degree with the mixing ratio and molecular weight of lignosulfonate, but in most cases compact complexes with a sulfonate/amino ratio close to 1.0 were formed, suggesting that all sulfonate groups are accessible for interactions with chitosan. The influence of the ionic strength and temperature on the complex formation and the behavior of the precipitated complexes were in agreement with that expected for classical polyelectrolyte complexes where the associative phase separation is primarily governed by the increase in entropy due to the release of counterions. Introduction Oppositely charged polyelectrolytes are generally known to form stable interpolymer complexes.1-4 Such polyelectrolyte complexes are of high practical relevance in industrial applications as flocculants, coatings, and binders, as well as in biological systems and in biomedical applications. Most insoluble polyelectrolyte complexes seem to exhibit 1:1 charge stoichiometry, independent of the charge density on the macromolecules and the structure of their backbones,1 provided that all charged groups are accessible for electrostatic interactions. Much of the literature focusing on polyelectrolyte complexes is based on studies of flexible, synthetic polymers (predominantly -C-C- backbones). The formation of such complexes depends on the structure of the two polymers in addition to the influence of added salt, the nature of the solvent, and the temperature, and the actual structure of the polyelectrolyte complex formed may vary. For flexible polyelectrolytes the “scrambled-salt” model (random process) and the “ladder-like” model (regular process) have been proposed by Michaels.5 Another type of polyelectrolyte complex is formed when DNA, which is a very stiff polymer often used in a circular form (plasmid), is compacted by polycations for applications in gene therapy.6 * Corresponding author. E-mail:
[email protected]. † Present address: Borregaard LignoTech, P.O. Box 162, NO-1701 Sarpsborg, Norway.
The general phase behavior of oppositely charged polymers is often described in terms of an associative phase diagram.7 In this work the interaction between two biopolymers, lignosulfonate (polyanion) and chitosan (polycation), is studied. Lignosulfonates are sulfonated lignins. The lignins themselves are insoluble polymers consisting of cross-linked phenylpropanoid monomers (Figure 1). The lignosulfonates are produced in the sulfite pulping process as a byproduct in the production of cellulose. In the process, the lignin is fragmented and sulfonated, rendering these hydrophobic molecules water soluble. The sulfonic acids are strong acids, and the lignosulfonates are therefore negatively charged at all practical pH values. However, the charge density varies somewhat with pH since lignosulfonates also contain phenolic hydroxyl groups (0.1-0.3 per monomer, pKa ) 6.211 depending on the substitution pattern8,9) and carboxyl groups (0-0.1 per monomer, pKa ca. 2.710,11). Lignosulfonates are used in several industrial fields, mainly as dispersants and binders. Commercial lignosulfonates are known to have broad molecular weight distributions (Mw ) 5000-60000 g/mol, Mw/Mn ) 3-12), and the degree of sulfonation (DS) varies from 0.4 to 0.7 sulfonate groups per phenylpropane unit.12,13 Lignosulfonates are very compact polymers as reflected by intrinsic viscosities in the range 2-12 mL/g, for molecular weights in the range 5000 to 400000 g/mol.13,14 Lignosulfonates are soluble in water over the entire pH range,15,16 provided DS is above 0.4. Modified
10.1021/bm020091n CCC: $25.00 © 2003 American Chemical Society Published on Web 01/31/2003
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Figure 1. The chemical structure of chitosans (a) and lignosulfonates (b). In chitosans the two monomers (GlcN and GlcNAc) are randomly distributed. In lignosulfonates the relative content of phenylpropanoid monomers, linkage types, and sulfonate groups is known, but their relative location (sequence) as well as the detailed branching pattern and position of charged groups are less well understood. The lignosulfonate fragment shown above is meant to illustrate general features rather than specifically identified structures.
(desulfonated) lignosulfonates with DS < 0.2 are insoluble below pH 2.5. Lignosulfonates have generally been presented as compact and spherical structures where the sulfonic acid and carboxylic acids are positioned mainly at the surface of a hydrophobic hydrocarbon core,16,17 although it cannot be ruled out that some of the sulfonate groups also may be positioned within the interior of the lignosulfonate particle.14 Such internal sulfonate groups may be inaccessible for interactions with other macromolecules. It should also be noted that the aromatic phenolpropane monomers of lignosulfonates may provide a basis for hydrophobic interactions with other molecules. The hydrophobicity is indeed reflected in the surfactant properties of lignosulfonates.18 Some studies have described interactions between lignosulfonates and polycations such as polyethyleneimine19 and proteins.20 The lignosulfonate/chitosan system has apparently only been studied by Tartakovsky et al.21 These authors claim that the formation of sulfonylamide linkages is responsible for the complex formation, as opposed to a purely electrostatic interaction, which perhaps should be expected. In this work we thus challenge this conclusion by investigating the lignosulfonate-chitosan system from a classical “polyelectrolyte complex” point of view. Chitosans are linear polysaccharides containing two sugar residues, N-acetyl-D-glucosamine (GlcNAc) and D-glucosamine (GlcN), respectively, which are both β-1,4-linked (Figure 1). Chitosans are commercially prepared by partial
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de-N-acetylation of chitin. The chemical composition of chitosans is generally described in terms FA, which is defined as the molar fraction of remaining N-acetylated (GlcNAc) residues. These residues are randomly distributed along the chitosan chains,22 and the average composition of chitosans chains can therefore be described statistically with FA as the sole parameter.22 The physical properties of chitosans are primarily governed by FA and the molecular weight. Chitosans are normally polydisperse, and the molecular weight of commercial products usually ranges from 10000 to 1000000 g/mol.23 The polymer has the shape of an expanded random coil in solution. The intrinsic viscosity is therefore about 2 orders of magnitude higher than that for lignosulfonates at comparable molecular weights.24 Commercial chitosans usually contain FA values between 0 and 0.2 and are only soluble in aqueous solutions at low pH since the pKa value is in the range 6.2-7.0.25 On the other hand, chitosans with FA between 0.4 and 0.6 possess full neutral solubility.26,27 The enhanced solubility is attributed to the combination of a high FA combined with the random distribution of acetyl groups along the chains, which effectively prevents alignment of structurally homogeneous regions (blocks) to form chain-chain associations.27 The major commercial application of chitosan is currently wastewater treatment.28,29 Dyes, suspended solids, heavy metals, pesticides, and other toxic compounds including humic substances may all be efficiently removed by chitosan. Well-characterized lignosulfonates may serve as useful model compounds for anionic polymers, especially aromatic polymers of the humic acid type, which interact with chitosans. Chitosans and lignosulfonates, with their well-characterized, but at the same time very different, structures and physical properties, offer a quite unique system for studies of the possible interactions. The main interest of our work focuses on the stoichiometry (charge ratio) needed for complex formation and the overall composition of the complexes. The influence of the ionic strength, pH, and temperature on the complex formation is also explored. The fact that chitosan with FA ) 0.50 is soluble also above the pKa value allows us to study complex formation with lignosulfonates as a function of pH without changing the solubility of the chitosan itself. This may provide a clearer answer to the role of electrostatic interactions in the complex formation with lignosulfonates. Materials and Methods Chitosans. Two chitosans with different fraction of acetylated units (FA) were used in this study. Chitosan A (Sea Cure, FA ) 0.10) was obtained from FMC Biopolymer AS, Norway. Chitosan B (FA ) 0.50) was prepared from chitin by heterogeneous deacetylation.26,30 Both samples were converted into water-soluble hydrocloride salts (chitosanHCl). FA was determined with 1H NMR spectroscopy as described by Vårum et al.31 Solutions of chitosan were prepared by dissolving the dry chitosan in MilliQ-grade water by gently shaking at 20-23 °C overnight. The ionic strength was adjusted to 0.1 M by adding NaCl, and pH was adjusted to 4.5 by adding dilute
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Table 1. Characteristic Properties of the Chitosan and Lignosulfonate Samples Applied (Taken from References 12 and 14)
sample chitosan A HCl form chitosan B HCl form lignosulfonate (unfractionated) fraction F-70 fraction F-60 fraction F-55 fraction F-50 fraction F-45 fraction F-40
fraction of acetylated units (FA)
amt of sulfur (mol/g)
degree of sulfonation (SO3/C9.95)
0.1 0.5
intrinsic viscosity (η, mL/g) 910 825
1.9 × 10-3 2.4 × 10-3 2.1 × 10-3 1.9 × 10-3 1.8 × 10-3 1.8 × 10-3 1.6 × 10-3
HCl. Only freshly prepared solutions ( 0.999). Chitosan A contained 8.50 ( 0.02% nitrogen (n ) 3) and the Na lignosulfonate sample contained 6.19 ( 0.07% sulfur (n ) 5).
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Figure 2. SEC-MALLS elution profiles (RI detector) and plots of log M versus volume (calibration curves) for chitosan A ([) and chitosan B (9).
Results and Discussion Sample Characteristics. The characteristics for the two chitosan samples, the lignosulfonate sample, and six lignosulfonate fractions used in this study are summarized in Table 1. The intrinsic viscosities [η] of chitosan A (FA ) 0.1) and chitosan B (FA ) 0.5) were determined to 910 and 825 mL/ g, respectively (at an ionic strength of 0.1 M, pH 4.5 and 20 °C). The weight average molecular weights for the chitosan samples were determined with SEC-MALLS to 545000 g/mol for chitosan A and 345000 g/mol for chitosan B. Figure 2 shows elution profiles of the chitosan samples. The calculated plots of log Mw versus elution volume (calibration plots) are included in the figure, and we obtain basically the same calibration plot for both samples. The polydispersities (Mw/Mn) were 3.2 for chitosan A and 2.4 for chitosan B, respectively. The lignosulfonate samples have molecular weights in the range 4600-398000 g/mol. By comparison of the chitosan sample (Mw ) 345000-545000 g/mol, [η] ) 825-910 mL/ g) and the highest molecular weight fractions of lignosulfonate (Mw ) 398000 g/mol, [η] ) 12.1 mL/g), the difference in chain shape and extension between the two classes of biopolymers are clearly demonstrated. The extended random coil of chitosan as opposed to the very compact lignosulfonate molecule can explain the marked difference in the intrinsic viscosity. The charge content is also different between the two polymers. The charge density of the chitosan (defined as the average degree of protonisation of the GlcN monomers) is determined by the fraction of GlcN monomers which equals 1 - FA, and the standard equation for an acid base equilibrium pH ) pKa - R/(1 - R)
(1)
where R is the degree of dissociation of the -NH3+ groups and Ka is the corresponding dissociation constant. Chitosan is a weak polyelectrolyte, with a pKa of 6.2-7. It follows that 90% of the monomer units in chitosan A have positive charges at pH 4.5. The charge density of lignosulfonates corresponds directly to the degree of sulfonation for all practical pH values due to the low pKa value of the sulfonate
groups. Thus, 40-60% of the monomers possess negative charges in neutral or slightly acidic solutions. It may be expected that the positive charges on the chitosan molecules are more accessible to other molecules as compared to the negative charges of the lignosulfonate, which possibly partly reside within the interior of the molecules.14 Complex Formation. Initial Observations. Initial experiments were performed using chitosan A (FA ) 0.10) and the high molecular weight (Mw ) 64000 g/mol), unfractionated Na-lignosulfonate (DS ) 0.52). The pH was 4.5, where the amino groups of the chitosan are fully positively charged and the sulfonate groups of the lignosulfonate are fully negatively charged as described above. When lignosulfonate was added to chitosan in a stoichiometric charge ratio (sulfonate groups to amino groups (SO3-/NH3+)) between 0.04 and 42.7 threadlike, fluffy aggregates were immediately formed. By centrifugation at 10000 rpm, the precipitated material settled. The total amount of precipitated material was determined gravimetrically after drying. A negligible (
