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Langmuir 2002, 18, 4536-4538
Colloidal Flocculation with Poly(ethylene oxide)/Polypeptide Complexes
Table 1. Structure and Molecular Weight of L-Polypeptidesa
Chen Lu, Robert Pelton,* John Valliant, Stuart Bothwell, and Karin Stephenson McMaster Centre for Pulp & Paper Research, Department of Chemical Engineering, McMaster University, Hamilton, Ontario L8S 4L7, Canada Received November 26, 2001. In Final Form: March 14, 2002
Introduction High molecular weight poly(ethylene oxide) (PEO) is used to flocculate fines and fillers in the papermaking process. However, flocculation is only achieved in the presence of cofactors, which are water-soluble, low molecular weight phenolic polymers capable of forming complexes with PEO.1-4 Commercial cofactors are waterborne phenolic resins.5 Although cost-effective for industry, commercial cofactors are poorly defined polymer mixtures that are not suitable for mechanistic studies. This note is a preliminary report of our investigation into polypeptides as PEO flocculation cofactors. From a scientific perspective, polypeptide chemistry offers narrow dispersity polymers in a broad range of well-defined structures. Furthermore, techniques such as circular dichroism, NMR, and isothermal titration calorimetry have been developed to probe polypeptide structure and conformation. From a practical perspective this work suggests a new, biotechnology-based, formaldehyde-free route to effective cofactors. Finally, from a broader perspective, these results indicate the types of protein segments that might bind strongly to PEO, thus impacting biocompatibility.6 Experimental Section The structures and molecular weights of the peptides are summarized in Table 1. The oligomer peptide (Glu,Tyr)4 with structure HO-Glu-(Tyr-Glu)3-Tyr-NH2 was synthesized on an Advanced Chemtech 348O automated synthesizer by standard FMOC solid-phase peptide chemistry7 on Wang resin (100-200 mesh) preloaded with FMOC-Glu (0.55 mmol/g). Coupling reactions were performed with a 5-fold excess of FMOC-protected L-amino acid in the presence of HBTU/HOBt/DIPEA. All compounds were prepared in >98% purity, as shown by HPLC, and the results of mass spectral analysis (electrospray) were consistent with the calculated masses of the target peptides. The polypeptides were compared to a commercial cofactor, Oxirez, supplied by Ciba (Allied Colloids) Canada.8 Precipitated calcium carbonate, PCC (Albacar HO, Specialty Minerals Inc.), consists of aggregates of scalenohedral needles with a mean particle size of 1.34 µm (Brookhaven Disk Centri* To whom correspondence should be addressed: phone (905) 529-7070, ext 27045; fax (905) 528-5114; e-mail peltonrh@ mcmaster.ca. (1) Pelton, R. H.; Allen, L. H.; Nugent, H. M. Sven. Papperstidn. 1980, 83 (9), 25. (2) Carrard, J. P.; Pummer, H. U.S. Patent 4,070,236, 1973. (3) Pelton R. H. In Colloid-Polymer Interactions; Farinato, R. S., Dubin, P. L., Eds.; John Wiley and Son: New York, 1999; p 51. (4) Pelton, R. H.; Xiao, H.; Brook, M. A.; Hamielec, A. Langmuir 1996, 12, 5756. (5) Carignan, A.; Garnier, G.; van de Ven, T. G. M. J. Pulp Paper Sci. 1998, 24 (3), 94. (6) Harris, J. M. Poly(ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications; Plenum Press: New York, 1992. (7) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161. (8) Satterfield, B.; Stockwell, J. Eur. Pat. Appl. WO 95/21296, 1995.
designation
MW (kDa)
poly(Glu,Tyr) (1:1) poly(Glu,Tyr) (4:1) poly(Glu,Ala,Tyr) (1:1:1) poly(Lys,Tyr) (1:1) poly(Lys,Phe) (1:1) (Glu,Tyr)4
36.1 31.3 28.9 128 47.2 1.12
a The poly samples were random copolymers purchased from Sigma-Aldrich with Glu present as the sodium salt and Lys as the hydrobromide salt. (Glu,Tyr)4 was prepared at McMaster University.
fuge), and a specific surface area of 12.0 m2/g (nitrogen adsorption). Dextran sulfate (MW ) 10 000) was purchased from Sigma as the sodium salt with 2.3 sulfate groups/glucose residue. Two PEO samples, one with a weight-average molecular weight of 8 × 106 Da (Polyox 309) and the other 1 × 106 Da (Polyox N-12K), were obtained from Union Carbide. PEO stock solutions were prepared by dissolving polymer (0.5 or 1 g/L) in water with mild agitation for 24 h. Buffer solution of pH 8 was prepared by mixing tris(hydroxymethyl)aminomethane (Tris) (Boehringer, Mannheim) and HCl. All work was performed with water from a Millipore Milli-Q system fitted with one Super C carbon cartridge, two ion-exchange cartridges, and one Organex Q cartridge. Flocculation measurements were made with a continuous turbidity sensor. The instrument and procedures have been described previously.9 All the experiments were carried out in 200 mL of 0.001 mol/L NaCl solution. In a typical flocculation experiment, 6.25 g of PCC was premixed with 0.125 g of dextran sulfate to give a dilute aqueous suspension of 125 mL. PCC/ dextran sulfate suspension was added to the flocculation system first to give a final concentration of 0.5 g/L, and pH was adjusted with HCl (0.1 mol/L) to 7.8. Polypeptide and PEO were added consecutively at 60 s intervals. The suspension was stirred at 475 rpm with a three-blade propeller (55 mm diameter) and the extent of flocculation was monitored by circulating the suspension through the turbidity sensor at a rate of 45 mL/min. The relative turbidity is approximately proportional to the concentration of unflocculated particles. Thermal titrations were carried out in an ultrasensitive isothermal titration calorimeter (VP-ITC) from MicroCal, LLC (Northampton, MA). In a typical experiment, 0.4 g/L (or 0.6 g/L) peptide solution and 0.6 g/L PEO solution were first prepared by dissolving peptide and PEO into buffer containing 0.001 mol/L CaCl2 and 0.01 M Tris (pH ) 8), respectively. Before the titration, all the samples were degassed by use of MicroCal Thermo-Vac for 5 min. During the titration, 28 10-µL portions of PEO solution were injected into polypeptide solution at 30 °C and stirred at 300 rpm. The duration of injection was set at 20 s and the time interval between each two injections was 240 s. The differential power (baseline) between sample cell and reference cell was adjusted to 5 µcal/s. The heat of diluting PEO solution into buffer, measured in a control experiment, was insignificant. Titration data were processed by MicroCal Origin version 5.0 software.
Results and Discussion Water-soluble polypeptides were prepared or purchased and their structures are summarized in Table 1. Two (9) Gregory, J. J. Colloid Interface Sci. 1985, 105 (2), 357.
10.1021/la015697w CCC: $22.00 © 2002 American Chemical Society Published on Web 05/03/2002
Notes
Figure 1. Flocculation of colloidal precipitated calcium carbonate treated with dextran sulfate (PCC + DS) by PEO 309 in the presence of polypeptides or Oxirez. Experimental conditions: [PCC] ) 0.5 g/L, [DS] ) 0.01 g/L, [polypeptide] or [Oxirez] ) 0.005 g/L, [PEO 309] ) 0.0025 g/L, [NaCl] ) 0.001 M, and temperature ) 25 °C. The pH was adjusted to 7.8 by the addition of 1.4 mL of 0.1 M HCl. PEO was introduced approximately 60 s after the addition of polypeptides or Oxirez.
colloidal systems were employed to evaluate the flocculation efficiencies of PEO/polypeptide mixtures. One consists of precipitated calcium carbonate (PCC), a paper filler that is a positively charged, electrostatically stabilized colloid, which is readily flocculated by PEO/cofactor mixtures.10,11 The second is PCC pretreated with dextran sulfate (PCC + DS) to give an anionic, electrosterically stabilized colloid that is difficult to flocculate and thus is a good model for industrial colloids.12 Figure 1 shows turbidity curves from flocculation experiments. At the beginning of the experiment, the relative turbidity of water was 0. The relative turbidity of the system increased to 1 upon the addition of PCC + DS. Polypeptide was introduced next; none of the polypeptides induced any change in turbidity. PEO was added last, and in those cases where flocculation occurred, the relative turbidity immediately dropped. Note that absolute time values in Figure 1 are arbitrary; the key feature is whether turbidity drops upon PEO addition. Furthermore, PEO alone cannot flocculate PCC + DS.12 The greatest extent of flocculation (i.e., the largest turbidity decrease) was observed when PEO (MW 8 × 106) was added to poly(Glu,Tyr) (1:1), whereas only 30% reduction of relative turbidity was observed when PCC + DS was flocculated with Oxirez, a good commercial cofactor. Note that the poly(Glu,Tyr) (1:1)/PEO complex is negatively charged so flocculation occurs despite electrostatic replusion between the complex and the negatively charged, dextran sulfate-coated PCC; a similar result has been reported for traditional cofactors. The poly(Glu,Tyr) (4:1) curve in Figure 1 is an example of no flocculation. Similar negative results were obtained with poly(Glu,Ala,Tyr) (1:1:1) and (Glu, Tyr)4. The inability of poly(Glu,Tyr) (4:1) and poly(Glu,Ala,Tyr) (1:1:1) to give flocculation may indicate too low a phenolic content to give complexes that can withstand the hydrodynamic forces in the flocculation experiments. Experiments for the cationic, amine-based peptides in PCC + DS are not reported because the results were complicated by the presence of excess dextran sulfate in solution, which interacted with the cationic peptides. (10) van de Ven, T. G. M. J. Pulp Paper Sci. 1997, 23 (9), 447. (11) Gibbs, A.; Pelton, R. H. J. Pulp Paper Sci. 1999, 25 (7), 267. (12) Cong, R.; Smith-Palmer, T.; Pelton, R. J. Pulp Paper Sci. 2001, 27 (11), 379.
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Figure 2. Flocculation of PCC by PEO 309 in the presence of polypeptides. Experimental conditions: [PCC] ) 0.5 g/L, [polypeptide] ) 0.005 g/L, [PEO 309] ) 0.0025 g/L, [NaCl] ) 0.001 mol/L, and temperature ) 25 °C. pH was adjusted to 7.2 by the addition of 3 mL of 0.1 mol/L HCl. PEO was added approximately 60 s after the addition of polypeptides.
Figure 3. Isothermal calorimetric titration of peptides with 0.6 g/L PEO N-12K (MW ) 106 Da) at 30 °C. All the samples were prepared in buffer containing 0.01 mol/L Tris (pH ) 8) and 0.001 mol/L CaCl2. [Poly(Glu,Tyr) (1:1)] ) 0.4 g/L, [poly(Glu,Ala,Tyr) (1:1:1)] ) 0.6 g/L, [(Glu,Tyr)4] ) 0.4 g/L.
To further illustrate the role of the phenolic tyrosine moieties, the flocculation of cationic PCC (no added DS) with poly(Lys,Tyr) (1:1) was compared to that with peptide based on phenylalanine (i.e., pendant benzene groups). The results were summarized in Figure 2. The tyrosinebased peptide flocculated cationic PCC, whereas the phenylalanine peptide showed no flocculation whatsoever. This result emphasizes the importance of tyrosine in PEO/ polypeptide complex formation. Isothermal calorimetric titrations of polypeptides with PEO (MW ) 106 Da) were employed to characterize complex formation. The peptides were dissolved in 0.001 mol/L CaCl2 and 0.01 mol/L Tris (pH ) 8) to mimic the solution conditions in the flocculation experiments.12 The titration results are summarized in Figure 3. The curve for poly(Glu,Tyr) (1:1), which gave the best flocculation, was initially exothermic and became endothermic. The titration of poly(Glu,Ala,Tyr) (1:1:1), which gave no flocculation of PCC + DS, showed a strong endothermic heat effect indicating complex formation. Thus, some PEO/ polypeptides form complexes that are not effective flocculants. Finally, the oligomeric peptide (Glu,Tyr)4 gave no flocculation and showed no heat effects when titrated with PEO. Presumably the oligomer was too short for complex formation.
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Conclusions (1) Water-soluble polypeptides with high tyrosine contents (i.e., phenolic moieties) form soluble complexes with high molecular weight PEO in 0.001 M CaCl2. (2) The PEO/peptide complexes can be good flocculants; however, flocculation efficiency is sensitive to polypeptide structure. PEO complexes with poly(Glu,Tyr) (1:1) gave good flocculation, whereas complexes with poly(Glu,Tyr) (4:1), with a lower tyrosine content, and the more hydrophobic poly(Glu,Ala,Tyr) (1:1:1) did not. Thus, PEO/ polypeptide complex formation is a necessary, but not sufficient, criterion for flocculation. (3) Polypeptide molecular weight is important. Poly(Glu,Tyr) (1:1) with MW 36.1 kDa caused PCC + DS
Notes
flocculation upon the addition of PEO, whereas (Glu,Tyr)4 with MW 1.1 kDa did not. Acknowledgment. This work was supported by the ONDEO-Nalco Company and the Canadian Government NSERC CRD program. The polypeptide synthesizer was funded by the Canadian Foundation for Innovation and the Ontario Innovation Trust. Acknowledged are Rongjuan Cong, Dr. Richard Epand, and Ms. Raquel Epand for useful discussions and the access to VP-ITC. C.L. also thanks the Ontario Graduate Scholarship Program and Shell Canada for scholarships. LA015697W
