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Langmuir 2003, 19, 9538-9541
Poly(N-isopropylacrylamide-co-2-methacryloamidohistidine) Copolymers and Their Interactions with Human Immunoglobulin-G Emel Kalaycıogˇlu,† Su¨leyman Patır,‡ and Erhan Pis¸ kin*,† Department of Chemical Engineering and Bioengineering Division, Department of Science-Education, Hacettepe University, 06532 Beytepe, Ankara, Turkey Received December 20, 2002. In Final Form: August 22, 2003
Introduction In recent years, thermosensitive polymers have been promoted as useful tools in biotechnological applications such as enzyme immobilization,1-4 thermal affinity separation,1,5,6 controlled drug release,1,7 immunodiagnostics,1,8 gene therapy,9-12 and so forth. Most of these polymers have been produced in the form of homopolymers or copolymers of N-isopropylacrylamide (NIPA). It is known that poly(NIPA) shows a volume phase transition in aqueous solution around its lower critical solution temperature (LCST);13 a small increase of this temperature provided a unique coil-to-globular conformation change of its macromolecules, which transferred from a soluble to a nonsoluble state as a result of a balance of hydrophilic (H-bonding)-hydrophobic interactions. This balance may be controlled by using various functional copolymers of NIPA.14-26 * Corresponding author. Tel./fax: +90 312 297 7400/+90 312 299 21 24. E-mail address:
[email protected]. † Department of Chemical Engineering and Bioengineering Division. ‡ Department of Science-Education. (1) Galaev, I. Y.; Mattiason, B. Tibtech 1999, 17, 335. (2) Chen, G.; Hoffman, A. S. Macromol. Chem. Phys. 1995, 196, 1251. (3) Chen, J. P.; Hsu, M. Sh. J. Mol. Catal. B: Enzym. 1997, 2, 233. (4) Kondo, A.; Imura, K.; Nakama, K.; Higashitani, K. J. Ferment. Bioeng. 1994, 78, 241. (5) Anastase-Ravion, S.; Ding, Z.; Hoffman, A. S.; Letourneur, D. J. Chromatogr., B 2001, 761, 247. (6) Kondo, A.; Kaneko, T.; Higashitani, K. Biotechnol. Bioeng. 1994, 44, 1. (7) Dilgimen, A. S.; Mustafaeva, Z.; Denchenko, M.; Kaneko, T.; Osada, Y.; Mustafaev, M. Biomaterials 2001, 22, 2383. (8) Leroux, J. C.; Roux, E.; Le Garrec, D.; Hong, K.; Drummond, D. C. J. Controlled Release 2001, 72, 71. (9) Hinrichs, W. L. J.; Schuurmans-Nieuwenbroek, N. M. E.; Wetering, P. V.; Hennink, W. E. J. Controlled Release 1999, 60, 249. (10) Bulmus¸ , V.; Patır, S.; Tuncel, A.; Pis¸ kin, E. J. Controlled Release 2001, 76, 265. (11) Dinc¸ er, S.; I˙ bris¸ ogˇlu, T.; Pis¸ kin, E. Abstr. NATO/ASI on Polymer Based Systems Tissue Engineering, Replacement and Regeneration; Algarve: Alvor, Portugal, 2001. (12) Dinc¸ er, S.; Tuncel, A.; Pis¸ kin, E. Macromol. Chem. Phys. 2002, 203, 1460. (13) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163. (14) Huglin, M. B.; Liu, Y.; Velada, J. L. Polymer 1997, 38, 5785. (15) Velada, J. L.; Liu, Y.; Huglin, M. B. Macromol. Chem. Phys. 1998, 199, 1127. (16) Brazel, C. S.; Perras, N. A. Macromolecules 1995, 28, 8016. (17) Brazel, C. S.; Perras, N. A. J. Controlled Release 1996, 39, 57. (18) Dinc¸ er, S.; Ko¨seli, V.; Kesim, H.; Rzaev, Z. M. O.; Pis¸ kin, E. Eur. Polym. J. 2002, 38, 2143. (19) Kesim, H.; Rzaev, Z. M. O.; Dinc¸ er, S.; Pis¸ kin, E. Polymer 2002, in press. (20) Chen, J. P.; Chu, D. H.; Sun, D. M. J. Chem. Technol. Biotechnol. 1997, 69, 421. (21) Erbil, C.; Akpinar, F. D.; Uyanık, N. Macromol. Chem. Phys. 1999, 2000, 2448. (22) Erbil, C.; Sarac¸ , A. S. Eur. Polym. J. 2002, 38, 1305. (23) Katime, I.; Valderruten, N.; Quintana, J. R. Polym. Int. 2001, 50, 869.
Monoclonal antibodies (i.e., immunoglobulins, IgGs) offer exciting potential as diagnostic and therapeutic substances and also serve as bioaffinity ligands for purifiying other high-value proteins of pharmaceutical importance.27 Several affinity separation techniques are used for the purification of antibodies. Pseudospecific ligands can be used for the bioaffinity separation of antibodies.28 They have low binding constants and, consequently, belong to the family of weak affinity ligands. Recently, it has been found that amino acids as pseudospecific ligands may hold certain advantages for industrial bioaffinity separations because they are not likely to cause an immune response in the case of leakage into the product. These ligands are also much more stable than protein ligands because they do not require a specific tertiary structure for maintaining biological activity.29 They offer additional advantages over biological ligands in terms of economy, ease of immobilization, and high adsorption capacity. Histidine has been used as a ligand in the affinity chromatography of proteins.30-33 Histidine interacts through its carboxyl, amino, and imidazole groups with several proteins at near their isoelectric points and has shown particular efficacy in separating IgG subclasses from human plasma and in the purification of monoclonal antibodies from cell cultures or ascite fluids.34 For example, calf chymosine, myxaline, and acid protease from Aspergillus niger, as well as catechol-2,3-dioxygenease, have been purified on histidine-immobilized supports.35 Histidine-linked matrixes were used for the purification of IgG from human plasma by us and also by others.36,37 Recently, we have attempted to use water-soluble thermo- and pH-sensitive NIPA-based copolymers for the affinity separation/identification of proteins in aqueous media, as an alternative to the approaches in which solid supports carrying the ligand molecules are usually used. In our previous study, we reported the use of NIPA and 2-(dimethylamino)propyl methacrylamide copolymers for albumin determination/separation.38 In the present study, NIPA was copolymerized with a histidine (as a pseudospecific ligand)-carrying monomer to obtain a temperature-sensitive copolymer for the bioaffinity separation of human immunoglobulin-G (HIgG) from aqueous media. This paper reports the synthesis/characterization of these copolymers and their interaction with HIgG. (24) Hirotsu, S.; Hirokawa, Y.; Tanaka, T. J. Chem. Phys. 1987, 87, 1302. (25) Wang, C.; Cao, W. Polym. Int. 1996, 41, 449. (26) Ko¨seli, V.; Rzaev, Z. M. O.; Pis¸ kin, E. Macromol. Chem. Phys., submitted for publication. (27) Duffy, S. A.; Moellering, G. M.; Prior, G. M.; Doyle, K. R.; Prior, C. P. BioPharm. 1989, September/October, 46. (28) Bueno, S. M. A.; Legallais, C.; Haupt, K.; Vijayalakshmi, M. A. J. Membr. Sci. 1996, 117, 45. (29) Huang, P. Y.; Carbonell, R. G. Biotechnol. Bioeng. 1999, 63, 633. (30) Vijayalakshmi, M. A. Trends Biotechnol. 1989, 7, 71. (31) Haupt, K.; Vijayalakshmi, M. A. J. Chromatogr. 1993, 644, 289. (32) Alvarez, C. I.; Strumia, M. C.; Bertorello, H. E. React. Funct. Polym. 1997, 34, 103. (33) Legallais, C.; Anspach, F. B.; Bueno, S. M. A.; Haupt, K.; Vijayalakshmi, M. A. J. Chromatogr., B 1997, 691, 33. (34) El-Kak, A.; Vijayalakshmi, M. A. J. Chromatogr., B 1992, 604, 29. (35) Bueno, S. M. A.; Haupt, K.; Vijayalakshmi, M. A. J. Chromatogr., B 1995, 667, 57. (36) Ventura, R. C. A.; Zollner, R.; Legallais, C.; Vijayalakshmi, M. A.; Bueno, S. M. A. Biomol. Eng. 2001, 17, 71. (37) Garipcan, B.; Denizli, A. Macromol. Biosci. 2002, 2, 135. (38) Tuncel, A.; Demirgo¨z, D.; Patır, S.; Pis¸ kin, E. J. Appl. Polym. Sci. 2002, 84, 2060.
10.1021/la027042g CCC: $25.00 © 2003 American Chemical Society Published on Web 09/30/2003
Notes
Langmuir, Vol. 19, No. 22, 2003 9539
Figure 1. Chemical structure of poly(NIPA-co-MAH).
Experimental Section Materials. NIPA monomer (Aldrich) was purified before use by recrystallization from n-hexane: bp 91.5 °C, mp 61.6 °C. The initiator, R,R′-azoisobutyronitrile (AIBN; Fluka) was recrystallized twice from methanol: mp 102.5 °C. Methanol and diethyl ether, both supplied from Merck, were used as the solvent and precipitant, respectively, in the purification of the copolymers. The monomer 2-methacryloamidohistidine (MAH) was synthesized from L-histidine (Biological Industries). HIgG was obtained from Sigma (St. Louis, U.S.A.). All other reagents were analytical grade and used as obtained. Synthesis of MAH. For the synthesis of MAH, the following experimental procedure was applied: 5.0 g of L-histidine and 0.2 g of hydroquinone were dissolved in 100 mL of CH2Cl2 solution. This solution was cooled to 0 °C. A total of 12.74 g of triethylamine was added to the solution. 5.0 mL of methacryl chloride was poured slowly into this solution under a nitrogen atmosphere, and then this solution was stirred magnetically at room temperature for 2 h. At the end of this chemical reaction period, unreacted methacryl chloride was extracted with 10% NaOH. The aqueous phase was evaporated in a rotary evaporator. The residue (i.e., MAH) was dissolved in ethanol. The Fourier transform infrared (FTIR) spectrum of MAH monomer has the characteristic streching vibration band and amide I and amide II absorption bands at 1647 and 1514 cm-1, carbonyl band at 1750 cm-1, and hydroxyl band at 3678 cm-1. 1H NMR spectra of MAH monomer (in D O at room temper2 ature), ppm: (1) 2H, CH2 1.21-1.65; (2) 3H, CH3 1.99; (3) 2H, CH2 4.62; (4) 1H, CH 5.21; (5) 1H, NH 5.79; (6) 2H, CH 6.81; (7) 1H, NH 7.53; (8) 1H, COOH 9.9. Synthesis of Poly(NIPA-co-MAH) Copolymers. A series of NIPA and MAH copolymers were synthesized by solution copolymerization by varying both the “NIPA/MAH” feed ratio (between 100/0.00 and 90.76/9.24 mol/mol) and the initiator concentration (between 0.5 and 2 mol %). A typical procedure was as follows: NIPA (2.0 g) and MAH (0.1 g) were dissolved in methanol (30 mL) and placed in a sealed cylindrical polymerization reactor (total volume, 100 mL), and AIBN was added to this homogeneous solution. After the complete dissolution of AIBN (0.06 g), the medium was purged with nitrogen for 5 min. The sealed reactor was placed into a shaking bath equipped with a temperature-control system. The copolymerization was conducted at 70 °C for 24 h at a shaking rate of 120 cpm under a nitrogen atmosphere. The unreacted monomers were removed by precipating the copolymerization mixture with diethyl ether. The precipitate was filtered through a sintered glass filter, washed repeatedly with diethyl ether, and dried in a vacuum at 40 °C for 48 h. The dissolution/precipitation procedure was repeated two times for the removal of impurities in the precipitated copolymer. Characterization of Poly(NIPA-co-MAH) Copolymers. The chemical structure of the NIPA-co-MAH random copolymer is shown in Figure 1. The chemical structures of the copolymers were analyzed by FTIR and 1H NMR. The FTIR spectra of the copolymers (KBr pellets) were recorded with a FTIR spectrophotometer (Schimadzu, DR8101, Japan) in the 4000-400-cm-1 range at room temperature. The 1H NMR spectra of the copolymers were recorded on a JEOL 6X-400 (400 MHz) spectrometer with D2O as the solvent at room temperature.
FTIR spectra (KBr pellet), cm-1: 3400-3100 (w) broad bands for NH secondary amide, 2960 (m) CH3 streching and 1380 (s) CH streching of sCH(CH3)2 group for NIPA unit, 3524 (w) OH streching, 1645 (w) CdO streching of amide I band, 1513 (m) NH streching of amide II band, 1550 (m) CdO streching, 710 (s) imidazole ring deformation for MAH unit. 1H NMR spectra (in D O at room temperature), ppm: (1) 6H, 2 CH3 3.2; (2) 2H, CH2 3.4; (3) 1H, CH 3.6; (4) 1H, CH 4.1; (5) 1H, NH 7.9 for NIPA unit; (6) 2H, CH2 1.1-1.4; (7) 3H, CH3 1.8; (8) 2H, CH2 5.5; (9) 1H, NH 5.9; (10) 1H, CH 6.2; (11) 2H, CH 6.8; (12) 1H, NH 7.4; (13) 1H, COOH 9.5 for MAH unit. The MAH content of the copolymer was obtained by back titration. A typical procedure was as follows: 0.2 g of copolymer was dissolved in 15 mL of a 0.1 M NaOH solution. Then, this mixture was back-titrated with a 0.2 M HCl solution. During the titration, the changes in the pH of the copolymer solution were determined by using a pH meter (pH-900, NEL). After titration, ∆pH/∆V versus Vtitrant was plotted to determine the equivalence point. By using the value of the used HCl volume at the equivalence point, the content of carboxyl group of the copolymer was calculated. Each MAH unit consists of one carboxyl group; therefore, the calculated value of the carboxyl group content is assumed equal to the MAH content of the copolymer. The conversions were determined by gravimetric analysis of the copolymer obtained at the end of polymerization and the amount of total monomer used, as given in eq 1.
polymerization yield % ) amount of copolymer obtained (g) × 100 (1) amount of total monomer used (g) The number-average molecular weights of the copolymers were determined by using an Ubbelohde viscosimeter (Schott Gerate, Germany). The viscosities of copolymer solutions (in methanol) prepared in the concentration range of 0.25-1.0 g/dL were measured at 25 °C. The viscosity-average molecular weights were calculated using eq 2.39
[η] ) 3.0 × 10-4 × Mη0.64
(2)
LCST measurements were performed in a UV-visible spectrophotometer (V-530 Jasco) equipped with a heating system and a thermocouple. An aqueous 0.5% (w/w) solution of poly(NIPA-co-MAH) copolymer was put into a polystrene cuvette with a volume of 2 mL. The temperature of the solution was increased at a rate of 1 °C/min, starting from room temperature, and the absorbance of the solution was perodically recorded at a wavelength of 540 nm. The LCST values of the copolymers were calculated from the absorbance-temperature curves by using the method described in the literature.40 These measurements were performed at pH values of 4.0, 7.4, and 9.0. The pH was adjusted to the desired value by adding 0.1 N HCl or 0.02 N NaOH to the freshly prepared copolymer solutions. Interaction of Copolymers with HIgG. NIPA-co-MAH random copolymers produced with different NIPA/MAH feed ratios were interacted with HIgG solutions with different concentrations (0-4000 µg/mL) at pH 7.4. The poly(NIPA-coMAH) concentration was kept constant at 1% (w/v). LCST values were obtained by measuring absorbances of the solutions at a wavelength of 540 nm as a function of temperature in a UV-vis spectrophotometer.40 The interaction kinetics of HIgG molecules with poly(NIPAco-MAH) copolymers were investigated in aqueous media containing different amounts of HIgG (0.25, 0.5, 1.0, and 2.0 mg HIgG/ml) and a constant amount of copolymer (9.24:90.76 (mol/ mol) MAH/NIPA). These interactions were followed at room temperature and at pH 7.4. The change of HIgG concentration with time was obtained by using the initial HIgG concentrations and also measuring the concentration of HIgG left in the interaction medium after the conjugates were separated from the medium at selected time intervals. HIgG concentrations were (39) Ganachaud, F.; Monteiro, M. J.; Gilbert, R. G.; Dourges, M. A.; Thang, S. H.; Rizzardo, E. Macromolecules 2000, 33, 6738. (40) Buutris, C.; Chatzi, E. G.; Kiparissides, C. Polymer 1997, 38, 2567.
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Notes
Table 1. MAH Contents of Copolymers Obtained by Back Titration feed composition
copolymer composition
polymer no.
NIPA (mol)
MAH (mol)
NIPA MAH MAH/copolymer (mol) (mol) (mmol/g copolymer)
1 2 3 4 5 6
98.74 97.52 95.15 90.76 95.15 95.15
1.26 2.48 4.85 9.24 4.85 4.85
98.80 97.39 95.22 91.12 94.97 95.58
1.20 2.61 4.78 8.88 5.03 4.42
0.125 0.225 0.405 0.725 0.425 0.375
Table 2. Polymerization Yields and Viscosity-Avarage Molecular Weights of Poly(NIPA) and Its Copolymers with MAH polymer NIPA/MAH AIBN copolymerization viscosity-average no. (mol/mol) (mol %) yield (%) molecular weight 0 1 2 3 4 5 6
100.00/0.00 98.74/1.26 97.52/2.48 95.15/4.85 90.76/9.24 95.15/4.85 95.15/4.85
2.00 2.00 2.00 2.00 2.00 1.00 0.05
85 89 82 91 81 87 86
82 945 62 376 62 790 62 082 62 541 68 142 77 590
determined with an UV-vis spectrophotometer by measuring the absorbances of the solutions at a wavelength of 280 nm.
Results and Discussion Poly(NIPA-co-MAH) Copolymers. A series of NIPA and MAH copolymers were synthesized by changing both the NIPA/MAH feed ratio (100:0.00-90.76:9.24 mol/mol) and the initiator (AIBN) concentration (0.5-2.0 mol %). The MAH contents of the copolymers obtained by back titration are given in Table 1. Note that MAH incorporated to the polymer chains during the copolymerization of NIPA and MAH used in the initial recipe in most of the polymerizations are very similar. Poly(NIPA-co-MAH) Copolymers: Polymerization Yields and Molecular Weights. The polymerization yields achieved are given in Table 2. As seen here, satisfactorily high polymer yields (81 and 91%) were achieved in all cases. The molecular weights of both the homopolymer and copolymers synthesized with different recipes were measured with a viscosimeter. Note that, as mentioned in the literature, there have been significant controversies in the molecular weight determination of poly(NIPA) by gel permeation chromatography (GPC).39,41,42 Some authors reported that the GPC method could not be used to obtain molecular weight information for this polymer because of filtration problems.39,42,43 Recently, the molecular weight characterization of poly(NIPA) was extensively investigated by Ganachaud et al.39 In their study, the molecular weights determined by GPC were compared with those obtained by matrix-assisted laser desorption/ionization mass spectrophotometry time-of-flight (MALDI-TOF) and theoretically predicted values. This comparison indicated that MALDI-TOF and GPC provided similar molecular weight distributions, at least for the low molecular weight range, whereas discrepancies arose for the higher molecular weights. Note that in the previous studies we also tried to measure the molecular weights of poly(NIPA) and its copolymers with other comonomers by GPC with (41) Wu, X. Y.; Pelton, R. H.; Tam, K. C.; Woods, D. R.; Hamielec, A. E. J. Polym. Sci., Part A: Polym. Chem. 2000, 31, 957. (42) Yasui, M.; Shiroya, T.; Fujimoto, K.; Kawaguchi, H. Colloids Surf., B 1997, 8, 311. (43) Haupt, K.; Bueno, S. M. A.; Vijayalakshmi, M. A. J. Chromatogr., B 1995, 667, 57.
Figure 2. Stimuli-responsive behavior of the copolymer synthesized with a MAH/NIPA feed ratio of 4.85:95.15 mol/ mol. Table 3. LCST Values of Poly(NIPA) and Its Copolymers with MAH at Three Different pH Values LCST (°C) polymer no.
pH 4.0
pH 7.4
pH 9.0
0 1 2 3 4 5 6
29.0 29.9 30.8 31.3 31.6 31.1 30.9
29.0 31.2 32.7 33.4 34.1 32.7 32.5
30.6 31.3 31.7 32.0 31.5 31.4
tetrahydrofuran as the eluent. However, we could not obtain meaningful results. As a consequence, we prefer to measure the molecular weight by the viscosimetric method in which the Mark-Houwink-Sakaruda parameters were taken as the values determined for poly(NIPA) in the study performed by Ganachaud et al.39 In our studies, the MAH concentration was kept lower than 10 mol %. By considering the low MAH contents of the copolymer samples, this approach was selected for the molecular weight determination.38 The viscosity-average molecular weight of the poly(NIPA) homopolymer was about 83 × 103. The molecular weights of the copolymers synthesized by using the same amount of initiator but with different NIPA/MAH ratios were about 62 × 103 and did not change with the MAH content. However, there were noticeable increases in the molecular weights when the initiator concentration was decreased, which is expected. The stimuli-responsive behavior of both the poly(NIPA) homopolymer and its copolymers with MAH was studied at pH values of 4.0, 7.4, and 9.0. To obtain the LCSTs, absorbances of aqeous solutions containing 0.5% (w/w) homo- or copolymer were measured at 540 nm. A typical absorbance-temperature curve for a copolymer synthesized with a MAH/NIPA ratio of 4.85:95.15 mol/mol and with an initiator concentration of 2 mol % is given in Figure 2. As seen here, there was a sharp increase in the absorbance when the phase transition was achieved. Note that at this point the transparent copolymer solution became turbid. The LCST values were determined from these curves as the temperature at which 10% of the absorbance plateau value was reached.2,38 The LCST values of both the poly(NIPA) homopolymer and its copolymers with MAH at three different pH values are given in Table 3. Note that the LCST value of the poly(NIPA) homopolymer was 29 °C and did not change with pH, which means that there is no pH sensitivity of the homopolymer. The
Notes
Figure 3. Changes in the LCST values of the copolymer with the concentration of HIgG in the medium. Determined spectrophotometrically at pH 7.4.
Figure 4. HIgG adsorption capacity values of the copolymer (0.725 mmol MAH/g copolymer). Determined at pH 7.4 and at room temperature.
incorporation of MAH in the copolymer chains resulted in an increase in the LCST value, which is also dependent on the pH of the medium, and has a maximum at pH 7.4. Note also that the increase in the LCST value is more when the MAH content is higher. As mentioned in the literature, an increase in the pH of the copolymer solution ionizes the COOH groups of the MAH units. The ionized carboxyl groups make the copolymer chains more hydrophilic, and this hardens the phase transition of the copolymer so that it increases the LCST value.44 It was also observed that there were decreases in the LCST values, not very significant but noticeable, when the molecular weights of the copolymers are increased. From the literature, it is seen that, when the molecular weights of the copolymers increase, as a result of the temperature increase between the copolymer chains, hydrophobic interactions increase. And this causes a decrease in the LCST values.44-46 (44) Bulmus¸ , V. Ph.D. Dissertation, Hacettepe University, Ankara, Turkey, 2000. (45) Feil, H.; Bae, Y. H.; Feijen, J.; Kim, S. W. Macromolecules 1993, 26, 2496.
Langmuir, Vol. 19, No. 22, 2003 9541
Interactions of HIgG with Poly(NIPA-co-MAH) Copolymers. Poly(NIPA-co-MAH) copolymers having different MAH contents were interacted with HIgG solutions at pH 7.4. The temperatures of these solutions (containing 1% (w/w) copolymer) were increased, and the absorbance-temperature curves were obtained. Then, the LCST values of the conjugates were evaluated as described before. Figure 3 shows the changes of LCST values with the HIgG concentration for three different copolymers with different MAH contents (indicated on the graph). Note that these interactions are most probably through the imidazole ring of histidine (on the copolymer chains) and the Fab region of the HIgG molecule. Note that most probably the change in the hydrophobicity-hydrophilicity balance of the copolymer chains due to the increase in the hydrophobicity of conjugation with the HIgG molecules caused the decrease in LCST. The kinetics of interaction of poly(NIPA-co-MAH) copolymers with HIgG were followed by measuring the changes in the absorbances of the solutions at a wavelength of 280 nm as a function of time. Figure 4 shows the interaction curves obtained with poly(NIPA-co-MAH) containing 0.725 mmol MAH/g copolymer. The HIgG initial concentrations are indicated on the graph. These experiments were done at room temperature at pH 7.4 with 1% (w/w) copolymer solution. As seen here, the reactions almost reached equilibrium in about 90-120 min, which is very similar to that reported in the literature with solidphase carriers. As a result of the interaction of copolymer chains with HIgG molecules through the MAH components of the copolymers, the equilibrium adsorption time was found to be 90 min, which shows good agreement with the literature.43,47-49 Conclusion In this study, random copolymers of NIPA and MAH were synthesized. The yields in the copolymerizations were successful and were in the range of 81-91 mol %. Chemical analysis of the products proved the formation of copolymers. The copolymer was both temperature- and pHsensitive. The produced copolymers were also sensitive to HIgG in the aqueous media. In a certain range of HIgG concentrations, the LCST of the copolymers showed a linear decrease with the increase in the HIgG concentration at different pH values. After the interaction of the copolymer chains with the HIgG molecules through the MAH components of the copolymers, the equilibrium adsorption time was found to be 90 min, which shows good agreement with the literature. The results indicated that the NIPA-co-MAH copolymer could be utilized as a new reagent for the determination of HIgG concentration in an aqueous medium. LA027042G (46) Dinc¸ er, S. M. Sc. Dissertation, Hacettepe University, Ankara, Turkey, 2000. (47) Denizli, A.; Arıca, Y. J. Biomater. Sci., Polym. Ed. 2000, 11, 367. (48) O ¨ zkara, S.; Yavuz, H.; Patır, S.; Arıca, M. Y.; Denizli, A. Sep. Sci. Technol. 2001, 37, 717. (49) El-Kak, A.; Vijayalakshmi, M. A. J. Chromatogr., B 1992, 604, 29.
