Novel Support Material with Polymeric Ligand for Ion Exchange Chromatography: Synthesis, Characterization and Chromatographic Performance

Öz

Within the scope of the study, polymeric ligands with weak cation exchange carboxyl (-COOH) groups formed on the surface, designed for High Performance Liquid Chromatography (HPLC), monodisperse and porous poly(2,3-dihydroxypropyl methacrylate-co-ethylene dimethacrylate), poly(DHPM- co-EDM) particles were tested in Ion Exchange Chromatography (IEC) mode for chromatographic separation of protein mixtures as a weak cation exchange column-material. The synthesis of the particles obtained for the design of the column support material, which is the first step of the study carried out in three stages, takes place in four different sections. Firstly, monodisperse polystyrene microgels to form the skeletal structure were synthesized by “Dispersion Polymerization”, then monodispersed-porous poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate), poly(GMA-co-EDM) microspheres with epoxy groups were produced by using “multi-stage microsuspension polymerization”. The particles were hydrolyzed in acidic medium to form the surface in diol form and poly(2,3-dihydroxypropyl methacrylate-co-ethylene dimethacrylate), poly(DHPM-co-EDM) particles were obtained which were suitable for chromatographic use. The particles obtained in the last step were used in the “Free Radical Grafting Polymerization” (FRGP) method for the production of polymeric ligand containing carboxyl group. Poly (DHPM-co-EDM) particles with –OH groups on the surface are silanated with 3-trimethoxysilylpropyl methacrylate (TMSPM) and derivatized with methacrylic acid (MAA) to form poly(MAA) ligand on the surface of poly(DHPM-c-EDM) particles were obtained for ion-exchange chromatography. The characterization and chromatographic performance of the particles were investigated in the second and third stages of the study. For particles synthesized by the FRGP method, the average-pore size was 33 nm and the specific-surface area was 21 m²/g. Theoretical plate height values ​​between 200-320 µm were obtained for weak cation exchange column. The peak resolution values ​​in the chromatogram were found to be above 1.4 for separation as short as 15 minutes. Protein recovery values ​​were found to be between 90-100% for all proteins. For the produced poly(MAA)-grafted-poly(DHPM-co-EDM) column, the BSS values ​​of the analytes from analysis to analysis and from day to day reproducibility tests were below 1%. These results showed that synthesized column packed materials can be successfully used in ion exchange chromatography. 

Anahtar Kelimeler

• Free Radical Grafting Polymerization (FRGP),High Performance Liquid Chromatograpy (HPLC),Ion Exchange Chromatography (IEC),Monodisperse and porous polymeric particles

Kaynakça

1. [1]. T. Ellingsten, O. Aune, J. Ugelstad, S. Hagen, “Monosized Stationary Phases For Chromatography”, Journal of Chromatography, vol. 535, pp. 147-161. 1990.
2. [2]. Q.C. Wang, F. Svec, J.M.J. Frechet, “Fine Control of the Porous Structure and Chromatographic Properties of Monodispers Macroporous Poly(styrene-co-divinylbenzene) Beads Prepared Using Polymer Porogens”, Journal of Polymer Science: Part A: Polymer Chemistry, vol. 32, pp. 2577-2588. 1994.
3. [3]. M. Galia, F. Svec, J.M.J. Frechet, “Monodisperse Polymer Beads As Packing Material For High Performance Chromatography: Effect of Divinylbenzene Content on the Porous and Chromotagraphic Properties of Poly(styrene-co-divinylbenzene) Beads Prepared in the Presence of Lineer Polystyrene as a Porogen”, Journal of Polymer Science: Part A: Polymer Chemistry, vol. 32, pp. 2169-2175. 1994.
4. [4]. F. Svec, J.M.J. Frechet, “New Designs of Macroporous Polymers and Supports: From Seperation to Biocatalysis”, Science, vol. 273, pp. 205-211. 1996.
5. [5]. K. Hossoya, M. Teramachi, N. Tanaka, A. Kobayashi, T. Kanda, Y. Ohtsu, “Preparation Strategy for Uniformly Sized, Polymer-Beads HPLC Packing Materials Having Practically Acceptable Column Efficiency. 1. Copolymerization Technique”, Anal. Chem., vol. 73, pp. 5852-5857. 2001
6. [6]. B. Saraçoğlu Kaya, A. Tuncel, “Chromatographic Performance of Weak Cation Exchanger Synthesized by a Simple Method”, 1st International Symposium on Multidisciplinary Studies and Innovative Technologies, ISMSIT, 2017, Paper ID: 343, p. 318.
7. [7]. B. Saraçoğlu Kaya, A. Tuncel, “Chromatographic Performance of Weak Cation Exchanger Carrying Molecular Brushes Synthesized by Atom Transfer Radical Polymerization”, 1st International Symposium on Multidisciplinary Studies and Innovative Technologies, ISMSIT, 2017, Paper ID: 341, p. 310.
8. [8]. B. Saraçoğlu Kaya, A. Tuncel, “Chromatographic Performance of Weak Cation Exchanger Carrying Molecular Brushes Synthesized by Reversible Addition Fragmentation Chain Transfer Polymerization”, 1st International Symposium on Multidisciplinary Studies and Innovative Technologies, ISMSIT, 2017, Paper ID: 342, p. 314.

9. [9]. B. Saraçoğlu Kaya, E. Sağ, “Design of Reversed Phase Chromatography (RPC) Column Packing Material for Protein Separation: Part 1-Synthesis and Characterization”, Cumhuriyet Science Journal vol. 38 (4), pp. 788-797. 2017.
10. [10]. B. Saraçoğlu Kaya, E. Sağ, “Design of Reversed-Phase Chromatography Column Packing Material for Protein Separation: Part 2- Chromatographic Performance”, Cumhuriyet Science Journal vol. 40 (1), pp. 213-220. 2019.
11. [11]. B. Caglayan, E. Unsal, T. Çamlı, M. Tuncel, A. Tuncel, “Monodisperse porous poly(vinyl acetate-co-divinylbenzene) particles by single-stage seeded polymerization: A packing material for reversed phase HPLC”, Journal of Separation Science, vol. 29, pp. 936-934. 2006.
12. [12]. V. Smigol, F. Svec, J.M.J. Frechet, “2-Dimensional High-Performance Liquid-Chromatography Using Monodisperse Polymer Beads Containing Segregated Chemistries Prepared by Pore-Size Specific Functionalization -Single-Column Combinations of Size Exclusion or İon-Exchange With Reversed-Phase Chromatography”, Analytical Chemistry, vol. 66 (23), pp. 4308-4315. 1994.
13. [13]. V. Smigol, F. Svec, J.M.J. Frechet, “High-Performance Liquid Chromatography of Complex mixtures Using Monodisperse Dual-Chemistry Polymer Beads Prepared by a Pore-Size-Specific Functionalization Process. A Single Column Combination of Hydrophobic Interaction and Reversed-Phase Chromatography”, Analytical Chemistry, vol. 66, pp. 2129-2138. 1994.
14. [14]. K. Lewandowski, F. Svec, J.M.J. Frechet, “Polar, A Novel Polar Seperation Medium For The Size Exclusion Chromatography of Small Molecules: Uniformly Sized, Porous Poly(vinylphenol-co-divinylbenzene) Beads”, J. Liq. Chrom. & Rel. Technol., vol. 20, pp. 227-243. 1997.
15. [15]. K. Lewandowski, F. Svec, J.M.J. Frechet, “Polar, Monodisperse, Reactive Beads from Functionalized Metacrylate Monomers by staged Templated Suspension Polymerization”, Chem. Mater., vol. 10, pp. 385-391. 1998.
16. [16]. S.T. Camlı, S. Şenel, A. Tuncel, “Cibacron Blue F3G-A Attached Uniform and Macroporous Poly(styrene-divinlybenzene) Particles For Specific Albumin Adsorption”, J. Biomater. Sci. Polym., vol. 10, pp. 875-889. 1999.
17. [17]. E. Unsal, S.T. Camli, M. Tuncel, S. Senel, A. Tuncel, “Monodisperse-Porous Particles With Different Polarities By Modified Seeded Polymerization and Their Use As Chromatographic Packing In HPLC”, Reactive and Functional Polymers, vol. 61, pp. 353–368. 2004.
18. [18]. E. Unsal, B. Elmas, B. Çağlayan, M. Tuncel, S. Patır, A. Tuncel, “Preparation of An Ion Exchange Chromatographic Support By A New Grafting From Strategy Based on Atom Transfer Radical Polymerization”, Analytical Chemistry, vol. 78, pp. 1-8. 2006.
19. [19]. E. Unsal, T. Irmak, E. Durusoy, M. Tuncel, A. Tuncel, “Monodisperse porous particles with polyionic ligands for ion exchange separation of proteins”, Analytical Chimica Acta, vol. 570, pp. 240-248. 2006.
20. [20]. E. Ünsal, “Yüksek Performanslı Sıvı Kromatografisi İçin Farklı Polaritelere Sahip, Fonksiyonel Gruplu Kolon Dolgu Materyallerinin Sentezi ve Kromatografik Karakterizasyonu”, Doktora Tezi, Hacettepe Üniversitesi Fen Bilimleri Enstitüsü, Ankara, Türkiye, 2006.
21. [21]. M. Petro, F. Svec, , J.M.J. Frechet, “Monodisperse Hydrolized Poly(glycidylmethacrylate-co-ethylene dimethacrylate) Beads as a Stationary Phase for Normal-Phase HPLC”, Analytical Chemistry, vol. 69, pp. 3131-3139. 1997.
22. [22]. E. Unsal, E. Uguzdogan, S. Patir, A. Tuncel, “Ion-exchanger synthesis using reversible addition-fragmentation chain transfer polymerization”, Journal of Separation Science, vol. 32, pp. 1791-1800. 2009.