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 Institut für Polymerchemie

Lehrstuhl für Makromolekulare Stoffe und Faserchemie

Prof. Dr. Michael R. Buchmeiser

Monolithic Polymeric Stationary Phases for Analytical and Preparative-Scale Separations of Low and High Molecular
Weight Analytes Including Proteins and DNA, as Well as For Down-Stream Processing


Large-volume monolithic columns are prepared via transition metal-catalyzed ring-opening metathesis polymerization (ROMP). Since ROMP rapidly proceeds even at lower temperature with high initiation efficiency, this polymerization process is easier to control by external cooling of the polymerization mixture. As a result, the generated heat of polymerization can easily dissipate. In turn, the high control over polymerization temperature provides highly mechanically stable monoliths with uniform porous structure. For monolith synthesis, e.g., trimethylolpropane-tris-(5-norbornene-2-carboxylate) and norborn-2-ene are used as cross-linker and monomer, respectively. Monolithic media are synthesized, e.g., using the 1st-generation Grubbs initiator RuCl2(PCy3)2(CHPh) (Cy=cyclohexyl) in the presence of macro- and microporogens such as 2-propanol and toluene. Alternatively, Schrock catalysts can be used, however with different porogens. To prepare large-volume monoliths, bulk polymerizations can be completed within borosilicate or PEEK column formats with diameters in the range of 3 to 49 mm. The pore structure and the basic properties of the large-volume monoliths is investigated by electron microscopy and inverse size exclusion chromatography (ISEC). Typically, monoliths 100x10, 230 x 26, 460x26 and 230 x 49 mm i.d. in size are tested, e.g., for the separation of a mixture of five proteins, i.e., insulin, cytochrome C, lysozyme, conalbumin, and ß-lactoglobulin. Preparative separation of these proteins can be achieved within less than 12 min in a 433.5 mL monolithic column by applying gradient elution in the RP-HPLC mode. Furthermore, weak and strong-anion exchangers can be prepared via post-synthesis grafting of, e.g., bicyclo[2.2.1]hept-5-en-2-yl)-methyl-N,N-dimethylamine hydrochloride (4) and bicyclo[2.2.1]hept-5-en-2-yl)methyl-N,N,N-trimethylammonium iodide (5), respectively. Both the weak and strong anion exchangers can be used for the preparative-scale separation, e.g., of 5`-phosphorylated oligodeoxythymidylic acids fragments d[pT]12-18 at different pH ranges from 5 to 9.
Figure 1.
Separation of a mixture of five proteins on monolithic column (230 x 49 mm i.d., 433.5 mL).
Chromatographic conditions; mobile phase A: 0.1% TFA in H2O; mobile phase B: 0.1% TFA in ACN;
gradient: 25-48 % B in 12 min., flow rate 55 mL/min; sample, 7.2 mg/mL of insulin (1), 11.6 mg/mL of cytochrome C (2),
13.3 mg/ mL of lysozyme (3), 12 mg/mL of conalbumin (4) and 6.6 mg/mL of ß-lactoglobulin (5) dissolved in water;
injection volume, 500 μL; 25°C; detection: UV, 214 nm.
Figure 2.
Separation of 5'-phosphorylated oligodeoxythymidylic acids fragments on a ROMP-derived strong anion-exchange
monolithic column (100x10 mm i.d., 7.08 mL); mobile phase A (pH 7): phosphate buffer (0.05 mol/L) + 5% ACN, mobile phase B: A + NaCl (1.0 mol/L);
gradient, 20-60% B in 60 min then 90 % B in 80 min; flow-rate, 5.0 mL/min; T=30°C; sample, 21 μg of d(pT)12-18.


Currently, functionalized large-volume devices for down-steam processing applications e.g., for the purification of lectins, are developed. In parallel, we focus on large-volume, polymeric monolith-based bioreactors containing immobilized enzymes.

    

Selected Publications
[1] F. Sinner, M. R. Buchmeiser, Angew. Chem. 2000, 112, 1491-1494; Angew. Chem. Int. Ed. 2000, 39, 1433-1436.
[2] F. Sinner, M. R. Buchmeiser, Macromolecules 2000, 33, 5777-5786.
[3] M. R. Buchmeiser, Macromol. Rapid. Commun. 2001, 22, 1081-1094.
[4] B. Mayr, R. Tessadri, E. Post, M. R. Buchmeiser, Anal. Chem. 2001, 73, 4071-4078.
[5] S. Lubbad, B. Mayr, C. G. Huber, M. R. Buchmeiser, J. Chromatogr. A 2002, 959, 121-129.
[6] B. Mayr, G. Hölzl, K. Eder, M. R. Buchmeiser, C. G. Huber, Anal. Chem. 2002, 74, 6080-6087.
[7] R. Bandari, A. Prager-Duschke, C. Kühnel, U. Decker, B. Schlemmer, M. R. Buchmeiser, Macromolecules 2006, 39, 5222-5229.
[8] R. Bandari, W. Knolle, M. R. Buchmeiser, Macromol. Rapid Commun. 2007, 28, 2090-2094.
[9] K. Eder, C. G. Huber, M. R. Buchmeiser, Macromol. Rapid Commun. 2007, 28, 2029-2032.
[10] C. Gatschelhofer, A. Mautner, F. Reiter, T. R. Pieber, M. R. Buchmeiser, F. M. Sinner, J. Chromatogr. A 2009, 1216, 2651-2657.
[11] B. Schlemmer, R. Bandari, L. Rosenkranz, M. R. Buchmeiser, J. Chromatogr. A 2009, 1216, 2664-2670.
[12] S. H. Lubbad, M. R. Buchmeiser, J. Sep. Sci. 2009, 32, 2521-2529.
[13] B. Scheibitz, A. Prager, M. R. Buchmeiser, Macromolecules 2009, 42, 3493-3499.
[14] S. Beckert, F. Stallmach, R. Bandari, M. R. Buchmeiser, Macromolecules 2010, 43, 9441-9446.
[15] S. H. Lubbad, M. R. Buchmeiser, J. Chromatogr. A 2010, 1217, 3223-3230.
[16] S. Mavila, M. R. Buchmeiser, Macromolecules 2010, 43, 9601-9607.
[17] S. H. Lubbad, M. R. Buchmeiser, J. Chromatogr. A 2011, 1218, 2362-2367.
[18] S. H. Lubbad, R. Bandari, M. R. Buchmeiser, J. Chromatogr. A 2011, 1218, 8897-8902.
[19] R. Bandari, M. R. Buchmeiser, Analyst, in press 2012.

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Letzte Änderung 24.05.2012 (Mail to IconJP) | © Universität Stuttgart |  Impressum