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Ionomer and protein size analysis by analytical ultracentrifugation and electrospray scanning mobility particle sizer

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By combining analytical ultracentrifugation (AUC) in liquid phase and scanning mobility particle sizer (SMPS) in the gas phase, additional information on the particle size and morphology has been obtained for rigid particles. In this paper, we transfer this concept to soft particles, allowing us to analyze the size and molar mass of the short side chain perfluorosulfonic acid ionomer Aquivion® in a dilute aqueous suspension. The determination of the primary size and exact molar mass of this class of polymers is challenging since they are optically transparent and due to the formation of different aggregate structures depending on the concentration and solvent properties. First, validation of AUC and SMPS measurements was carried out using the well-defined biopolymers bovine serum albumin (BSA) and lysozyme (LYZ) to confirm the reliability of the results of the two unique and independent classifying methods. Then, the ionomer Aquivion® was studied using both techniques. From the mean molar mass of 185 ± 14 kDa obtained by AUC, a mean hydrodynamic diameter of 7.6 ± 0.5 nm was calculated. The particle size obtained from SMPS (7.1 nm) agrees very well with the results from AUC showing that the molecule was transferred into the gas phase without significantly changing its structure. In conclusion, the Aquivion® is molecularly dispersed in the used aqueous buffer solution without any aggregate formation in the investigated concentration range (< 2 g l−1).

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  1. Aldebert P, Dreyfus B, Pineri M (1986) Small-angle neutron scattering of perfluorosulfonated ionomers in solution. Macromolecules 19:2651

  2. Aldebert P, Dreyfus B, Gebel G et al (1988) Rod like micellar structures in perfluorinated ionomer solutions. J Phys France 49:2101

  3. Bailey M, Angley L, Perugini M (2009) Methods for sample labeling and meniscus determination in the fluorescence-detected analytical ultracentrifuge. Anal Biochem 390:218

  4. Bhattacharyya S, Maciejewska P, Börger L et al (2006) Development of a fast fiber based UV-vis multiwavelength detector for an ultracentrifuge. In: Wandrey C, Cölfen H (eds) Analytical ultracentrifugation, VIII edn. Springer, New York, p 9

  5. Brookes E, Cao W, Demeler B (2010) A two-dimensional spectrum analysis for sedimentation velocity experiments of mixtures with heterogeneity in molecular weight and shape. Eur Biophys J 39:405

  6. Canfield R (1963) The amino acid sequence of egg white lysozyme. J Biol Chem 238:2698

  7. Carney R, Kim J, Qian H et al (2011) Determination of nanoparticle size distribution together with density or molecular weight by 2D analytical ultracentrifugation. Nat Commun 2:335

  8. Cauchy A-L (1832) Mémoire sur la rectification des courbes et la quadrature des surfaces courbes. Oxford University, Oxford

  9. Cölfen H, Pauck T (1997) Determination of particle size distributions with angström resolution. Colloid Polym Sci 275:175

  10. Colvin J (1952) The size and shape of lysozyme. Can J Chem 30:831

  11. Demeler B (2005) UltraScan: a comprehensive data analysis software package for analytical ultracentrifugation experiments. In: Scott DJ, Harding SE, Rowe AJ (eds) Analytical ultracentrifugation: techniques and methods. Royal Society of Chemistry, London, p 210

  12. Demeler B, van Holde K (2004) Sedimentation velocity analysis of highly heterogeneous systems. Anal Biochem 335:279

  13. Demeule B, Shire S, Liu J (2009) A therapeutic antibody and its antigen form different complexes in serum than in phosphate-buffered saline: a study by analytical ultracentrifugation. Anal Biochem 388:279

  14. Elzey S, Grassian V (2010) Agglomeration, isolation and dissolution of commercially manufactured silver nanoparticles in aqueous environments. J Nanopart Res 12:1945

  15. Epstein P (1924) On the Resistance Experienced by Spheres in their Motion through Gases. Phys Rev 23:710

  16. Fasman G (1976) Handbook of biochemistry and molecular biology: proteins, 3rd edn. CRC Press, Cleveland

  17. Gebel G (2000) Structural evolution of water swollen perfluorosulfonated ionomers from dry membrane to solution. Polymer 41:5829

  18. Ghielmi A, Vaccarono P, Troglia C et al (2005) Proton exchange membranes based on the short-side-chain perfluorinated ionomer. J Power Sources 145:108

  19. Grot W (1986) Nafion as a separator in electrolyte cells in Nafion product bulletin. DuPont Co., Wilmington

  20. Harding S, Schuck P, Abdelhameed A et al (2011) Extended Fujita approach to the molecular weight distribution of polysaccharides and other polymeric systems. Methods 54:136

  21. Hirayama K, Akashi S, Furuya M et al (1990) Rapid confirmation and revision of the primary structure of bovine serum albumin by ESIMS and Frit-FAB LC/MS. Biochem Biophys Res Commun 173:639

  22. Kaddis C, Lomeli S, Yin S et al (2007) Sizing large proteins and protein complexes by electrospray ionization mass spectrometry and ion mobility. J Am Soc Mass Spectrom 18:1206

  23. Kaufman S, Kuchumov A, Kazakevich M et al (1998) Analysis of a 3.6-MDa hexagonal bilayer hemoglobin from Lumbricus terrestris using a gas-phase electrophoretic mobility molecular analyzer. Anal Biochem 259:195

  24. Kemptner J, Marchetti-Deschmann M, Siekmann J et al (2010) GEMMA and MALDI-TOF MS of reactive PEGs for pharmaceutical applications. J Pharm Biomed Anal 52:432

  25. Koestner R, Roiter Y, Kozhinova I et al (2011) AFM imaging of adsorbed Nafion polymer on mica and graphite at molecular level. Langmuir 27:10157

  26. Kratky O, Leopold H, Stabinger H (1973) The determination of the partial specific volume of proteins by the mechanical oscillator technique in part D: enzyme structure. Elsevier 27:98

  27. Ku B, de la Mora JF (2009) Relation between electrical mobility, mass, and size for nanodrops 1–6.5 nm in diameter in air. Aerosol Sci Technol 43:241

  28. Lebowitz J, Lewis S, Schuck P (2002) Modern analytical ultracentrifugation in protein science: a tutorial review. Protein Sci 11:2067

  29. Li H, Schlick S (1995) Effect of solvents on phase separation in perfluorinated ionomers, from electron spin resonance of VO2+ in swollen membranes and solutions. Polymer 36:1141

  30. Liu W-H, Yu T-Y, Yu T et al (2007) Static light scattering and transmission microscopy study of dilute Nafion solutions. e-Polymers 109:1

  31. Lousenberg R (2005) Molar mass distributions and viscosity behavior of perfluorinated sulfonic acid polyelectrolyte aqueous dispersions. J Polym Sci 43:421

  32. Mächtle W, Börger L (2006) Analytical ultracentrifugation of polymers and nanoparticles. Springer, Berlin Heidelberg

  33. Mauritz K, Moore R (2004) State of understanding of Nafion. Chem Rev 104:4535

  34. Moore R, Martin C (1988) Chemical and morphological properties of solution-cast perfluorosulfonate ionomers. Macromolecules 21:1334

  35. Mourey T, Slater L, Galipo R et al (2011) Size-exclusion chromatography of perfluorosulfonated ionomers. J Chromatogr A 1218:5801

  36. Ngo T, Yu T, Lin H-L (2013) Influence of the composition of isopropyl alcohol/water mixture solvents in catalyst ink solutions on proton exchange membrane fuel cell performance. J Power Sources 225:293

  37. Pavlov G, Perevyazko I, Schubert U (2010) Velocity sedimentation and intrinsic viscosity analysis of polystyrene standards with a wide range of molar masses. Macromol Chem Phys 211:1298

  38. Perrin F (1936) Mouvement Brownien d’un ellipsoide (II). Rotation libre et dépolarisation des fluorescences. Translation et diffusion de molécules ellipsoidales. J Phys Radium 7:1

  39. Pitschke M, Fels A, Schmidt B et al (1995) Polymeric fluorescent dyes for labeling of proteins and nucleic acids. Colloid Polym Sci 273:740

  40. Planken K, Cölfen H (2010) Analytical ultracentrifugation of colloids. Nanoscale 2:1849

  41. Saucy D, Ude S, Lenggoro I et al (2004) Mass analysis of water-soluble polymers by mobility measurement of charge-reduced ions generated by electrosprays. Anal Chem 76:1045

  42. Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys J 78:1606

  43. Schuck P, Zhao H, Brautigam C (2015) Basic principles of analytical ultracentrifugation. CRC Press, Boca Raton

  44. Shibayama M, Matsunaga T, Kusano T et al (2014) SANS studies on catalyst ink of fuel cell. J Appl Polym Sci.

  45. Siracusano S, Baglio V, Moukheiber E et al (2015) Performance of a PEM water electrolyser combining an IrRu-oxide anode electrocatalyst and a short-side chain Aquivion membrane. Int J Hydrogen Energy 40:14430

  46. Strauss H, Karabudak E, Bhattacharyya S et al (2008) Performance of a fast fiber based UV/Vis multiwavelength detector for the analytical ultracentrifuge. Colloid Polym Sci 286:121

  47. Tammet H (1995) Size and mobility of nanometer particles, clusters and ions. J Aerosol Sci 26:459

  48. Tenzer S, Docter D, Rosfa S et al (2011) Nanoparticle size is a critical physicochemical determinant of the human blood plasma corona: a comprehensive quantitative proteomic analysis. ACS Nano 5:7155

  49. Thajudeen T, Walter J, Srikantharajah R et al (2017) Determination of the length and diameter of nanorods by a combination of analytical ultracentrifugation and scanning mobility particle sizer. Nanoscale Horizons 2:253–260

  50. Uchiyama S, Arisaka F, Stafford WF, Laue T (eds) (2016) Analytical ultracentrifugation. Instrumentation software, and applications. Springer, Tokyo

  51. Uttinger M, Walter J, Thajudeen T et al (2017) Brownian dynamics simulations of analytical ultracentrifugation experiments exhibiting hydrodynamic and thermodynamic non-ideality. Nanoscale 9:17770

  52. van Holde K, Weischet W (1978) Boundary analysis of sedimentation-velocity experiments with monodisperse and paucidisperse solutes. Biopolymers 17:1387

  53. van Holde K, Johnson W, Ho P (2006) Principles of physical biochemistry, 2nd edn. Pearson/Prentice Hall, Upper Saddle River

  54. Voigt M, Klaumünzer M, Thiem H et al (2010) Detailed analysis of the growth kinetics of ZnO nanorods in methanol. J Phys Chem C 114:6243

  55. Walter J, Peukert W (2016) Dynamic range multiwavelength particle characterization using analytical ultracentrifugation. Nanoscale 8:7484

  56. Walter J, Löhr K, Karabudak E et al (2014) Multidimensional analysis of nanoparticles with highly disperse properties using multiwavelength analytical ultracentrifugation. ACS Nano 8:8871

  57. Walter J, Sherwood P, Lin W et al (2015) Simultaneous analysis of hydrodynamic and optical properties using analytical ultracentrifugation equipped with multiwavelength detection. Anal Chem 87:3396

  58. Walter J, Segets D, Peukert W (2016) Extension of the deep UV-capabilities in multiwavelength spectrometry in analytical ultracentrifugation: the role of oil deposits. Part Part Syst Charact 33:184

  59. Wang W, Damm C, Walter J et al (2016) Photobleaching and stabilization of carbon nanodots produced by solvothermal synthesis. Phys Chem Chem Phys 18:466

  60. Welch C, Labouriau A, Hjelm R et al (2012) Nafion in dilute solvent systems: dispersion or solution? ACS Macro Lett. 1:1403

  61. Wu X, Scott K, Puthiyapura V (2012) Polymer electrolyte membrane water electrolyser with Aquivion short side chain perfluorosulfonic acid ionomer binder in catalyst layers. Int J Hydrog Energy 37:13243

  62. Xu F, Zhang H, Ilavsky J et al (2010) Investigation of a catalyst ink dispersion using both ultra-small-angle X-ray scattering and cryogenic TEM. Langmuir 26:19199

  63. Yamaguchi M, Matsunaga T, Amemiya K et al (2014) Dispersion of rod-like particles of nafion in salt-free water/1-propanol and water/ethanol solutions. J Phys Chem B 118:14922

  64. Yeo R (1980) Dual cohesive energy densities of perfluorosulfonic acid (Nafion) membrane. Polymer 21:432

  65. Zhang C, Thajudeen T, Larriba C et al (2012) Determination of the scalar friction factor for nonspherical particles and aggregates across the entire knudsen number range by direct simulation monte carlo (DSMC). Aerosol Sci Technol 46:1065

  66. Zhang H, Li J, Tang H et al (2014) Hydrogen crossover through perfluorosulfonic acid membranes with variable side chains and its influence in fuel cell lifetime. Int J Hydrog Energy 39:15989

  67. Zhao H, Ghirlando R, Alfonso C et al (2015) A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation. PLoS ONE 10:e0126420

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The authors acknowledge HI ERN and Bavaria (Grant-no. DBF01253) as well as Greenerity GmbH for financial support of this work. The authors further acknowledge the funding of the Deutsche Forschungsgemeinschaft (DFG) through the Cluster of Excellence “Engineering of Advanced Materials” as well as DFG Project PE 427/28-2. TT acknowledges the fellowship from Alexander von Humboldt foundation. SEW acknowledges the travel grant from ARBRE-MOBIEU and COST for the 23. International Analytical Ultracentrifugation Workshop and Symposium.

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Correspondence to Wolfgang Peukert.

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Special Issue: 23rd International AUC Workshop and Symposium.

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Wawra, S.E., Thoma, M., Walter, J. et al. Ionomer and protein size analysis by analytical ultracentrifugation and electrospray scanning mobility particle sizer. Eur Biophys J 47, 777–787 (2018).

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  • Scanning mobility particle sizer
  • Analytical ultracentrifugation
  • Multiwavelength detector
  • Combined analysis