European Biophysics Journal

, Volume 47, Issue 7, pp 777–787 | Cite as

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

  • Simon E. Wawra
  • Martin Thoma
  • Johannes Walter
  • Christian Lübbert
  • Thaseem Thajudeen
  • Cornelia Damm
  • Wolfgang PeukertEmail author
Original Article


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).


Scanning mobility particle sizer Analytical ultracentrifugation Multiwavelength detector Combined analysis 



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.

Supplementary material

249_2018_1314_MOESM1_ESM.docx (2.1 mb)
Supplementary material 1 (DOCX 2136 kb)


  1. Aldebert P, Dreyfus B, Pineri M (1986) Small-angle neutron scattering of perfluorosulfonated ionomers in solution. Macromolecules 19:2651CrossRefGoogle Scholar
  2. Aldebert P, Dreyfus B, Gebel G et al (1988) Rod like micellar structures in perfluorinated ionomer solutions. J Phys France 49:2101CrossRefGoogle Scholar
  3. Bailey M, Angley L, Perugini M (2009) Methods for sample labeling and meniscus determination in the fluorescence-detected analytical ultracentrifuge. Anal Biochem 390:218CrossRefGoogle Scholar
  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 9Google Scholar
  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 39:405CrossRefGoogle Scholar
  6. Canfield R (1963) The amino acid sequence of egg white lysozyme. J Biol Chem 238:2698PubMedGoogle Scholar
  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 2:335CrossRefGoogle Scholar
  8. Cauchy A-L (1832) Mémoire sur la rectification des courbes et la quadrature des surfaces courbes. Oxford University, OxfordGoogle Scholar
  9. Cölfen H, Pauck T (1997) Determination of particle size distributions with angström resolution. Colloid Polym Sci 275:175CrossRefGoogle Scholar
  10. Colvin J (1952) The size and shape of lysozyme. Can J Chem 30:831CrossRefGoogle Scholar
  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 210Google Scholar
  12. Demeler B, van Holde K (2004) Sedimentation velocity analysis of highly heterogeneous systems. Anal Biochem 335:279CrossRefGoogle Scholar
  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 388:279CrossRefGoogle Scholar
  14. Elzey S, Grassian V (2010) Agglomeration, isolation and dissolution of commercially manufactured silver nanoparticles in aqueous environments. J Nanopart Res 12:1945CrossRefGoogle Scholar
  15. Epstein P (1924) On the Resistance Experienced by Spheres in their Motion through Gases. Phys Rev 23:710CrossRefGoogle Scholar
  16. Fasman G (1976) Handbook of biochemistry and molecular biology: proteins, 3rd edn. CRC Press, ClevelandGoogle Scholar
  17. Gebel G (2000) Structural evolution of water swollen perfluorosulfonated ionomers from dry membrane to solution. Polymer 41:5829CrossRefGoogle Scholar
  18. Ghielmi A, Vaccarono P, Troglia C et al (2005) Proton exchange membranes based on the short-side-chain perfluorinated ionomer. J Power Sources 145:108CrossRefGoogle Scholar
  19. Grot W (1986) Nafion as a separator in electrolyte cells in Nafion product bulletin. DuPont Co., WilmingtonGoogle Scholar
  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 54:136CrossRefGoogle Scholar
  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 173:639CrossRefGoogle Scholar
  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 18:1206CrossRefGoogle Scholar
  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 259:195CrossRefGoogle Scholar
  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 52:432CrossRefGoogle Scholar
  25. Koestner R, Roiter Y, Kozhinova I et al (2011) AFM imaging of adsorbed Nafion polymer on mica and graphite at molecular level. Langmuir 27:10157CrossRefGoogle Scholar
  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 27:98Google Scholar
  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 43:241CrossRefGoogle Scholar
  28. Lebowitz J, Lewis S, Schuck P (2002) Modern analytical ultracentrifugation in protein science: a tutorial review. Protein Sci 11:2067CrossRefGoogle Scholar
  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 36:1141CrossRefGoogle Scholar
  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 109:1Google Scholar
  31. Lousenberg R (2005) Molar mass distributions and viscosity behavior of perfluorinated sulfonic acid polyelectrolyte aqueous dispersions. J Polym Sci 43:421CrossRefGoogle Scholar
  32. Mächtle W, Börger L (2006) Analytical ultracentrifugation of polymers and nanoparticles. Springer, Berlin HeidelbergGoogle Scholar
  33. Mauritz K, Moore R (2004) State of understanding of Nafion. Chem Rev 104:4535CrossRefGoogle Scholar
  34. Moore R, Martin C (1988) Chemical and morphological properties of solution-cast perfluorosulfonate ionomers. Macromolecules 21:1334CrossRefGoogle Scholar
  35. Mourey T, Slater L, Galipo R et al (2011) Size-exclusion chromatography of perfluorosulfonated ionomers. J Chromatogr A 1218:5801CrossRefGoogle Scholar
  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 225:293CrossRefGoogle Scholar
  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 211:1298CrossRefGoogle Scholar
  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 7:1CrossRefGoogle Scholar
  39. Pitschke M, Fels A, Schmidt B et al (1995) Polymeric fluorescent dyes for labeling of proteins and nucleic acids. Colloid Polym Sci 273:740CrossRefGoogle Scholar
  40. Planken K, Cölfen H (2010) Analytical ultracentrifugation of colloids. Nanoscale 2:1849CrossRefGoogle Scholar
  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 76:1045CrossRefGoogle Scholar
  42. Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys J 78:1606CrossRefGoogle Scholar
  43. Schuck P, Zhao H, Brautigam C (2015) Basic principles of analytical ultracentrifugation. CRC Press, Boca RatonCrossRefGoogle Scholar
  44. Shibayama M, Matsunaga T, Kusano T et al (2014) SANS studies on catalyst ink of fuel cell. J Appl Polym Sci. CrossRefGoogle Scholar
  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 40:14430CrossRefGoogle Scholar
  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 286:121CrossRefGoogle Scholar
  47. Tammet H (1995) Size and mobility of nanometer particles, clusters and ions. J Aerosol Sci 26:459CrossRefGoogle Scholar
  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 5:7155CrossRefGoogle Scholar
  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 2:253–260CrossRefGoogle Scholar
  50. Uchiyama S, Arisaka F, Stafford WF, Laue T (eds) (2016) Analytical ultracentrifugation. Instrumentation software, and applications. Springer, TokyoGoogle Scholar
  51. Uttinger M, Walter J, Thajudeen T et al (2017) Brownian dynamics simulations of analytical ultracentrifugation experiments exhibiting hydrodynamic and thermodynamic non-ideality. Nanoscale 9:17770CrossRefGoogle Scholar
  52. van Holde K, Weischet W (1978) Boundary analysis of sedimentation-velocity experiments with monodisperse and paucidisperse solutes. Biopolymers 17:1387CrossRefGoogle Scholar
  53. van Holde K, Johnson W, Ho P (2006) Principles of physical biochemistry, 2nd edn. Pearson/Prentice Hall, Upper Saddle RiverGoogle Scholar
  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 114:6243CrossRefGoogle Scholar
  55. Walter J, Peukert W (2016) Dynamic range multiwavelength particle characterization using analytical ultracentrifugation. Nanoscale 8:7484CrossRefGoogle Scholar
  56. Walter J, Löhr K, Karabudak E et al (2014) Multidimensional analysis of nanoparticles with highly disperse properties using multiwavelength analytical ultracentrifugation. ACS Nano 8:8871CrossRefGoogle Scholar
  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 87:3396CrossRefGoogle Scholar
  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 33:184CrossRefGoogle Scholar
  59. Wang W, Damm C, Walter J et al (2016) Photobleaching and stabilization of carbon nanodots produced by solvothermal synthesis. Phys Chem Chem Phys 18:466CrossRefGoogle Scholar
  60. Welch C, Labouriau A, Hjelm R et al (2012) Nafion in dilute solvent systems: dispersion or solution? ACS Macro Lett. 1:1403CrossRefGoogle Scholar
  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 37:13243CrossRefGoogle Scholar
  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 26:19199CrossRefGoogle Scholar
  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 118:14922CrossRefGoogle Scholar
  64. Yeo R (1980) Dual cohesive energy densities of perfluorosulfonic acid (Nafion) membrane. Polymer 21:432CrossRefGoogle Scholar
  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 46:1065CrossRefGoogle Scholar
  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 39:15989CrossRefGoogle Scholar
  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:e0126420CrossRefGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2018

Authors and Affiliations

  1. 1.Institute of Particle Technology (LFG)Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)ErlangenGermany
  2. 2.Interdisciplinary Center for Functional Particle Systems (FPS)Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)ErlangenGermany
  3. 3.School of Mechanical SciencesIndian Institute of Technology GoaPondaIndia

Personalised recommendations