, Volume 21, Issue 1, pp 167–176 | Cite as

SEC-MALLS analysis of TEMPO-oxidized celluloses using methylation of carboxyl groups

  • Ryoya Hiraoki
  • Hayaka Fukuzumi
  • Yuko Ono
  • Tsuguyuki Saito
  • Akira Isogai
Original Paper


Two cellouronic acids [sodium (1 → 4)-β-polyglucuronates, CUAs] and one 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized wood cellulose (TOC) became soluble in 8 % lithium chloride/N,N-dimethylacetamide (LiCl/DMAc) after the methylation of C6 carboxyl groups in these samples using trimethylsilyldiazomethane (TMSD). The obtained solutions were diluted to 1 % LiCl/DMAc and subjected to size-exclusion chromatography combined with multi-angle laser-light scattering (SEC-MALLS). Neither depolymerization nor side reactions took place during methylation; this was confirmed by SEC-MALLS and nuclear magnetic resonance analyses, using CUAs as models. The SEC-MALLS analysis of the original wood cellulose and the carboxyl-methylated TOC prepared from it, using 1 % LiCl/N,N-dimethyl-2-imidazolidinone and 1 % LiCl/DMAc, respectively, as eluents, showed that the weight-average degree of polymerization of the original wood cellulose decreased from 3,100 to 2,210 through TEMPO-mediated oxidation. The molecular-mass distributions of the original wood cellulose and the TOC both consisted of one large peak with a small shoulder, indicating that some of the oxidized hemicelluloses remained in the TOC. The combination of methylation of carboxyl groups in polysaccharides using TMSD and subsequent SEC-MALLS analysis using 1 % LiCl/DMAc as an eluent may be applicable not only to TOCs, but also to other polysaccharides with carboxyl groups, for evaluation of their molecular-mass parameters.


TEMPO-oxidized cellulose Cellouronic acid Methyl ester Trimethylsilyldiazomethane SEC-MALLS LiCl/DMAc 



This research was supported by a Grant-in-Aid for Scientific Research (Grant Number 21228007) from the Japan Society for the Promotion of Science (JSPS).


  1. Bailey WF, Bobbitt JM (2007) Mechanism of the oxidation of alcohols by oxoammonium cations. J Org Chem 72:4504–4509CrossRefGoogle Scholar
  2. Bohrn R, Potthast A, Schiehser S, Rosenau T, Sixta H, Kosma P (2006) The FDAM method: determination of carboxyl profiles in cellulosic materials by combining group-selective fluorescence labeling with GPC. Biomacromolecules 7:1743–1750CrossRefGoogle Scholar
  3. Bragd PL, van Bekkum H, Besemer AC (2004) TEMPO-mediated oxidation of polysaccharides: survey of methods and applications. Top Catal 27:49–66CrossRefGoogle Scholar
  4. Ciucanu I, Kerek F (1984) A simple and rapid method for the permethylation of carbohydrates. Carbohydr Res 131:209–217CrossRefGoogle Scholar
  5. Da Silva Perez D, Montanari S, Vignon MR (2003) TEMPO-mediated oxidation of cellulose III. Biomacromolecules 4:1417–1425CrossRefGoogle Scholar
  6. de Nooy AEJ, Besemer C, van Bekkum H (1995) Highly selective nitroxyl radical-mediated oxidation of primary alcohol groups in water-soluble glucans. Carbohydr Res 269:89–98CrossRefGoogle Scholar
  7. Dupont AL (2003) Cellulose in lithium chloride/N,N,-dimethylacetamide, optimisation of a dissolution method using paper substrates and stability of the solutions. Polymer 44:4117–4126CrossRefGoogle Scholar
  8. Fujisawa S, Isogai T, Isogai A (2010) Temperature and pH stability of cellouronic acid. Cellulose 17:607–615CrossRefGoogle Scholar
  9. Fujisawa S, Okita Y, Fukuzumi H, Saito T, Isogai A (2011) Preparation and characterization of TEMPO-oxidized cellulose nanofibril films with free carboxyl groups. Carbohydr Polym 84:579–583CrossRefGoogle Scholar
  10. Fukuzumi H, Saito T, Isogai A (2010) Thermal stabilization of TEMPO-oxidized cellulose. Polym Degrad Stabil 95:1502–1508CrossRefGoogle Scholar
  11. Hashimoto N, Aoyama T, Shioiri T (1981) New methods and reagents in organic synthesis. 14. A simple efficient preparation of methyl esters with trimethylsilyl diazomethane (TMSCHN2) and its application to gas chromatographic analysis of fatty acids. Chem Pharm Bull 29:1475–1478CrossRefGoogle Scholar
  12. Henninges U, Okubayashi S, Rosenau T, Potthast A (2012) Irradiation of cellulosic pulps: understanding its impact on cellulose oxidation. Biomacromolecules 13:4171–4178Google Scholar
  13. Hirota M, Tamura N, Saito T, Isogai A (2009) Oxidation of regenerated cellulose with NaClO2 catalyzed by TEMPO and NaClO under acid-neutral conditions. Carbohydr Polym 78:330–335CrossRefGoogle Scholar
  14. Hirota M, Furihata K, Saito T, Kawada T, Isogai A (2010) Glucose/glucuronic acid alternating co-polysaccharides prepared from TEMPO-oxidized native celluloses by surface peeling. Angew Chem Int Ed 49:7670–7672CrossRefGoogle Scholar
  15. Isogai A, Kato Y (1998) Preparation of polyuronic acid from cellulose by TEMPO-mediated oxidation. Cellulose 5:153–164CrossRefGoogle Scholar
  16. Isogai T, Yanagisawa M, Isogai A (2009) Degrees of polymerization (DP) and DP distribution of cellouronic acids prepared from alkali-treated celluloses and ball-milled native celluloses by TEMPO-mediated oxidation. Cellulose 16:117–127CrossRefGoogle Scholar
  17. Isogai A, Saito T, Fukuzumi H (2011) TEMPO-oxidized cellulose nanofibers. Nanoscale 3:71–85CrossRefGoogle Scholar
  18. Iwamoto S, Weihua K, Isogai A, Iwata T (2009) Elastic modulus of single cellulose microfibrils from tunicate measured by atomic force microscopy. Biomacromolecules 10:2571–2576CrossRefGoogle Scholar
  19. Kühnel E, Laffan DDP, Lloyd-Jones GC, Martinez del Campo T, Shepperson IR, Slaughter JL (2007) Mechanism of methyl esterification of carboxylic acids by trimethylsilyldiazomethane. Angew Chem Int Ed 46:7075–7078CrossRefGoogle Scholar
  20. Milanovic J, Schiehser S, Milanovic P, Potthast A, Kostic M (2013) Molecular weight distribution and functional group profiles of TEMPO-oxidized lyocell fibers. Carbohydr Polym 98:444–450CrossRefGoogle Scholar
  21. Okita Y, Saito T, Isogai A (2010) Cellulose nanofibers prepared by TEMPO-mediated oxidation of native cellulose. Biomacromolecules 11:1696–1700CrossRefGoogle Scholar
  22. Potthast A, Rosenau T, Kosma P (2006) Analysis of oxidized functionalities in cellulose. Polysaccharides II 205:1–48CrossRefGoogle Scholar
  23. Presser A, Hüfner A (2004) Trimethylsilyldiazomethane: a mild and efficient reagent for the methylation of carboxylic acids and alcohols in natural products. Monatsh Chem 135:1015–1022CrossRefGoogle Scholar
  24. Röhring J, Lange T, Potthast A, Rosenau T, Sixta H, Kosma P (2001) Novel methods to determine carbonyl functions in cellulosic substances. In: Proceedings of the post-symposium 11th international symposium on wood pulping chemistry, Grenoble, June 18–49, pp 94–97Google Scholar
  25. Saito T, Isogai A (2004) TEMPO-mediated oxidation of native cellulose. The effect of oxidation conditions on chemical and crystal structures of the water-insoluble fractions. Biomacromolecules 5:1983–1989CrossRefGoogle Scholar
  26. Saito T, Yanagisawa M, Isogai A (2005) TEMPO-mediated oxidation of native cellulose: SEC-MALLS analysis of water-soluble and -insoluble fractions in the oxidized products. Cellulose 12:305–315CrossRefGoogle Scholar
  27. Saito T, Nishiyama Y, Putaux JL, Vignon M, Isogai A (2006) Homogeneous suspensions of individualized microfibrils from TEMPO-catalyzed oxidation of native cellulose. Biomacromolecules 7:1687–1691CrossRefGoogle Scholar
  28. Saito T, Kimura S, Nishiyama Y, Isogai A (2007) Cellulose nanofibers prepared by TEMPO-mediated oxidation of native cellulose. Biomacromolecules 8:2485–2491CrossRefGoogle Scholar
  29. Saito T, Kuramae R, Wohlert J, Berglund LA, Isogai A (2013) An ultrastrong nanofibrillar biomaterial: the strength of single cellulose nanofibrils revealed via sonication-induced fragmentation. Biomacromolecules 14:248–253CrossRefGoogle Scholar
  30. Schult T, Hjerde T, Optun OI, Kleppe PJ, Moe S (2002) Characterization of cellulose by SEC-MALLS. Cellulose 9:149–158CrossRefGoogle Scholar
  31. Shibata I, Yanagisawa M, Saito T, Isogai A (2006) SEC-MALS analysis of cellouronic acid prepared from regenerated cellulose by TEMPO-mediated oxidation. Cellulose 13:73–80CrossRefGoogle Scholar
  32. Shinoda R, Saito T, Okita Y, Isogai A (2012) Relationship between length and degree of polymerization of TEMPO-oxidized cellulose nanofibrils. Biomacromolecules 13:842–849CrossRefGoogle Scholar
  33. Smith DK, Bampton RF, Alexander W (1963) Use of new solvents for evaluating chemical cellulose for the viscose process. J Ind Eng Chem Process Des Dev 2:57–62CrossRefGoogle Scholar
  34. Striegel AM (1993) Theory and applications of DMAc/LiCl in the analysis of polysaccharides. Carbohydr Polym 34:267–274CrossRefGoogle Scholar
  35. Tot I, Müller Y, Werner C, Rosenau T, Potthast A (2009) A novel, mild and selective methylation of carboxyl groups in cellulosic pulps. Holzforschung 63:657–663CrossRefGoogle Scholar
  36. Yamamoto M, Kuramae R, Yanagisawa M, Ishii D, Isogai A (2011) Light-scattering analysis of native wood holocelluloses totally dissolved in LiCl–DMI solutions: high probability of branched structures in inherent cellulose. Biomacromolecules 12:3982–3988CrossRefGoogle Scholar
  37. Yanagisawa M, Isogai A (2005) SEC-MALS-QELS study on the molecular conformation of cellulose in LiCl/amide solutions. Biomacromolecules 6:1258–1265CrossRefGoogle Scholar
  38. Yanagisawa M, Isogai A (2007) Size exclusion chromatographic and UV–vis absorption analyses of unbleached and bleached softwood kraft pulps using LiCl/1,3-dimethyl-2-imidazolidinon as a solvent. Holzforschung 61:236–241CrossRefGoogle Scholar
  39. Yanagisawa M, Shibata I, Isogai A (2005) SEC-MALLS analysis of softwood kraft pulp using LiCl/1,3-dimethyl-2-imidazolidinone as an eluent. Cellulose 12:151–158CrossRefGoogle Scholar
  40. Yang D, Kumar V (2012) Preparation and characterization of novel oxidized cellulose acetate methyl esters. Carbohydr Polym 90:1486–1493CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Ryoya Hiraoki
    • 1
  • Hayaka Fukuzumi
    • 1
  • Yuko Ono
    • 1
  • Tsuguyuki Saito
    • 1
  • Akira Isogai
    • 1
  1. 1.Graduate School of Agricultural and Life SciencesUniversity of TokyoTokyoJapan

Personalised recommendations