Correlations of Apparent Cellulose Crystallinity Determined by XRD, NMR, IR, Raman, and SFG Methods

  • Christopher Lee
  • Kevin Dazen
  • Kabindra Kafle
  • Andrew Moore
  • David K. Johnson
  • Sunkyu ParkEmail author
  • Seong H. KimEmail author
Part of the Advances in Polymer Science book series (POLYMER, volume 271)


Although the cellulose crystallinity index (CI) is used widely, its limitations have not been adequately described. In this study, the CI values of a set of reference samples were determined from X-ray diffraction (XRD), nuclear magnetic resonance (NMR), and infrared (IR), Raman, and vibrational sum frequency generation (SFG) spectroscopies. The intensities of certain crystalline peaks in IR, Raman, and SFG spectra positively correlated with the amount of crystalline cellulose in the sample, but the correlation with XRD was nonlinear as a result of fundamental differences in detection sensitivity to crystalline cellulose and improper baseline corrections for amorphous contributions. It is demonstrated that the intensity and shape of the XRD signal is affected by both the amount of crystalline cellulose and crystal size, which makes XRD analysis complicated. It is clear that the methods investigated show the same qualitative trends for samples, but the absolute CI values differ depending on the determination method. This clearly indicates that the CI, as estimated by different methods, is not an absolute value and that for a given set of samples the CI values can be compared only as a qualitative measure.


X-ray diffraction Sum frequency generation spectroscopy Infrared spectroscopy Raman spectroscopy Nuclear magnetic resonance Crystallinity index Wood pulp 



This work was supported by the Center for Lignocellulose Structure and Formation (CLSF), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, and Office of Basic Energy Sciences under Award Number DE-SC0001090. This work was also supported by Subcontract No. XGB-3-23024-01 with the National Renewable Energy Laboratory (NREL), under Contract No. DE-AC36-08-GO28308 with the U.S. Department of Energy. Sample preparation and XRD and NMR data collection were carried out with funding from the NREL. IR, Raman, and SFG analyses as well as XRD simulation and data analyses were carried out with CLSF support.


  1. 1.
    Pauly M, Keegstra K (2008) Cell-wall carbohydrates and their modification as a resource for biofuels. Plant J 54(4):559–568CrossRefGoogle Scholar
  2. 2.
    Zugenmaier P (2008) Crystalline cellulose and cellulose derivatives: characterization and structures. Springer, BerlinGoogle Scholar
  3. 3.
    Cosgrove DJ (2005) Growth of the plant cell wall. Nat Rev Mol Cell Biol 6(11):850–861CrossRefGoogle Scholar
  4. 4.
    Paredez AR, Somerville CR, Ehrhardt DW (2006) Visualization of cellulose synthase demonstrates functional association with microtubules. Science 312(5779):1491–1495CrossRefGoogle Scholar
  5. 5.
    Somerville C, Bauer S, Brininstool G, Facette M, Hamann T, Milne J, Osborne E, Paredez A, Persson S, Raab T, Vorwerk S, Youngs H (2004) Toward a systems approach to understanding plant cell walls. Science 306(5705):2206–2211CrossRefGoogle Scholar
  6. 6.
    Thomas LH, Forsyth VT, Sturcova A, Kennedy CJ, May RP, Altaner CM, Apperley DC, Wess TJ, Jarvis MC (2013) Structure of cellulose microfibrils in primary cell walls from collenchyma. Plant Physiol 161(1):465–476CrossRefGoogle Scholar
  7. 7.
    Doblin MS, Kurek I, Jacob-Wilk D, Delmer DP (2002) Cellulose biosynthesis in plants: from genes to rosettes. Plant Cell Physiol 43(12):1407–1420CrossRefGoogle Scholar
  8. 8.
    Fernandes AN, Thomas LH, Altaner CM, Callow P, Forsyth VT, Apperley DC, Kennedy CJ, Jarvis MC (2011) Nanostructure of cellulose microfibrils in spruce wood. Proc Natl Acad Sci U S A 108(47):E1195–E1203CrossRefGoogle Scholar
  9. 9.
    Nishiyama Y, Langan P, Chanzy H (2002) Crystal structure and hydrogen-bonding system in cellulose 1 beta from synchrotron X-ray and neutron fiber diffraction. J Am Chem Soc 124(31):9074–9082CrossRefGoogle Scholar
  10. 10.
    Himmel ME, Ding S-Y, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD (2007) Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315(5813):804–807CrossRefGoogle Scholar
  11. 11.
    Pérez S, Samain D (2010) Structure and engineering of celluloses. In: Derek H (ed) Advances in carbohydrate chemistry and biochemistry, vol 64. Academic, New York, pp 25–116Google Scholar
  12. 12.
    Park S, Johnson DK, Ishizawa CI, Parilla PA, Davis MF (2009) Measuring the crystallinity index of cellulose by solid state 13C nuclear magnetic resonance. Cellulose 16(4):641–647CrossRefGoogle Scholar
  13. 13.
    Carroll A, Somerville C (2009) Cellulosic biofuels. Annu Rev Plant Biol 60:165–182CrossRefGoogle Scholar
  14. 14.
    Mittal A, Katahira R, Himmel ME, Johnson DK (2011) Effects of alkaline or liquid-ammonia treatment on crystalline cellulose: changes in crystalline structure and effects on enzymatic digestibility. Biotechnol Biofuels 4:41CrossRefGoogle Scholar
  15. 15.
    Atalla RH, Vanderhart DL (1984) Native cellulose – a composite of 2 distinct crystalline forms. Science 223(4633):283–285CrossRefGoogle Scholar
  16. 16.
    Jarvis M (2003) Cellulose stacks up. Nature 426(6967):611–612CrossRefGoogle Scholar
  17. 17.
    Horii F, Yamamoto H, Kitamaru R, Tanahashi M, Higuchi T (1987) Transformation of native cellulose crystals induced by saturated steam at high-temperatures. Macromolecules 20(11):2946–2949CrossRefGoogle Scholar
  18. 18.
    Langan P, Nishiyama Y, Chanzy H (2001) X-ray structure of mercerized cellulose II at 1 angstrom resolution. Biomacromolecules 2(2):410–416CrossRefGoogle Scholar
  19. 19.
    Ruan D, Zhang LN, Zhou JP, Jin HM, Chen H (2004) Structure and properties of novel fibers spun from cellulose in NaOH/thiourea aqueous solution. Macromol Biosci 4(12):1105–1112CrossRefGoogle Scholar
  20. 20.
    Wada M, Heux L, Sugiyama J (2004) Polymorphism of cellulose I family: reinvestigation of cellulose IVI. Biomacromolecules 5(4):1385–1391CrossRefGoogle Scholar
  21. 21.
    Atalla RH, VanderHart DL (1999) The role of solid state C-13 NMR spectroscopy in studies of the nature of native celluloses. Solid State Nucl Magn Reson 15(1):1–19CrossRefGoogle Scholar
  22. 22.
    Sugiyama J, Persson J, Chanzy H (1991) Combined infrared and electron diffraction study of the polymorphism of native celluloses. Macromolecules 24(9):2461–2466CrossRefGoogle Scholar
  23. 23.
    Nishiyama Y, Sugiyama J, Chanzy H, Langan P (2003) Crystal structure and hydrogen bonding system in cellulose Iα from synchrotron X-ray and neutron fiber diffraction. J Am Chem Soc 125(47):14300–14306CrossRefGoogle Scholar
  24. 24.
    Kim SH, Lee CM, Kafle K (2013) Characterization of crystalline cellulose in biomass: basic principles, applications, and limitations of XRD, NMR, IR, Raman, and SFG. Korean J Chem Eng 30(12):2127–2141CrossRefGoogle Scholar
  25. 25.
    Ruland W (1961) X-ray determination of crystallinity and diffuse disorder scattering. Acta Crystallogr 14(11):1180–1185CrossRefGoogle Scholar
  26. 26.
    Park S, Baker JO, Himmel ME, Parilla PA, Johnson DK (2010) Cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance. Biotechnol Biofuels 3:10CrossRefGoogle Scholar
  27. 27.
    Barnette AL, Bradley LC, Veres BD, Schreiner EP, Park YB, Park J, Park S, Kim SH (2011) Selective detection of crystalline cellulose in plant cell walls with sum-frequency-generation (SFG) vibration spectroscopy. Biomacromolecules 12(7):2434–2439CrossRefGoogle Scholar
  28. 28.
    Barnette AL, Lee C, Bradley LC, Schreiner EP, Park YB, Shin H, Cosgrove DJ, Park S, Kim SH (2012) Quantification of crystalline cellulose in lignocellulosic biomass using sum frequency generation (SFG) vibration spectroscopy and comparison with other analytical methods. Carbohydr Polym 89(3):802–809CrossRefGoogle Scholar
  29. 29.
    Lee CM, Mittal A, Barnette AL, Kafle K, Park YB, Shin H, Johnson DK, Park S, Kim SH (2013) Cellulose polymorphism study with sum-frequency-generation (SFG) vibration spectroscopy: identification of exocyclic CH2OH conformation and chain orientation. Cellulose 20(3):991–1000CrossRefGoogle Scholar
  30. 30.
    Lee CM, Kafle K, Park YB, Kim SH (2014) Probing crystal structure and mesoscale assembly of cellulose microfibrils in plant cell walls, tunicate tests, and bacterial films using vibrational sum frequency generation (SFG) spectroscopy. Phys Chem Chem Phys 16(22):10844–10853CrossRefGoogle Scholar
  31. 31.
    Lee CM, Mohamed NM, Watts HD, Kubicki JD, Kim SH (2013) Sum-frequency-generation vibration spectroscopy and density functional theory calculations with dispersion corrections (DFT-D2) for cellulose Iα and Iβ. J Phys Chem B 117(22):6681–6692CrossRefGoogle Scholar
  32. 32.
    Park YB, Lee CM, Koo B-W, Park S, Cosgrove DJ, Kim SH (2013) Monitoring meso-scale ordering of cellulose in intact plant cell walls using sum frequency generation spectroscopy. Plant Physiol 163(2):907–913CrossRefGoogle Scholar
  33. 33.
    Kafle K, Xi X, Lee CM, Tittmann BR, Cosgrove DJ, Park YB, Kim SH (2014) Cellulose microfibril orientation in onion (Allium cepa L.) epidermis studied by atomic force microscopy (AFM) and vibrational sum frequency generation (SFG) spectroscopy. Cellulose 21:1075–1086CrossRefGoogle Scholar
  34. 34.
    Schroeder LR, Gentile VM, Atalla RH (1986) Nondegradative preparation of amorphous cellulose. J Wood Chem Technol 6(1):1–14CrossRefGoogle Scholar
  35. 35.
    Segal L, Creely J, Martin A, Conrad C (1959) An empirical method for estimating the degree of crystallinity of native cellulose using the X-ray diffractometer. Text Res J 29(10):786–794CrossRefGoogle Scholar
  36. 36.
    Thygesen A, Oddershede J, Lilholt H, Thomsen AB, Stahl K (2005) On the determination of crystallinity and cellulose content in plant fibers. Cellulose 12:563–576CrossRefGoogle Scholar
  37. 37.
    Macrae CF, Bruno IJ, Chisholm JA, Edgington PR, McCabe P, Pidcock E, Rodriguez-Monge L, Taylor R, Streek JV, Wood PA (2008) Mercury CSD 2.0-new features for the visualization and investigation of crystal structures. J Appl Crystallogr 41(2):466–470CrossRefGoogle Scholar
  38. 38.
    French AD, Cintrón MS (2013) Cellulose polymorphy, crystallite size, and the segal crystallinity index. Cellulose 20(1):583–588CrossRefGoogle Scholar
  39. 39.
    Atalla RH (1999) Individual structures of native celluloses. In: Proceedings 10th international symposium on wood and pulping chemistry: main symposium, 7–10 June 1999, Yokohama, Japan. Tappi Press, Atlanta, pp 608–614Google Scholar
  40. 40.
    Nelson ML, O'Connor RT (1964) Relation of certain infrared bands to cellulose crystallinity and crystal lattice type. Part II. A new infrared ratio for estimation of crystallinity in celluloses I and II. J Appl Polym Sci 8(3):1325–1341CrossRefGoogle Scholar
  41. 41.
    Oh SY, Yoo DI, Shin Y, Kim HC, Kim HY, Chung YS, Park WH, Youk JH (2005) Crystalline structure analysis of cellulose treated with sodium hydroxide and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy. Carbohydr Res 340(15):2376–2391CrossRefGoogle Scholar
  42. 42.
    Agarwal UP, Reiner RS, Ralph SA (2010) Cellulose I crystallinity determination using FT–Raman spectroscopy: univariate and multivariate methods. Cellulose 17(4):721–733CrossRefGoogle Scholar
  43. 43.
    Bansal P, Hall M, Realff MJ, Lee JH, Bommarius AS (2010) Multivariate statistical analysis of X-ray data from cellulose: a new method to determine degree of crystallinity and predict hydrolysis rates. Bioresour Technol 101(12):4461–4471CrossRefGoogle Scholar
  44. 44.
    Terinte N, Ibbett R, Schuster KC (2011) Overview on native cellulose and microcrystalline cellulose I structure studied by X-ray diffraction (WAXD): comparison between measurement techniques. Lenzinger Ber 89:118–131Google Scholar
  45. 45.
    Driemeier C, Calligaris GA (2010) Theoretical and experimental developments for accurate determination of crystallinity of cellulose I materials. J Appl Crystallogr 44(1):184–192CrossRefGoogle Scholar
  46. 46.
    Driemeier C (2014) Two-dimensional Rietveld analysis of celluloses from higher plants. Cellulose 21(2):1065–1073CrossRefGoogle Scholar
  47. 47.
    Schenzel K, Fischer S, Brendler E (2005) New method for determining the degree of cellulose I crystallinity by means of FT Raman spectroscopy. Cellulose 12(3):223–231CrossRefGoogle Scholar
  48. 48.
    Agarwal UP, Reiner RR, Ralph SA (2012) Estimation of cellulose crystallinity of lignocelluloses using near-IR FT-Raman spectroscopy and comparison of the Raman and Segal-WAXS methods. J Agric Food Chem 61(1):103–113CrossRefGoogle Scholar
  49. 49.
    Ding SY, Himmel ME (2006) The maize primary cell wall microfibril: a new model derived from direct visualization. J Agric Food Chem 54(3):597–606CrossRefGoogle Scholar
  50. 50.
    Newman RH, Hill SJ, Harris PJ (2013) Wide-angle X-ray scattering and solid-state nuclear magnetic resonance data combined to test models for cellulose microfibrils in mung bean cell walls. Plant Physiol 163(4):1558–1567CrossRefGoogle Scholar
  51. 51.
    Zhang T, Mahgsoudy-Louyeh S, Tittmann B, Cosgrove DJ (2014) Visualization of the nanoscale pattern of recently-deposited cellulose microfibrils and matrix materials in never-dried primary walls of the onion epidermis. Cellulose 21:853–862CrossRefGoogle Scholar
  52. 52.
    Patterson A (1939) The Scherrer formula for X-ray particle size determination. Phys Rev 56(10):978CrossRefGoogle Scholar
  53. 53.
    Oliveira RP, Driemeier C (2013) CRAFS: a model to analyze two-dimensional X-ray diffraction patterns of plant cellulose. J Appl Crystallogr 46(4):1196–1210CrossRefGoogle Scholar
  54. 54.
    Fernandes AN, Thomas LH, Altaner CM, Callow P, Forsyth VT, Apperley DC, Kennedy CJ, Jarvis MC (2011) Nanostructure of cellulose microfibrils in spruce wood. Proc Natl Acad Sci 108(47):E1195–E1203CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Christopher Lee
    • 1
  • Kevin Dazen
    • 1
  • Kabindra Kafle
    • 1
  • Andrew Moore
    • 2
  • David K. Johnson
    • 3
  • Sunkyu Park
    • 2
    Email author
  • Seong H. Kim
    • 1
    Email author
  1. 1.Department of Chemical Engineering and Materials Research InstitutePennsylvania State UniversityUniversity ParkUSA
  2. 2.Department of Forest BiomaterialsNorth Carolina State UniversityRaleighUSA
  3. 3.National Renewable Energy LaboratoryGoldenUSA

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