# Dynamic Light Scattering Plus Scanning Electron Microscopy: Usefulness and Limitations of a Simplified Estimation of Nanocellulose Dimensions

^{1}

^{2}

^{3}

^{*}

## Abstract

**:**

## 1. Introduction

_{3}– moieties) or are stabilized by another component besides them and water, the detection of single crystals is not easily distinguished from that of their aggregates [14]. Third, the choice of the scatter angle and the concentration of the sample are seldom considered, even though they are of utmost importance when dealing with polydisperse samples that tend to agglomerate [16]. Interestingly, Boluk and Danuma [17] also supported microscopy with DLS to measure CNCs, while not relying on the reported hydrodynamic diameter, but on the diffusion coefficient.

## 2. Materials and Methods

#### 2.1. Materials

_{2}SO

_{4}(97 wt %) were received from Scharlab (Scharlab SL, Sentmenat, Spain). Sodium polyethylene sulfonate (PES-Na) was provided by BTG (BTG Instruments GmbH, Weßling, Germany), along with the surface charge analyzer.

#### 2.2. Production of CNFs

#### 2.3. Production of CNCs

_{2}SO

_{4}65 wt %. The suspension was kept at 50–55 °C and under agitation by means of a heating plate and a Teflon-coated magnet. After 30 min, 100 g of ice was added to stop the reaction. Nanocrystals were sedimented by three consecutive centrifugations (10,000× g, 15 min), discarding the free acid solution and adding distilled water after each of them. Then, the suspension of CNCs was wrapped in a nanofiltration membrane with a molecular cutoff of 10 kDa. The resulting bags were immersed in distilled water for 7 days, changing the washing water every two days, to remove the remnants of acid. Neutralized CNCs (pH 6–7) were stored in topaz bottles and under 4 °C.

#### 2.4. Other Characterization Techniques

_{W}), oven-dried at 105 °C, and weighed again (m

_{D}). WRV was calculated from Equation (1):

_{PES-Na}is its concentration, V

_{b}is the volume of the titrating agent spent in a blank experiment (without nanocellulose), and m is the weight of the sample on a dry basis.

#### 2.5. Dynamic Light Scattering

#### 2.6. Estimation of Particle Dimensions

_{m}is the diameter of the fibrils as measured from FE-SEM images, and d

_{H}is the hydrodynamic diameter reported from DLS assays.

## 3. Results and Discussion

#### 3.1. Definition of Proper Testing Conditions

_{H}values.

_{H}values, high polydispersity (0.8–1), and a lack of reproducibility, probably due to aggregation-induced sedimentation. In contrast, a concentration of 0.05 wt % or 0.075 wt % is optimal or near-optimal for all samples, both CNFs and CNCs. Concentrations up to 0.1 wt % are also appropriate.

^{–}groups) and nanocrystals (with SO

_{3}

^{2–}groups to a certain extent). It may vary for other kinds of nanocellulose. More specifically, DLS assays are not recommended for cellulosic particles with very low surface charge and d

_{H}> 100 nm, such as mechanical micro-/nanofibers [30]. In general, the consistency that is required for them to not settle over the course of the measurement lies above the maximum concentration advised by the manufacturer.

#### 3.2. Dimensions of CNCs

^{2}= 0.97), followed by Lorentzian (R

^{2}= 0.89), and lastly Gaussian (R

^{2}= 0.76). The fitted log-normal function is presented in Equation (4):

^{4}nm

^{–1}in this case), and d

_{m}is to be expressed in nanometers.

_{H}was lower than 30 nm.

_{H}value, 544 nm, is of the order of magnitude of what is commonly found in the literature by performing DLS on neutral aqueous suspensions. Some examples follow: 301 nm for CNCs from microcrystalline cellulose [31], 190 nm for CNCs from non-wood pulps, 370 nm for softwood CNCs, and 430 nm for hardwood CNCs [32].

#### 3.3. Dimensions of CNFs with Different Oxidation Degrees

^{2}= 0.94 for 5 mmol/g, 0.93 for 10 mmol/g, and 0.96 for 15 mmol/g) or, to a lesser extent, Lorentzian functions (R

^{2}= 0.87, 0.82, and 0.98, respectively). Equation (5) (CNFs5), Equation (6) (CNFs10), and Equation (7) (CNFs15) express these log-normal distribution functions:

_{m}is to be expressed in nanometers.

#### 3.4. Relating Key Properties of Nanocellulose to Its Dimensions

_{2}SO

_{4}selectively hydrolyzes the least crystalline regions of cellulose, as long as its concentration is not excessive (concentrations beyond 65 wt % are not recommended for this purpose [38]).

^{−}and BrO

^{−}[39]. In the context of nanofiber dimensions, we want to highlight that the larger the removal of amorphous parts, the shorter the fibril (Figure 5b). Selective depolymerization is one of the factors leading to smaller structures. Indeed, the degree of polymerization and the length of nanofibers are tightly correlated [40]. In any case, electrostatic repulsion and the hydration of carboxylate groups, which prevents them from hydrogen-bonding with nearby cellulose chains, are also significant in what pertains to nanocellulose dimensions.

^{–}groups introduced. The low charge density of nanocrystals reveals a low degree of sulfation, which explains their tendency to aggregate.

## 4. Conclusions

^{2}= 0.97). Geometrical relationships between this width and the d

_{H}distribution provided by DLS allow us to estimate the average length of CNC clusters as 1.9 µm. For CNFs, the results could be correlated to the ratio of oxidizing agent to pulp: 178 nm × 3.37 μm (5 mmol NaClO/g pulp), 121 nm × 1.67 μm (10 mmol NaClO/g pulp), and 92 nm × 1.23 μm (15 mmol NaClO/g pulp). Although this trend was expected, the width was probably overestimated due to the absence of single nanofibers whose diameter was lower than 30 nm.

## Author Contributions

## Funding

## Data Availability Statement

## Acknowledgments

## Conflicts of Interest

## References

- Fraschini, C.; Chauve, G.; Le Berre, J.-F.; Ellis, S.; Méthot, M.; O’Connor, B.; Bouchard, J. Critical Discussion of Light Scattering and Microscopy Techniques for CNC Particle Sizing. Nord. Pulp Pap. Res. J.
**2014**, 29, 31–40. [Google Scholar] [CrossRef] - Gamelas, J.A.F.; Pedrosa, J.; Lourenço, A.F.; Mutjé, P.; González, I.; Chinga-Carrasco, G.; Singh, G.; Ferreira, P.J.T. On the Morphology of Cellulose Nanofibrils Obtained by TEMPO-Mediated Oxidation and Mechanical Treatment. Micron
**2015**, 72, 28–33. [Google Scholar] [CrossRef] - Sanchez-Salvador, J.L.; Balea, A.; Negro, C.; Monte, M.C.; Blanco, A. Gel Point as Measurement of Dispersion Degree of Nano-Cellulose Suspensions and Its Application in Papermaking. Nanomaterials
**2022**, 12, 790. [Google Scholar] [CrossRef] - Aguado, R.; Tarrés, Q.; Pèlach, M.À.; Mutjé, P.; de la Fuente, E.; Sanchez-Salvador, J.L.; Negro, C.; Delgado-Aguilar, M. Micro- and Nanofibrillated Cellulose from Annual Plant-Sourced Fibers: Comparison between Enzymatic Hydrolysis and Mechanical Refining. Nanomaterials
**2022**, 12, 1612. [Google Scholar] [CrossRef] [PubMed] - Hotaling, N.A.; Bharti, K.; Kriel, H.; Simon, C.G. DiameterJ: A Validated Open Source Nanofiber Diameter Measurement Tool. Biomaterials
**2015**, 61, 327–338. [Google Scholar] [CrossRef] [Green Version] - Sanchez-Salvador, J.L.; Campano, C.; Balea, A.; Tarrés, Q.; Delgado-Aguilar, M.; Mutjé, P.; Blanco, A.; Negro, C. Critical Comparison of the Properties of Cellulose Nanofibers Produced from Softwood and Hardwood through Enzymatic, Chemical and Mechanical Processes. Int. J. Biol. Macromol.
**2022**, 205, 220–230. [Google Scholar] [CrossRef] - Campano, C.; Balea, A.; Blanco, Á.; Negro, C. A Reproducible Method to Characterize the Bulk Morphology of Cellulose Nanocrystals and Nanofibers by Transmission Electron Microscopy. Cellulose
**2020**, 27, 4871–4887. [Google Scholar] [CrossRef] - Radakisnin, R.; Abdul Majid, M.S.; Jamir, M.R.; Jawaid, M.; Sultan, M.T.; Mat Tahir, M.F. Structural, Morphological and Thermal Properties of Cellulose Nanofibers from Napier Fiber (Pennisetum purpureum). Materials
**2020**, 13, 4125. [Google Scholar] [CrossRef] [PubMed] - Aguado, R.; Lourenço, A.F.; Ferreira, P.J.; Moral, A.; Tijero, A. Cationic Cellulosic Derivatives as Flocculants in Papermaking. Cellulose
**2017**, 24, 3015–3027. [Google Scholar] [CrossRef] - Zakharov, P.; Scheffold, F. Advances in Dynamic Light Scattering Techniques BT—Light Scattering Reviews 4: Single Light Scattering and Radiative Transfer; Kokhanovsky, A.A., Ed.; Springer Berlin Heidelberg: Berlin/Heidelberg, Germany, 2009; pp. 433–467. ISBN 978-3-540-74276-0. [Google Scholar]
- Fotie, G.; Gazzotti, S.; Ortenzi, M.A.; Piergiovanni, L. Implementation of High Gas Barrier Laminated Films Based on Cellulose Nanocrystals for Food Flexible Packaging. Appl. Sci.
**2020**, 10, 3201. [Google Scholar] [CrossRef] - Gallardo-Sánchez, M.A.; Diaz-Vidal, T.; Navarro-Hermosillo, A.B.; Figueroa-Ochoa, E.B.; Ramirez Casillas, R.; Anzaldo Hernández, J.; Rosales-Rivera, L.C.; Soltero Martínez, J.F.; García Enríquez, S.; Macías-Balleza, E.R. Optimization of the Obtaining of Cellulose Nanocrystals from Agave Tequilana Weber Var. Azul Bagasse by Acid Hydrolysis. Nanomaterials
**2021**, 11, 520. [Google Scholar] [CrossRef] - Metzger, C.; Drexel, R.; Meier, F.; Briesen, H. Effect of Ultrasonication on the Size Distribution and Stability of Cellulose Nanocrystals in Suspension: An Asymmetrical Flow Field-Flow Fractionation Study. Cellulose
**2021**, 28, 10221–10238. [Google Scholar] [CrossRef] - Bao, C.; Chen, X.; Liu, C.; Liao, Y.; Huang, Y.; Hao, L.; Yan, H.; Lin, Q. Extraction of Cellulose Nanocrystals from Microcrystalline Cellulose for the Stabilization of Cetyltrimethylammonium Bromide-Enhanced Pickering Emulsions. Colloids Surfaces A Physicochem. Eng. Asp.
**2021**, 608, 125442. [Google Scholar] [CrossRef] - Delepierre, G.; Eyley, S.; Thielemans, W.; Weder, C.; Cranston, E.D.; Zoppe, J.O. Patience Is a Virtue: Self-Assembly and Physico-Chemical Properties of Cellulose Nanocrystal Allomorphs. Nanoscale
**2020**, 12, 17480–17493. [Google Scholar] [CrossRef] - Khlebtsov, B.N.; Khlebtsov, N.G. On the Measurement of Gold Nanoparticle Sizes by the Dynamic Light Scattering Method. Colloid J.
**2011**, 73, 118–127. [Google Scholar] [CrossRef] - Boluk, Y.; Danumah, C. Analysis of Cellulose Nanocrystal Rod Lengths by Dynamic Light Scattering and Electron Microscopy. J. Nanopart. Res.
**2013**, 16, 2174. [Google Scholar] [CrossRef] - Kohl, K. Comparison of Dynamic Light Scattering and Rheometrical Methods to Determine the Gel Point of a Radically Polymerized Hydrogel under Mechanical Shear. Micromachines
**2020**, 11, 462. [Google Scholar] [CrossRef] - Leong, S.S.; Ng, W.M.; Lim, J.; Yeap, S.P. Dynamic Light Scattering: Effective Sizing Technique for Characterization of Magnetic Nanoparticles. In Handbook of Materials Characterization; Sharma, S.K., Ed.; Springer International Publishing: Cham, Switzerland, 2018; pp. 77–111. ISBN 978-3-319-92955-2. [Google Scholar]
- Aguado, R.; Murtinho, D.; Valente, A.J.M. Association of Antioxidant Monophenolic Compounds with β-Cyclodextrin-Functionalized Cellulose and Starch Substrates. Carbohydr. Polym.
**2021**, 267, 118189. [Google Scholar] [CrossRef] - Haddad, G.; Fontanini, A.; Bellali, S.; Takakura, T.; Ominami, Y.; Hisada, A.; Hadjadj, L.; Rolain, J.M.; Raoult, D.; Bou Khalil, J.Y. Rapid Detection of Imipenem Resistance in Gram-Negative Bacteria Using Tabletop Scanning Electron Microscopy: A Preliminary Evaluation. Front. Microbiol.
**2021**, 12, 658322. [Google Scholar] [CrossRef] [PubMed] - Serra-Parareda, F.; Tarrés, Q.; Sanchez-Salvador, J.L.; Campano, C.; Pèlach, M.À.; Mutjé, P.; Negro, C.; Delgado-Aguilar, M. Tuning Morphology and Structure of Non-Woody Nanocellulose: Ranging between Nanofibers and Nanocrystals. Ind. Crops Prod.
**2021**, 171, 113877. [Google Scholar] [CrossRef] - Tarrés, Q.; Aguado, R.; Pèlach, M.À.; Mutjé, P.; Delgado-Aguilar, M. Electrospray Deposition of Cellulose Nanofibers on Paper: Overcoming the Limitations of Conventional Coating. Nanomaterials
**2022**, 12, 79. [Google Scholar] [CrossRef] [PubMed] - Noguchi, Y.; Homma, I.; Matsubara, Y. Complete Nanofibrillation of Cellulose Prepared by Phosphorylation. Cellulose
**2017**, 24, 1295–1305. [Google Scholar] [CrossRef] - Tarrés, Q.; Boufi, S.; Mutjé, P.; Delgado-Aguilar, M. Enzymatically Hydrolyzed and TEMPO-Oxidized Cellulose Nanofibers for the Production of Nanopapers: Morphological, Optical, Thermal and Mechanical Properties. Cellulose
**2017**, 24, 3943–3954. [Google Scholar] [CrossRef] - Serra-Parareda, F.; Aguado, R.; Tarrés, Q.; Mutjé, P.; Delgado-Aguilar, M. Potentiometric Back Titration as a Robust and Simple Method for Specific Surface Area Estimation of Lignocellulosic Fibers. Cellulose
**2021**, 28, 10815–10825. [Google Scholar] [CrossRef] - Filipova, I.; Serra, F.; Tarrés, Q.; Mutjé, P.; Delgado-Aguilar, M. Oxidative Treatments for Cellulose Nanofibers Production: A Comparative Study between TEMPO-Mediated and Ammonium Persulfate Oxidation. Cellulose
**2020**, 27, 10671–10688. [Google Scholar] [CrossRef] - Eckelt, J.; Knopf, A.; Röder, T.; Weber, H.K.; Sixta, H.; Wolf, B.A. Viscosity-Molecular Weight Relationship for Cellulose Solutions in Either NMMO Monohydrate or Cuen. J. Appl. Polym. Sci.
**2011**, 119, 670–676. [Google Scholar] [CrossRef] - Segal, L.; Creely, J.J.; Martin, A.E.; Conrad, C.M. An Empirical Method for Estimating the Degree of Crystallinity of Native Cellulose Using the X-Ray Diffractometer. Text. Res. J.
**1959**, 29, 786–794. [Google Scholar] [CrossRef] - Velásquez-Cock, J.; Gañán, P.; Posada, P.; Castro, C.; Serpa, A.; Gómez H., C.; Putaux, J.-L.; Zuluaga, R. Influence of Combined Mechanical Treatments on the Morphology and Structure of Cellulose Nanofibrils: Thermal and Mechanical Properties of the Resulting Films. Ind. Crops Prod.
**2016**, 85, 1–10. [Google Scholar] [CrossRef] - Taheri, A.; Mohammadi, M. The Use of Cellulose Nanocrystals for Potential Application in Topical Delivery of Hydroquinone. Chem. Biol. Drug Des.
**2015**, 86, 102–106. [Google Scholar] [CrossRef] - Verma, C.; Chhajed, M.; Gupta, P.; Roy, S.; Maji, P.K. Isolation of Cellulose Nanocrystals from Different Waste Bio-Mass Collating Their Liquid Crystal Ordering with Morphological Exploration. Int. J. Biol. Macromol.
**2021**, 175, 242–253. [Google Scholar] [CrossRef] - Vallejos, M.E.; Felissia, F.E.; Area, M.C.; Ehman, N.V.; Tarrés, Q.; Mutjé, P. Nanofibrillated Cellulose (CNF) from Eucalyptus Sawdust as a Dry Strength Agent of Unrefined Eucalyptus Handsheets. Carbohydr. Polym.
**2016**, 139, 99–105. [Google Scholar] [CrossRef] - Sanchez-Salvador, J.L.; Campano, C.; Lopez-Exposito, P.; Tarrés, Q.; Mutjé, P.; Delgado-Aguilar, M.; Monte, M.C.; Blanco, A. Enhanced Morphological Characterization of Cellulose Nano/Microfibers through Image Skeleton Analysis. Nanomaterials
**2021**, 11, 2077. [Google Scholar] [CrossRef] - Ang, S.; Haritos, V.; Batchelor, W. Cellulose Nanofibers from Recycled and Virgin Wood Pulp: A Comparative Study of Fiber Development. Carbohydr. Polym.
**2020**, 234, 115900. [Google Scholar] [CrossRef] - Saito, T.; Kimura, S.; Nishiyama, Y.; Isogai, A. Cellulose Nanofibers Prepared by TEMPO-Mediated Oxidation of Native Cellulose. Biomacromolecules
**2007**, 8, 2485–2491. [Google Scholar] [CrossRef] [PubMed] - Baati, R.; Magnin, A.; Boufi, S. High Solid Content Production of Nanofibrillar Cellulose via Continuous Extrusion. ACS Sustain. Chem. Eng.
**2017**, 5, 2350–2359. [Google Scholar] [CrossRef] - Ioelovich, M. Study of Cellulose Interaction with Concentrated Solutions of Sulfuric Acid. ISRN Chem. Eng.
**2012**, 2012, 428974. [Google Scholar] [CrossRef] [Green Version] - Spier, V.C.; Sierakowski, M.R.; Reed, W.F.; de Freitas, R.A. Polysaccharide Depolymerization from TEMPO-Catalysis: Effect of TEMPO Concentration. Carbohydr. Polym.
**2017**, 170, 140–147. [Google Scholar] [CrossRef] - Shinoda, R.; Saito, T.; Okita, Y.; Isogai, A. Relationship between Length and Degree of Polymerization of TEMPO-Oxidized Cellulose Nanofibrils. Biomacromolecules
**2012**, 13, 842–849. [Google Scholar] [CrossRef] - Ensor, D.S.; Pilat, M.J. The Effect of Particle Size Distribution on Light Transmittance Measurement. Am. Ind. Hyg. Assoc. J.
**1971**, 32, 287–292. [Google Scholar] [CrossRef]

**Figure 1.**Size distribution of nanocellulose samples (consistency: 0.05 wt %), quantifying the intensity for every interval of hydrodynamic diameters (d

_{H}), setting the device either for forward angle scattering or for backscattering.

**Figure 2.**Intensity-weighted average hydrodynamic diameter of CNCs (

**a**) and CNFs (

**b**), as a function of concentration. The amplitude of the error bars is twice the standard deviation. Inset of (

**a**) is the general shape of the size distribution (0.075 wt %).

**Figure 3.**Cellulose nanocrystals and their aggregates: micrograph of a CNC cluster (

**a**), outlined particles after binarization in ImageJ (

**b**), and width histogram by counts, including fitting to Gaussian, Lorentzian, and log-normal distributions (

**c**).

**Figure 4.**Width histograms of CNFs with different doses of hypochlorite during oxidation, namely 5 mmol/g (

**a**), 10 mmol/g (

**b**), and 15 mmol/g (

**c**). The relative frequency is fitted to Lorentzian and log-normal distributions. Inset figures show outlined particles after binarization and isolation.

**Figure 5.**Shifted and normalized X-ray diffraction patterns of CNCs and CNFs, following a two-point baseline correction (

**a**). The scheme (

**b**) does not imply that depolymerization of amorphous domains is the only cause for a decrease in CNF length.

Dimension | FE-SEM | DLS | Calculated | ||
---|---|---|---|---|---|

Average | Mode | Average | Mode | ||

Diameter (nm) | 237 | 125–150 | 544 ± 6 | 459–571 | -- |

Length (μm) | 1.29 | 1.80–2.00 | -- | 1.91 |

Sample | d_{m} (nm) | d_{H} (nm) | Calculated Length (μm) | ||
---|---|---|---|---|---|

Average | Mode | Average | Mode | ||

CNFs5 | 178 | 100–150 | 543 ± 20 | 459–571 | 3.37 |

CNFs10 | 121 | 50–100 | 332 ± 49 | 295–342 | 1.67 |

CNFs15 | 92 | 50–100 | 250 ± 5 | 220–255 | 1.23 |

**Table 3.**Key characteristics of CNCs and CNFs: degree of polymerization (DP), water retention value (WRV), charge density (CD), and transmittance at 600 nm.

Sample | DP | WRV (g/g) | CD (meq/g) | Transmittance (%) |
---|---|---|---|---|

CNCs | -- | -- | 0.07 | 71.8 |

CNFs5 | 260 | 13.3 | 1.36 | 89.8 |

CNFs10 | 135 | 11.8 | 1.70 | 94.0 |

CNFs15 | 117 | 13.5 | 1.99 | 99.9 |

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |

© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

## Share and Cite

**MDPI and ACS Style**

Tarrés, Q.; Aguado, R.; Zoppe, J.O.; Mutjé, P.; Fiol, N.; Delgado-Aguilar, M.
Dynamic Light Scattering Plus Scanning Electron Microscopy: Usefulness and Limitations of a Simplified Estimation of Nanocellulose Dimensions. *Nanomaterials* **2022**, *12*, 4288.
https://doi.org/10.3390/nano12234288

**AMA Style**

Tarrés Q, Aguado R, Zoppe JO, Mutjé P, Fiol N, Delgado-Aguilar M.
Dynamic Light Scattering Plus Scanning Electron Microscopy: Usefulness and Limitations of a Simplified Estimation of Nanocellulose Dimensions. *Nanomaterials*. 2022; 12(23):4288.
https://doi.org/10.3390/nano12234288

**Chicago/Turabian Style**

Tarrés, Quim, Roberto Aguado, Justin O. Zoppe, Pere Mutjé, Núria Fiol, and Marc Delgado-Aguilar.
2022. "Dynamic Light Scattering Plus Scanning Electron Microscopy: Usefulness and Limitations of a Simplified Estimation of Nanocellulose Dimensions" *Nanomaterials* 12, no. 23: 4288.
https://doi.org/10.3390/nano12234288