Cross-Linking Cellulosic Fibers with Photoreactive Polymers

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Cross-linking cellulosic fibers with photo-reactive polymers: visualization with confocal Raman and fluorescence microscopy Marek Janko, Michael Jocher, Alexander Böhm, Laura Babel, Steven Bump, Markus Biesalski, Tobias Meckel, and Robert W Stark Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00565 • Publication Date (Web): 23 Jun 2015 Downloaded from http://pubs.acs.org on July 2, 2015

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Cross-linking cellulosic fibers with photo-reactive polymers: visualization with confocal Raman and fluorescence microscopy

Marek Janko1,2, Michael Jocher3, Alexander Boehm2,3, Laura Babel4, Steven Bump4, Markus Biesalski2,3,*, Tobias Meckel4*, Robert W. Stark1,2*

1

Physics of Surfaces, Institute of Materials Science, Technische Universität Darmstadt, Alarich-WeissStraße 2, 64287 Darmstadt, Germany 2

Center of Smart Interfaces, Technische Universität Darmstadt, Alarich-Weiss-Straße 10, 64287 Darmstadt, Germany

3

Macromolecular Chemistry and Paper Chemistry, Department of Chemistry, Technische Universität Darmstadt, Alarich-Weiss-Straße 8, D-64287 Darmstadt, Germany

4

Membrane Dynamics, Department of Biology, Technische Universität Darmstadt, Schnittspahnstraße 3-5, 64287 Darmstadt, Germany

* Author for correspondence: Markus Biesalski, [email protected] * Author for correspondence: Tobias Meckel, [email protected] * Author for correspondence: Robert W. Stark, [email protected]

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ACS Paragon Plus Environment

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Abstract

The properties of paper sheets can be tuned by adjusting the surface or bulk chemistry using functional polymers that are applied during (online) or after (offline) papermaking processes. In particular, polymers are widely used to enhance the mechanical strength of the wet state of paper sheets. However, the mechanical strength depends not only on the chemical nature of the polymeric additives but also on the distribution of the polymer on and in the lignocellulosic paper. Here, we analyze the photochemical attachment and distribution of hydrophilic polydimethylacrylamide-co-methacrylate-benzophenone P(DMAA-co-MABP) copolymers with defined amounts of photo-reactive benzophenone moieties in model paper sheets. Raman microscopy was used for the unambiguous identification of P(DMAA-coMABP) and cellulose specific bands and thus the copolymer distribution within the cellulose matrix. Two-dimensional Raman spectral maps at the intersections of overlapping cellulose fibers document that the macromolecules only partially surround the cellulose fibers, favors to attach to the fiber surface and connects the cellulose fibers at crossings. Moreover, the copolymer appears to accumulate preferentially in holes, vacancies, and dips on the cellulose fiber surface. Correlative brightfield, Raman, and confocal laser scanning microscopy finally reveal a reticular three-dimensional distribution of the polymer and show that the polymer is predominately deposited in regions of high capillarity, i.e., in proximity to fine cellulose fibrils. These data provide deeper insights into the effects of paper functionalization with a copolymer and aid in understanding how these agents ultimately influence the local and overall properties of paper.

Keywords: Paper, wet strength, Raman spectroscopy, confocal laser scanning microscopy, photoreactive polymers, polymer distribution.

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Introduction Cellulosic paper products with tailored functionalities offer considerable economic and ecological potential because of their versatile material properties. Paper has low density and an anisotropic sheet structure due to its molecular and hierarchical composition 1, and therefore exhibits tremendous tensile strength in the dry state. However, in the wet state, the H-bonds between individual fibers are loosened, and the tensile strength of paper is considerably lowered, which can lead to the disintegration of the sheet. Structurally, cellulose is a polysaccharide composed of a linear chain of glucose molecules covalently linked via glycosidic oxygen bridges

2, 3

. These cellulose chains feature abundant hydroxyl

groups on their surfaces, which form hydrogen bonds with neighboring chains. As approximated in the Page equation 4, the tensile strength of paper sheets can be understood in terms of individual fiber strength, H-bonding and van der Waals interactions between the fibers in the sheet and the relative bonding area of fiber-crossings

5, 6

. Additionally, fibrils acting as bridging elements play an important

role 7. Failure occurs at the weakest point, which is the fiber-to-fiber connection. A high wet tensile strength is essential for cellulosic paper products and is required for many different paper grades progressing from packaging materials, filter applications, to tissues hydrophobic or microfluidic papers

10-12

8, 9

and

. If no wet-strengthening agents are applied, the wet tensile

strength of non-modified paper sheets is typically on the order of only 1-3% of the tensile strength of the same paper sample under dry conditions. Thus, polymer additives (resins) are often used to enhance cross-linking of the fibers and to increase the wet tensile strength of paper

13-15

. Typical examples of

such resins range from polyamidoamine-epichlorohydrine resins and urea- or melamine-formaldehyde resins to glyoxalated polyacrylamide. The common feature of these additives is that cross-linking of the polymer itself (homo-crosslinking) or with the lignocellulosic fibers within the sheet (co-crosslinking) occurs during the drying process, i.e., by heat. Thus, large amounts of energy are required to ensure the structural integrity of paper products during wetting. Less energy-intensive processes are based on well controllable photochemical reactions, which are used to enhance the wet-strength of paper sheets 3


16

or

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to chemical ligate defined functions to the interface of cellulosic fibers 17. In particular, photo-reactive, hydrophilic polymers can be applied to the fiber interface. Such photo-crosslinkable polymers yield wet strength properties of the paper sheet similar to those produced by commercially available additives

18

. The key features of photo-reactive polymers as wet-

strengthening agents are simplicity of processing and covalent bonding to the cellulosic fibers. Using electron and fluorescence microscopy, we showed that the polymers bind to the paper sheets and are physisorbed, so that non cross-linked polymers can be removed by solvent extraction

17

. We also

showed that the amount of fiber-attached polymers can be varied over a wide range (from a few mg polymer per g fiber to about hundred mg polymer per g fiber)

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in a dip-coating process by adjusting

the concentration of the polymer in the coating-solution. However, the exact location of the polymers in the paper sheet, and whether fiber crossings are predominately coated or whether the polymer coats the complete individual fiber are open questions. Particularly, with paper sheets having cavities and holes of various sizes between the fibers, as well as on and in the fibers, the distribution of the polymer affects the local and overall properties of the cellulose network. Therefore, it is important to analyze and determine the distribution of the macromolecules within the paper matrix. To this end, modern optical microscopy such as confocal fluorescence microscopy

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or FT-IR

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and Raman

microspectroscopy 21 are powerful tools. In this study, we demonstrate that correlative confocal Raman and fluorescence microscopy helps to understand the mechanism of photo wet-strengthening of paper sheets with photo-linked polymers. Direct chemical identification and direct determination of the distribution of P(DMAA)-based photolinked polymers in cellulose fiber networks are shown. Specifically, using Raman imaging scans, we investigated where the photo-reactive polymer accumulates inside the paper sheets. Analyzing the chemical nature of the fiber-bound polymer and correlating such results to confocal laser scanning fluorescence microscopy images thus highlights the contribution of the photo-reactive polymer to the stabilization of such fiber networks in wet conditions. 4


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Materials and Methods 2.1 Materials The 2,2’-azobis (2-methylpropionitrile) (AIBN, >98%, Fluka), 4-hydroxybenzophenone (98%, Alfa Aesar), triethylamine (99%, Grüssing GmbH), acetone (>99.5%, Roth), dichloromethane (p.a., Biesterfeld), methanol (>99.5%, BASF), diethyl ether (99.5%, Sigma-Aldrich), dimethylformamide (DMF, >99.8%, Sigma Aldrich), tetrahydrofuran (THF, >99.5%, Roth), methacryloxyethyl thiocarbamoyl rhodamine B (RhBMA, PolySciences), Calcofluor White (Sigma-Aldrich) and pulp (fiber source: cotton linters; curl: 17.4%; fibrillation degree: 4.9%; fines content: 6.5%; lignin content < 1%) were used as received. The N,N’-dimethylacrylamide (DMAA, 99%, Sigma-Aldrich) and methacryloyl chloride (97%, Sigma-Aldrich) were passed through a basic alumina column, distilled under reduced pressure and stored under nitrogen prior to use.

2.2 Monomer and polymer synthesis The synthesis of the photo-reactive monomer 4-metharyloyloxybenzophenone (MABP) was carried out as reported by Schlemmer et al.

22

. The polymers used in the current study were prepared via free

radical polymerization and characterized with respect to their molar composition as well as molar mass using 1H-NMR spectroscopy and size exclusion chromatography (SEC; PSS GRAM VS was used as the column, DMF/LiCl was used as the eluent, and the system was calibrated using narrow dispersed polymethyl methacrylate standards).

2.2.1 Poly(dimethylacrylamide-co-4-benzoylphenyl-2-methacrylate (P(DMAA-co-MABP)) The DMAA (1.9 ml, 19.0 mmol) was added to MABP (266.0 mg, 1.0 mmol) in 1,4-dioxane in a Schlenk flask under nitrogen atmosphere. The initiator AIBN (9.8 mg, 0.06 mmol) was added, and the solution was degassed using four freeze-pump-thaw cycles. The flask was placed in a bath at 60°C for 16 h for polymerization. Subsequently, the polymer was precipitated in diethyl ether and purified by re5


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precipitation with THF and diethyl ether. The polymer was obtained as a white solid (0.73 g, 35%, Mn=53.000 gmol-1). The 1H-NMR analysis yielded a molar composition of approximately 5.3 mol% of the photo-reactive benzophenone group in the copolymer.

2.2.2 Poly(dimethylacrylamide-co-4-benzoylphenyl-2-methacrylate-co-rhodamineBmethacrylic acid (P(DMAA-co-MABP-co-RhBMA)) For the synthesis of P(DMAA-co-MABP-co-RhBMA), DMAA (1.8 g, 19.48 mmol) was added to 4methacryloyloxybenzophenone (133.4 mg, 0.5 mmol) and RhBMA (13.7 mg, 0.02 mmol) in DMF (20 mL) in a Schlenk flask under nitrogen atmosphere. The initiator AIBN (9.6 mg, 0.06 mmol) was added, and the solution was degassed by four freeze-pump-thaw cycles. The flask was placed in a bath at 60°C for 16 h for polymerization. After this time, the polymer was precipitated in 300 mL of diethyl ether and dried in vacuum. The polymer was purified by re-precipitation with acetone (12 mL) and diethyl ether (200 mL). The polymer was obtained as a slightly pink solid (1.28 g, 64%, Mn = 25.000 g/mol). The 1H-NMR analysis yielded a molar composition of approximately 3.0% mol of the photo-reactive benzophenone group in the copolymer.

2.3 Preparation of lab-engineered paper Bleached dry cotton linters were used for the preparation of lab-engineered paper substrates. The pulp was refined in a Voith LR 40 lab refiner at an effective specific energy of 200 kWh/t. After refining, the cotton linters pulp had a °SR (Schopper-Riegler freeness) of 37. A series of paper sheets with grammages of 23.5 g m-2 or 28.3 g m-2 were produced on a conventional Rapid-Koethen hand sheet maker according to DIN 54358 and ISO 5269/2. The prepared hand sheets were free of any additives or fillers. The grammage values of the lab-engineered paper sheets were obtained as recently described 17.

2.4 Preparation of polymer-modified paper hand sheets 6


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For the modification of the lab-engineered cellulose sheets, either unlabeled P(DMAA-co-MABP) or fluorescently labeled P(DMAA-co-MABP-co-RhBMA) was dissolved in THF at a constant concentration of approximately 30 mg ml-1. Paper substrates with a grammage of 23.5 g m-2 were cut into pieces (2.5 x 2.5 cm), and the desired functional polymer was absorbed onto the cellulose fibers by submerging the paper into the polymer solution for approximately 20 s (dip-coating method). After airdrying for 30 min at room temperature and humidity, cross-linking was performed via UV light exposure. To covalently attach the physisorbed polymer to the cellulose fibers, the samples were transferred to a Newport 1000 W Oriel Flood Exposure source (λ = 365 nm) and illuminated. Therefore, all samples were exposed to an irradiance of E = 16 J cm-2 at which approximately 94% of the benzophenone groups reacted

23

. The samples were exposed for approximately 16 min. After

exposure, non-bound polymer residues were removed by a 150-min solvent extraction in a Soxhletapparatus using THF as the solvent. As shown previously, this solvent extraction procedure removes the non-bound polymer. More than 90% of the transferred polymer mass remains covalently attached to the fiber surface.

17

Because the polymers are attached by a photo-chemical process, the amount of

photo-reactive benzophenone-moieties determines the cross-link density. As long as at least two benzophenone groups per polymer chain have reacted in such cross-links, the macromolecules are firmly attached to the cellulosic fiber. Thus, already a 0.5 mol% fraction of benzophenone groups is sufficient to chemically bind the macromolecules to the cellulosic fibers. 18 Here, we used copolymers carrying about 3 mol% benzophenone groups. Further details regarding the preparation technique, the transferred mass of polymer, and the influence of the relative benzophenone content in the polymers on the chemical attachment are given elsewhere. 17, 18 After preparation, all samples were left to dry in air at norm-climate conditions (23°C, 50% relative humidity) for at least 15 h prior to further characterization.

2.5 Characterization of polymer-modified paper hand sheets by confocal Raman, brightfield and 7


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confocal fluorescence microscopy Polymer-modified cellulose sheets were examined using brightfield and confocal laser scanning microscopy (CLSM). The untreated cellulose sheets were stained with Calcofluor White before they were subjected to chemical modification with photo-reactive polymers. To this end, the lab-engineered paper sheets were submerged into a 0.1 µM Calcofluor White solution (pH 10.6, adjusted by potassium hydroxide) for 20 min in the dark at 23°C. This concentration of Calcofluor White (as evaluated in a previous dilution series; data not shown) was identified as the minimum concentration that still allowed collection of fluorescence images with reasonable signal-to-noise ratio (SNR). After air-drying, the samples were modified with the photo-reactive P(DMAA-co-MABP) copolymer as described above. Brightfield and confocal fluorescent images were recorded on a Leica TCS SP5II Confocal Laser Scanning Microscope (CLSM, Leica Microsystems GmbH, Mannheim, Germany) with an HCX PL APO20xNA 0.7 Immor HCX PL APO 63x NA 1.2 W CORR objective. Brightfield images were obtained using a 488-nm laser for illumination and detecting the transmitted light on a photomultiplier tube (PMT). Fluorescent samples were imaged by sequentially exciting each pixel line of the confocal scan with 405-nm and 561-nm lasers, corresponding to the excitation wavelengths of Calcofluor White and Rhodamine B, respectively. Emission was detected between 420 nm and 470 nm for Calcofluor White and 575 nm and 630 nm for Rhodamine-labeled P(DMAA) on sensitive Leica HyD™ detectors. The sequential detection scheme led to negligible crosstalk of Caclofluor White emission into the Rhodamine B detection channel. Raman measurements were collected in air and at room temperature (20°C) with a confocal Raman microscope system (WiTec alpha 300 R, Ulm, Germany). The microscope was equipped with a piezoscanner (PI, Karlsruhe, Germany) and a frequency-doubled Nd:YAG laser (laser wavelength λ = 532 nm) with a polarization ratio of 100:1. The scattered light was collected by a standard air lens (Nikon, Japan) (20× magnification, NA = 0.40) and focused to a 50-µm multimode fiber acting as the pinhole of the confocal setup. The confocal resolution was approximately 7 µm in focal depth, and the 8


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diffraction-limited spot size was approximately 800 nm in diameter. Spectra were recorded at a maximum laser power of 3.0 mW to avoid sample damage. Reference spectra were averaged, representing a mean of ten single spectra, and integrated for 10 seconds each. The distribution of chemical species on the cellulose sample was determined in Raman spectral imaging mode. In these processes, a predefined sample area was scanned, and the Raman spectra (integration time per spectrum > 1 second) were acquired at every imaging point. As a result, a 2D array of Raman spectra was obtained by collecting the spectra in a pixel-by-pixel and line-by-line manner. By extracting the peak height of specific Raman bands from the data, a Raman image was calculated (for details, see supporting information). The specific position of the Raman peaks was previously defined by Gaussian fits. All spectra were subject to cosmic ray removal and a second-order polynomial background subtraction, excluding the position of the specific peaks, to reduce the fluorescence background. The samples analyzed were pre-scanned to reduce auto-fluorescence. The backscattered light from the sample was detected with a vacuum-sealed high-sensitivity backilluminated CCD camera cooled to -56°C. The CCD spectrometer was operated with a 600 1/mm grating resulting in a spectral resolution of approximately 4 cm-1 (0 – 3600 cm-1) per CCD pixel.

Results and Discussion Figure 1 depicts a schematic and idealized illustration of the photochemical cross-linking of the copolymer in the paper sheet. A detailed description of the chemical reactions is given elsewhere 17, 18. To investigate the photochemical attachment and distribution of hydrophilic polydimethylacrylamideco-methacrylate-benzophenone P(DMAA-co-MABP) copolymers in model paper sheets, reference Raman spectra were taken of the chemical agents used for synthesis of the copolymer coating, i.e., the pure homopolymer polydimethylacrylamide (PDMAA) and the pure benzophenone (BP). In particular, the BP with its phenyl rings showed multiple distinct Raman peaks. The strongest peaks were found at approximately 997, 1149, 1592, 1648, and 3062 cm-1 and are assigned to the in-plane deformation 9


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vibration of the phenyl ring, the stretching vibration of C-C-C, the phenyl ring stretching mode of C=C and C-C, a stretching mode of the carbonyl group C=O, and the C-H stretching vibration, respectively 24-26

. The most intense Raman bands in the homopolymer PDMAA spectra are found at approximately

1416, 1448, 1619, and 2934 cm-1 and correspond to the symmetric and asymmetric bending modes of methyl groups (CH3), the vinyl stretching mode (C=C), and the C-H symmetric stretching mode of methyl groups, respectively 27, 28. The Raman spectra of the non-modified and P(DMAA-co-MABP)-modified paper sheets show sample specific Raman bands as given in Table S1 (supporting information) and Figure 2. The P(DMAA-coMABP) copolymer features multiple bands within the fingerprint region of the polymer chain (between 300 and 1700 cm-1) and a peak distinct for the polymer at approximately 2934 cm-1. The most characteristic copolymer bands, and the most distinct from the Raman bands of the paper sheets, are those at approximately 1596, 1655, and 2934 cm-1, which correspond well to the characteristic bands reported for the reference spectra of the PDMAA polymer and the BP. Within the spectrum of the non-modified paper samples, specific peaks occur at approximately 1095, 1120, 1335, 1378, and 2897 cm-1 and can be assigned to the paper-sheet component cellulose. These bands are related to asymmetric and symmetric C-O-C stretch modes, C-H2 bending modes, and to C-H / C-H2 stretch modes of methine-/methylene-groups within the cellulose backbone, as reported by Wiley et al., Edwards et al., and Gierlinger et al. 29-31. Accordingly, the spectrum of a P(DMAA-co-MABP)-modified cellulose sheet is a superposition of the copolymer and the cellulose spectrum. Within this superposition spectrum, the intensity of the cellulose-specific C-O-C stretch band at 1095 cm-1 is weakened, whereas the band at 2897 cm-1 widens due to the P(DMAA-co-MABP) polymer-specific Raman contribution. These Raman signal changes are well defined and allow for discrimination between the polymer and the paper. Following the measurement of the reference materials, two-dimensional Raman scans of P(DMAA-co-MABP) copolymer-treated lab-engineered paper sheets (cotton linters pulp) were 10


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collected to map the polymer distribution within the paper. In particular, the areas of overlapping cellulose fibers were analyzed because these fiber intersections increase the mechanical stability and thus the cohesion of the entire cellulose network (Figure 3). Filtering of the two-dimensional Raman scans for P(DMAA-co-MABP) copolymer and cellulose specific Raman bands enables the identification of the polymer domains in the fiber network. The data displayed in Figures 3(b) and 4 associate the Raman scattering intensity with a color-coded Raman map. The sample regions colored in yellow, cyan or magenta show strong Raman scattering, and areas with low intensity are displayed in black. The scans show that areas with an increased P(DMAA-co-MABP) Raman signal have low CH and CH2 (2852-2914 cm-1) Raman signals and vice versa. The intensity of the Raman signals within an image thereby depends on the intensity of the excitation laser, the change in polarizability of the exited molecules, the frequency of the excitation laser and the number of scattering molecules within the sample. Because the intensity and frequency of the laser were held constant and the orientation of the cellulose fibers was not changed during the measurements, the differences in the intensity of the Raman signals can be ascribed to the number of scattering molecules. Thus, in the case of the cellulose specific Raman maps, the intensity at the fiber crossing is high at locations where a high amount of cellulose is sampled. Accordingly, the low C-H Raman signals at the same fiber crossing result from the lower amounts of cellulose molecules, which are likely in holes and surface irregularities. A similar effect is observed in the polymer-specific Raman maps. Therefore, it appears that the P(DMAA-co-MABP) copolymer accumulates preferentially in holes, vacancies, and dips, filling these cellulose surface irregularities. Because the paper sheets are modified using a solution of photo-reactive copolymers, the data also suggest that evaporation of the fluid phase after the transfer to the fiber network leads to capillary transport of the polymer into those areas of the sheet. Thus, the polymer could block pores on the cellulose fiber surface, changing the wetting of the paper and the mechanical properties. Similar to other patterning agents such as the sizing reagent alkyl ketene dimer (AKD)

32

, P(DMAA-

co-MABP) chemically modifies the fiber surface and forms covalent bonds with the cellulosic fibers. 11


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The P(DMAA-co-MABP) is photo-linked to the cellulose by its photo-reactive BP functional group because the exposure of the functionalized paper to UV light (254 nm or 365 nm) leads to a π, π*- or an n, π*-transition within the carbonyl group of the BP and results in the formation of a reactive triplet species. This change is directly identified in the reference Raman spectra (Figure 2). Comparing the spectrum of the unexposed copolymer with the spectrum of the UV-light exposed copolymer paper, the complete disappearance of the BP specific stretching mode of the carbonyl group at approximately 1648 cm-1 is observed. This observation indicates the post-exposure transition of the BP carbonyl group. Subsequent to the formation of the reactive triplet species, the biradical species can abstract a proton (hydrogen) radical from a neighboring aliphatic C-H group, i.e., either from the cellulose or the backbone of an adjacent P(DMAA-co-MABP) polymer, leading to the formation of a new OH group at the BP and two remaining carbon radicals 17, 33, 34. The two resulting radicals eventually recombine and could create a covalent C-C bond between the polymer chain and the cellulose fiber surface (Figure 1). Note that, because this reaction is considered to be non-specific, the radicals formed at the BP group may also react with aliphatic groups of the copolymer itself forming a fiber-attached polymer network. As such, the illustration in Figure 1 resembles one of the possible cross-links in an idealized picture. The superposition image (Figure 4(c)) of the cellulose characteristic Raman intensity map between 2852 and 2914 cm-1 (Figure 4(a)) and the characteristic P(DMAA-co-MABP) copolymer Raman intensity map for the 2926 to 2987 cm-1 band (Figure 4(b)) clearly illustrate the spatial distribution of the polymer on the cellulose fiber network. A similar observation is obtained by filtering the two-dimensional scans for the cellulose specific symmetric and asymmetric C-O-C stretch modes between 1076 and 1134 cm-1 and comparing with the P(DMAA-co-MABP) characteristic C=C and phenyl ring C=C/C-C stretching modes between 1546 and 1697 cm-1 (Figure 4(d-f)). Nonetheless, minor differences exist in the intensity distribution between the two Raman spectral maps of the cellulose peaks at approximately 2897 cm-1 (Figure 4(a)) and 1095 cm-1 (Figure 4(d)). In particular, in the center of the figures, where the polymer crosses the cellulose fiber, the Raman intensity of the 12


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2897 cm-1 band in certain regions is considerably higher than the intensity of the 1095 cm-1 band. This result suggests that a rather faint (if any) reduction in the cellulose Raman signal is caused by the polymer layer. Because the sampling plane (focal depth) and thus the excited volume of the sample is the same for both images, the differences in the intensities of the characteristic cellulose Raman bands at approximately 2897 and 1095 cm-1 are mainly polarization dependent. The anisotropy in the Raman scattering is attributed to the polarization of the incident laser and the orientation of the scattering C-OC and the C-H/C-H2 groups in the cellulose backbone, as similar to other highly ordered fibrillar macromolecular arrangements, such as collagen

35

. The band intensity typically varies if the electrical

field is applied parallel or perpendicular to the molecular axis. Intensity differences in the Raman spectra thus imply that the preferential polarizability of the C-O-C molecular subunit is perpendicular to that of the C-H/C-H2 groups. In fact, Gierlinger et al. showed that the intensity of almost all significant cellulose bands changes depending on the orientation of the molecules in the plant cell wall cellulose backbone relative to the polarization of the excitation laser 29, 36, 37. Finally, the peak intensity at approximately 1095 cm-1 also depends on the crystalline and amorphous regions in the cellulose 38. However, because these regions are small compared with the diffraction limited laser spot size of approximately 800 nm in diameter, this effect has only a minor influence here and is thus neglected. In contrast to the cellulose Raman maps, the polymer intensity maps for different characteristic polymer bands (Figure 4(b) and (e)) are similar in distribution to one another and independent of the orientation of the sample. The polymer structures revealed are across and along the cellulose fibers and apparently attach to adjacent and overlapping cellulose fibers to form additional connections. This observation is in accordance with further investigations in which larger area polymer coatings were also found. As observed previously, these coatings occurred primarily at the cellulose fiber surface and at the interface between connecting fibers. The polymer covered part of the surface of the underlying fiber, particularly at the intersection region of the fibers. Such polymer accumulations at the cellulose fiber crossing points might originate from the offline process of polymer-modification of the paper 13


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sheets. The unmodified paper sheets are immersed into the polymer solution. The solution is driven into the cellulose network by capillary transport and the polymer adsorbs at the fiber surface. During the subsequent drying process, i.e. once the paper sheet is taken out of the polymer solution, capillary forces drive mass transport towards intersection areas (predominately fiber-crossings). Consequently, such polymer coating not only might result in the functionalization of the paper but also could improve the fiber-fiber interaction and enhance the mechanical stability of the network, particularly in the wet state. Hence, a considerable increase in the wet tensile strength for P(DMAA-co-MABP)–modified cellulose sheets was observed and reported recently

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. Paper sheets modified with cross-linked and

fiber-attached copolymers exhibited a remarkable increase in the wet tensile strength compared with that of unmodified lab sheets. The resulting wet strength, which was controlled and modulated by adjusting the molar composition of the copolymer, and the mass of the polymer relative to that of the dry fiber or the wavelength used for the purposes of cross-linking, were attributed to both homocrosslinking (i.e., the polymer cross-links with itself to form a polymer network surrounding the cellulose fibers) and to co-crosslinking (i.e., the polymer reacts with itself as well as with the present cellulose fibers). Co-crosslinking was considered as the dominant process because the photochemical reaction of the BP group is unspecific, and a much higher number of aliphatic C-H groups is available on the surface of the cellulose fibers than in the polymer 18. The Raman measurements presented in this work indicate the distribution of the polymer within the cellulose fiber network. However, due to the limited resolution of the system, the Raman measurements were not sufficiently sensitive to detect the subtle changes such as those between the polymer layer and the cellulose fiber-surface. The net variation in the 2900 cm-1 region caused by the mechanism of BP radical reactions with the cellulose C-H groups could not be unambiguously resolved. Methods with higher spectral and optical resolution such as tip enhanced Raman spectroscopy or super-resolved fluorescence techniques, could offer the required sensitivity. With these techniques, it should be possible to further clarify the question of whether co-crosslinking with the cellulose fibers or 14


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homo-crosslinking is the dominant process. However, by combining the chemically sensitive Raman spectral maps with scanned transmission light and confocal laser scanning microscopy images obtained from the same area of the paper fiber network, an in-depth distribution of the polymer along the fiber surface and further information on the origin of the crosslinking of the polymer-modified paper can be given (Figure 5, Figure 6). The correlation of information obtained from the two different imaging methods can be visualized by superimposing both images. Figure 5 shows the laser-scanned brightfield transmission images of the cellulose fiber network (Figure 5, gray images) overlaid with the total spectral intensity Raman maps of the same area (Figure 5, yellowish images). The overview image illustrates that the polymer spreads only slightly across the pores of the paper sheet and in particular between individual cellulose fibers, thus retaining the overall porous paper network, as also shown in other studies

17

.

Similar observations have been noted when traces of glue were detected on formerly bonded fiber surfaces 39. Additionally, the image overlays enable a vivid depiction of the fiber orientation, and as given in Figure 5(c) and (d), clearly display the distribution of the Raman specific cellulose and polymer bands. Again, the cellulose shows the orientation-dependent intensity variations due to the change in polarizability. The fiber oriented from top to the bottom features a more pronounced Raman signal (2852-2914 cm-1) of CH and CH2 stretch vibrations compared with that of the fiber arranged from left to right. Nonetheless, it must be mentioned that the scattering intensity is also affected by the depth position of the cellulose fiber. Because cellulose fibers in the network are not always in the same focal plane, the excitation intensity varies, and thus the Raman scattering intensity changes. Figure 5(d) indicates a moderate polymer distribution outside the fibers, with a higher concentration at the fiber crossings and no or only small amount of polymer found inside the cellulose. These results are consistent with Raman depth-scans and CLSM measurements 15


19

, which

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indicate that the polymer only partially surrounds the cellulose fibers, favors to attach to the fiber surface, and remains absent from the inside of the fibers (Figure 6 and Figure 7). Moreover, the most intense polymer signals were always found in proximity to fine cellulose fibrils that bridge the adjacent fibers at the connection points of fiber crossings. Hence, the polymer is preferably deposited at areas of high capillarity. This finding is clearly validated by the confocal fluorescence images of the polymer distribution on the cellulose network (Figure 6). Figure 6 confirms that (i) the polymer is deposited preferably on the fine fibrillar extensions protruding from the main cellulose fibers and (ii) that Calcofluor White does not stain the polymer. For larger cellulose fibers, the Rhodamine-B-labeled P(DMAA) shows a disconnected and reticulate distribution on the surface of the cellulose. Thus, the extensive polymer coating of the thin cellulose fibrils especially affects the overall properties of the functionalized paper sheet, e.g., the wetting properties or the paper fiber network stability, due to additional cross-links and connections.

Conclusion Owing to their tremendous design flexibility, polymers are essential for the functionalization of paperbased materials. Applications range from polymers used to tailor the wettability of paper to polymers that enhance the strength of this non-woven material, particularly in the wet state. The polymers used commonly feature molecule specific Raman fingerprints that allow the determination and mapping of the chemical additive within a paper sample, which fosters a fundamental understanding of the structure-property relationships of polymer-modified paper. Our investigations illustrate the application of a correlative microscopy approach using scanning Raman microscopy in combination with brightfield and confocal laser scanning microscopy to image the distribution of a hydrophilic, polydimethylacrylamide P(DMAA)-based copolymer in cellulose networks. Two-dimensional Raman scans at the intersections of overlapping cellulose fibers show that the polymer accumulates predominantly in holes, vacancies, and dips on the cellulose surface. At these positions, polymer fibrils 16


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and larger area polymer coatings are formed along and across the cellulose fibers. However, depth scans indicate that the polymer only partially surrounds the cellulose fibers and favors to attach to the fiber-crossings. Moreover, the superposition of Raman spectral images with the CLSM images directly allows identification of the overall distribution of the polymer in the paper fiber network. Although the CLSM images show cellulose fibers and fine fibrils of cellulose, the Raman data verify that the fine fibrils were primarily well coated with the polymer. Formation of such coatings with the polymer not only functionalizes the paper but also can increase the fiber-fiber interaction and enhance the mechanical wet-strength of the network

18

. These results show that high resolution confocal images

obtained with the CLSM complement the information obtained from Raman imaging. Together, our results offer a deeper insight into the effects of paper functionalization with polymers and contribute to an understanding of how the agents ultimately influence local and overall properties. The correlative analysis of polymer-modified paper is independent of the functional properties of the polymer and is therefore also suitable for extension to many different scientific questions related to “functional papers” as well as for optimization of processes used to functionalize paper sheets for various emerging applications such as microfluidic papers, electrically conductive papers, or papers used in biomedical applications.

Acknowledgements We thank Franz Carstens and Samuel Schabel from the Faculty of Paper Technology and Mechanical Process Engineering of the Technische Universität Darmstadt for providing cotton linters pulp as well as lab-engineered paper substrates. In addition, we gratefully acknowledge the Center of Smart Interfaces of the Technische Universität Darmstadt for financial support. MB acknowledges financial support from the Verband der Deutschen Papierindustrie (VDP) within the INFOR programme (MAP NV164) and from the Bundesministerium für Wirtschaft und Energie (BMWi) under AiF-IGF-grant No. 17919N “PhoreNast”. 17


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Supporting information Raman data analysis and image generation. The image generation from the Raman spectra is explained and the peak assignments in the Raman spectra of benzophenone, PDMAA, P(DMAA-co-MABP), and paper sheet samples are given. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figures

Figure 1. Proposed mechanism for the photo-crosslinking of (PDMAA-co-MABP) copolymers and the lignocellulosic network. Note that the depicted C-H insertion of the benzophenone (BP) into aliphatic groups of the lignocellulosic fibers resembles one of the possible reactions an excited BP group will undergo. Details of the photochemical reactions have been outlined elsewhere 17, 18.

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Figure 2. Raman spectra of pure BP (dark yellow, bottom), P(DMAA-co-MABP 5%) copolymer (red), pure PDMAA (orange), cellulose (black), and the superposition spectrum of cellulose functionalized with the P(DMAA-co-MABP) copolymer (blue, top). The gray box indicates the BP specific stretching mode of carbonyl groups at approximately 1648 cm-1.

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Figure 3. Brightfield image (a) and two-dimensional Raman scan (b) of a cellulose fiber intersection. The 30 µm x 30 µm black square in (a) indicates the area of the two-dimensional Raman scan in (b). The Raman scan displays the integrated spectral intensity distribution between 120-1700 cm-1 and 2700-3700 cm-1.

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Figure 4. Two-dimensional Raman scan (30 µm x 30 µm) of two overlapping cellulose fibers. Images show the integrated intensity maps for (a) the characteristic cellulose band (spectral range = 28522914 cm-1) colored in magenta, the characteristic P(DMAA-co-MABP) polymer Raman band (spectral range = 2926-2987 cm-1) (b) colored in cyan, and (c) the overlay image of both. Image (d) shows the intensity map (30 µm x 30 µm) of the cellulose-specific symmetric and asymmetric C-O-C stretch modes (spectral range = 1076-1134 cm-1) and (e) the P(DMAA-co-MABP) characteristic C=O and phenyl ring C-C stretch modes (spectral range = 1546-1697 cm-1). Image (f) is the superposition of (d) and (e).

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Figure 5. Brightfield microscopy image (a) of a cellulose fiber network obtained on a confocal setup superimposed with the Raman map (b) of the same area (60 µm x 60 µm). The superposition image (c) displays the Raman scan with the spectral intensity distribution for a characteristic cellulose band (at approximately 2897 cm-1, spectral range used for the filter = 2852-2914 cm-1), and the intensity map (d) for a characteristic polymer band (at approximately 2934 cm-1, spectral range = 2926-2987 cm-1) is shown.

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Figure 6. CLSM image (a) of a Calcofluor-White-labeled cellulose fiber network and a Raman spectral map (b) of the band intensity at approximately 2930 cm-1 (spectral range 2926-2987 cm-1) of the same area. The superposition image (c) displays the Raman map with the spectral intensity distribution for the characteristic polymer band at approximately 2934 cm-1 wavenumbers and the CLSM image of the cellulose fiber network. Thin polymer-coated cellulose fibrils crossing between the cellulose fibers can be clearly observed.

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Figure 7. Distribution of fluorescently labeled PDMAA on a cellulose fiber network imaged by confocal fluorescence microscopy. The images show the cellulose fibers stained by Calcofluor White (a), PDMAA labeled by Rhodamine B (b), an overlay of the latter (c), a brightfield image (d), an overlay of the PDMAA and brightfield images (e), and an overlay of all channels (f). The RhodamineB-labeled PDMAA shows a disconnected and reticulate distribution on the surface of the cellulose fibers. In particular, the polymer is preferably deposited at areas of high capillarity, i.e., in proximity to fine cellulose fibrils (arrow) and at the connection points of fiber crossings.

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Cross-Linking Cellulosic Fibers with Photoreactive Polymers

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