In addition to natural cellulosic I rich fibres such as cotton, man-made lyocell fibres were also examined. Lyocell is a cellulose II rich fibre made from reconstituted cellulose from eucalyptus wood pulp. Lyocell fibres are made up of highly ordered linear chains of β-(1-4)-glucan polymers and as a result the degree of crystallinity is very high (≤80%). These high degrees of crystallinity are generated during the manufacturing process, in which the extraction and stretching of the reconstituted fibres cause the microfibrils to more likely orientate in a parallel fashion. One characteristic of lyocell fibres is the microfibrillar structures where a portion of the cellulose chains aggregate to form micro-crystals, while the remainder of the chains exist in an amorphous phase. Like natural cellulose fibres, such as cotton, alkali treatments have a significant impact on the supramolecular, molecular and morphological properties of the cellulose II polymers. This translates as changes in the properties of crystallinity, microfibre orientation, lyocell pore structure and accessibility (Crawshaw and Cameron, 2000; Široký et al., 2009). As mentioned previously, X-ray diffraction and ATR- FTIR only give overall measurements of the changes in a large sample size. Carbohydrate-binding modules can effectively define changes seen in crystalline and amorphous cellulose at a spatial context (Blake et al., 2006; Kljun et al., 2011).
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Figure 5.6: Indirect immunofluorescence detection of amorphous and crystalline cellulose carbohydrate-binding modules labelling transverse-sections (thickness of 0.5 µm) of resin-embedded lyocell fibres to highlight the different proposed areas through the fibre.
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CBMs were used on resin-embedded cross sections of untreated lyocell fibres in Figure 5.6. From the CBM labelling, it can be seen that the lyocell semi- permeable fibre skin is present through the very strong binding of all CBMs at the outer surface layer. The presence of a ‗skin‘, approximately 100 nm in thickness and composed of mostly amorphous cellulose II, is discussed in Bredereck 2000 (Bredereck and Hermanutz, 2005). In Biganska (2002), there are three proposed regions of cellulose in lyocell fibres. However, using CBMs only two regions (outer skin and inside the fibres) were visibly detected. Additionally, CBM17 binds very well to the inside areas of the untreated lyocell fibres, where high levels of amorphous cellulose are expected. In contrast, CBM28 binds to most of the transverse section with the exception of a segment area in which the binding of CBM28 is very weak. This may support the hypothesis of the dominance of detection of amorphous, non-ordered regions over crystalline regions with the lyocell fibre and its skin-core differences (Biganska et al., 2002; Široký et al., 2012).
CBM3a and CBM28 (Figure 5.7 and Figure 5.8) are shown binding to crystalline and amorphous cellulose after varying treatment conditions of NaOH, temperature and fabric tension. Of all the regenerated cellulosic fibres, lyocell is known to have high degrees of crystallinity. This can be seen with the stronger levels of binding of CBM3a, which binds to crystalline cellulose, compared to CBM28, which is much weaker. Further work carried out by Široký (2012), showed that in addition to NaOH, temperature and fabric tension also have an effect on the crystalline and amorphous supramolecular structures of the lyocell cellulose skin (Široký et al., 2012). For CBM3a (Figure 5.7), most crystalline cellulose was detected under the conditions: 3.33 mol dm-3 NaOH, 49 Nm-1 fabric tension at
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25°C and 4.48 mol dm-3 NaOH, 147 Nm-1 fabric tension at 40°C (Lyocell tension, temperature and alkali treatments varied out by Dr Jan Široký, The University of Leeds). In the case of CBM28 binding (Figure 5.8) there were significant variations seen, which can potentially indicate that amorphous regions of cellulose are more sensitive and dependent upon thermal, mechanical and chemical treatments when compared to crystalline regions. These results from CBM28 on lyocell can be linked to the data shown in Boraston (2003), in which chains of amorphous or ‗shapeless‘ cellulose are predicted to exist in at least two or more physical substructures. As a result, CBM28 may have strong affinity or perhaps discrimination for these different physical forms of amorphous cellulose (Boraston et al., 2003).
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Figure 5.7: In situ fluorescence analysis of CBM3a labelling to lyocell fibres in response to NaOH under varying temperatures (25°C and 40°C) and fabric tensions (49 Nm-1 and 147 Nm-1). Samples provided by Dr Jan Široký (Široký et al., 2012). Scale: 10 µm.
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Figure 5.8: In situ fluorescence analysis of CBM28 labelling to lyocell fibres in response to NaOH under varying temperatures (25°C and 40°C) and fabric tensions (49 Nm-1 and 147 Nm-1). Samples provided by Dr Jan Široký (Široký et al., 2012). Scale: 10 µm.
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After quantifying the fluorescence in CBM3a and CBM28, Široký et al. (2012) compared the values with ATR-FTIR LOI data (Široký et al., 2009, 2012). Generally the image analysis of CBM3a binding correlates closely with the ATR- FTIR LOI and HBI readings (Figure 5.9) respectively. Tension of the fabric under 49 Nm-1 had a stronger effect on the supramolecular crystallinity than 147 Nm-1, while the effects of temperature were seen to be more pronounced on amorphous cellulose than crystalline. With CBM28 for amorphous cellulose, the binding also generally follows in that as HBI decreases, so does the intensity of the immunofluorescence (Figure 5.10).
Figure 5.9: Relative intensity of CBM3a fluorescence with lateral order index readings (Široký et al., 2009) with increasing NaOH concentrations under
varying tension and temperatures (49-147 N m-1 and 25-40°C respectively). (a) 49 N m-1/25°C; (b) 49 N m-1/40°C; (c) 147 N m-1/25°C; (d) 147 N m-1/40°C. Figure provided by Dr Jan Široký (Široký et al., 2012).
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Figure 5.10: Relative intensity of CBM28 fluorescence compared with hydrogen bond intensity readings (Široký et al., 2009) with increasing NaOH concentrations, under an applied tension strength of 147 N m-1 at 25°C. Figure provided by Dr Jan Široký (Široký et al., 2012).
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5.5 Preliminary studies of fibre cell wall crystallinity and