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Cellulose (2019) 26:959–970
Table 1 The determined amount of different sugar units in the three different kraft pulps after acid methanolysis (uncertainty ± 5%)
Polysaccharide film Eucalyptus kraft (mg/g) Birch kraft (mg/g) Softwood kraft (mg/g)
Arabinose
0.2
0.6
7.8
Galactose
5.1
3.0
8.3
Glucose
102.5
94.0
120.2
Mannose
1.2
8.2
47.2
Rhamnose
0.6
0.3
0.1
Xylose
135.1
173.9
68.6
Galacturonic acid
0.2
0.3
1.1
Glucuronic acid
0.8
0.8
0.5
4-O–Me-glucuronic acid 7.6
7.4
2.5
Sum
253.2
288.6
256.3
intensity in the green (512–542 nm), orange (575–605 nm), and red (615–645 nm) spectra. Fil- tered distilled water was used as sheath fluid in the analyses. Nile red (Tamro, Vantaa, Finland) was used as a fluorescent dye to stain the particles prior to FCM analysis, as in previously published studies (Va¨ha¨salo et al. 2003; Va¨ha¨salo and Holmbom 2005; Strand et al. 2013, 2018). Nile red is an environment-sensitive fluorophore that exhibits a blue-shift proportional to the hydrophobicity of its environment (Greenspan and Fowler 1985). It was expected that the added nile red mainly stained the surface of the CNCs, since the solubility of nile red in water is negligible. The CNC suspensions were diluted 4 9 or 15 9 with filtered distilled water prior to FCM analysis, depending on the concentration of detectable particles in the sample. 20 l L of methanol solution of nile red (10 ppm) was added to stain the CNCs, and the fluorescence intensity in the red spectrum was used as an indicator of particle hydrophobicity. FCM analysis was used to count the number of particles in the sample, and group the particles together as populations based on their recorded light scattering properties. Particle populations were gated using Partec FloMax software, in order to calculate the average scattering of particles in side and forward directions, as well as particle hydrophobicity (Va¨ha¨salo et al. 2003).
The diluted suspension was concentrated by centrifu- gation at 3400 g for 15 min. The supernatants were removed using a suction flask. The bottom phases were collected, diluted with distilled water, and again centrifuged at 3400 g for 30 min. The supernatants were again removed in order to concentrate the suspension. The collected cellulose fraction was diluted to 1 L with distilled water for washing. The suspension was again centrifuged at 3400 g for 30 min, and the supernatant was removed. The washing procedure was repeated one additional time. The collected cellulose fraction was dialyzed against distilled water for 4–5 days in a dialysis membrane with 12–14 kDa cut-off (Medicell International, UK). The dialysis water was replaced daily until the pH of the dialysis water remained unchanged over the course of 1 day. After dialysis, the CNC suspension was homoge- nized at 20,000 rpm using a Polytron PT3000 (GWB) for 5 min, and with ultrasonic impulses for 2 9 10 min. The CNC suspension was allowed to sediment, in order let large residual fiber fragments sink to the bottom of the flask. Only the non- sedimenting particles in the supernatant was used for further analyses.
Flow cytometry (FCM)
Analysis of extractives
Flow cytometry (FCM) was used to analyze the CNC suspensions, using a Partec CyFlow Blue apparatus, equipped with a blue Argon laser (488 nm). The light scattered by the particles were recorded in forward direction (FSC) and side direction (SSC, 90 ). The apparatus was also equipped with three different fluorescence channels, which recorded the scattering
Grinding of the different kraft pulps was performed with a laboratory-sized mill (IKA MF 10). The kraft pulp powders were freeze-dried for a few days. Roughly 4 o.d. g of the different kraft pulp powders were packed into stainless steel extraction cells
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