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different fluorescence channels, which recorded the scattering 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). Nile red is an environment-sensitive fluo- rophore 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 PEC particles, since the migration of Nile red into the PECs, and the solubility of Nile red in water, are both negligible effects. PEC samples were diluted 3–100 times with filtered distilled water prior to FCM analysis, depending on the concentration of particles in the sample. Methanol solution of Nile red (10 ppm) was added to stain the PEC particles, 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 popula- tions were gated using Partec FloMax software, in order to calculate the average scattering of particles in side and forward direction, as well as particle hydrophobicity (Va¨ha¨salo et al. 2003).
The amount of titrant needed for charge neutralization was used to calculate the total charge of the analyzed polyelectrolyte (Tables 1 and 2).
Formation of polyelectrolyte complexes
Different volumes of 0.1% polycation solution were pipetted into glass bottles. A fixed volume of 0.1% polyanion solution was pipetted into the polycation solution under vigorous magnetic stirring. To simplify and standardize the procedure, the polyanion was always added to the polycation. Polyelectrolyte com- plexes (PECs) started forming immediately when the polyanion came in contact with the polycation, which was evident as increasing opacity of the mixture upon visual inspection. The PEC mixtures were diluted to a total volume of 30 mL with distilled water within a few seconds of contact time, prior to analyses. All experiments were performed at room temperature.
Turbidity measurements
The initial turbidity of the PEC mixture was analyzed within 1 min after PEC formation using a turbimeter (Hach 2100AN IS, ISO 7027) equipped with a LED light source (870 ± 30 nm). In order to assess the long-term stability of the PECs, the mixture was stored in closed glass bottles at room temperature for 24 h before the turbidity was measured again using the same device. The mixtures were stirred for a short time prior to the turbidity measurements.
Results and discussions
Flow cytometry
Turbidity measurements
Flow cytometry (FCM) was used to analyze the PEC mixtures, using a Partec CyFlow Blue apparatus, equipped with a blue Argon laser (488 nm). The light scattered by the PEC particles were recorded in forward direction (FSC) and side direction (SSC, 90 ). The apparatus was also equipped with three
Solutions of polycations were mixed together with solutions of polyanions, forming nearly clear solutions or turbid suspensions depending on the cation/anion ratio (Fig. 1). The increase in turbidity was caused by
Table 1 Properties of the polycations used in the experimental series
Charge density [ l eq/g]
Polycation
Molar mass [kDa]
Source
Raifix 01015 SW
? 831
Low
Waxy maize
Raisabond 15
? 882
Medium
Potato
Raifix 25035
? 1870
Medium
Potato
PolyDADMAC
? 3978
100–200
–
PolyDADMAC
? 4949
200–350
–
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