Characterisation of the Core-Shell Microgels

6.3.1 Dynamic Light Scattering (DLS)

The hydrodynamic radius of the microgel particles was determined using dynamic light scattering.

DLS measures fluctuations of the scattering intensity of highly diluted samples. The intensity fluctuations are related to the Brownian motion of the particles since the interference of the scattered light waves depends on the positions of the particles relative to each other. In thermal equilibrium, smaller particles show higher rate constants of diffusion then larger ones. By analysing the field autocorrelation function and the relaxation rate, respectively, the diffusion coefficient of the particles is obtained. [307] For analysing the time-dependent intensity fluctuations, high sensitive detection systems, such as photomultipliers, are required to resolve the incoming intensity at short times.

The DLS measurements were performed using either a Peters-ALV goniometer setup or a Zetasizer Nano ZS instrument (ZEN 3500, Malvern Instruments, Herrenberg, Germany). Measurements performed with both instruments show that these instruments are equally good to analyse the size of spherical microgel particles of narrow size distribution. The Peters-ALV instrument is equipped with a 50 mV He-Ne laser (λ = 632.8 nm), focusing lenses, a measuring cell, and an ALV-500 correlator and is suitable for measuring samples having sizes in the range between 1 nm and 1 µm. The temperature of the samples was adjusted using a tempered bath containing toluene as index matching substance.

All experiments with this apparatus were done at a scattering angle of 90°. The Zetasizer Nano ZS instrument consists of a 532 nm laser and can be used for the analysis of particle sizes ranging from 0.6 nm and 6 µm. In this case the scattering experiments were performed at a scattering angle of 173°.

For the analysis of the microgels dispersed in protein free buffer solution, the microgels were diluted to a concentration of 5x10^-2 g L^-1 using the respective buffer solution. Perturbation of the measurement by the presence of dust particles was avoided by filtering the buffer solution through a syringe filter (0.2 µm pore width, polyethersulfone, VWR). To study the influence of protein adsorption on the microgel size, a microgel dispersion of 0.1 wt-% was mixed with the respective protein solution to a given molar ratio n(protein)/n(microgel) and incubated for 30 min. Afterwards the microgel/protein mixture was diluted to the concentration of 5x10^-2 g L^-1 required for the DLS analysis. The DLS experiments were executed at temperatures between 283 and 333 K. Before running the experiment, the microgel dispersion was incubated at the desired temperature for 20 min to attain thermal equilibration. For each sample and temperature, a set of 3 measurements was performed each consisting of 10 single runs. The measured intensity correlation functions were analysed using the methods of cumulants which provides the z-averaged translational diffusion coefficient <D>. [308]

The hydrodynamic radius is obtained from the diffusion coefficient using the Stokes-Einstein equation which reads: [81]

〈𝐷𝐷〉= 𝑘𝑘B𝑅𝑅

6𝜋𝜋𝜋𝜋𝑅𝑅_h (6.2)

where kB is the Boltzmann constant, T is the absolute temperature, and η is the viscosity of the solution.

6.3.2 ζ-Potential Measurements

The electrophoretic mobility µE of the microgels was determined using a Zetasizer Nano ZS instrument (ZEN 3500, Malvern Instruments, Herrenberg, Germany). The measurements were performed under a scattering angle of 17° at 298 K with folded capillary cells (Malvern Instruments).

Before using the cells for the experiment, the inner part of the cells was carefully rinsed with ethanol, water, and subsequently with the buffer solution, which was used to dilute the microgel particles, several times. For the sample preparation, the concentration of the microgel particles was adjusted to a concentration of 0.001 wt-% by immersing the microgel into the respective buffer solution. The microgel dispersion and the cells were allowed to equilibrate to the required temperature for 10 minutes. For each sample a series of 5 measurements was performed each consisting of 20-25 single runs. Therein, the determination of the electrophoretic mobility by this apparatus is based on the combination of the laser doppler velocimetry method and phase analysis light scattering.

The ζ-potential of spherical particles is directly correlated to µE via the Henry equation which reads:

𝜇𝜇_E=𝜈𝜈

𝐸𝐸=2𝜀𝜀r𝜀𝜀0𝜁𝜁

3𝜋𝜋 𝑓𝑓¹(𝜅𝜅𝑎𝑎) (6.3)

where υ is the particle velocity in presence of the electric field E, εr and ε0 is the dielectric constant of the solvent and vacuum, respectively, η is the viscosity of the solution, and f1(κa) is the Henry formula with κ as reciprocal Debye screening length, and a as particle radius. The Henry formula depends on the ionic strength of the solution as well as on the particle radius and is expressed by the following equation:

𝑓𝑓₁(𝜅𝜅𝑎𝑎) = 1 +�2 +�1 + 2.5��1 + 2exp(−𝜅𝜅𝑎𝑎)�𝜅𝜅𝑎𝑎�⁻¹�³�

−1 (6.4)

For systems which are described by κa >>1 the Smoluchowski approximation can be used which sets the Henry formula to 1.5. Although this approximation is valid for the microgel systems, the ζ-potential of the microgels studied was calculated using the exact definition given by equation (6.4) with a set to Rh.

6.3.3 Conductometric and Potentiometric Titration

Simultaneous conductometric and potentiometric measurements were performed using the 809 Titrando system (Metrohm Ion analysis, Herisau, Switzerland) equipped with an external dosing device (807 Dosing Unit, Metrohm Ion analysis), a stirrer (801 Stirrer, Metrohm Ion analysis), pH electrode (pH 0-14, Metrohm Ion analysis), and a conductivity module (856 Conductivity Module, Metrohm Ion analysis). The Titrando system was controlled by the tiamo 2.0 software (Metrohm Ion analysis). For the titration experiment, the microgel dispersion was diluted with water to a total concentration of 0.5 wt-%. in case of CSM-10 and of 1 wt-% in case of CSM-0-2. Then, 20 mL of the microgel suspension were titrated with 0.01 M NaOH. The titrations were run in a thoroughly cleaned 50 mL beaker fitted with the pH and the conductivity electrode. The NaOH solution was slowly added to the microgel dispersion (0.1 mL min^-1 for the titration of CSM-10 and 0.05 mL min^-1 for the titration

of CSM-0-2), because the relaxation time required for attaining equilibrium between the aqueous and gel phase is considerably longer than for the corresponding low molecular weight acid. [309-310]

The potentiometric and conductometric titration curve of CSM-10 are shown in Figure 4.3 a. From the equivalent point of both titration curves the amount of acrylic acid polymerised into the microgel particles is determined. Additionally, the dissociation degree αdiss of the carboxylic acid functional groups can be calculated as a function of the amount of added NaOH and, thus, as a function of the pH according to [309]

𝛼𝛼diss=[𝑉𝑉]_pH

[𝑉𝑉]_eq (6.5)

where [V]pH and [V]eq is the volume of added NaOH at a given pH value and at the equivalent point, respectively. To calculate the apparent pKa value of the carboxylic acid functional groups as a function of the dissociation degree, the following equation is used: [310]

𝑝𝑝𝐾𝐾_a=𝑝𝑝𝐻𝐻 −log� 𝛼𝛼_diss

1− 𝛼𝛼_diss� (6.6)

6.3.4 Electron Microscopy

6.3.4.1 Field Emission Scanning Electron Microscopy (FE-SEM)

The FE-SEM micrographs were acquired using a LEO gemini microscope (LEO 1530, Zeiss, Germany) equipped with a field emission cathode. The electron high tension was set to 3 keV. The PS core latex was diluted to a concentration of 0.01 wt-% and put on a cleaned silica wafer. After drying at room temperature the silica wafer was fixed by using an aluminium foil. In order to provide for electric conductivity a platinum layer of 0.9 nm thickness was sputtered onto the samples before the measurement using the Cressington Sputter Coater208HR.

6.3.4.2 Cryogenic Transmission Electron Microscopy (Cryo-TEM)

Samples for Cryo-TEM were prepared by placing a small drop of the microgel dispersion with a concentration of 0.2-wt-% onto a TEM copper grid (Agar, G 600HH, Polysciences, Eppelheim, Germany). Excess of the microgel solution was removed by blotting with filter paper. The samples were kept at room temperature and rapidly vitrified in liquid ethane at its freezing point in a cryo-box.

The samples were inserted into a cryo-transfer holder (CT3500, Gatan, Munich, Germany) and transferred to a Zeiss EM922EFTEM (Zeiss NTS GmbH, Oberkochen, Germany). Examination was carried out at temperatures around 90 K. The Cryo-TEM was operated at an acceleration voltage of 200 kV. The images were recorded digitally by a bottom-mounted CCD camera system (Ultra Scan 1000, Gatan, Munich, Germany) and processed with a digital imaging processing system (Digital Micrograph 3.9 for GMS 1.4, Gatan, Munich, Germany).