PMMA particles

PMMA particles of three different types were used within this work. From previous experiments a re-maining stock was available with particles synthesized by A. Schofield at the University of Edinburgh, which were labelled with Fluorescein (sample names start with FL). Preliminary tests and some later measurements were done with those. More particles were received from M. K. Klein, who synthesized particles in the collaborating chemistry group of Prof. Zumbusch [85]. Particularly their particles with a near-infrared (NIR) fluorophore had very useful properties (sample names tart with NIR). They did not suffer from bleaching even after days of non-stop measurements. After a statement in [67], that fluores-cent dyes might be the reason for electrostatic repulsion, attempts were made to load undyed particles synthesized by M. K. Klein with the maximum possible amount of Rhodamine 6G, in the hope to obtain particles with more electrical charges. This dye is widely used in fluorescence microscopy (sample names starting with RP). While the rhodamine dye was simply adsorbed in the polymer network of the particles, NIR and fluorescine dyes were covalently linked already during the particle synthesis. Table5.1gives an overview of the PMMA particles that were finally used in the experiments presented here.

sample diameter diameter hydrodynamic poly- dye producer

dry microscope diameter dispersity

FL13 1.7µm 1.3µm 1.9µm 6 % fluorescine Schofield

FL2 2.2µm 2.1µm 2.3µm 13 % fluorescine Schofield

FL4 2.4µm 2.2µm 2.6µm 7 % fluorescine Schofield

NI2 1.0µm 0.9µm 1.1µm 7 % NIR Klein

NI4 2.0µm 1.8µm 2.1µm 6 % NIR Klein

RP1 1.1µm 1.0µm n.a. 6 % rhodamine Klein

RP44 2.6µm 2.5µm 2.7µm 5 % rhodamine Klein

RP45 1.8µm 1.7µm 1.9µm 8 % rhodamine Klein

Table 5.1:Particles used in the confocal microscopy experiments

5.1 Sample preparation Size determination

The diameters of dried particles were obtained with a standard scanning electron microscope (SEM).

For the determination of “microscope” diameters and polydispersity the particle detection algorithm de-scribed in Chapter3was used to obtain the visible diameters from confocal microscopy images up to an error of a few percent. They are usually somewhat smaller than the dry diameters since the outer shell of the particles is usually not that bright in the images. This is partly because some of the fluorescent dye is washed away in the particle production and partly because the intensity emitted from the center is higher.

The reason is that for the voxel at the center of a given particle, a larger fraction of the volume of that particle is in the focus of the microscope. About the determined polydispersity values one should men-tion that the results here are only upper limits. The particle sizing with the detecmen-tion algorithm already introduces an error of about1.5%for big particles (diameter of∼10px and more) and about5% for small particles (4-5px) (see Fig.3.19and the text below). The hydrodynamic diameter was obtained from dilute samples (below0.1 volume fraction). This was done either with dynamic light scattering (DLS) by measuring intensity correlation functions, or by computing the mean square displacements (MSDs) directly from particle trajectories obtained with confocal microscopy. Both techniques only yield the diffusion constant𝐷and one has to use the Stokes-Einstein relation

𝐷= 𝑘_𝐵𝑇

6𝜋𝜂𝑅, (5.1)

with temperature𝑇, Boltzmann’s constant𝑘_𝐵, dynamic viscosity of the solvent𝜂and particle radius𝑅. The viscosity is given by𝜂= 2.2mPa⋅s at𝑇 = 22^◦C, as obtained from [86] for a CHB-decaline mixture of85 %∕15 %by weight, which is close to the ratio in density matching mixtures for these particles. An error of about5 − 10 %has to be accepted for these measurements. Note that both, particle sizes and viscosity do change with temperature. For instance𝜂changes by more than1 %per degree Celsius.

Incorporation of rhodamine

For the incorporation of rhodamine in PMMA particles the procedure used here is similar to the one described in [67] and [77]. A powder of dry PMMA-PHSA particles is dispersed in dodecane, at a particle volume fraction of10 %. Additionally, a colouring solution with0.93mg∕ml of rhodamine perchlorate in a mixture of 25 vol.% acetone and 75 vol.% cyclohexanone is prepared by stirring and ultrasound sonication (∼ 10min). Afterwards this rhodamine solution is filtered by means of an0.2µm cellulose filter. An amount of1ml of the particle suspension is heated to40^◦C and, during continuous stirring with an agitator,0.5ml of the rhodamine solution is slowly poured into the suspension. PMMA particles swell when they are dispersed in a solvent that contains acetone, which allows the dye to diffuse slowly into the particles. After 10 minutes stirring at40^◦C one can observe a colour change of the sample from purple to a lighter red. The particles are then immediately transferred to decaline, in order to avoid the destruction of the PHSA stabilizer in acetone. Decaline is added to the suspension, and the sample is centrifuged at a moderate speed of950rpm (∼ 100𝑔) for 5 minutes. Afterwards the solvent on top of the sedimented particles is removed. This washing step is repeated 5 times (increasing to1100rpm resp.∼ 130𝑔) in order to obtain particles in almost pure decaline. The parameters used in this colouring procedure are optimized to load the particle with the highest possible amount of the dye. More rhodamine results in the production of small rhodamine crystallites in the sample. A considerably longer exposure to the rhodamine solution destroys the particles. E.g. after 20 minutes pairs and triplets of merged particles were observed. With a too short exposure (only 5 min) the dye does not diffuse into the center of the particles so that the particles appear as rings in the confocal microscope.

Regrafting the stabilizer

While the PHSA stabilizer is covalently bound to the particle surface for NIR and fluorescine particles, this is not the case for the rhodamine particles that were used here. After colouring, many aggregates and smaller clusters were observed in the sample, indicating that the stabilizer was partly washed away.

In order to regraft the stabilizer a mixture of 1% PHSA, 66% ethyl acetate and 33% butyl acetate (by weight) is prepared and diluted by further adding 4g of decaline to 1g of the mixture. The particles are transferred to decaline by doing 5 washing steps (same processing as after the colouring). In the last washing step, instead of decaline, 5g of the prepared PHSA solution is added to approximately 300mg PMMA particles. After 15 min of sonication (80% Elmasonic P) the particles are again washed with decaline to remove the remaining PHSA and the acetates.

Storage, washing and drying of particles

For storage the particles are usually dispersed in decaline (Sigma-Aldrich 98 %mixture of cis+trans), where they are stable for years. Washing with decaline to get rid of other solvents (e.g. from the colouring procedure) can be done by centrifuging the sample for 5-10 minutes at a moderate speed of 1000 up to 1500 rpm (∼100 to 200𝑔) and replacing the supernatant solvent. Another good solvent for washing is dodecane. With its lower viscosity and lower density (0.75g∕ml compared to 0.88g∕ml for decaline) most of the particles with sizes of between1 and2µm sediment to the bottom of the sample vial within 10 minutes, without centrifugation. One can use this relatively slow sedimentation to remove smaller particles and dirt from the sample, by carefully withdrawing the supernatant with a Pasteur pipette. Before drying particles several washing cycles with dodecane (Alfa Aesar 99+%) were applied for an additional cleaning. At room temperature the vapour pressure of dodecane is more than ten times higher compared to that of decaline. This allows one to dry the particles by exposing the wet sediment in a sample vial (usually 0.5ml of the suspension in a 4ml vial) to a constant nitrogen flow for 30-60 minutes. Dry particles should be stored in a desiccator protecting them from humidity. This is important since water in the sample decreases the screening length for electrical charges and thus the repulsion between colloids becomes weaker.

Origin of particle charges

Since there are no functional groups at the surface of PMMA-PHSA particles that could dissociate, it is not clear yet why they exhibit an electric charge when dispersed in CHB. Leunissen [87] found no signs for electric charges in apolar solvents like cis-decalin or cyclohexane. They argue that the charges are a result of a very dilute concentration of decomposed CHB leading to a small concentration of H⁺ and Br⁻ ions. The protons would associate with the slightly polar carboxyl groups of the PHSA stabilizer and lead to an effective positive charge. They mention further that the protons could also associate to polar groups of the PMMA polymer network in the particle cores. Upon adding a certain amount of the salt TBAC (tetrabutylammonium chloride) they could even observe a charge reversal, which could be explained with Cl⁻ ions being adsorbed on the particle surfaces that first neutralize and then overrule the positive charges. Unlike Dinsmore et al. [67], Leunissen [87] did not find the charge to be dependent on whether the particles were dyed or not. As mentioned above, attempts were made within this work to load particles with the highest possible amount of rhodamine. However, the repulsion of the particles did not become stronger with the use of higher concentrations of rhodamine in the colouring process.

Micro-electrophoresis, estimation of the charge number

In order to estimate the number of charges on the PMMA particles sample cells (similar to the sandwich cells shown below,5.1) were prepared with two platinum wires as electrodes, so that one could observe

5.1 Sample preparation them in the confocal microscope under the influence of an electric field. These measurements were done with the help of Markus Endermann, a student assistant. Particles were dispersed in the typical CHB/decaline mixture but at very low volume fractionsΦ<0.005. During the observation of the sample in the microscope, an electric voltage of30V was applied to the two electrodes that were separated by a distance of𝑑= 2mm. Every5s the electric field was reversed in order to keep the particles in the field of view. A main problem, which might be due to the simple design of the cell with platinum wires, was that in some of our cells the electric field produced disturbing fluid currents that lead to a chaotic motion of the particles. A solution might be to separate the electrodes by a larger distance, but this requires higher voltages since the electric field decreases with1∕𝑑.

It was confirmed that regardless of the type of dye the particles had a positive charge. Even a mixture of rhodamine and fluorescine particles showed only positive charges although particles of different dyes attracted each other. Only for one particle batch (FL2) quantitative measurements were performed. Al-most perfectly uniform back-and-forth motion in the alternating field was observed in the corresponding sample cell. Using the particle detection and tracking methods (chapters3,4) the average absolute ve-locity of about 200 particles was determined to be𝑣= 3µm∕s. With a dielectric constant of𝜖_𝑟 = 7for the CHB/decaline mixture the electrophoretic mobility can be estimated as𝜇_𝑒 =𝑣∕𝐸= (𝑣⋅𝑑⋅𝜖_𝑟)∕𝑈 ≃ 1.4 × 10⁻⁹m²V⁻¹s⁻¹. This is a value comparable to those for other PMMA particles of similar sizes in the literature [87]. Looking at the distribution of the mean values of𝜇_𝑒 for different particles a relative standard deviation of19 %is obtained for that sample. This is not directly the charge polydispersity since 𝜇_𝑒 still depends on𝑅, but it can be interpreted as a hint that the charge polydispersity is usually higher than the size polydispersity (13 %for FL2).

Using the balance between friction and electrostatic forces one obtains a formula for the number of charges𝑍_ep:

𝑍_ep𝑒 𝐸= 6𝜋𝜂𝑅𝑣 ⇒ 𝑍_ep=𝜇_𝑒6𝜋𝜂𝑅

𝑒 , (5.2)

with the elementary charge𝑒, the particle radius 𝑅 = 2.2µm and the viscosity 𝜂 = 2.2mPa⋅s. With the electrophoretic mobility𝜇_𝑒from above one obtains a number of𝑍_ep ≃ 350elementary charges. It is clear that this is only a rough estimate since the conditions for a measurement in a dilute suspension with an applied electric field are totally different to the situation in a dense suspension without electric field. As discussed later in this chapter, it was observed that these charged systems are very sensitive.

Small changes in the system can change the repulsive forces dramatically. For instance the unintentional additions of ions via dirt, or a solvent that was exposed to air for a too long time. With these considerations in mind, there was no need for doing more electrophoretic measurements. They cannot yield precise information about the effective charge in a dense sample.