Centrifugation and Cloud Point Extraction

Centrifugation and particularly ultracentrifugation is a common approach to separate or pre-concentrate nanomaterials in water samples. Generally, centrifugation techniques use enhanced sedimentation to separate solid particles in a suspension according to particle size and density. Particles of higher density or larger size settle at a faster rate and are thus separated from particles with lower density and smaller size. The settling velocity is determined by the size, shape, by density and viscosity of the surrounding media.

The displacement of nanomaterials in water is mostly influenced by the Brownian motion of water molecules which typically overcomes the gravitational force. Ultracentrifugation is, therefore, employed which enhances the gravitational force by up to 1000 000 times. Care should be taken, as prolonged ultracentrifugation may press the settling solids into a pellet that would be difficult to re-disperse. On the other hand, a significant loss of nanomaterials to the supernatant can occur when insufficient centrifugation force is applied, resulting in a low yield of nanomaterial separation. Ultracentrifugation can be classified into two subgroups: analytical and preparative ultracentrifugation. Analytical ultracentrifugation incorporates an optical detection system (UV/Vis) for real-time monitoring of the physical properties of the sedimenting particles. Preparative ultracentrifugation is used to separate particles based on estimations of their densities and subsequent analysis.

Density gradient centrifugation (ultracentrifugation) allows separation of many components of the sample. It was originally developed for the separation of cellular organelles. There are two forms of density gradient centrifugation. Isopycnic centrifugation, where a density gradient is created by gently overlaying lower concentrations of, e.g. sucrose on higher concentrations in a centrifuge tube from 10% to about 70% sucrose, then sample is placed on the top of the gradient and centrifuged. Particles travel through the gradient until they reach the point in the gradient in which their density matches that of surrounding sucrose (isopycnic point). Generally, isopycnic separation in a density gradient is not possible for nanomaterials with densities higher than 3.1 g cm^-3 (sodium polytungstate), a value much lower than the density of most inorganic nanomaterials such as Au (19.1 g cm^-3) and Ag (10.5 g cm^-3).

Rate-zonal centrifugation involves layering of the sample on top of a preformed liquid density gradient, with density continuously increases toward the bottom of the sample. Under centrifugal forces, the particles will sediment through the gradient column in separate zones, each zone consisting of particles characterized by their size and sedimentation rate. To achieve a rate-zonal separation, the density of the sample particles must be greater than the density of the gradient column at any position along the column which the particles must travel. The run must be terminated before any of the separated zones reach the bottom of the centrifuge tube.

Cloud point extraction (CPE) is a technique for pre-concentrating nanomaterials by amending the aqueous medium with a surfactant (e.g. Triton X-114) at a concentration that exceeds the critical micelle concentration, followed by heating to a certain temperature called cloud point temperature. Above that temperature, the surfactant separates from the aqueous phase and forms a surfactant phase. The CPE procedure consists of three steps (Figure 4): first, addition of the surfactant to the sample, then increase of the temperature to the cloud point, and finally a phase separation which is accomplished by centrifugation.

Figure 4. Scheme of the cloud point extraction for separation and enrichment of nanomaterials

Nanomaterials are thus concentrated into the surfactant-rich phase and can be further analyzed. CPE has been successfully applied to separate and concentrate TiO₂ and SiO₂ nanoparticles from natural waters prior to TEM analysis. The method was also used to enrich AgNPs from wastewater. However, the specificity of CPE toward certain nanomaterials and its advantages compared to filtration methods and regular centrifugation remain under discussion.