Ultracentrifugation is a specialized technique used to spin samples at exceptionally high speeds. Current ultracentrifuges can spin to as much as 150 000 rotations per minute (rpm) (equivalent to 1 000 000 g) (Biocompare, 2019b). However, extreme centrifugal forces may cause overheating, so to avoid sample damage, ultracentrifuges are equipped with vacuum systems that keep a constant temperature in the centrifuge’s rotor (Biocompare, 2019b).
Centrifugation, and ultracentrifugation, is nowadays, at the core of the laboratory routine. Benchtop centrifuges are essential devices in any biology or chemistry laboratory, and they are used on a day-to-day basis in a wide range of experimental protocols, from concentrating solutions to isolating cells and subcellular components. Ultracentrifugation widened the applications of benchtop centrifugation, allowing the isolation of smaller sized particles, and the study of purified molecules and molecular complexes (Ohlendieck & Harding, 2017). In biology, the development of ultracentrifugation in the early 1900s, widened the possibilities of scientific research to the subcellular level, allowing for the differential separation of cellular components, such as organelles, lipid membranes, and even to purify proteins and ribonucleic acids (DNA and RNA).
The basis of ultracentrifugation is the same as normal centrifugation: to separate the components of a solution based on their size and density, and the density (viscosity) of the medium (solvent) (Ohlendieck & Harding, 2017). As a general principle, (ultra)centrifugation abides by the following rules (Ohlendieck & Harding, 2017):
The use of ultracentrifugation over centrifugation (and vice-versa) denotes basic differences between the two techniques. Fundamental differences between centrifugation and ultracentrifugation include:
There is, currently on the market, a wide variety of ultracentrifuges. The choice among different brands and models must consider the type of experimental applications to be performed, the availability of different rotors (making it possible to adapt the ultracentrifuge to different experimental settings), and the temperature range. Some ultracentrifuges further offer remote monitoring and control, and password-protection (Biocompare, 2019b).
It is important to ask: For which experimental setting do I need an ultracentrifuge? In that sense, two types of ultracentrifuges are available: analytical and preparative. Analytical ultracentrifugation is used in the study of purified macromolecules or supramolecular assemblies, while preparative ultracentrifugation is used in the actual separation of tissues, cells, subcellular components, and other biochemically interesting particles (Ohlendieck & Harding, 2017).
Analytical ultracentrifuges are equipped with optical detection systems that allow the researcher to follow the centrifugation process in real-time. These systems may use ultraviolet (UV) light absorption or refracting index interference (RII) optical detection systems (ultracentrifuges may be equipped with one or both types of optical systems) (Cole, 2009). While UV detection directly measures the absorbance (abs) of a substance at a specific wavelength, RII measures changes in the refraction index (radiation direction) of a given substance, compared to the solvent it is dissolved in (Ohlendieck & Harding, 2017). The purpose of analytical centrifugation is different from other types of centrifugation. Although component isolation is possible with analytical centrifugation, the goal of this technique is to obtain data to characterize the sample that is spun (sedimentation velocity, viscosity, concentration, etc.). With analytical centrifugation, it is possible to follow the variations in sample concentration as a function of the applied centrifugal force. This technique is used in two main experimental settings: sedimentation velocity and sedimentation equilibrium studies, which are key in macromolecular characterization (Ohlendieck & Harding, 2017). Results from sedimentation velocity experiments provide data that are used to calculate the molecular size (molecular weight), shape, and molar masses of new chemical molecules, ribonucleic acids, proteins, and others (Cole, 2009; Ohlendieck & Harding, 2017).
Preparative ultracentrifuges are mostly used to process biological samples for further analysis. The most common application of preparative ultracentrifugation is in tissue and subcellular fractionation, to isolate increasingly smaller components of the biological samples (Jasinski, Schwartz, Haque, & Guo, 2015; Martin et al., 2018; Momen-Heravi, 2017; Ohlendieck & Harding, 2017; Wasan, Cassidy, Kennedy, & Peteherych, 2001). For that, two main centrifugation methods are used: differential and density-gradient centrifugation.
Figure 1: General principle of differential centrifugation, applied to subcellular fractionation (image credit: (Kumar))
Density gradient centrifugation goes further in particle separation than differential centrifugation. It is ideal when the goal is to isolate particles of similar sizes, but different densities. In this case, it is possible to establish density gradient solutions with increasing concentrations of specific materials, in the spinning tubes (Figure 2). Cesium salt gradients are used in the separation of DNA, and sucrose gradients are used in subcellular fractionation to isolate organelles and multiprotein complexes, like ribosomes (Dumetre & Darde, 2004; Perper, Zee, & Mickelson, 1968). Today, there are several commercial gradient solution kits to isolate specific particles. Roughly, there are two types of density gradient centrifugation: rote-zonal centrifugation and isopycnic centrifugation (also called equilibrium centrifugation), which differ in the way particles are separated across the gradient (see Figure 2).
Figure 2: Types of preparative centrifugation. A – Rote-zonal centrifugation B – Isopycnic centrifugation (image credit: (Frei))
Rote-zonal centrifugation – particle separation depends mostly on particle mass. Zones, or bands, are generated, each containing a particle fraction of a specific mass. However, care must be taken when performing rote-zonal centrifugation. Because the mass of the particles is higher than the density of the solvent, if they are centrifuged for too long, all particles will eventually deposit in the bottom of the tube (Frei).
Isopycnic (equilibrium) centrifugation – particle separation depends solely on their density. In isopycnic separation, particles are mixed with the gradient solution, and during centrifugation, they will move until they reach the gradient phase which equals their density (isopycnic or equilibrium point). Because the density of the gradient medium is always higher than the density of particles, these will never sediment, independently of the centrifugation time. Continuous gradients may be used in isopycnic centrifugation, however, discontinuous gradients in which particles form bands at the interface between the density gradient layers are more suitable for the separation of some biological samples, like the separation of lymphocytes from whole blood (Frei; Ohlendieck & Harding, 2017).
Due to their intrinsic differences, analytical and preparative ultracentrifugation are used for different purposes:
Analytical ultracentrifugation
Preparative ultracentrifugation
Modern ultracentrifuges are heavy, sturdy equipment that requires certain know-how for proper usage and care.
From the development of the first ultracentrifuge in the 1920s by Svedberg, up to today, the scientific advances that resulted from the application of ultracentrifugation to biology, chemistry, material science, and others, are countless. In its most obvious approaches, ultracentrifugation extended the limits of biology research to the subcellular level, by allowing the isolation of particles as small as ribosomes, subcellular organelles, membranes, and ribonucleic acids. With the advent of analytical ultracentrifugation, research took another step further towards the understanding of the submicroscopic world, with the ability to further characterize molecular size, shape, and structure. However, ultracentrifugation has its own limitations, like any other laboratory technique. These include:
To date, ultracentrifugation, in its most varied forms and protocols is used routinely and continues to be a fundamental approach not only in academic research but also in industry and the medical context. Nevertheless, it is fundamental to keep in mind how to properly use ultracentrifuges, to keep the sample and device user safe, and to ensure long-term functionality of the devices.
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