Preparative and analytical are the two types of centrifugation techniques.

## Preparative Centrifugation

Preparative centrifugation leads to separation, isolation, and purification of various cellular constituents for biochemical investigations. Large amounts of cellular components are isolated to study their morphology, composition and biological activities. It is also used in separation of whole cell, plasma membrane, lipoproteins, chromatin, nucleic acids, and viruses.

## Analytical centrifugation

Analytical centrifugation is used to study pure compounds and macromolecules to determine their shape, molecular weight, and purity. It is primarily concerned with the study of sedimentation characteristics of biological macromolecules rather than fractions as done in preparative centrifugation technique. This technique requires small amount of sample, specific type of centrifuge and detector system to monitor process of centrifugation continuously.

Some particles are extremely small in size. They do not separate in solution, unless they are subjected to high centrifugal force. When a suspension is rotated at a certain speed or revolutions per minute (rpm), centrifugal force causes particles to move radially away from axis of rotation. The force applied on particles (in comparison to gravity) is called Relative Centrifugal Force (RCF). For example, an RCF of 500x g indicates that the centrifugal force applied is 500 times greater than the gravitational force.

Rate of centrifugation depends upon centrifugal field (G), which is square root of angular velocity of the rotor (ω, in radians per second) and the radial distance (r, in cm) of the particle from the axis of rotation.

G = ω2r

The angular velocity (in radians per second) is equal to,

ω = 2π rev min-1 /60

Therefore, centrifugal field (G) is equal to,

G = 4π2 rev min-1/3600

So, the relative centrifugal field (RCF) i.e. the ratio of the weight of the particle in the centrifugal field to the weight of the same particle when acted on by the gravity (g = 980 cms-2) alone is equal to,

RCF = 4π2 (rev min-1 ) 2 r/3600 ×980

Rate of sedimentation depends upon the centrifugal force applied, density and viscosity of the medium in which particles get sedimented, density, size and the extent to which the particle is deviating from the spherical shape.

Sedimentation coefficient

Sedimentation coefficient characterizes a behavior of a particle type in sedimentation processes. It is expressed in Svedberg unit (S), which is named after Theodor Svedberg (Swedish scientist), who invented ultracentrifuge. It is defined as the ratio of sedimentation velocity of particles to the acceleration that is applied to it. He described ultracentrifuge of producing colloid particles and validated it with theory on Brownian movements. Svedberg determined molecular weights of substances including proteins and viruses.

Sedimentation coefficient (s) is mathematically represented as,

s = vt /a

Where, vt = sedimentation speed

a = acceleration

The sedimentation speed (in ms-1) is also known as terminal velocity. The sedimentation coefficient is a rate per unit centrifugal field. The S values for most proteins range between 10−13 sec and 10−11 sec. The sedimentation coefficient has the dimension unit of time and is expressed in Svedberg.

One Svedberg = 10−13 s

S values for most proteins range between 1 × 10−13 sec to 2 × 10−11 sec. Sedimentation coefficients of two molecules joined together are not additive. If they are measured separately then they have Svedberg values. These values may not add up to that of the bound particle, as there is loss of surface area when two molecules bind together and their sedimentation rates become independent of their mass.

### Types of Preparative Centrifugation

Preparative Centrifugation: This is divided into four types.

1. Differential Centrifugation
3. Rate Zonal Centrifugation
4. Isopycnic Centrifugation

#### Differential Centrifugation

In differential centrifugation, separation of various components is achieved primarily based on the size of the particles. It is commonly used in simple pelleting and in obtaining partially pure preparation of subcellular organelles and macromolecules (Figure 1).

In this process, tissue sample is first homogenized to break cell membrane called homogenate. The homogenate is then subjected to repeated centrifugation. In each step, pellet is removed and centrifugal force is increased. Finally, purification is done through equilibrium sedimentation, and desired layer is extracted for further analysis. Obtaining partially purified organelles by differential centrifugation serves as preliminary step for analysis and further purification using other types of centrifugal separation methods.

Separation process is based on size and density of the particles. Larger and denser particles get pelleted out at lower centrifugal forces. For example, unbroken whole cells will pellet at low speeds and short intervals such as 1,000g for 5 minutes. Smaller cell fragments and organelles remain in the supernatant and require more force and time to get pelleted out. Depending upon their separating order, cells can be derived for further application (Figure 1). The order in which cells get separated is shown below.

• Whole cell and Nuclei
• Mitochondria, lysosomesand peroxisomes
• Microsomes
• Ribosomes and cytosol

Homogenization of tissue sample is the first step before doing centrifugation. The tissue is homogenized in a blender to break the cell membrane and mix all cell constituents. The mixture obtained after homogenization is called homogenate. A buffer solution is added to the homogenate. The buffer (generally sucrose is used) added must be dense and inert so there is no damage of the sample due to chemical reactions or osmosis. The homogenized sample is ready for centrifugation. Samples are placed in tubes within or attached to the rotor in ultracentrifuge. The speed of rotation is increased at each stage that leads to the separation of cellular components. Different fragments of a cell have different sizes and density, thus each fragment settles into pellet with different minimum centrifugal forces.

Samples are separated into different layers by first centrifuging homogenate under weak forces, removing pellet, and then exposing subsequent supernatants to the sequentially greater centrifugal fields. Each time, a portion having different density gets sedimented at the bottom of tube and later extracted. Repeated steps produce layers, which include different parts of original sample. Each step of homogenization process can be monitored using phase-contrast microscope to check for cellular breakup.

Sedimentation of various components depends upon solvent density, mass, shape and partial specific volume of molecule, and rotor size and rate of rotation of the centrifuge. Sedimentation velocity can be monitored and subsequently sedimentation coefficient (corresponds to molecular weight) is calculated. Equilibrium sedimentation uses gradient of cesium chloride and sucrose. It separate particles based on their individual densities (mass/volume). It is used as purifying process for differential centrifugation. This method uses fixed angle rotor. Pellets are analyzed for chemical and enzyme markers and protein. This allows determination of the recovery of particles of interest and contaminants, which may be difficult to remove. This method is very useful for studying mammalian tissues and in cancer research

 Subcellular Particle Markers Nucleus DNA Mitochondria Succinate dehydrogenase Lysosomes Acid Phosphatase, β-galactosidase Peroxisome Catalase Endoplasmic Reticulum (ER) NADPH-cytochrome c reductase, Rotenone-insensitive NADH-cytochrome c reductase Rough Endoplasmic Reticulum (RER) RNA, NADPH-cytochrome c reductase, Rotenone-insensitive NADH-cytochrome c reductase Golgi UDP-galactose galactosyl transferase Plasma Membrane 5’-nucleotidase, Na+/K+-A TPase, leucine amino peptidase, alkalinephosphatase

Composition of the pellets

Composition of various fractions produced by differential centrifugation is defined for commonly used tissues such as mammalian or rat liver. Generally, various constituents in the pellet are as shown below.

Nuclear Pellet

This basically contains nuclei. Other components present in the pellet are mitochondria, and sheets of plasma membrane. Unbroken cells and debris (including connective tissue) are also present in unfiltered homogenate. Formation of this pellet is sometimes carried out at 500g rather than 1000g.

Heavy Mitochondrial Pellet

This pellet predominantly contains mitochondria with few contaminants commonly used in the respiratory system studies. Minor components such as lysosomes, peroxisomes, golgi membranes and various membrane vesicles are present (as they are entrapped during pelleting process). Some plasma membrane fragments may also be present.

Light Mitochondrial Pellet

This fraction contains mitochondria, lysosomes, peroxisomes, golgi membranes and some portion of endoplasmic reticulum. This is most variable of all differential centrifugation fractions.

Microsomal Pellet

This fraction only contains the membrane vesicles. Some of those vesicles will have been present in cell before homogenization (e.g. endosomes, secretory vesicles and vesicles from the trans-Golgi network), others come from plasma membrane, Golgi, and smooth and rough endoplasmic reticulu

Density gradient centrifugation is a method to purify subcellular organelles and macromolecules having different densities and sizes. Density gradients are generated by placing layer over layer of gradient media such as, sucrose in a tube with the heaviest layer at the bottom and the lightest at the top in either a discontinuous or continuous mode and then centrifuged. Although the particles in suspension are themselves denser than the liquid at the top of the gradient, average density for a sample (i.e. particles plus suspending liquid) is lower. Only under this condition, the sample zone is supported by density gradient (top). The two main types of density gradient centrifugation are rate-zonal and isopynic separation (Figure 3).

#### Rate Zonal Centrifugation

Rate-zonal separation takes advantage of particle size and the mass (sedimentation rates) instead of particle density for sedimentation. So, particles migrate through the gradient accordingly and get separated into distinct zones or bands (if they were layered as a thin zone onto the top of the gradient). It is time dependent.

This technique involves a careful layering of sample solution on top of preformed liquid density gradient (highest density of the liquid density gradient must not exceed the density of the densest particle to be separated). This technique is useful for separation of proteins, macromolecules, antibodies, viruses, DNA-RNA hybrids, hormones, enzymes, ribosomal subunits, and organelles. For successful rate zonal centrifugation, density of the sample solution must be less than that of lowest density portion of the gradient, density of sample particle must be greater than that of highest density portion of the gradient, the pathlength of the gradient must be sufficient for the separation to occur and time factor is important.

#### Isopycnic Centrifugation

This technique is used to separate molecules on the basis their of density (isopycnic means equal density). Fixed angle rotor or swinging bucket rotors can be used for this separation. A self-generating density gradient is established due to equilibrium sedimentation, and then analyte molecules gets concentrated as bands where the molecule density matches the surrounding solution, i.e. a particle of particular density will sink during centrifugation until a position is reached where the density of the surrounding solution is exactly the same as the density of the particle.

The time of centrifugation does not matter once the equilibrium is reached. One of the most common applications of this method is separation of nucleic acids in CsCl gradient. Caesium chloride is used because at a concentration of 1.6 to 1.8 g/mL, as it is similar to the density of DNA. A gradient is formed after sometime due to centrifugal force as well as diffusion. The sedimenting particles (caesium ions) sediment away from the rotor, and become more concentrated near the bottom of the tube. The diffusive force arises due to the concentration gradient of caesium chloride that is always directed towards the center of the rotor. The balance between these two forces generates a stable density gradient in the caesium chloride solution, which is denser near the bottom of the tube, and less dense near the top.

Isopycnic gradient ultracentrifugation is generally used to separate plasma lipoproteins. For successful isopycnic centrifugation density of the sample particle must fall within the limits of the gradient densities, the run time must be sufficient for the particles to band at their isopycnic point, excessive run times, and any gradient lengths have no adverse effect.

Table 2. Comparison between rate zonal and isopycnic centrifugation

 Rate Zonal Centrifugation Isopycnic Centrifugation The particles are separated based on their size and mass. The particles are separated based on their density. Useful for small particles and is time dependent. Useful for large particles, as runs can be longer for prolonged particle viability or liability. Rate zonal runs are usually carried out in shallower density gradients (useful in reducing damage of osmotically active particles). Isopycnic runs are usually carried out in deeper gradients making it unsuitable for osmotically active particle. Rate zonal runs are carried out by in lower RFC. It is useful when separation of fragile particles comes into account as reduced hydrodynamic shear that occurs during sedimentation aids in maintaining particle integrity. Isopycnic runs are carried out in higher RFC. Not useful for the separation of fragile particles The duration of centrifugation is critical in rate zonal run and lesser number of particles can be separated by this method in comparison to isopycnic density gradient centrifugation. The duration of centrifugation is not critical in an isopycnic run and large number of particles can be separated by isopycnic density gradient centrifugation.

### Analytical Centrifugation

This method uses analytical ultracentrifuges for separation of molecules. It is re-emerging as versatile tool to study proteins. It is a most versatile, rigorous and accurate means for determining molecular weight and hydrodynamic and thermodynamic properties of the proteins and other macromolecules. Determination of sedimentation coefficients allows for the modeling of hydrodynamic shape of proteins and protein complexes. It uses small and relatively pure sample.

Instrumentation

An ability to measure a distribution of the sample while it is spinning makes analytical ultracentrifuge different from other preparative centrifuges. At accurately controlled speed and temperature, concentration distribution of a sample is recorded at known time in analytical ultracentrifugation. Rotors of analytical ultracentrifuge is typically capable of rotating at speeds up to 60,000 rpm and generates 250,000 × g of centrifugal field. In order to minimize frictional heating and aerodynamic turbulence, the rotors are usually spun in an evacuated chamber. The instability of rotors can cause convection and stirring of the cell contents, especially at low concentration and concentration gradient of the solute, and may lead to uncertainty in the concentration distribution in regions of high concentration gradient.

Ultracentrifuge cells are also designed in such a way that it can stand stress generated by centrifugal field. Boundary forming cells allow solvent to be layered over the sample of a solution while the cell is spinning at moderately low speed. These cells are useful for preparing sharp artificial boundary to measure boundary spreading for the measurements of diffusion coefficients, and for examining sedimentation velocity of small molecules. The band forming cells, layer small volume of solution on top of a supporting density gradient in band sedimentation and an active enzyme sedimentation studies. For data collection, a set of concentration measurements at different radial positions and at a given time is done.

## Applications of Centrifugation

Different type of centrifuges have different applications.

• Current research (in the field of biochemistry, biology, chemistry, molecular biology, biotechnology or pharmaceutical sciences) and clinical applications rely on centrifugation for isolation of cells, subcellular organelles and macromolecules for various analysis.
• To study the effects of centrifugal forces on cells
• Developing embryos, and protozoa
• To determine certain properties about cells, including surface tension relative viscosity of the cytoplasm, and spatial and functional interrelationship of cell organelles in cells.
• In food, chemical, and mineral industries centrifuges are used to separate water from all sorts of solids, separation of cream from milk, and water treatment.
• Centrifuges are essential device in clinical laboratories and small medical facilities. They separate substances with different densities and are also used for removing chylomicrons.
• Centrifugation studies have been very important in development of manned space flight programs. Human volunteers are placed into very large centrifuges and then spun at high speeds to feel same centrifugal field they feel during launch of space vehicles.
• Commercial uses of centrifugation include
• Separation and purification of wine
• Separating textiles
• Enriching uranium
• As human centrifuge for astronauts
• In-flight robot
• Water treatment to dry sludge
• Oil industry to remove solids from drilling fluid
• Geotechnical centrifugation for simulating blast and earthquake phenomenon.