8/10/2019 expo jose de la hoz

1/3

10.2.2 Centrifugation

Centrifugation is an alternative method when the filtration is ineffective, such as in the case of

small particles. Centrifugation requires more expensive equipment than filtration and typically

cannot be scaled to the same capacity as filtration equipment. Two basic types of large-scale

centrifuges are the tubular and the disk centrifuge as shown schematically in Figure 10.2. The

tubular centrifuge consists of a hollow cylindrical rotating element in a stationary casing. The

suspension is usually fed through the bottom and clarified liquid is removed from the top leaving

the solid deposit on the bowl's wall. The accumulated solids are recovered manually from the

bowl. A typical tubular centrifuge has a bowl of 2 to 5 in. in diameter and 9 to 30 in. in height with

maximum rotating speed of 15,000 to 50,000 rpm (Ambler, 1979). The disk centrifuge is the type

of centrifuge used most often for bioseparations. It has the advantage of continuous operation. It

consists of a short, wide bowl 8 to 20 in. in diameter that turns on a vertical axis (Figure 10.2b).

The closely spaced cone-shaped discs in the bowl decrease the distance that a suspended particle

has to be moved to be captured on the surface and increases the collection efficiencies. In

operation, feed liquid enters the bowl at the bottom, flows into the channels and upward past the

disks. Solid particles are thrown outward and the clear liquid flows toward the center of the bowland is discharged through an annular slit. The collected solids can be removed intermittently or

continuously.

When a suspension is allowed to stand, the particles will settle slowly under the influence of

gravity due to the density difference between the solid and surrounding fluid, a process known as

sedimentatiqn. The velocity of a particle increases as it falls and reaches a constant velocity

(known as terminal velocity) at which.

Weight force - Buoyancy force Drag force

8/10/2019 expo jose de la hoz

2/3

The expression for the terminal velocity can be derived from the balance of the forces acting on a

particle as,

In the case of a sedimentation process, the acceleration term in Eq. (10.5) is equal to the

acceleration due to gravity. Due to the small difference in density between the cells and the broth,

simple settling can take along time unless cells are large or the cells form a large aggregate. Under

the centrifugal force, the acceleration term in Eq. (10.5) becomes

Where (w) is the angular velocity and r is the radial distance from thecenter of a centrifuge to a particle. Therefore, the increase inacceleration by the centrifugal force speeds up the settling process.

Once the cellular materials are separated, those with intracellularproteins need to be ruptured to release their products. Disruption ofcellular materials is usually difficult because of the strength of thecell walls and the high osmotic pressure inside. The cell rupturetechniques have to be very powerful, but they must be mild enough

so that desired components are not damaged. Cells can be rupturedby physical, chemical, or biological methods.

Physical Methods Physical methods include mechanicaldisruption by milling, homogenization, or ultrasonication. Typicalhigh-speed bead mills are composed of a grinding chamber filled withglass or steel beads which are agitated with disks or impellersmounted on a motor-driven shaft. The efficiency of cell disruption ina bead mill depends on the concentration of the cells, the amount andsize of beads, and the type and rotation speed of the agitator. Theoptimum wet solid content for the cell suspension for a bead mill istypically somewhere between 30 percent to 60 percent by volume.'The amount of beads in the chamber is 70 percent to 90 percent by

volume (Keshavarz et al., 1987). Small beads are generally moreefficient, but the smaller the bead, the harder it is to separate themfrom ground solids. Cell disruption by bead mills is inexpensive andcan be operated on a large scale.

A high-pressure homogenizer is a positive displacement pump withan adjustable orifice valve. It is one of the most widely used methodsfor large-scale cell disruption. The pump pressurizes the cell

8/10/2019 expo jose de la hoz

3/3

suspension (about 50 percent wet cell concentration) toapproximately 400 to 550 bar and then rapidly releases it through aspecial discharge valve, creating very high shear rates. Refrigeratedcooling to 4 or 5C is necessary to compensate for the heat geIleratedduring the adiabatic compression and the homogenization steps(Cliffe, 1988).

An ultrasonicator generates sound waves above 16 kHz, whichcauses pressure fluctuations to form oscillating bubbles that implodeviolently generating shock waves. Cell disruption by anultrasonicator is effective with most cell suspensions and is widelyused in the laboratory. However, it is impractical to be used on a largescale due to its high operating cost.

Chemical methods of cell rupture include thetreatment of cells with detergents (surfactants), alkalis, organicsolvents, or by osmotic shock. The use of chemical methods requiresthat the product be insensitive to the harsh environment created bythe chemicals. After cell disruption, the chemicals must be easily

separable or they must be compatible with the products.Surfactants disrupt the cell wall by solubilizing the lipids in thewall. Sodium dodecylsulfate (SDS), sodium sulfonate, Triton X-IOO,and sodium taurocholate are examples of the surfactants oftenemployed in the laboratory.Alkali treatment disrupts the cell walls ina number of ways including the saponification of lipids. Alkalitreatment is inexpensive and effective, but it is so harsh that it maydenature the protein products. Organic solvents such as toluene canalso rupture the cell wall by penetrating the cell wall lipids, swellingthe wall. When red blood cells or a number of other animal cells aredumped into pure water, the cells can swell and burst due to theosmotic flow of water into the cells.

Enzymatic digestion of the cell wall is a goodexample of biological cell disruption. It is an effective method that isalso very selective and gentle, but its high cost makes it impractical tobe used for large-scale operations.