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Centrifugation, Centrifuge, Gravitational Force, Separation, Centrifugal Force

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Centrifugation, Centrifuge, Gravitational Force, Separation, Centrifugal Force American Journal of Biomedical Engineering 2015, 5(2): 67-78 DOI: 10.5923/j.ajbe.20150502.03 A Review on Centrifugation in the Pharmaceutical Industry Stephen Olaribigbe Majekodunmi Department of Pharmaceutics and Pharmaceutical technology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria Abstract The importance of centrifugation in the pharmaceutical industry has rarely been studied. Centrifugation is one of the most important and widely applied research techniques in biochemistry, cellular and molecular biology and in evaluation of suspensions and emulsions in pharmacy and medicine. This review focuses on the basics and principle of centrifugation, classes of centrifuges, types of centrifuge separations, different types of density gradient media, classification of industrial centrifuges, types of industrial centrifuges and applications of centrifugation in the pharmaceutical industry. Centrifugation, clearly, plays an important role in the pharmaceutical industry in the production of bulk drugs, biological products, determination of molecular weight of colloids and in the evaluation of suspensions and emulsions. Keywords Centrifugation, Centrifuge, Gravitational force, Separation, Centrifugal force away from the axis of the centrifuge, while less-dense 1. Introduction components of the mixture migrate towards the axis. Pharmacists, chemists and biologists may increase the Centrifugation is one of the most important and widely effective gravitational force on a test tube so as to move applied research techniques in biochemistry, cellular and rapidly and completely cause the precipitate ("pellet") to molecular biology, evaluation of suspensions and emulsions gather on the bottom of the tube. The remaining solution is in pharmacy and in medicine. Current research and clinical properly called the "supernate" or "supernatant liquid". The applications rely on isolation of cells, subcellular organelles, supernatant liquid is then either quickly decanted from the and macromolecules, often in high yields. A centrifuge uses tube without disturbing the precipitate, or withdrawn with a centrifugal force (g- force) to isolate suspended particles Pasteur pipette [2]. from their surrounding medium on either a batch or a As enunciated by Sir Isaac Newton in his first law of continuous – flow basis [1]. motion, a freely moving body (such as a ball) tends to travel Many particles or cells in a liquid suspension, given time, in a straight line, and if directed along a curved path by some will eventually settle at the bottom of a container due to restraining force (such as would result were a hand-held gravity. However the length of time required for such string tied to it) it will exert a force against the directing or separations is impractical. Other particles, extremely small in restraining force in its continual effort to fly off onto a size, will not separate at all in solution, unless subjected to straight tangential course. It is a familiar observation that an high centrifugal force. When a suspension is rotated at a object revolving in a circle exerts a force away from the certain speed or revolutions per minute (RPM), centrifugal centre of rotation. This force, which is the outward pull of the force causes the particles to move radially away from the ball on its string, is the centrifugal force. Also, there is axis of rotation. The force on the particles (compared to general appreciation of the fact that the amount of this force gravity) is called Relative Centrifugal Force (RCF). For can be increased by increasing either the angular velocity of example, an RCF of 500 x g indicates that the centrifugal rotation, the mass of the object, or the radius of the circle force is 500 times greater than earthly gravitational force. through which the object moves. Perhaps not so generally Centrifugation is a process which involves the use of the appreciated is the fact that whereas the centrifugal force is centrifugal force for the sedimentation of heterogeneous directly proportional to the radius and to the mass, it is mixtures with a centrifuge, used in industry and in laboratory proportional to the square of the angular velocity. For settings. This process is used to separate two immiscible example, doubling the mass of the rotating object will liquids. More-dense components of the mixture migrate increase the centrifugal force by a factor of 2, but doubling the number of revolutions per minute (rpm) will increase the * Corresponding author: [email protected] (Stephen Olaribigbe Majekodunmi) centrifugal force by a factor of 4 (equals 2 times 2); similarly, Published online at http://journal.sapub.org/ajbe increasing the speed by a factor of 10 will increase the force Copyright © 2015 Scientific & Academic Publishing. All Rights Reserved by a factor of 100 (equals 10 times 10). Centrifugal force is 68 Stephen Olaribigbe Majekodunmi: A Review on Centrifugation in the Pharmaceutical Industry expressed by the basic relation F = mν2 / R = 4π2mn2R; F is Table 1. Classes of centrifuges and their applications the centrifugal force, m the mass, R the radius, v the speed, Centrifuge Classes and n the number of revolutions per second [3]. Low – High – Ultra/micro - Centrifuge is any device that applies a sustained speed speed ultra centrifugal force; that is, a force due to rotation. Effectively, Maximum Speed 10 28 100 / 150 the centrifuge substitutes a similar, stronger, force for that of (rpm x 103) gravity. Every centrifuge contains a spinning vessel; there Maximum RCF 7 100 800 / 900 are many configurations, depending on use. A perforated (x103) rotating drum in a laundry that throws off excess water from Pelleting Applications clothes, for example, is a type of centrifuge. A similar type is Bacteria Yes Yes (Yes) used in industry to separate fluids from solid matter after Animal and plant Yes Yes (Yes) crushing. cells Nuclei Yes Yes (Yes) Precipitates Some Most (Yes) 2. Basics of Centrifugation Membrane Some Some Yes The earth's gravitational force is sufficient to separate fractions Ribosomes / many types of particles over time. A tube of anticoagulated - - Yes whole blood left standing on a bench top will eventually Polysomes separate into plasma, red blood cell and white blood cell Macromolecules - - Yes fractions. However, the length of time required precludes Viruses - Most Yes this manner of separation for most applications. In practice, ( - ) Can be done but not necessarily used for this purpose centrifugal force is necessary to separate most particles. In addition, the potential degradation of biological compounds RCF is dependent on the speed of rotation in rpm and the during prolonged storage means faster separation techniques distance of the particles from the center of rotation. When the are needed. speed of rotation is given in rpm (Q) and the distance (r) is The rate of separation in a suspension of particles by way expressed in centimeters, RCF can be calculated by using the of gravitational force mainly depends on the particle size and formula in Figure 2. density. Particles of higher density or larger size typically RCF = 11.17 x Rmax (RPM/1000)2 (2) travel at a faster rate and at some point will be separated from Formula for relative centrifugal force (RCF). particles less dense or smaller. This sedimentation of particles, including cells, can be explained by the Stokes equation, which describes the movement of a sphere in a 3. Principles of Centrifugation gravitational field. Equation (1) calculates the velocity of µ sedimentation utilizing five parameters [4]. Particles having a size above 5 m sediment at the bottom due to gravitation force. Such a suspension can be separated V = d2 (p–L) 3 g (1) by simple filtration techniques. If the size of particles is less 18 n than 5µm they undergo Brownian motion. In such v = sedimentation rate or velocity of the sphere suspension a stronger centrifugal force is applied to separate d = diameter of the sphere the particles. p = particle density Considering a body of mass m rotating in a circular path of L = medium density radius r at a velocity of v. The force acting on the body in a n = viscosity of medium radial direction is given by: g = gravitational force F = mv2/r From the Stokes equation five important behaviors of Where F = centrifugal force, m = mass of body, v = particles can be explained: velocity of the body, r = radius of circle of rotation. 1. The rate of particle sedimentation is proportional to the The gravitational force acting upon the same body G = mg particle size. Where, G = gravitational force 2. The sedimentation rate is proportional to the difference g = acceleration due to gravity in density between the particle and the medium. The centrifugal effect is the ratio of the centrifugal force 3. The sedimentation rate is zero when the particle density and gravitational forces so that is the same as the medium density. C = F/G = mv2/mgr = v2/gr (3) 4. The sedimentation rate decreases as the medium Since, v = 2π r n where n = speed of rotation (r.p.m.) viscosity increases. π 2 π2 2 2 5. The sedimentation rate increases as the gravitational C = F/G = (2 r n) /g r = 4 r n 2 2 2 force increases. = 2π /g D n = kD n (4) American Journal of Biomedical Engineering 2015, 5(2): 67-78 69 where, k = 2π2/g = constant particles. D = maximum diameter of the centrifuge Differential pelleting is commonly used for harvesting cells or producing crude subcellular fractions from tissue homogenate. For example, a rat liver homogenate containing nuclei, mitochondria, lysosomes, and membrane vesicles that is centrifuged at low speed for a short time will pellet mainly the larger and more dense nuclei. Subsequent centrifugation at a higher centrifugal force will pellet particles of the next lower order of size (e.g., mitochondria) and so on. It is unusual to use more than four differential centrifugation cycles for a normal tissue homogenate.
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