What is centrifugation?

INTRODUCTION

Centrifugation is the use of the centrifugal forces generated in a spinning rotor to separate particles, living components such as cells, viruses, sub-cellular organelles, macromolecules such as proteins and nucleic acids and macromolecular complexes such as ribonucleoproteins and lipoproteins. The three main methods of separation by centrifugation are differential pelleting, rate-zonal centrifugation, and isopycnic centrifugation. The first two methods separate particles primarily based on size. The latter method isopycnic centrifugation separates particles based on their density. The choice of centrifugation method depends on the nature of the particles and often when more than one separation technique is required. For example, membrane fractionation often involves first making an enriched fraction from a cell homogenate by differential pelleting followed by isopycnic centrifugation to obtain purified fractions.

Centrifugation is one of the most important and widely applied research techniques in biochemistry, cellular and molecular biology, and the evaluation of suspensions and emulsions in pharmacy and medicine. Centrifugation plays an important role in the pharmaceutical industry in the production of bulk drugs, and biological products, and determination of the molecular weight of colloids, and the evaluation of suspensions and emulsions.

The earth's gravitational force is sufficient to separate many types of particles over time. A tube of anticoagulated whole blood left standing on a bench top will eventually separate into plasma, red blood cell, and white blood cell fractions. However, the length of time required precludes this manner of separation for most applications. In practice, centrifugal force is necessary to separate most particles. In addition, the potential degradation of biological compounds during prolonged storage means faster separation techniques are needed. The rate of separation in a suspension of particles by way of gravitational force mainly depends on particle size and density. Particles of higher density or larger size typically travel at a faster rate and some points will be separated from 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 gravitational field. Equation (1) calculates the velocity of sedimentation utilizing five parameters.

`v\=\frac{d^2(\rho\-\rho\_0)g}{18\eta}   ...(1)`

Where,

V = Sedimentation rate or velocity of settling the sphere

d = Diameter of the sphere

p = Particle density

L = Medium density

n = Viscosity of medium

g = Gravitational force

OBJECTIVES

Centrifugation is a technique of separating substances that involves the application of centrifugal force. The particles are separated from a solution according to their size, shape, density, viscosity of the medium, and rotor speed. Separation is achieved by spinning a vessel containing material at high speed; the centrifugal force pushes heavier materials to the outside of the vessel. A centrifuge is a laboratory device used for the separation of immiscible fluids, gas, or liquid.

PRINCIPLE

Centrifugation is used to separate all types of particle-based on their sedimentation properties. The sedimentation properties of particles depend on several different factors including size, density, and shape. However, both density and shape vary significantly depending on the composition of the solution in which the particles are suspended. Particles are separated primarily based on either their density (isopycnic separations) or size (differential pelleting and rate-zonal separations). The density affects sedimentation to a much lesser extent than size. In centrifugation, it is important to differentiate between the speed of centrifugation (r.p.m.) and the relative centrifugal force (RCF or G) since these are often confused.

Fig.1: Principle of Centrifugation

The centrifugal force generated by a centrifuge can easily be calculated from the equation

`RCF\=11.18\times R\times\left(\frac{r.p.m}{1000}\right)^2  ...(2)`

where, R is the distance from the center of rotation in centimeters; that is, the centrifugal force increases as the particles move down the centrifuge tube. As a general rule, the greater the centrifugal force the shorter the separation time. However, centrifugation also generates hydrostatic forces within the solution and so excessive centrifugal forces can disrupt some biological particles such as ribosomes.

The other very important aspect in optimizing centrifugal separations is the choice of the rotor. Centrifuge rotors can be divided up into five different types and of these the most frequently used are fixed-angle and swinging bucket rotors.

As a general guide, fixed-angle rotors are used for efficient pelleting and isopycnic centrifugation of macro-molecules, while swinging bucket rotors are primarily used for isopycnic gradients for cells and organelles and rate-zonal centrifugation. The other three types of the rotor are vertical rotors that can be used for gradient separations where no pelleting occurs, zonal rotors that are used for large-scale gradient separations, and analytical rotors where instead of tubes the samples are centrifuged in sector-shaped cells with transparent sides. The efficiency of rotors for pelleting particles is expressed in terms of their k-factors with the most efficient rotors having the smallest k-factors.

Applications of Principle:

High centrifugal force is required if the particles in suspensions are very small. To separate such small particles from suspensions the centrifuge is designed to be small in size that can operate at a very high speed of rotation. If a large amount of material is to be separated and a low centrifugal effect is sufficient to separate the suspension then the diameter of the centrifuge is increased and speed is kept low.

APPLICATIONS

(i) Bulk drugs production: In the synthetic chemical reactions the product formed is crystal. The crystallized drugs are separated from the mother liquor by the process of centrifugation.

For example, traces of mother liquor are separated from aspirin crystals by the use of centrifugal force.

(ii) Production of biological products: During manufacturing, most of the biological products are either proteinaceous or macromolecules which remain as colloidal dispersion in water. A normal filtration method is not suitable to separate these colloid particles and thus in those cases, centrifugal methods are used. The examples include:

(a) Insulin purification from other precipitates of protein materials by centrifugation.

(b) Blood cell separation from plasma by the centrifugal method.

(c) Bacterial enzymes are separated from the bacterial culture medium by sedimenting the bacterial cells by centrifugation.

(d) Dirt and water are separated from olive oil and fish-liver oils.

1. It has applications in the field of medicine such as DNA/ RNA separation.
2. In the purification of mammalian cells, fractionation of sub-cellular organelles and fractionation of membrane vesicles.
3. Used in diagnostic laboratories for blood and urine test.

(iii) Stability testing of the dispersed system: Suspensions and emulsions have problems of sedimentation and creaming, respectively over the storage periods. These problems are not observed immediately after the preparation of a suspension or emulsion. To investigate the stability of these preparations rates of sedimentation and creaming in suspension or emulsion, respectively, is determined using a centrifuge by rotating at 200 to 3000 r.p.m. The absence of sedimentation and creaming is an indication of the stability of those preparations.

(iv) Determination of molecular weight of colloids: Polymers, proteins, and such macromolecules often exist in colloidal dispersions. The molecular weights of those substances can be determined by ultra-centrifugation. The larger molecules get arranged at the periphery and the lighter molecules near the center.

(v) Other applications:

(i) Centrifugation is used to separate skim milk from whole milk and to separate two miscible substances.

(ii) Separating particles from an airflow using cyclonic separation.

(iii) Separation of urine components and blood components in forensic and research laboratories.

(iv) Aids in the separation of proteins using purification techniques such as salting out, for example, ammonium sulfate precipitation.

(v) Other medical applications include the separation of blood cells from blood (plasma) and the removal of fibrinogen (serum). They are also used to determine the hematocrit and to separate urinary components.

(vi) Centrifugation is used to analyze the hydrodynamic properties of macromolecules.

EQUIPMENTS USED FOR CENTRIFUGATION

There are two types of centrifugal techniques for separating particles, differential centrifugation, and density gradient centrifugation. Density gradient centrifugation can further be divided into rate-zonal and isopycnic centrifugation. An industrial centrifuge is a machine used for fluid/particle separation. Industrial centrifuges can be classified into 3 main types as given the Table.1.

Table.1: Classification of Industrial Centrifuges

Type	Principle	Examples
Filtration centrifuge	Using perforated baskets, which perform a filtration-type operation (work like a spin-dryer)	Perforated basket centrifuge
Sedimentation centrifuge	With a solid walled vessel, where particles sediment towards the wall under the influence of the centrifugal force.	Tubular bowl centrifuge
Continuous centrifuge	A continuous process or very high capacity is required	Continuous flow centrifuge

 

Perforated Basket Centrifuge

There are three types of perforated basket centrifuge namely; batch type (top-driven or bottom-driven), semi-continuous type, and continuous type.

Principle: Centrifugal force generates a pressure that forces the liquid through the caked solids, the filter cloth, the backing screen, and the basket perforations. The filter cloth retains the solid particles inside the rotating basket.

(A) Batch Type Top Driven Centrifuge: This centrifuge is used for chemical and pharmaceutical manufacturing for separation purposes due to easy discharge from the bottom. This centrifuge is used when the concentration of slurry is high.

Construction: This centrifuge consists of a rotating basket suspended on a vertical shaft and driven by a motor from the top. This centrifuge is called a top-driven or bottom discharge basket centrifuge, Fig.2. The sides of the basket are perforated and are also covered with a screen on the side. Surrounding the basket is a stationary casing that collects the filtrate. The basket diameter is ranging from 20 to 48 inches with 1800 to 800 r.p.m. speed of rotation. The larger the diameter lowest is the speed. The basket is made up of stainless steel covered with Monel. The size of perforations may be 3 mm but for very fine particles basket is lined with fine mesh or cloth supported on a coarse gauge. Unbalanced basket cause strain and vibrations. The machine picks its top speed in a short time to avoid longer operation at a critical speed which is undesirable for cake and its removal.

Fig.2: Vertical Basket Top Driven Centrifuge

Working:

(i) Feeding: The slurry is introduced to the rotating basket having a filter cloth. The filter cloth captures the solids. Centrifugal force drives the liquid through the caked solids and the mother liquor is discharged through perforations in the basket circumference.

(ii) Washing: A washing liquid is introduced and is driven through the caked solids. The plug flow action of the wash liquid purifies the solids and removes residual mother liquor.

(iii) Spinning: Residual liquor is driven from the caked solids and is discharged through the basket perforations to achieve maximum cake dryness.

(iv) Scraping: A scraper knife advances into the rotating basket to discharge the solids to downstream equipment. The solids are discharged through openings in the basket bottom.

(v) Residual heel removal: After scraping, a 6-10 mm layer remains inside the rotating basket. With the scraper in an advanced position, high-pressure nitrogen or air is used to dislodge this residual heel. This step can be performed after several centrifuge cycles or after each cycle.

(B) Batch Type Bottom Driven Centrifuge:

Construction: Batch type is driven centrifuge consists of a rotating basket placed on a vertical shaft and driven by a motor from the bottom called a driven or top-discharge basket centrifuge, Fig.3. The sides of the basket are perforated and are also covered with a screen on the inside. Surrounding the basket is a stationary casing that collects the filtrate.

Fig.3: Vertical Basket Bottom Driven Centrifuge

Working: When the basket rotates, the product enters centrally and is thrown outside by centrifugal force and held by a filter cloth. The filtrate is forced through the cloth and is removed through the liquid outlet. The solid material remains on the cloth. The cake can be washed off with water to get filtrate. The stepwise operation is given below.

(a) Feeding: The slurry is introduced to the rotating basket having a filter bag. The filter bag captures the solids. Centrifugal force drives the liquid through the caked solids and the mother liquor is discharged through perforations in the basket circumference.

(b) Washing: A washing liquid is introduced and is driven through the caked solids. The plug flow action of the wash liquid purifies the solids and removes residual mother liquor.

(c) Spinning: Residual liquor is driven from the caked solids and is discharged through the basket perforations to achieve maximum cake dryness.

(d) Solids Discharge: The filter bag containing the solids is removed from the basket and the filter bag inverts to discharge the solids into a receiver, or the solids are removed manually by scooping them from the filter bag.

Advantages:

1. The initial cost is considerably high but after it is cheaper to use.
2. The perforated basket centrifuge is rapid in operation.
3. The centrifuge is very compact and hence can be accommodated in a small floor place.
4. It can handle concentrated slurries up to paste-like consistency.
5. The final product has a very low moisture content.
6. The dispersed solids are separated as cake.

Disadvantages:

1. Being this centrifuge involves several steps the entire cycle is complex,
2. It is a batch process and speed is required to be controlled thus involving considerable labor costs.
3. Lengthy operation results in the formation of a hard cake due to the continuous, application of centrifugal force. This makes cake removal a difficult task.
4. Being it has moving components there is considerable wear and tear on the equipment.

Applications:

1. It is applicable when very fine particles are to be separated from the mixture.
2. These centrifuges are used to separate crystals from the mother liquor.
3. Liquids can be clarified by removing unwanted solid dirt from oils.
4. Sugar crystals are separated using a perforated centrifuge.
5. Used for removing precipitated proteins from insulin.
6. It is extensively used for separating crystalline drugs such as aspirin and sulfamethoxazole from the mother liquor.

Non-Perforated Basket Centrifuge

This is a sedimentation centrifuge. This type of centrifuge is used when resistance to the flow of liquid offered by cake is high. The non-perforated basket centrifuge is used to separate suspension whose solid content is higher. The principle involved in the separation of solid particles in this machine is density difference as they move away from the axis of rotation. It consists of a simple drum-shaped basket or bowl, usually rotating around a vertical axis. The solids accumulate and compress as the effect of the centrifugal force, but they are not dewatered. The residual liquid is drained out when the rotation of the bowl is stopped. The layer of solids is removed manually by scraping. Unloading can be achieved semi-automatically first by the use of a skimmer pipe to remove the residual liquid and then by lowering a knife blade into the solid and so cutting it out from the bowl. This allows for avoiding the switching-off off of the machine.

The separation is based on the difference in the densities of solid and liquid phases without a porous barrier.

Construction:

It consists of a metallic basket. The basket is suspended on a vertical shaft and is driven by a motor using a suitable power system. The basket contains a non-perforated side wall that retains a solid phase on its sides during centrifugation, Fig.4. The liquid that remains at the top is removed by a skimming tube.

Fig.4: Non-Perforated Basket Centrifuge

Working:

The suspension is fed continuously into the basket. During centrifugation, the solid phase is retained on the sides of the basket, while the liquid remains on the top. The liquid is removed over a weir or through a skimming tube. When a suitable depth of solids has been deposited on the walls of the basket, the operation is stopped. The solids are then scraped off by hand or using a scraper blade.

Applications:

1. The non-perforated basket centrifuge is useful when the deposited solids offer high resistance to the flow of liquid.
2. It allows for avoiding switching off the machine.

Semi-Continuous Centrifuge

Principle: Semicontinuous centrifuge is a filtration centrifuge. The separation is done based on the difference in the densities of the solid and liquid. This separation occurs through a perforated wall. The bowl contains a perforated side wall, Fig.5. During centrifugation, the liquid phase passes through the perforated wall, while the solid phase remains in the bowl. The solid is washed and removed by cutting the sediment using a blade.

Fig.5: Semi-Continuous Centrifuge

Construction: It consists of a rotating basket placed on a horizontal shaft and driven by a motor from the side. The side of the basket is perforated. Surrounding the basket is a stationary casing that collects the filtrate. The slurry is introduced through a pipe that enters the basket through the center. To wash the crystal the wash pipe is also introduced through the center of the basket. The layer of cake is removed by a chute fitted with a knife. The knife cuts down the cake within the basket. The knife-chute assembly is raised with the help of a hydraulic apparatus.

Working: The basket is rotated horizontally by a motor. The slurry is introduced through the slurry entry pipe. The liquid passes out through the perforated side. The crystals remain within the basket. When the cake height is about 2 - 3 inches the slurry entry is stopped by a "feeler-diaphragm valve assembly". The basket rotates at a predetermined time then the cake is washed with water. The basket is rotated for another predetermined time. After that, the hydraulic apparatus raises the knife-chute assembly to cut the cake. The cake is collected through the chute.

Advantages:

1. It is a short-cycle centrifuge.
2. It works automatically reducing labor costs.
3. It can be batch operated when solids can be drained fast from the bowl.

Disadvantage:

1. It has problems during drain discharge.
2. Its high speed considerably breaks crystals.
3. Many moving parts are involved making the construction and functioning more complicated.

Applications:

The semi-continuous type centrifuge has the following applications :

1. Crystals can be separated from the mother liquor.
2. Liquids can be clarified by removing unwanted solid dirt from oils.
3. It is used to purify mononuclear cells from human peripheral blood.

Super Centrifuge

Principle: It is a solid bowl-type continuous centrifuge used for separating two immiscible liquid phases. It is a sedimentation type of centrifuge in which during centrifugation the heavier liquid is thrown against the wall of the bowl while the lighter liquid remains as an inner layer. The two layers are simultaneously separated.

Construction: It consists of a long, hollow, cylindrical bowl with a diameter between 15 - 50 cm which rotates at high speed to generate a settling acceleration of up to about 18,000 g (where g is the acceleration due to gravity) for industrial models and 65,000 g for laboratory models. The bowl is suspended from a flexible spindle at the top and the bottom is fitted loosely in a bush, Fig.6. It is rotated on its vertical axis. Feed is introduced through the bottom through a nozzle. Two liquid outlets are provided at different heights. Inside the bowl, there are three baffles (not shown in the figure) to catch the liquid and force it to travel at the same speed of rotation as the bowel wall.

Fig.6: Super-centrifuge

Working: The centrifuge is allowed to rotate on its vertical axis at about 2000 r.p.m. The feed is introduced at the bottom through the bowl neck using a nozzle under pressure. A distributor disperses the feed to prevent traveling too far along the bowl length. During centrifugation, two liquid phases separate based on their densities. The heavier liquid moves towards the periphery and the lighter liquid forms an inner layer. Both the liquids climb up to the top of the vertical bowl. These two layers are simultaneously separated and removed from different heights through modified outlets. The centrate discharges from the top of the bowl by overflowing into a collecting cover.

Application:

1. A Super centrifuge is used for separating liquid phases of emulsions in foods and pharmaceuticals.

Continuous Centrifuges

Continuous centrifuges constitute the second and third generations. In second-generation centrifuges, there is the continuous discharge of both liquid and solids from the basket, and are the pusher and conical screen types. Examples of third-generation centrifuges include the screen decanter, baffle ring, screen baffle, and siphon centrifuges.

Conical screen centrifuges: These centrifuges are available in a wide variety of designs. The first classification of these is the angle of the screen (a) wide-angle (slip discharge and guide channel centrifuges) and (b) small-angle screens. Small-angle screen centrifuges are subdivided according to the method of solids discharge used: vibration (vibration centrifuge); oscillation (oscillating and tumbler centrifuges); metering (worm or scroll screen centrifuges); and pushing (pusher conical screen centrifuge). Conical screen centrifuges may rotate about either the vertical or the horizontal axis.

Pusher centrifuge: They consist of a rotating perforated drum lined with a slot screen and a push plate reciprocating with a frequency of about 1 Hz, with a variable advance of between 30 and 60 mm. At first sight, the mechanical design of a tumbler appears complicated, but the construction is simple in comparison with other types of the continuous centrifuge. This is reflected in low capital and running costs and its applications, which have covered freely filtering materials such as iron ore, coal fines, and coarser crystalline materials. Although the shape of the drum is conical like that of a conical basket centrifuge, the tumbler typically enables longer residence times.

The geometry and complex motions of many continuous centrifuges make them extremely difficult to formulate. The third generation represents improvements in process technique being built-in into the continuous centrifuges. In the screen decanter centrifuge, sedimentation and filtration are combined to reduce the total time for separation and limit their application to easily filterable products.

In the baffle ring centrifuge, the particles bounce against rotating rings to effect a further release of liquid. In the screen baffle centrifuge, the particles bounce against the screens. Applications of both the ring and screen baffle machines are limited to granular particles. The siphon centrifuge is an adaptation of peeler and pendulum machines. The siphoning action downstream of the filter medium increases the pressure difference across the basket, leading to an increase in capacity.

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