What is centrifugation? Definition and principle of the method. Centrifugation. Its use in various areas of biology. The centrifugation method makes it possible to determine

Date of writing: 27.04.2024

Reading time: 47 minutes

Lecture No. 5

The separation of liquid heterogeneous mixtures is effectively carried out by the centrifugation method, based on the use of centrifugal force. Devices in which liquid heterogeneous mixtures are separated under the action of centrifugal force are called centrifuges.

The centrifugation method is widely used in various fields of technology; The number of types and designs of centrifuges is very large.

The main part of the centrifuge is a drum (a rotor with solid or perforated walls), rotating at high speed on a vertical or horizontal shaft. The separation of heterogeneous mixtures in centrifuges can be carried out either by the principle of settling or by the principle of filtration. In the first case, drums with solid walls are used, in the second - with holes; drums with holes are covered with a filter. If the walls of the drum are solid, then the material, under the influence of centrifugal force, is arranged in layers according to its specific gravity, and a layer of material with a high specific gravity is located directly next to the walls of the drum. If the walls of the drum have holes and are equipped on the inner surface with a filter partition, for example a filter cloth, then the solid particles of the mixture remain on the filter partition, and the liquid phase passes through the pores of the solid sediment and the filter partition and is removed from the drum. The liquid phase separated in a centrifuge is called centrate.

Centrifugal force; separation factor. When the centrifuge drum and the liquid in it rotate, centrifugal force arises as an inertial force.

C=m W 2 / r (1)

m-weight of a rotating body (fluid) in kgf;

r - radius of rotation in m

W - peripheral rotation speed in m/s;

The peripheral rotation speed is defined as:

W=ω r = 2 π n r/60 (2)

P- number of revolutions per minute;

ω-angular velocity of rotation in radians

g-gravity acceleration in m/sec 2, if m=G/g, then centrifugal force WITH, acting on a rotating body with mass m and weight G, is equal to C= G(2π n r/60) 2 /rg Or C ≈ G n 2 r/900 (3)

Equation (2.3) shows that an increase in centrifugal force is more easily achieved by increasing the number of revolutions than by increasing the diameter of the drum. Small-diameter drums with a high number of revolutions can develop greater centrifugal force than large-diameter drums with a low number of revolutions.

Thus, the centrifugal force acting on a particle can be greater than the force of gravity as many times as the acceleration of the centrifugal force is greater than the acceleration of gravity. The ratio of these accelerations is called separation factor and denote Kr:

W 2 / r – acceleration of centrifugal force.

Taking G=1n, we get: Kr=n 2 r /900

For example, for a centrifuge with a rotor with a diameter of 1000 mm (r=0.5 m) rotating at a speed of n=1200 rpm, the separation factor will be 800. The separating effect of the centrifuge increases in proportion to the value of Kp.

The value of K for cyclones is on the order of hundreds. And for centrifuges - about 3000, thus, the driving force of the sedimentation process in cyclones and centrifuges is 2-3 orders of magnitude greater than in settling tanks. Thanks to this, the productivity of cyclones and centrifuges is higher than the productivity of settling tanks, and small particles can be effectively separated in them: in centrifuges with a size of about 1 micron. In cyclones - about 10 microns.

From a comparison of the equations it is clear that the separation factor K p is numerically equal to the centrifugal force that develops during the rotation of a body weighing 1 kg.

Characteristics of centrifugation processes . As mentioned above, centrifugation can be carried out using the settling principle (in solid drums) or the filtration principle (in perforated drums). In their physical essence, both processes differ from each other. In addition, there are separate varieties of each of these processes, which are determined by the content of the solid phase and the degree of its dispersion, as well as the physical properties of the suspension.

Centrifugation in settling drums is carried out both to purify liquids from contaminants contained in small quantities (liquid clarification) and to separate suspensions containing a significant amount of solid phase (settling centrifugation).

Centrifugation in settling drums generally consists of two physical processes: sedimentation of the solid phase (the process follows the laws of hydrodynamics) and compaction of the sediment; The basic laws of soil mechanics (dispersed media) apply to the latter process.

Up to a certain concentration limit of the solid phase (equal to approximately 3-4% by volume), its deposition in the settling drum occurs without the formation of an interface between the solid and the liquid. With increasing concentration, such a surface is formed due to the enlargement and sedimentation of solid particles in the liquid.

The centrifugation process in settling drums is fundamentally different from the separation process in settling tanks. In the latter, the deposition rate can practically be considered constant, since the process occurs in a gravitational field, the acceleration of which does not depend on the coordinates of the falling particle.

Acceleration of the field of centrifugal forces is a variable quantity and depends, at a constant angular velocity, on the radius of rotation of the particle. In addition, the lines of force of the centrifugal field are not parallel to each other and, therefore, the direction of action of the centrifugal forces will be different for different particles (not lying on the same radius of rotation).

Therefore, the laws of settling processes cannot be extended to the centrifugation process in settling drums.

The separation capacity of settling centrifuges is characterized by the performance index (sigma) Σ, which is the product of the area of the cylindrical settling surface F in the rotor and the separation factor Kp.

Σ=F Kr (1), Kr= W2/rg ≈n2 r/900, whence Σ /F=Kr (2)

Considering that the separation factor expresses the ratio of the settling rates of particles in the settling centrifuge and settling tank, in accordance with equality (2), the value of Σ should be considered equal to the area of the settling tank, equivalent in performance for a given suspension to the centrifuge in question. The performance index reflects the influence of all design features of the precipitation centrifuge that determine its separation ability.

When determining the productivity of batch settling centrifuges, it is necessary to take into account the time spent on starting, braking and unloading the centrifuge. Determining the productivity of a filter centrifuge is as difficult as determining the productivity of any filter.

Even more complex is the process of centrifugation in filter drums. The process occurs in three stages:

formation of sediment, compaction of sediment, and finally removal from the pores of the sediment of liquid retained by capillary and molecular forces.

As a result, the entire process of centrifugal filtration cannot be identified with conventional filtration that occurs under the influence of gravity. Only its first period is fundamentally close to conventional filtration and differs from it only in the magnitude of the hydraulic pressure of the liquid flowing through the sediment layer under the influence of centrifugal forces. During this period, moisture in the sediment is in free form and is removed from it most intensively. The second period is similar to the corresponding period during settling centrifugation and, finally, the third is characterized by the penetration of air into the compacted sediment, i.e. mechanical drying of the sediment

The duration of the above periods depends on the physical properties and concentration of the suspensions, as well as on the characteristics of the centrifuge.

The complexity and diversity of centrifugation processes makes it difficult to develop a theory of the process (especially its kinetics) and precise methods for calculating centrifuges.

Centrifuge performance. Typically, the productivity of centrifuges is expressed by the volume of suspension entering the centrifuge per unit time (l/hour), or the weight of the sediment obtained after centrifugation (kg/hour).

Course work

Centrifugation

1. Principle of the method

The separation of substances using centrifugation is based on the different behavior of particles in a centrifugal field. A suspension of particles placed in a test tube is loaded into a rotor mounted on the centrifuge drive shaft.

In a centrifugal field, particles having different densities, shapes or sizes settle at different rates. The sedimentation rate depends on the centrifugal acceleration, which is directly proportional to the angular velocity of the rotor and the distance between the particle and the axis of rotation:

and the centrifugal acceleration will then be equal)

Since one revolution of the rotor is 2p radians, the angular speed of the rotor in revolutions per minute can be written as follows:

Centrifugal acceleration is usually expressed in units g and is called relative centrifugal acceleration, i.e.

When listing the conditions for particle separation, indicate the rotation speed and radius of the rotor, as well as the centrifugation time. Centrifugal acceleration is usually expressed in units g, calculated from the average radius of rotation of a liquid column in a centrifuge tube. Based on the equation, Dole and Kotzias compiled a nomogram expressing the dependence of the OCP on the rotor rotation speed and radius r.

The rate of sedimentation of spherical particles depends not only on centrifugal acceleration, but also on the density and radius of the particles themselves and on the viscosity of the suspension medium. The time required for the sedimentation of a spherical particle in a liquid medium from the liquid meniscus to the bottom of the centrifuge tube is inversely proportional to the sedimentation rate and is determined by the following equation:

where t - sedimentation time in seconds, rj - medium viscosity, g h - particle radius, r h - density of the particle, p - density of the medium, g m - distance from the axis of rotation to the meniscus of the liquid, g d - distance from the axis of rotation to the bottom of the test tube.

As follows from the equation, at a given rotor speed, the time required to settle homogeneous spherical particles is inversely proportional to the square of their radii and the difference in the densities of the particles and the medium and is directly proportional to the viscosity of the medium. Therefore, a mixture of heterogeneous, approximately spherical particles, differing in density and size, can be separated either due to different times of their deposition to the bottom of the test tube at a given acceleration, or due to the distribution of sedimenting particles along the test tube, established after a certain period of time. When separating substances, it is necessary to take into account such important factors as the density and viscosity of the medium. Using the described methods, it is possible to separate cellular organelles from tissue homogenates. The main components of the cell are deposited in the following sequence: first, whole cells and their fragments, then nuclei, chloroplasts, mitochondria, lysosomes, microsomes, and finally ribosomes. The settling of non-spherical particles does not follow an equation, so particles of the same mass but different shapes settle at different speeds. This feature is used when studying the conformation of macromolecules using ultracentrifugation.

consists of isolating biological material for subsequent biochemical studies. In this case, it is possible to take large quantities of initial biological material, for example, seeding microbial cells from batch or continuous cultures, as well as seeding plant and animal cells from tissue cultures and blood plasma. Using preparative centrifugation, large numbers of cellular particles are isolated to study their morphology, structure and biological activity. The method is also used to isolate biological macromolecules such as DNA and proteins from pre-purified preparations.

Analytical centrifugation used primarily for the study of pure or essentially pure preparations of macromolecules or particles, such as ribosomes. In this case, a small amount of material is used, and the sedimentation of the particles under study is continuously recorded using special optical systems. The method allows you to obtain data on the purity, molecular weight and structure of the material. In workshops for students, preparative centrifugation is used much more often than analytical centrifugation, so we will dwell on it in more detail, although both methods are based on general principles.

2. Preparative centrifugation

2.1 Differential centrifugation

This method is based on differences in the sedimentation rates of particles that differ in size and density. The material to be separated, for example tissue homogenate, is centrifuged with a stepwise increase in centrifugal acceleration, which is selected so that at each stage a certain fraction is deposited at the bottom of the tube. At the end of each step, the precipitate is separated from the supernatant and washed several times to ultimately obtain a pure precipitate fraction. Unfortunately, it is almost impossible to obtain an absolutely pure sediment; To understand why this happens, let's look at the process that occurs in a centrifuge tube at the beginning of each centrifugation stage.

At first, all homogenate particles are distributed evenly throughout the volume of the centrifuge tube, so it is impossible to obtain pure preparations of sediments of the heaviest particles in one centrifugation cycle: the first sediment formed contains mainly the heaviest particles, but, in addition, also a certain amount of all the original components. A sufficiently pure preparation of heavy particles can be obtained only by re-suspension and centrifugation of the original sediment. Further centrifugation of the supernatant with a subsequent increase in centrifugal acceleration leads to sedimentation of particles of medium size and density, and then to sedimentation of the smallest particles having the lowest density. In Fig. Figure 2.3 shows a diagram of the fractionation of rat liver homogenate.

Differential centrifugation is probably the most common method for isolating cellular organelles from tissue homogenates. This method is most successfully used to separate cellular organelles that differ significantly from each other in size and density. But even in this case, the resulting fractions are never absolutely homogeneous, and other methods described below are used for their further separation. These methods, based on differences in organelle density, provide more efficient separations by performing centrifugation in solutions with a continuous or stepwise density gradient. The disadvantage of these methods is that it takes time to obtain a solution density gradient.

2.2 Zone-speed centrifugation

The velocity-zonal method, or, as it is also called, s-zonal centrifugation, consists of layering the test sample on the surface of a solution with a continuous density gradient. The sample is then centrifuged until the particles are distributed along the gradient in discrete zones or bands. By creating a density gradient, the mixing of zones resulting from convection is avoided. The speed zone centrifugation method is used to separate RNA-DNA hybrids, ribosomal subunits and other cellular components.

2.3 Isopycnic centrifugation

Isopycnic centrifugation is carried out both in a density gradient and in the usual way. If centrifugation is not carried out in a density gradient, the preparation is first centrifuged so that particles whose molecular weight is greater than that of the particles being studied settle. These heavy particles are discarded and the sample is suspended in a medium whose density is the same as that of the fraction to be isolated, and then centrifuged until the particles of interest settle to the bottom of the tube and particles of lower density float to the surface of the liquid ..

Another method is to layer the sample on the surface of the solution with a continuous density gradient covering the range of densities of all components of the mixture. Centrifugation is carried out until the buoyant density of particles is equal to the density of the corresponding zones, i.e., until the particles are separated into zones. The method is called zonal-isopycnal, or resonant centrifugation, since the main point here is the buoyant density, and not the size or shape of the particles. The density at which particles form isopycnal bands is influenced by the nature of the suspension medium; particles can be permeable to some compounds in the solution and impermeable to others, or they can attach molecules of the solution. When using a zonal rotor, mitochondria, lysosomes, peroxisomes and microsomes are concentrated in bands with 42%, 47%, 47% and 27% sucrose, corresponding to densities of 1.18, 1.21, 1.21 and 1.10 g-cm -3 accordingly. The density of subcellular organelles also depends on their selective absorption of certain compounds. Administration of the detergent Triton WR-1339, which does not cause hemolysis, to rats leads to an increase in the size and decrease in the density of liver lysosomes; the density of mitochondria and peroxisomes remains unchanged. Despite the fact that the sedimentation properties of lysosomes, as a rule, do not change, their equilibrium density in the sucrose gradient decreases from 1.21 to 1.1, which leads to a corresponding separation of the lysosomal-peroxisomal fraction. This feature is used in the quantitative separation of lysosomes, mitochondria and peroxisomes, based on the removal from a homogeneous medium of all particles with a density greater than that of microsomes and subsequent isopycnal centrifugation of the precipitated heavy particles.

2.4 Equilibrium density gradient centrifugation

To create a density gradient, salts of heavy metals, such as rubidium or cesium, as well as sucrose solutions are used. The sample, such as DNA, is mixed with a concentrated solution of cesium chloride. Both the solute and the solvent are initially distributed uniformly throughout the volume. During centrifugation, an equilibrium distribution of concentration and, consequently, density of CsCl is established, since cesium ions have a large mass. Under the influence of centrifugal acceleration, DNA molecules are redistributed, collecting in the form of a separate zone in a part of the test tube with a corresponding density. The method is used primarily in analytical centrifugation and was used by Meselson and Stahl to study the mechanism of DNA replication E. coli . Equilibrium density gradient centrifugation is also one of the methods for separating and studying lipoproteins in human blood plasma.

2. 5 Generating and Extracting Gradients

2.5.1 Nature of gradients

To create density gradients in solutions, sucrose solutions are most often used, sometimes with a fixed pH. In some cases, good separation is obtained when using D 2 0 instead of ordinary water. In the table. Table 2.1 shows the properties of some sucrose solutions.

The choice of gradient is dictated by specific fractionation objectives. For example, Ficol, produced by Pharmacia Fine Chemicals, can replace sucrose in cases where it is necessary to create gradients with high density and low osmotic pressure. Another advantage of Ficol is that it does not pass through cell membranes. To create gradients of higher density, salts of heavy metals, such as rubidium and cesium, are used, however, due to the corrosive effect of CsCl, such gradients are used only in rotors made of resistant metals, such as titanium.”

2.5.2 Method for creating a step density gradient

To create a density gradient, several solutions with successively decreasing density are carefully pipetted into a centrifuge tube. Then the sample is layered onto the topmost layer, which has the lowest density, in the form of a narrow zone, after which the tube is centrifuged. Smooth linear gradients can be obtained by smoothing step gradients when the solution sits for a long time. The process can be speeded up by gently stirring the contents of the tube with a wire or by gently shaking the tube.

2.5.3 Method for creating a smooth density gradient

In most cases, a special device is used to create a smooth density gradient. It consists of two cylindrical vessels of strictly defined identical diameter, communicating with each other at the bottom using a glass tube with a control valve, which allows you to regulate the proportions in which the contents of both vessels are mixed. One of them is equipped with a stirrer and has an outlet through which the solution flows into centrifuge tubes. The denser solution is placed in the mixer; the second cylinder is filled with a solution of lower density. The height of the solution column in both cylinders is set so that the hydrostatic pressure in them is the same. The denser solution is gradually released from the mixer into centrifuge tubes and is simultaneously replaced by an equal volume of a solution of lower density entering the mixer from the second cylinder through the control valve. The homogeneity of the solution in the mixer is ensured by constantly stirring the solution using a stirrer. As the solution is poured into centrifuge tubes, its density decreases and a linear density gradient is created in the tubes. Nonlinear gradients can be created using a system consisting of two cylinders of unequal diameter.

To form density gradients of varying steepness, a system of two mechanically controlled syringes is used, which are filled with solutions of unequal density. Different gradients can be created by changing the relative speed of the pistons.

2.5.4 Removing gradients from centrifuge tubes

After centrifugation and particle separation are complete, the resulting zones must be removed. This is done in several ways, most often by displacement. The centrifuge tube is pierced at the base and a very dense medium, for example a 60-70% sucrose solution, is slowly introduced into its lower part. The solution on top is displaced, and fractions are collected using a syringe, pipette or a special device connected through a tube to the fraction collector. If the tubes are made of celluloid or nitrocellulose, the fractions are removed by cutting the tube with a special blade. To do this, a centrifuge tube secured in a stand is cut directly under the desired area and the fraction is sucked out with a syringe or pipette. With a suitable cutting device design, solution loss will be minimal. Fractions are also collected by piercing the base of the tube with a thin hollow needle. The droplets flowing from the tube through the needle are collected in a fraction collector for further analysis.

2.5.5 Preparative centrifuges and their applications

Preparative centrifuges can be divided into three main groups: general purpose centrifuges, high-speed centrifuges and preparative ultracentrifuges. General purpose centrifuges give a maximum speed of 6000 rpm -1 and an overall speed of up to 6000 g . They differ from each other only in capacity and have a number of replaceable rotors: angular and with hanging cups. One of the features of this type of centrifuge is their large capacity - from 4 to 6 dm 3, which allows them to be loaded not only with centrifuge tubes of 10.50 and 100 cm 3, but also with vessels with a capacity of up to 1.25 dm 3. In all centrifuges of this type, the rotors are rigidly mounted on the drive shaft, and the centrifuge tubes, together with their contents, must be carefully balanced and differ in weight by no more than 0.25 g. An odd number of tubes must not be loaded into the rotor, and if the rotor is not fully loaded, the tubes should be placed symmetrically, one against the other, thus ensuring an even distribution of the tubes relative to the axis of rotation of the rotor.

High speed centrifuges give a maximum speed of 25,000 rpm -1 and an overall speed of up to 89,000g. The rotor chamber is equipped with a cooling system that prevents heat that occurs due to friction when the rotor rotates. As a rule, high-speed centrifuges have a capacity of 1.5 dm 3 and are equipped with replaceable rotors, both angular and with hanging cups.

Preparative ultracentrifuges give a maximum speed of up to 75,000 rpm -1 and a maximum centrifugal acceleration of 510,000 g . They are equipped with both a refrigerator and a vacuum unit to prevent the rotor from overheating due to friction with the air. The rotors of such centrifuges are made of high-strength aluminum or titanium alloys. Rotors made of aluminum alloys are mainly used, but in cases where particularly high speeds are required, rotors made of titanium are used. To reduce vibration resulting from rotor imbalance due to uneven filling of centrifuge tubes, ultracentrifuges have a flexible shaft. Centrifuge tubes and their contents must be carefully balanced to the nearest 0.1 g. Similar requirements must be observed when loading the rotors of general purpose centrifuges.

2.6 Rotor design

2.6.1 Angle rotors and rotors with suspended bowls

Preparative centrifuge rotors are usually of two types - angular and with hanging bowls. They are called angular because the centrifuge tubes placed in them are always at a certain angle to the axis of rotation. In rotors with hanging beakers, the test tubes are installed vertically, and when rotated under the action of the resulting centrifugal force, they move to a horizontal position; the angle of inclination to the axis of rotation is 90°.

In right-angle rotors, the distance traveled by the particles to the corresponding wall of the test tube is very small, and therefore sedimentation occurs relatively quickly. After colliding with the walls of the test tube, the particles slide down and form a sediment at the bottom. During centrifugation, convection currents arise, which greatly complicate the separation of particles with similar sedimentation properties. Nevertheless, rotors of a similar design are successfully used to separate particles whose sedimentation rates vary quite significantly.

In rotors with suspended cups, convection phenomena are also observed, but they are not so pronounced. Convection is the result of the fact that, under the influence of centrifugal acceleration, particles settle in a direction not strictly perpendicular to the axis of rotation, and therefore, as in angular rotors, they strike the walls of the test tube and slide to the bottom.

Convection and vortex effects can be avoided to some extent by using sectorial tubes in hanging bowl rotors and adjusting the rotor speed; The density gradient centrifugation method also lacks the disadvantages listed above.

2.6.2 Continuous rotors

Continuous rotors are designed for high-speed fractionation of relatively small quantities of solid material from large volume suspensions, for example for isolating cells from culture media. During centrifugation, a suspension of particles is added continuously to the rotor; The throughput of the rotor depends on the nature of the deposited drug and varies from 100 cm 3 to 1 dm 3 per minute. The peculiarity of the rotor is that it is an insulated chamber of a special design; its contents do not communicate with the external environment, and therefore do not become polluted or dispersed.

2.6.3 Zone rotors or Anderson rotors

Zonal rotors are made of aluminum or titanium alloys, which are capable of withstanding very significant centrifugal accelerations. They usually have a cylindrical cavity that is closed with a removable lid. Inside the cavity, on the axis of rotation, there is an axial tube onto which a nozzle with blades is placed, dividing the rotor cavity into four sectors. The blades or baffles have radial channels through which a gradient is forced from the axial tube to the periphery of the rotor. Thanks to this design of the blades, convection is reduced to a minimum.

The rotor is filled when it rotates at a speed of about 3000 rpm -1. A pre-created gradient is pumped into the rotor, starting from a layer of the lowest density, which is evenly distributed along the periphery of the rotor and is held at its outer wall perpendicular to the axis of rotation due to centrifugal force . As gradient layers of higher density are subsequently added, there is a continuous shift toward the center of the less dense layers. After the entire gradient has been pumped into the rotor, it is filled to its full volume with a solution called a “cushion”, the density of which matches or slightly exceeds the highest density of the preformed gradient.

Then, through the axial tube, the test sample is layered , which is forced out of the tube into the rotor volume using a solution of lower density, while the same volume of the “cushion” is removed from the periphery. After all these procedures, the rotor rotation speed is brought to operating speed and either zonal-velocity or zonal-isopycnal fractionation is carried out for the required period of time. . The extraction of fractions is carried out at a rotor speed of 3000 rpm -1 . The contents of the rotor are displaced by adding a “cushion” from the periphery; less dense layers are displaced first . Thanks to the special design of the axial channel of the Anderson rotor, mixing of zones when they are displaced does not occur. The output gradient is passed through a recording device, for example the cell of a spectrophotometer, with which the protein content can be determined by absorbance at 280 nm, or through a special radioactivity detector, after which fractions are collected.

The capacity of zonal rotors used at medium speeds varies from 650 to 1600 cm 3, which makes it possible to obtain a fairly large amount of material. Zone rotors are used to remove protein impurities from various preparations and to isolate and purify mitochondria, lysosomes, polysomes and proteins.

2.6.4 Analysis of subcellular fractions

The properties of the subcellular particles obtained during fractionation of the drug can be attributed to the properties of the particles themselves only if the drug does not contain impurities. Therefore, it is always necessary to evaluate the purity of the resulting preparations. The effectiveness of homogenization and the presence of impurities in the preparation can be determined using microscopic examination. However, the absence of visible impurities is not yet reliable evidence of the purity of the drug. To quantify the purity, the resulting preparation is subjected to chemical analysis, which makes it possible to determine its protein or DNA content, its enzymatic activity, if possible, and its immunological properties.

Analysis of the distribution of enzymes in fractionated tissues is based on two general principles. The first of these is that all particles of a given subcellular population contain the same set of enzymes. The second assumes that each enzyme is localized at a specific location within the cell. If this position were true, then enzymes could act as markers for the corresponding organelles: for example, cytochrome oxidase and monoamine oxidase would serve as marker enzymes for mitochondria, acid hydrolases as markers for lysosomes, catalase as a marker for peroxisomes, and glucose-6-phosphatase - a marker of microsomal membranes. It turned out, however, that some enzymes, such as malate dehydrogenase, R-glucuronidase, NADP H-cytochrome c reductase, are localized in more than one fraction. Therefore, the selection of marker enzymes for subcellular fractions in each specific case should be approached with great caution. Moreover, the absence of a marker enzyme does not mean the absence of corresponding organelles It is likely that during fractionation the enzyme is lost from the organelles or is inhibited or inactivated; therefore, at least two marker enzymes are usually determined for each fraction.

Fraction	Volume, cm"	General breeding	Exnumination, 660 nm	Enzyme activity units	The output of activity in the faction,%

2.7 Fractionation by differential centrifugation

2.7.1 Presentation of results

The results obtained from tissue fractionation are most conveniently presented in the form of graphs. Thus, when studying the distribution of enzymes in tissues, the data are best presented in the form of histograms, which make it possible to visually evaluate the results of the experiments.

Enzymatic activity protein content in the sample is determined both in the original homogenate and in each isolated subcellular fraction separately. The total enzymatic activity and protein content in the fractions should not differ greatly from the corresponding values in the original homogenate.

Then the enzymatic activity and protein content in each fraction are calculated as a percentage of the total yield, on the basis of which a histogram is drawn up. The relative amount of protein in each fraction in the order of their isolation is sequentially plotted along the abscissa axis, and the relative specific activity of each fraction is plotted along the ordinate axis. Thus, the enzymatic activity of each fraction is determined by the area of the columns.

2.7.2 Analytical ultracentrifugation

Unlike preparative centrifugation, the purpose of which is to separate substances and purify them, analytical ultracentrifugation is used mainly to study the sedimentation properties of biological macromolecules and other structures. Therefore, in analytical centrifugation, rotors and recording systems of a special design are used: they allow continuous monitoring of the sedimentation of the material V centrifugal field.

Analytical ultracentrifuges can reach speeds of up to 70,000 rpm -1, while creating a centrifugal acceleration of up to 500,000 g . Their rotor, as a rule, has the shape of an ellipsoid and is connected through a string to a motor, which allows you to vary the speed of rotation of the rotor. The rotor rotates in a vacuum chamber equipped with a refrigeration device and has two cells, analytical and balancing, which are installed strictly vertically in the centrifuge, parallel to the axis of rotation. The balancing cell serves to balance the analytical cell and is a metal block with a precision system. It also has two index holes, located at a strictly defined distance from the axis of rotation, with the help of which the corresponding distances in the analytical cell are determined. The analytical cell, whose capacity is usually 1 cm 3, has a sectorial shape. When properly installed in the rotor, despite the fact that it stands vertically, it works on the same principle as a rotor with hanging cups, creating almost ideal sedimentation conditions. At the ends of the analytical cell there are windows with quartz glasses. Analytical ultracentrifuges are equipped with optical systems that allow observation of particle sedimentation throughout the entire centrifugation period. At specified intervals, the sedimented material can be photographed. When fractionating proteins and DNA, sedimentation is monitored by absorption in the ultraviolet, and in cases where the solutions under study have different refractive indices - using the Schlieren system or the Rayleigh interference system. The last two methods are based on the fact that when light passes through a transparent solution consisting of zones with different densities, light refraction occurs at the boundary of the zones. During sedimentation, a boundary is formed between zones with heavy and light particles, which acts as a refractive lens; in this case, a peak appears on the photographic plate used as a detector. During sedimentation, the boundary moves, and, consequently, the peak, by the speed of which one can judge the rate of sedimentation of the material. Interferometric systems are more sensitive than schlieren systems. Analytical cells are single-sector, which are most often used, and two-sector, which are used for the comparative study of solvent and solute.

In biology, analytical ultracentrifugation is used to determine the molecular weights of macromolecules, check the purity of the resulting samples, and also to study conformational changes in macromolecules.

2.8 Applications of analytical ultracentrifugation

2.8.1 Determination of molecular weights

There are three main methods for determining molecular weights using analytical ultracentrifugation: sedimentation rate determination, sedimentation equilibrium method, and sedimentation equilibrium approximation method.

Determination of molecular weight by sedimentation rate - this is the most common method. Centrifugation is carried out at high speeds, so that the particles, initially evenly distributed throughout the entire volume, begin to orderly move along a radius from the center of rotation. A clear interface is formed between the region of the solvent, already free of particles, and the part that contains them. This boundary moves during centrifugation, which makes it possible to determine the rate of sedimentation of particles using one of the above methods, recording this movement on a photographic plate.

The sedimentation rate is determined by the following relationship:

Where X - distance from the axis of rotation in cm,

t - time in s,

w - angular velocity in rad-s -1,

s - sedimentation coefficient of the molecule.

The sedimentation coefficient is the speed per unit acceleration, it is measured in Seedberg units ; 1 Svedberg unit is equal to 10_13 s. The numerical value of s depends on the molecular weight and shape of the particles and is a value characteristic of a given molecule or supramolecular structure. For example, the sedimentation coefficient of lysozyme is 2.15 S; catal aza has a sedimentation coefficient of 11.35S, bacterial ribosomal subunits range from 30 to 50S, and eukaryotic ribosomal subunits range from 40 to 60S.

Where M - molecular weight of the molecule, R - gas constant, T - absolute temperature, s - molecule sedimentation coefficient, D - diffusion coefficient of the molecule, v - partial specific volume, which can be considered as the volume occupied by one gram of dissolved substance, p - density of the solvent.

Sedimentation equilibrium method. Determination of molecular weights by this method is carried out at relatively low rotor speeds, on the order of 7,000-8,000 rpm -1, so that molecules with a large molecular weight do not settle to the bottom. Ultracentrifugation is carried out until the particles reach equilibrium, which is established under the influence of centrifugal forces, on the one hand, and diffusion forces, on the other, i.e., until the particles stop moving. Then, from the resulting concentration gradient, the molecular weight of the substance is calculated according to the formula

Where R - gas constant, T - absolute temperature, ω - angular velocity, p - solvent density, v - partial specific volume, With X And With 2 - solute concentration over distances G G and g 2 from the axis of rotation.

The disadvantage of this method is that to achieve sedimentation equilibrium it takes a long time - from several days to several weeks with continuous operation of the centrifuge.

The method of approaching sedimentation equilibrium was developed in order to get rid of the disadvantages of the previous method associated with the large amount of time required to establish equilibrium. Using this method, molecular weights can be determined when the centrifuged solution is in a state of approaching equilibrium. First, the macromolecules are distributed throughout the entire volume of the analytical cell, then, as centrifugation proceeds, the molecules settle, and the density of the solution in the area of the meniscus gradually decreases. The change in density is carefully recorded, and then, through complex calculations involving a large number of variables, the molecular weight of a given compound is determined using the formulas:

Where R - gas constant, T - absolute temperature, v - partial specific volume, p - solvent density, dcldr - concentration gradient of the macromolecule, g m and g d - distance to the meniscus and the bottom of the test tube, respectively, s m and s d - concentration of macromolecules at the meniscus and at the bottom of the test tube, respectively, M m And M R - molecular weight values determined from the distribution of the concentration of the substance at the meniscus and the bottom of the test tube, respectively.

2.8.2 Evaluation of drug purity

Analytical ultracentrifugation is widely used to evaluate the purity of DNA, virus, and protein preparations. The purity of preparations is undoubtedly very important in cases where it is necessary to accurately determine the molecular weight of a molecule. In most cases, the homogeneity of a preparation can be judged by the nature of the sedimentation boundary, using the method of determining the sedimentation rate: a homogeneous preparation usually gives one sharply defined boundary. Impurities present in the preparation appear as an additional peak or shoulder; they also determine the asymmetry of the main peak.

2.8.3 Study of conformational changes in macromolecules

Another area of application of analytical ultracentrifugation is the study of conformational changes in macromolecules. A DNA molecule, for example, can be single- or double-stranded, linear or circular. Under the influence of various compounds or at elevated temperatures, DNA undergoes a number of reversible and irreversible conformational changes, which can be determined by changes in the sedimentation rate of the sample. The more compact the molecule, the lower its coefficient of friction in solution and vice versa: the less compact it is, the greater the coefficient of friction and, therefore, the slower it will sediment. Thus, differences in the rate of sedimentation of a sample before and after various influences on it make it possible to detect conformational changes occurring in macromolecules.

In allosteric proteins, such as aspartate transcarbamoylase, conformational changes occur as a result of their binding to the substrate and small ligands. Dissociation of the protein into subunits can be caused by treating it with substances such as urea or parachloromercuribenzoate. All these changes can be easily monitored using analytical ultracentrifugation.

Forming tubular products using the method centrifugation. Under centrifugation in the building materials industry... which carry out such an impact are called centrifugation. In the industry of the Republic of Belarus horizontal centrifuges are used...

Particle Deposition

Laboratory work >> Chemistry

Cells already released by low-speed centrifugation from the nucleus, mitochondria and... ultracentrifugation Features of this type centrifugation reflected in it itself... for us an example of use centrifugation in a sucrose density gradient, ...

Using a centrifuge

Coursework >> Industry, production

Various operations in batch centrifuges centrifugation– loading, separation, unloading – occur... distinguish between preparative and analytical centrifugation. With preparative centrifugation the starting biological material is taken...

Description of the presentation Centrifugation. Its use in various areas of biology. by slides

Centrifugation. Its use in various areas of biology. Completed by: Levikov, D. A.

Centrifugation This is the separation of mechanical mixtures into their component parts by the action of centrifugal force. The devices used for this purpose are called centrifuges. The main part of the centrifuge is the rotor with nests for centrifuge tubes mounted in it. The rotor rotates at high speed, as a result of which significant centrifugal forces are created, under the influence of which mechanical mixtures are separated, for example, particles suspended in the liquid are settled.

Processes occurring in a centrifuge The following processes are divided into centrifuges: 1) Centrifugal filtration. 2) Centrifugal settling. 3) Centrifugal clarification.

Centrifugal filtration Centrifugal filtration is the process of separating suspensions in centrifuges with perforated drums. The inner surface of such a drum is covered with filter cloth. The suspension is thrown toward the walls of the drum by centrifugal force, while the solid phase remains on the surface of the fabric, and the liquid, under the influence of centrifugal force, passes through the sediment layer and the fabric is removed out through the holes in the drum. Centrifugal filtration usually consists of three sequential physical processes: 1) filtration with the formation of sediment; 2) sediment compaction; 3) removal from the sediment of liquid held by molecular forces;

Centrifugal sedimentation Centrifugal sedimentation is the process of separating suspensions in centrifuges with drums with solid walls. The suspension is introduced into the lower part of the drum and, under the influence of centrifugal force, is thrown towards the walls. A layer of sediment forms at the walls, and the liquid forms an inner layer and is displaced from the drum by the suspension entering the separation. The liquid rises upward, overflows over the edge of the drum and is removed out. In this case, two physical processes occur: 1) Sedimentation of the solid phase. 2) Sediment compaction.

Centrifugal clarification Centrifugal clarification is the process of separating fine suspensions and colloidal solutions. It is also carried out in solid drums. In its physical essence, centrifugal clarification is a process of free sedimentation of solid particles in a field of centrifugal forces. In drums with solid walls, emulsions are also separated. Under the action of centrifugal force, the components of the emulsion, in accordance with their density, are arranged in the form of delimited layers: an outer layer of liquid with a higher density and an inner layer of lighter liquid. Liquids are discharged separately from the drum.

In clinical and sanitary laboratories, centrifugation is used to separate red blood cells from blood plasma, blood clots from serum, dense particles from the liquid part of urine, etc. For this purpose, either manual centrifuges or electrically driven centrifuges are used, the rotation speed of which can be adjusted. Ultracentrifuges, whose rotor speed exceeds 40,000 rpm, are usually used in experimental practice to separate cell organelles, separate colloidal particles, macromolecules, and polymers.

Centrifugation method in cytology The differential centrifugation method is used for cell fractionation, i.e., stratification of their contents into fractions depending on the specific gravity of various organelles and cellular inclusions. To do this, finely ground cells are rotated in a special apparatus - an ultracentrifuge. As a result of centrifugation, cell components precipitate out of solution, arranged according to their density. More dense structures are deposited at lower centrifugation speeds, and less dense structures are deposited at high speeds. The resulting layers are separated and studied separately.

Centrifugation in botany and plant physiology Centrifugation makes it possible to obtain different fractions of subcellular particles and to study the properties and functions of each fraction separately. For example, chloroplasts can be isolated from spinach leaves, washed from cell fragments by repeated centrifugation in an appropriate medium, and their behavior can be studied under various experimental conditions or their chemical composition can be determined. Then, using various modifications of the technique, it is possible to destroy these plastids and isolate their constituent elements through differential centrifugation (re-sedimentation of particles at different acceleration values). In this way, it was possible to show that plastids contain structures characterized by a very ordered structure - the so-called grana; All grana are located within the chloroplast limiting membrane (chloroplast envelope). The advantages of this method are simply invaluable, since it allows us to identify the existence of functional subunits that are part of larger subcellular particles; in particular, using the method of differential centrifugation, it was possible to show that grana are the main structural element of the chloroplast.

Centrifugation method in virology The Bracquet density gradient centrifugation method can be used both to isolate and obtain quantitative characteristics of plant viruses. As it turned out, this method is fraught with many possibilities and is currently widely used in the field of virology and molecular biology. When conducting studies using density gradient centrifugation, the centrifuge tube is partially filled with a solution, the density of which decreases in the direction from the bottom to the meniscus. Sucrose is most often used to create a gradient in the fractionation of plant viruses. Before centrifugation begins, virus particles can either be distributed throughout the entire volume of the solution or applied to the top of the gradient. Brakke proposed three different techniques for density gradient centrifugation. With isopycpic (equilibrium) centrifugation, the process continues until all particles in the gradient reach a level where the density of the medium is equal to their own density. Thus, fractionation of particles occurs in this case in accordance with differences in their density. Sucrose solutions are not dense enough for isopycnic separation of many viruses. In high-speed zonal centrifugation, the virus is first applied to a previously created gradient. Particles of each type are sedimented through a gradient in the form of a zone, or band, at a rate depending on their size, shape and density. Centrifugation is completed when the particles still continue to sediment. Equilibrium zonal centrifugation is similar to high-speed zonal centrifugation, but in this case centrifugation continues until the isopycnal state is reached. The role of the density gradient in high-speed centrifugation is to impede convection and fix different types of molecules in certain zones. The theory of density gradient centrifugation is complex and not entirely understood. In practice, this is a simple and elegant method that is widely used when working with plant viruses.

Difficulties in using the centrifugation method The use of the differential centrifugation method is associated with many methodological difficulties. Firstly, when particles are released, their structure can be damaged. Therefore, it was necessary to develop special methods for destroying cells that would not cause damage to the structure of subcellular fractions. Secondly, since subcellular particles have membranes, various osmotic effects can occur during their release. Consequently, in order to ensure that the ultrastructure of the objects under study is not destroyed even during their isolation, it is necessary to carefully select the composition of the medium in which the destruction of cells and the deposition of particles occurs. And finally, washing subcellular particles (resuspension of them in the medium and subsequent repeated centrifugation) can lead to the loss of some substances contained in them, which, under the influence of diffusion forces, pass into solution. In this regard, it is sometimes difficult to understand which of the small molecules are actually elements of the structures under study, and which were simply adsorbed on their surface during the isolation process. This situation makes it difficult to accurately determine some functional properties of the selected objects.

2.5.1 Nature of gradients

2.5.2 Method for creating a step density gradient

2.5.3 Method for creating a smooth density gradient

2.5.4 Removing gradients from centrifuge tubes

2.5.5 Preparative centrifuges and their applications

2.6 Rotor design

2.6.1 Angle rotors and rotors with suspended bowls

2.6.2 Continuous rotors

2.6.3 Zone rotors or Anderson rotors

2.6.4 Analysis of subcellular fractions

Fraction	Volume, cm"	General breeding	Exnumination, 660 nm	Enzyme activity units	The output of activity in the faction,%

2.7 Fractionation by differential centrifugation

2.7.1 Presentation of results

2.7.2 Analytical ultracentrifugation

2.8 Applications of analytical ultracentrifugation

2.8.1 Determination of molecular weights

The sedimentation rate is determined by the following relationship:

Where X - distance from the axis of rotation in cm,

t - time in s,

w - angular velocity in rad-s -1,

s - sedimentation coefficient of the molecule.

The disadvantage of this method is that to achieve sedimentation equilibrium it takes a long time - from several days to several weeks with continuous operation of the centrifuge.

2.8.2 Evaluation of drug purity

2.8.3 Study of conformational changes in macromolecules

Particle Deposition

Laboratory work >> Chemistry

Using a centrifuge

Coursework >> Industry, production

Preparative centrifugation is one of the methods for isolating biological material for subsequent biochemical research. Allows you to isolate a significant number of cellular particles for a comprehensive study of their biological activity, structure and morphology. The method is also applicable for isolating basic biological macromolecules. Field of use: medical, chemical and biochemical research.

Classification of preparative centrifugation methods

Preparative centrifugation is carried out using one of the following methods:

Differential. The method is based on the difference in the rate of sedimentation of particles. The material under study is centrifuged with a gradual increase in centrifugal acceleration. At each stage, one of the medium fractions is deposited at the bottom of the test tube. After centrifugation, the resulting fraction is separated from the liquid and washed several times.
Zone-speed. The method is based on layering the test medium on a buffer solution with a known continuous density gradient. The sample is then centrifuged until the particles are distributed along the gradient, forming discrete bands (zones). The density gradient allows you to eliminate mixing of zones and obtain a relatively pure fraction.

Isopycnic. It can be carried out in a density gradient or in the usual way. In the first case, the processed material is layered onto the surface of a buffer solution with a continuous density gradient and centrifuged until the particles are separated into zones. In the second case, the medium under study is centrifuged until a sediment of particles with a high molecular weight is formed, after which the particles under study are isolated from the resulting residue.
Equilibrium. It is carried out in a density gradient of heavy metal salts. Centrifugation allows you to establish the equilibrium distribution of the concentration of the dissolved test substance. Then, under the influence of centrifugal acceleration forces, the particles of the medium are collected in a separate zone of the test tube.

The optimal methodology is selected taking into account the goals and characteristics of the environment being studied.

Classification of preparative laboratory centrifuges

Depending on the design features and operational characteristics, preparative centrifuges can be divided into 3 main groups:

General purpose. Maximum speed – 8,000 rpm with relative centrifugal acceleration up to 6,000 g. Universal laboratory centrifuges are equipped with angular rotors or rotors with hanging containers for storing biological material. They are distinguished by a large capacity from 4 dm 3 to 6 dm 3, which allows the use of standard centrifuge tubes with a volume of 10-100 dm 3 and vessels with a capacity of no more than 1.25 dm 3. Due to the peculiarities of fastening the rotor to the drive shaft, the tubes or vessels must be balanced and differ in weight by a maximum of 0.25 g. It is not permissible to operate the centrifuge with an odd number of tubes. When the rotor is partially loaded, containers with the test medium should be placed symmetrically relative to each other, thereby ensuring their uniform distribution relative to the axis of rotation of the rotor.

Express. Maximum speed – 25,000 rpm with relative centrifugal acceleration up to 89,000 g. To prevent heating due to friction forces arising during rotation of the rotor, the working chamber is equipped with a cooling system. They are equipped with angular rotors or rotors with hanging containers for placing biological material. Capacity of high-speed preparatives
centrifuges – 1.5 dm 3 .
Ultracentrifuges. Maximum speed – 75,000 rpm with relative centrifugal acceleration up to 510,000g. To prevent heating due to friction forces arising during rotation of the rotor, they are equipped with a cooling system and a vacuum unit. Ultracentrifuge rotors are made of ultra-strong titanium or aluminum alloys. To reduce vibrations due to uneven filling, the rotors have a flexible shaft.

A separate category should include specially designed preparative centrifuges designed to carry out certain types of research and solve specific problems. This group includes centrifuges with heating jackets, refrigerated centrifuges and other similar equipment.

Features of the rotor design in preparative centrifuges

Preparative centrifuges are equipped with angular or horizontal rotors:

Angled rotors - test tubes are located at an angle of 20-35° to the axis of rotation during centrifuge operation. The distance traveled by the particles to the corresponding wall of the test tube is small, and therefore their sedimentation occurs quite quickly. Because of the convection currents that occur during centrifugation, fixed-angle rotors are rarely used to separate particles whose size and properties cause significant differences in settling rates.
Horizontal rotors – tubes in this type of rotor are mounted vertically. During the rotation process, under the influence of centrifugal force, the vessels with the processed material move to a horizontal position. These design and operation features make it possible to reduce convection phenomena, so rotors of this type are optimal for separating particles with different sedimentation rates. The use of sectorial tubes allows for an additional reduction in the effects of vortex and convection phenomena.

The type of rotor determines the scope of use of the equipment. The ability to change the rotor allows you to use the same centrifuge model to solve diverse problems. Medical centrifuges for the Centurion laboratory are available in floor-standing or tabletop versions, which makes it possible to use the equipment in any room, regardless of the available space.