Sedementation When particles are forced through a solution, they experience resistance to movement which depends on properties of the particle such as

Dr. Nikhat Siddiqi 1

Sedementation

• When particles are forced through a solution, they experience resistance to movement which depends on properties of the particle such as mass, shape and density and properties of the solvent such as its temperature, viscosity, density and composition.

• A commonly-used experimental format where this occurs is sedimentation in a centrifugal field which is also called centrifugation.

• This can be used as a preparative strategy to separate complex mixtures present in biological samples.

• Alternatively, it can also be used analytically to determine the mass, shape or density of particles.


Physical Basis of Centrifugation

• Consider a particle of mass, mo, suspended in a solvent of density, ρ.

• This particle would experience an upthrust equivalent to the weight of displaced liquid in accordance with Archimedes’s principle.

• The buoyant mass, m, of the particle is therefore mo minus a correction factor for this upthrust;

m = mo − mo · ν · ρ


• where ν is the partial specific volume of the particle. • This is equal to the volume displaced by the particle and is

approximately equal to the reciprocal of the density of the solute although the degree of particle hydration can affect it.

• For example,charged particles (which attract solvent molecules, compacting them in its vicinity) give smaller values of ν than might be anticipated from particle density alone.

• If this particle is exposed to a centrifugal field (Figure 7.5), it experiences a centrifugal force, F;


F = m · ω2 · r• where ω is the angular velocity of rotation (in

units of radians/sec. with one revolution = 2π radians) and r is the distance of the particle from the centre of rotation (cm).

• Combining Equations gives us;F = mo(1 − ν · ρ) · ω2 · r



• This equation means that, as the mass, angular velocity or distance from the centre of rotation increases, so does the centrifugal force experienced by a particle.


• As the particle passes through the solvent it experiences resistance due to a frictional force, F , operating in the ∗ opposite direction;

F = f · v∗


• where f is a frictional coefficient dependent on the shape and mass of the particle and the viscosity of the solvent and v is the velocity of the particle.

• At the beginning of centrifugation, the particle accelerates through the solvent but eventually the frictional force decreases this acceleration such that a constant velocity called the sedimentation velocity is achieved.

• At this point;• F = F∗• i.e. mo(1 − ν · ρ) · ω2 · r = f · v


• The sedimentation velocity is given by;v = mo(1 − ν · ρ) · ω2 · r/f

• Sedimentation velocity is dependent on the shape of the particle but, for a perfect sphere, this quantity is related to physical properties of the particle and solvent by;

υ = a2[ρp - ρm].ω2.r/18η


• where a is the particle diameter, ρp and ρm are the densities of the particle and solvent, respectively and η is the viscosity of the solvent.

• The inverse relationship with viscosity means that sedimentation velocity decreases markedly with increase in solvent viscosity and (since viscosity is dependent on temperature) with temperature.

• For this reason, sedimentation velocities are usually corrected for different solvent composition and temperature to standard conditions of pure water at 20 ◦C.


• It is difficult to determine ν, ρ and f accurately so a useful measure of differential behavior in a centrifugal field is provided by the sedimentation coefficient, s;


Principle



• This equation relates RCF to RPM.• The RCF is defined as number of times gravity

g.• Centrifuged particles migrate at rate that

depends on mass, shape and density of the particle and density of the medium.





• The unit for s is second.• The sedimentation coefficient is defined as the

ratio of a particle’s sedimentation velocity to the acceleration applied to it.

• The sedimentation coefficient is expressed as svedberg unit.


• The more massive particle tends to move faster than a less massive one.

• The denser particle moves faster than a less dense one.

• The denser the solution the more slowly a particle will move.

• The greater the frictional coefficient, the more slowly the particle.


Instrumentation for Centrifugation



Low Speed Centrifuges







High Speed Centrifuge

• For sensitive biochemical operations.• High speed and temperature control chamber

are essential.• Temperature is maintained at 40C.







Ultracentrifuge

• Most sophisticated and used for analytical and preparative work.

• Because of enormous heat generated, the rotor chamber is refrigerated and placed in vacuum to avoid friction.


Preparative Ultracentrifuges

• Produce centrifugal field of 600000 g.• The rotor chamber is refrigerated, sealed and

evacuated (80000 rpm).


Analytical Ultracentrifuges

• Speed up to 70000 rpm (500000 g).• Consist of rotor which is refrigerated and

evacuated.• Optical system to observe the sendimenting

material.


Applications of Analytical Ultracentrifugation

• Determination of protein homogeneity in solution (or a mixture of forms, e.g. monomer/dimer or aggregates).

• Determination of conformational changes associated with oligomerization or binding of another component

• Determination of molecular weights or subunit stoichiometry (monomer, dimer, trimer etc) in solution using sedimentation equilibrium.

• The analytical ultracentrifuge provides information about the oligomeric state of a protein and is more accurate than gel filtration.




Sample containers

• Centrifuge tubes or bottles of different sizes.• Made of glass, cellulose esters, polycarbonate,

polyethylene, kynar, nylon, stainless steel etc.


APPLICATIONS OF CENTRIFUGATION


Cell Fractionation

• Velocity sedimentation centrifugation separates particles ranging from coarse precipitates to subcellular organelles.




Analytical Measurements



Density Gradient Centrifugation


Zonal Centrifugation Prepared in Density Gradient


• After centrifugation the contents of the tube are fractionated by drop collection from the bottom of the tube.

• In analytical centrifugation optical techniques are used to detect the molecules.

• The fractions obtained can be assayed by radioactivity, chemical tests, enzymatic actvity etc or a combination of these.




Uses

• Separation of enzymes, hormones, RNA-DNA hybrids, ribosomal subunits, subcellular organelles, for the analysis of size distribution of samples of polysomes and lipoprotein fractions.


Isopycnic Centrifugation


Isopycnic Centrifugation


Uses

• Separation of nucleic acids.• Both DNA and RNA are classified according to

their s values.• It is used in analytical centrifuge to determine

the base composition of DNA and to separate linear DNA from circular DNA.


Applications of Analytical Cnetrifugation

• Determine the relative molecular mass.• Estimation of purity of a macromolecule.• Determination of conformational changes in

molecules like DNA and proteins on denaturation.


Reference

• Modern Experimental Biochemistry by Rodney Boyer, third edition, 2000.


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Sedementation When particles are forced through a solution, they experience resistance to movement which depends on properties of the particle such as