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. Dr. Nikhat Siddiqi

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 Dr. Nikhat Siddiqi

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; Dr. Nikhat Siddiqi

Combining Equations gives us; F = mo(1 − ν · ρ) · ω2 · r 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 Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

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. Dr. Nikhat Siddiqi

As the particle passes through the solvent it experiences resistance due to a frictional force, F∗, operating in the opposite direction; F∗ = f · v Dr. Nikhat Siddiqi

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 Dr. Nikhat Siddiqi

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 Dr. Nikhat Siddiqi

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. Dr. Nikhat Siddiqi

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; Dr. Nikhat Siddiqi

Principle Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

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. Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

The sedimentation coefficient is expressed as svedberg unit. 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. Dr. Nikhat Siddiqi

The denser particle moves faster than a less dense one. 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. Dr. Nikhat Siddiqi

Instrumentation for Centrifugation Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Low Speed Centrifuges Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

High Speed Centrifuge For sensitive biochemical operations. High speed and temperature control chamber are essential. Temperature is maintained at 40C. Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

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. Dr. Nikhat Siddiqi

Preparative Ultracentrifuges Produce centrifugal field of 600000 g. The rotor chamber is refrigerated, sealed and evacuated (80000 rpm). Dr. Nikhat Siddiqi

Analytical Ultracentrifuges Speed up to 70000 rpm (500000 g). Consist of rotor which is refrigerated and evacuated. Optical system to observe the sendimenting material. Dr. Nikhat Siddiqi

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. Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Sample containers Centrifuge tubes or bottles of different sizes. Made of glass, cellulose esters, polycarbonate, polyethylene, kynar, nylon, stainless steel etc. Dr. Nikhat Siddiqi

Applications of Centrifugation Dr. Nikhat Siddiqi

Cell Fractionation Velocity sedimentation centrifugation separates particles ranging from coarse precipitates to subcellular organelles. Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Analytical Measurements Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Density Gradient Centrifugation Dr. Nikhat Siddiqi

Zonal Centrifugation Prepared in Density Gradient Dr. Nikhat Siddiqi

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. Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

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. Dr. Nikhat Siddiqi

Isopycnic Centrifugation Dr. Nikhat Siddiqi

Isopycnic Centrifugation Dr. Nikhat Siddiqi

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. Dr. Nikhat Siddiqi

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. Dr. Nikhat Siddiqi

Reference Modern Experimental Biochemistry by Rodney Boyer, third edition, 2000. Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi