Centrifugation Calculators

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Centrifugation uses centrifugal force to separate particles, cells, or macromolecules based on their sedimentation rates — determined by size, density, and shape. The applied centrifugal force is expressed as RCF (relative centrifugal force, in × g): RCF = 1.118 × 10⁻⁵ × r × RPM². Differential centrifugation applies a series of increasing speeds to sequentially pellet different cellular components. Density gradient centrifugation (sucrose or cesium chloride gradients) separates particles by buoyant density. Centrifugation is one of the most fundamental laboratory techniques in cell biology, biochemistry, molecular biology, and clinical diagnostics.

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RCF Formula

RCF (× g) = 1.118 × 10⁻⁵ × r (cm) × RPM²

r = rotor radius (cm); RPM = revolutions per minute. Always express centrifuge speed as RCF (× g) in protocols for reproducibility.

Differential Centrifugation

Separates cellular components by sequential centrifugation at increasing speeds: 300–600 × g, 5 min: pellets intact cells and cell debris. 3,000 × g, 15 min: pellets nuclei and heavy mitochondria. 10,000–20,000 × g, 15 min: pellets mitochondria, lysosomes, peroxisomes. 100,000 × g, 60 min: pellets microsomes (ER fragments), ribosomes; supernatant = cytosol.

Density Gradient Centrifugation

Sucrose gradient: layers of increasing sucrose concentration; particles separate based on sedimentation coefficient (Svedberg units, S). Rate-zonal (velocity): separates by size/shape (same density but different sizes). Isopycnic (equilibrium): separates by buoyant density; particles float at density = medium density; CsCl gradients for DNA (native DNA ≈ 1.70 g/mL; denatured ≈ 1.72 g/mL). Percoll gradient: colloidal silica; separates cells, organelles.

Ultracentrifugation

> 100,000 × g; analytical ultracentrifuge: sedimentation velocity and equilibrium analyses; determines molecular weight, shape, purity. Preparative: isolation of ribosomes, membranes, viruses.

Glossary

Centrifugation
Separation of particles by size and density using centrifugal force; RCF = 1.118×10⁻⁵ × r × RPM²; differential centrifugation uses sequential increasing speeds to pellet different cellular components.
Differential Centrifugation
Sequential centrifugation at increasing RCF to pellet different cellular components: cells (300×g), nuclei (3,000×g), mitochondria (10,000×g), microsomes (100,000×g); yields impure but enriched fractions.
Isopycnic Centrifugation
Density gradient centrifugation where particles band at their buoyant density in a CsCl or sucrose gradient; used for DNA isolation (native CsCl ≈ 1.70 g/mL) and high-purity organelle isolation.

Frequently Asked Questions

Centrifugation applies centrifugal force to separate particles based on their sedimentation rate. The centrifugal force 'mimics' gravity but is much stronger (hundreds to millions of × g). Sedimentation depends on: particle size — larger particles sediment faster; particle density — denser particles sediment faster than less dense ones (in a medium of intermediate density); medium viscosity — higher viscosity slows sedimentation; centrifugal force applied — higher RCF = faster sedimentation. Stokes' law (for spherical particles): sedimentation rate = (2r²(ρ_particle − ρ_medium)×g) / (9η). Different cellular components (intact cells, nuclei, mitochondria, microsomes) have different sizes and densities → sequential pelleting at increasing RCF.

Differential centrifugation: sequential application of increasing centrifugal force to separate particles of different sizes/densities. Each speed pellets progressively smaller/less dense particles. Quick and simple but yields impure fractions (e.g., mitochondrial pellet contains some lysosomes). Used for: crude cell fractionation; isolating nuclei, mitochondria, microsomes; concentrating viruses. Density gradient centrifugation: particles move through a gradient of increasing density (sucrose, CsCl, Percoll) until they reach their equilibrium density. Rate-zonal: separate by sedimentation coefficient (size); incomplete separation based on density differences. Isopycnic: complete separation by buoyant density; particles band at the position where their density equals the gradient density. Used for: purifying DNA by CsCl gradient; separating different viruses; isolating organelles with high purity.

Common protocols: Mammalian cells: 200–400 × g, 5 min → cell pellet for passaging or downstream work. Bacteria: 3,000–5,000 × g, 10 min → bacterial pellet. Platelet-rich plasma: 200 × g, 10 min (platelets remain in supernatant). Plasma isolation from blood: 1,500 × g, 10 min → plasma supernatant; 2,000 × g removes platelets. RNA/DNA extraction: 12,000–16,000 × g, 10 min in microcentrifuge → phase separation in TRIzol. Protein precipitation: 10,000–20,000 × g, 10 min → protein pellet. Virus concentration: 100,000 × g, 2h → virus pellet. Ribosome isolation: 100,000–200,000 × g, 2h → ribosome pellet.

An ultracentrifuge generates RCF values above 100,000 × g (up to ~800,000 × g for analytical instruments). Types: Preparative ultracentrifuge: isolates ribosomes, membrane vesicles, viruses, large protein complexes; uses swinging-bucket, fixed-angle, or vertical rotors; requires vacuum and refrigeration. Analytical ultracentrifuge (AUC): measures sedimentation coefficient and diffusion coefficient with real-time optical detection (absorbance, interference); determines: molecular weight (from sedimentation + diffusion coefficients or sedimentation equilibrium); sample homogeneity; self-association/protein-protein interactions; complex stoichiometry. Applications of ultracentrifugation: CsCl density gradient for DNA banding (classical experiment by Meselson and Stahl, 1958, proving semiconservative DNA replication); ribosome subunit separation (70S → 30S + 50S; 80S → 40S + 60S); isolation of exosomes (100,000 × g, 1–2h).