Centrifugation and Ultracentrifugation

Principles of Centrifugation - Spin Basics

• Core Principle: Separates substances based on density, size, and shape using centrifugal force.
• Centrifugal Force: Generated by spinning; expressed as multiples of gravity (g) or Relative Centrifugal Force (RCF).
  • RCF Calculation: $RCF = 1.118 \times 10^{-5} \times r \times (RPM)^2$, where 'r' is rotor radius (cm).
• Sedimentation: Particles move based on their properties; denser/larger particles sediment faster.

⭐ Sedimentation Coefficient: Measured in Svedberg units (S); $1S = 10^{-13}$ seconds. Indicates sedimentation velocity per unit of centrifugal field.

Types of Centrifuges & Rotors - Machine Menagerie

Centrifuges:

• Low-Speed (Benchtop): < 10,000 rpm; < 6,000 $g$. Pellets cells, nuclei.
• High-Speed: Up to 25,000 rpm; up to 60,000 $g$. Refrigerated. Pellets organelles, microbes.
• Ultracentrifuge: Up to 80,000 rpm (prep) or 150,000 rpm (analytical); > 600,000 $g$. Refrigerated, vacuum. Isolates viruses, ribosomes, macromolecules.

  ⭐ Analytical ultracentrifuges determine molecular weight & purity.

Rotors:

• Fixed-Angle: Tubes at 14°-40°. Efficient pelleting. For differential centrifugation.
  • Pellet compact, on side/bottom.
• Swinging-Bucket (Horizontal): Tubes swing to 90°. For density gradients (rate-zonal, isopycnic).
  • Bands well-resolved; pellet at bottom.
• Vertical: Tubes vertical. Shortest path. For isopycnic DNA separation (e.g., CsCl). ​

Centrifugation Techniques - Separation Strategies

Separates components by size, shape, density using centrifugal force. Sedimentation rate ($v$) depends on particle properties (e.g., radius $r$, particle density $\rho_p$) and medium properties (e.g., medium density $\rho_m$, viscosity $\eta$).

• Differential Centrifugation (DC)

  • Basis: Different sedimentation rates (primarily size & density).
  • Method: Sequential pelleting at increasing g-forces (↑ g-force).
  • Yields: Pellets of decreasing size/density (e.g., Nuclei → Mitochondria/Lysosomes → Microsomes → Ribosomes).

  ⭐ Mnemonic for pelleting order (largest to smallest): "Never Make Little Mice Run" (Nuclei, Mitochondria/Lysosomes, Microsomes, Ribosomes) 📌

• Density Gradient Centrifugation (DGC)

  • Uses a density gradient medium (e.g., Sucrose, CsCl, Percoll).
  • Rate-Zonal (Size/Shape based):
    • Sample layered on pre-formed shallow gradient.
    • Separates particles by differences in sedimentation rate through the gradient.
    • Particles form distinct zones based on size/shape.
    • E.g., Separation of ribosomal subunits (40S, 60S).
  • Isopycnic (Density based):
    • Particles migrate to a point where their buoyant density equals the gradient density.
    • Equilibrium method; separation independent of time once equilibrium is reached.
    • E.g., Separation of DNA isoforms (plasmid vs chromosomal), organelles.

    💡 Isopycnic: Particles stop when their density matches the gradient's density.

Ultracentrifugation & Applications - Super Spin & Uses

• Achieves very high speeds (>20,000 rpm) & g-forces (>100,000 x g).
• Operates under vacuum & refrigeration to manage heat & air friction.
• Types & Primary Uses:
  • Analytical (AUC):
    • Determines molecular weight, purity, shape of macromolecules.
    • Measures sedimentation coefficient ($S$). Optical detection systems monitor particle movement.
  • Preparative:
    • Isolates/purifies subcellular organelles (e.g., ribosomes, mitochondria), viruses, macromolecules.
    • Used in differential & density gradient (rate-zonal, isopycnic) methods.
• Key Applications:
  • Lipoprotein fractionation (e.g., HDL, LDL, VLDL).
  • Virus isolation & characterization.
  • Studying protein-DNA interactions & macromolecular assembly.
• Svedberg unit ($S$): Unit of sedimentation rate; $1 S = 10^{-13}$ seconds.

⭐ Ultracentrifugation is essential for the separation and characterization of plasma lipoproteins (chylomicrons, VLDL, LDL, HDL) based on their differing densities, which is vital in dyslipidemia studies.

High‑Yield Points - ⚡ Biggest Takeaways

• Differential centrifugation separates by size and density through increasing g-forces.
• Rate-zonal centrifugation separates by sedimentation rate (S value), reflecting mass and shape.
• Isopycnic centrifugation separates particles based solely on their buoyant density.
• Svedberg unit (S) measures sedimentation rate, not directly molecular weight.
• Ultracentrifugation employs very high g-forces for separating macromolecules and organelles.
• Analytical ultracentrifugation determines molecular weight, shape, and purity of macromolecules.