Ultracentrifuge – Principle, Types, Parts, Working Procedure, Applications, Advantages & Precautions

Ultracentrifuge – Principle, Types, Parts, Working Procedure, Applications, Advantages & Precautions

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Introduction to Ultracentrifuge

  • An ultracentrifuge is a highly advanced centrifuge that operates at extremely high speeds (60,000–150,000 rpm).
  • It is used to separate very small biological and chemical molecules that cannot be separated by regular centrifuges.
  • Widely used in biotechnology, molecular biology, microbiology, and pharmaceutical labs.
  • Most ultracentrifuges are refrigerated to avoid heat damage caused by high-speed spinning.
  • Can work in batch mode or continuous flow mode, depending on research needs.
Ultracentrifuge
Figure: Ultracentrifuge

*In short: The ultracentrifuge is essential for studying proteins, DNA, RNA, viruses, ribosomes, and organelles with high precision.

Principle of Ultracentrifuge

  • Works on the principle of sedimentation: particles with higher density settle faster than lighter ones when exposed to force.
  • Under normal gravity, this process is slow, so ultracentrifuge applies centrifugal force thousands of times greater than gravity.
  • When sample rotates:
    • Larger & denser molecules → move outward quickly, form pellets at tube bottom.
    • Smaller & lighter molecules → remain suspended in the supernatant.
  • Thus, molecules can be separated based on size, mass, and density.

Parts of an Ultracentrifuge

  1. Rotor – Core part where tubes are placed. Types:
    • Swinging bucket rotor – Most common; gives high concentration of particles.
    • Fixed-angle rotor – Holds tubes at an angle.
    • Vertical rotor – Aligns tubes with centrifugal force; good for density separation.
  2. Drive Motor – Provides power to spin the rotor at very high speeds.
  3. Temperature Control System – Maintains temperature (0–40°C) to prevent heat damage.
  4. Optical Systems (in analytical ultracentrifuge) – Absorbance, interference, and fluorescence systems for real-time observation.
  5. Gradient-Forming Device – Helps in density gradient preparation (sucrose, cesium chloride).
  6. Safety Lid & Chamber – Protects operator, ensures safe operation.
  7. Fraction Collector & Spectrophotometer (in some models) – Collects separated fractions for analysis.

Types of Ultracentrifuges

Ultracentrifuges are mainly of two types:

1. Analytical Ultracentrifuge (AUC)

  • Used for studying and analyzing molecules in real-time.
  • Has detection systems (optical methods) to monitor sedimentation.
  • Can determine:
    • Sedimentation coefficient
    • Molecular mass
    • Size and shape of proteins/nucleic acids
  • Two main approaches:
    • Sedimentation Velocity – Studies how fast molecules move.
    • Sedimentation Equilibrium – Studies balance of forces (density-based).
  • Applications: Studying protein-protein interactions, DNA conformations, macromolecule structures.

2. Preparative Ultracentrifuge

  • Used for separation and isolation of biomolecules.
  • Does not provide real-time analysis (samples are analyzed later).
  • Techniques used:
    • Differential centrifugation – Based on size (large particles pellet at low speed; smaller need higher speed).
    • Density gradient centrifugation – Based on density differences.
    • Isopycnic centrifugation – Particles stop at their matching density point.
  • Applications: Separation of organelles, ribosomes, DNA, RNA, viruses.

Working Procedure of Ultracentrifuge

A. Analytical Ultracentrifuge

  1. Prepare small sample (20–120 mm³) in specialized cells.
  2. Place inside ultracentrifuge.
  3. Rotor spins → molecules migrate outward.
  4. Position tracked by Schlieren optical system.
  5. Data plotted as solute concentration vs radial distance.
  6. Molecular mass and other properties calculated.

B. Preparative Ultracentrifuge

1. Density Gradient Centrifugation

  • Prepare sucrose gradient (high concentration bottom, low top).
  • Place sample on top and load into rotor.
  • Spin at high speed.
  • Molecules move until they reach density equilibrium.
  • Bands collected for analysis.

2. Differential Centrifugation

  • Homogenize sample in buffer.
  • Spin at low speed → large particles (nuclei, cell debris) pellet.
  • Transfer supernatant → spin at higher speed.
  • Repeated until all organelles (mitochondria, ribosomes, etc.) are separated.

Applications of Ultracentrifuge

  • Molecular Biology – DNA, RNA, and protein isolation.
  • Cell Biology – Separation of organelles (mitochondria, nuclei, ribosomes).
  • Virology – Virus purification and density studies.
  • Biochemistry – Analysis of protein-protein interactions.
  • Genetics – DNA banding using CsCl gradients.
  • Pharmaceuticals – Purification of vaccines, enzymes, hormones.
  • Clinical Diagnostics – Detecting biomolecular complexes.
  • Environmental Studies – Analyzing macromolecules in soil/water.

Precautions While Using Ultracentrifuge

  • Always follow manufacturer’s manual.
  • Check rotors for cracks/corrosion regularly.
  • Balance tubes with equal sample volume.
  • If fewer samples, balance with distilled water.
  • Never exceed recommended rotor speed.
  • Keep lid closed during operation.
  • Follow proper rotor care and cleaning guidelines.

Advantages of Ultracentrifuge

  • Can separate very small molecules (proteins, nucleic acids, viruses).
  • Provides quantitative analysis (analytical ultracentrifuge).
  • High reproducibility.
  • Maintains sample integrity with temperature control.
  • Wide applications in biology, medicine, and industry.

Limitations of Ultracentrifuge

  • Very expensive (purchase and maintenance).
  • Requires trained personnel.
  • Risk of rotor damage at very high speeds.
  • Samples must be carefully prepared and balanced.
  • Not suitable for very large sample volumes.

FAQs on Ultracentrifuge

Q1. What is the maximum speed of an ultracentrifuge?
Typically up to 150,000 rpm.

Q2. Which is better: analytical or preparative ultracentrifuge?
Depends on use – analytical for study/analysis, preparative for isolation/separation.

Q3. Why are ultracentrifuges refrigerated?
To prevent heat damage during high-speed rotation.

Q4. Which biomolecules are studied using ultracentrifuge?
DNA, RNA, proteins, viruses, ribosomes, cell organelles.

Conclusion

The ultracentrifuge is one of the most powerful tools in biology and research. Its ability to separate and study macromolecules at nanoscale level makes it vital for molecular biology, genetics, virology, and pharmaceuticals.

By choosing the correct type (analytical or preparative) and following proper procedures, scientists can obtain precise results that are not possible with traditional centrifuges.

In short: Without ultracentrifuges, modern molecular biology and biomedical research would not be possible.

References and Sources

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