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Ultracentrifuge Principles


The ultracentrifuge operates based on the principles of centrifugation, applying additional forces to enhance sedimentation.

  • It relies on the sedimentation principle, where denser particles settle faster than less dense ones under gravity.
  • The ultracentrifuge rotates the sample, creating a centrifugal force perpendicular to the axis, causing particles to move differently.
  • Larger, denser molecules move faster and towards the periphery of the tubes.
  • Smaller, less dense molecules move slower and may remain suspended or float toward the center.
  • After centrifugation, larger and denser particles form pellets at the bottom, while smaller and less dense particles remain in the supernatant or float on the surface.

Ultracentrifuge Essentials: Principles, Types, and Applications | Zoology Optional Notes for UPSC

Types of Ultracentrifuge


Ultracentrifuges serve various purposes and come in two main types:

1. Analytical Ultracentrifuge (AUC)

  • Analytical ultracentrifuges are used for particle analysis.
  • AUC quantitatively analyzes macromolecules in solutions.
  • It determines sedimentation coefficients, offering insights into particle properties like shape, size, and mass.
  • AUC employs three optical systems for real-time monitoring (absorbance, interference, fluorescence).
  • Frequently used for characterizing biomolecules such as proteins and nucleic acids.

2. Preparative Ultracentrifuge

  • Preparative ultracentrifuges isolate and separate particles in a sample through centrifugation.
  • Analysis is performed after centrifugation, distinguishing them from analytical ultracentrifuges.
  • These ultracentrifuges handle processes like density gradient centrifugation, differential centrifugation, and isopycnic centrifugation.
  • Particles can be separated based on their density or size, helping determine their properties.

Ultracentrifuge Components


Ultracentrifuges consist of several essential components:

  • Rotors: Ultracentrifuges use three types of rotors: vertical rotors, swinging bucket rotors, and fixed-angle rotors. Swinging bucket rotors are common in ultracentrifuges.
  • Drive: The drive powers the rotor holding cells or tubes with the sample. It also includes features like temperature control.
  • Optical Systems: Analytical ultracentrifuges feature optical systems (absorbance, interference, fluorescence) for real-time analysis.
  • Gradient-Forming Device: Found in preparative ultracentrifuges, this device helps create density gradients.
  • Refractometers: These devices measure the refractive index and are useful for specific ultracentrifuge applications.
  • Fraction Collector: Used in preparative ultracentrifuges for collecting separated particles.

Ultracentrifuge Operating Procedure


The operation of an ultracentrifuge varies based on the type, whether analytical or preparative:
Analytical Ultracentrifuge:

  • Small samples are placed in analytical cells within the ultracentrifuge.
  • The ultracentrifuge operates, causing biomolecules to migrate radially from the center.
  • The Schlieren optical system is used to determine the distance of molecules from the center.
  • A graph is created to calculate molecular mass.
Preparative Ultracentrifuge:
  • Density Gradient Centrifugation:

    • A density gradient is prepared using sucrose layers.
    • Samples are placed over the gradient, and the centrifuge is operated.
    • Particles separate based on matching density with the medium.
    • Fractions are collected.
  • Differential Centrifugation:

    • The sample is homogenized in a buffer.
    • After centrifugation, pellets form at the tube bottom, separated from supernatant.
    • The supernatant is centrifuged at varying speeds to further separate particles.
    • Particles are identified based on specific indicators.

Uses of Ultracentrifuge


Ultracentrifuges find applications in various fields:

  • Preparative Ultracentrifuges: Used for pelleting cell organelles, nucleic acid separation, and more.
  • Density Gradient Centrifugation: Separates nucleic acids.
  • Analytical Ultracentrifuges: Detect and characterize macromolecules, assess molecular properties, and differentiate between molecular complexes.
  • Determination of Particle Densities: Used to measure the densities of macromolecules.
  • Purification: Prepares biological extracts by separating different components.

Ultracentrifuge Precautions


Ultracentrifuges are delicate instruments and require careful handling:

  • Always follow the manufacturer's manual when operating the ultracentrifuge.
  • Regularly inspect rotors for signs of corrosion and cracks.
  • Ensure the volume of sample in tubes is equal when adding them to the rotor.
  • Balance the number of tubes in the ultracentrifuge.
  • Do not exceed the specified rotor speed.
  • Keep the chamber lid closed during operation.
  • Adhere to the manufacturer's rotor care guidelines to prevent damage.
The document Ultracentrifuge Essentials: Principles, Types, and Applications | Zoology Optional Notes for UPSC is a part of the UPSC Course Zoology Optional Notes for UPSC.
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