Overview of Differential Centrifugation: RPM Importance in Various Types

Centrifugation is a technique used to separate components of a mixture based on their density, size, and shape by applying centrifugal force. A centrifuge is a machine that spins samples at high speeds, generating centrifugal force to induce sedimentation of particles.

Overview of Differential Centrifugation: RPM Importance in Various Types

1. Fixed-Angle Centrifuge:

  • In a fixed-angle centrifuge, the sample tubes are held at a fixed angle, typically between 20 and 40 degrees from the vertical axis. When the centrifuge spins, particles in the sample move towards the bottom of the tube.
  • These centrifuges are suitable for applications where the particles have significantly different densities, such as separating cells from a liquid medium. They are commonly used in biological research, clinical diagnostics, and pharmaceutical industries.
  • Overview of Differential Centrifugation: RPM Importance in Various Types

2. Swinging-Bucket Centrifuge:

  • In swinging-bucket centrifuges, the sample tubes are held in swinging buckets that swing outward as the centrifuge spins. This swinging motion helps to maintain the sample at a consistent angle relative to the centrifugal force.
  • They are particularly useful for applications where the sample might separate into distinct layers during centrifugation. Swinging-bucket centrifuges are widely utilised in biochemistry, microbiology, and genetic engineering research.

3. Ultracentrifuge:

  • Ultracentrifuges are high-speed centrifuges capable of reaching speeds of up to 100,000 revolutions per minute (rpm) or more. They are used for applications requiring extremely high levels of centrifugal force, such as separating biomolecules like proteins and nucleic acids. Ultracentrifuges are indispensable tools in fields like biochemistry, molecular biology, and biophysics.
  • Ultracentrifugation techniques such as density gradient centrifugation are crucial for isolating subcellular organelles and studying macromolecular interactions .

4. High-Speed Centrifuge:

  • High-speed centrifuges operate at speeds lower than ultracentrifuges but higher than conventional centrifuges. They are commonly used in laboratories for a wide range of applications, including separating cellular components, isolating DNA, and purifying proteins.
  • High-speed centrifuges are versatile and can accommodate various types of sample tubes and rotors. They find applications in medical diagnostics, food science, and environmental analysis. They are also used in the pharmaceutical industry for drug discovery and development.

5. Refrigerated Centrifuge:

  • Refrigerated centrifuges are equipped with cooling systems that maintain the temperature of the samples during centrifugation. This is crucial for applications involving temperature-sensitive samples, such as enzymes or live cells.
  • Refrigerated centrifuges are often used in molecular biology, cell biology, and biochemistry laboratories. They play a vital role in studying biological processes, drug discovery, and vaccine development. These centrifuges are also utilised in clinical diagnostics for analysing blood samples and separating plasma or serum.

6. Preparative Centrifuge:

  • Preparative centrifuges are designed for large-scale purification and isolation of biomolecules or particles. They typically have a higher capacity and can process larger volumes of samples compared to analytical centrifuges.
  • Preparative centrifuges are commonly used in biopharmaceutical production, where large quantities of purified biomolecules are required. They are also employed in industrial settings for the separation of chemicals, minerals, and food products. Preparative centrifugation techniques are crucial for obtaining highly purified samples for downstream analysis and applications.

Sucrose is often used in differential centrifugation as a density gradient medium. By layering different concentrations of sucrose solutions, samples can be centrifuged, allowing particles of different densities to sediment at different levels within the gradient. This enables the separation of various components based on their density.

In summary, centrifugation is a powerful technique used in various scientific fields for separating components of mixtures. Centrifuges play a crucial role in achieving this separation by generating centrifugal force, and they come in various types, each with specific applications and capabilities. Sucrose is frequently employed in differential centrifugation as a density gradient medium to aid in the separation of components based on density differences.

Practical Application of Differential Centrifugation

Isolation of Subcellular Organelles

In cell biology research, scientists utilise sucrose density gradients and differential centrifugation techniques to isolate specific organelles from cell lysates.

Organelle Separation Process

Sequential rounds of centrifugation at increasing speeds, coupled with sucrose density gradients, facilitate the separation of organelles based on their buoyant density.

Analysis and research

Isolated organelles provide valuable material for studying their structure, function, biochemical composition, and involvement in cellular processes.

Experimental Utilisation

Purified organelles obtained through centrifugation are employed in enzyme assays, protein purification, electron microscopy studies, and other experiments, contributing to biomedical research and drug discovery.

🚀 MCQs on Differential Centrifugation & Types of Centrifuges (Maximum Set)

(With Answers & Explanations)

📘 SECTION 1 — Straight / Direct-Fact MCQs

1. Differential centrifugation separates cellular components mainly on the basis of:

a) Color
b) Density and size
c) Temperature
d) pH
Answer: b
Explanation: Centrifugation separates particles based on density, size, and sedimentation rate.

2. Swinging-bucket rotors hold tubes:

a) Vertically fixed
b) Horizontally fixed
c) At variable angles during spinning
d) At a constant angle of 90°
Answer: c
Explanation: Buckets swing outward to align with centrifugal force.

3. Typical speed range of an ultracentrifuge is:

a) 1,000–5,000 rpm
b) 5,000–20,000 rpm
c) 20,000–40,000 rpm
d) 60,000–100,000+ rpm
Answer: d
Explanation: Ultracentrifuges operate at extremely high rpm for molecular separations.

4. Sucrose is used in density gradient centrifugation because it:

a) Is colored
b) Forms layers of different densities
c) Freezes easily
d) Increases viscosity
Answer: b
Explanation: Sucrose solutions can be layered to allow separation by buoyant density.

5. Fixed-angle rotors typically hold tubes at:

a) 0–10°
b) 10–20°
c) 20–40°
d) 50–80°
Answer: c
Explanation: Most fixed-angle rotors maintain 20–40° tilt.

6. Refrigerated centrifuges are essential when working with:

a) Metals
b) Enzymes and live cells
c) Plastics
d) Gases
Answer: b
Explanation: Enzymes and cells are heat-sensitive.

7. The purpose of increasing rpm stepwise in differential centrifugation is to isolate:

a) Entire organisms
b) Organelles in sequence
c) DNA only
d) Only proteins
Answer: b
Explanation: Low speeds pellet nuclei, while higher speeds pellet mitochondria, microsomes, etc.

8. High-speed centrifuges typically operate at:

a) <5,000 rpm
b) 10,000–30,000 rpm
c) 60,000–100,000 rpm
d) >150,000 rpm
Answer: b
Explanation: High-speed centrifuges bridge between standard lab units and ultracentrifuges.

9. Preparative centrifuges are mainly used for:

a) Analytical measurements
b) Small sample volumes
c) Large-scale purification
d) Nuclear magnetic resonance
Answer: c
Explanation: Designed for high-capacity sample processing.

10. Differential centrifugation begins with:

a) The highest speed
b) Very low speed
c) Intermediate speed
d) Random speed selection
Answer: b
Explanation: Start low to pellet heavier organelles first (nuclei).

📗 SECTION 2 — Conceptual / Application MCQs

11. If mitochondria fail to pellet at 12,000 rpm, the most probable reason is:

a) Rotor is too cold
b) Sample is too viscous
c) Inadequate g-force
d) Tube color is wrong
Answer: c
Explanation: Pelleting depends on g-force, not tube color or temperature.

12. Which rotor type gives the sharpest band separation in density gradients?

a) Swinging-bucket
b) Fixed-angle
c) Vertical tube rotor
d) None
Answer: a
Explanation: Swinging buckets allow layers to remain horizontal → sharper bands.

13. A researcher wants to isolate ribosomes. Which centrifuge is essential?

a) Microcentrifuge
b) High-speed centrifuge
c) Ultracentrifuge
d) Preparative centrifuge
Answer: c
Explanation: Ribosomes require >100,000 × g.

14. Best method for separating plasma from blood cells:

a) Ultracentrifugation
b) Refrigerated centrifuge at moderate rpm
c) Density gradient with cesium chloride
d) None
Answer: b
Explanation: Blood cells pellet easily at 2,000–5,000 rpm; sample requires cooling.

15. If a sample heats up during centrifugation, what could happen?

a) Increased enzyme activity
b) Denaturation of proteins
c) Increased pelleting efficiency
d) Better separation
Answer: b
Explanation: Heat damages biomolecules; hence refrigerated units are used.

16. Differential centrifugation sequence is based on:

a) Buoyant density only
b) Size + density + sedimentation rate
c) Color difference
d) Temperature difference
Answer: b
Explanation: All three physical parameters influence sedimentation.

17. Which combination correctly pairs organelle with typical g-force?

a) Nuclei – 100,000 × g
b) Mitochondria – 10,000 × g
c) Ribosomes – 1,000 × g
d) Lysosomes – 500 × g
Answer: b
Explanation: Nuclei pellet at low g; mitochondria at mid g; ribosomes at high g.

18. Sucrose density gradients are especially useful when separating:

a) Particles with almost equal density
b) Very large debris
c) Insoluble pigments
d) Metals
Answer: a
Explanation: Gradient sharply resolves small density differences.

19. In which scenario is a fixed-angle rotor superior?

a) When sharp bands are required
b) When pelleting is the main objective
c) When sample layering must remain horizontal
d) When working with live cells
Answer: b
Explanation: Fixed angles pellet faster and more efficiently.

20. A lab wants to purify viruses; which centrifugation method is best?

a) Low-speed centrifugation
b) High-speed centrifugation only
c) Density gradient ultracentrifugation
d) Preparative centrifugation
Answer: c
Explanation: Virus purification uses sucrose/CsCl gradients at ultrahigh speeds.

📕 SECTION 3 — Assertion–Reason MCQs

21. Assertion (A): Ultracentrifuges require vacuum systems.

Reason (R): High rpm produces friction and heat, which vacuum reduces.
a) A and R both true; R explains A
b) A true; R false
c) A false; R true
d) Both false
Answer: a
Explanation: Vacuum decreases friction and prevents overheating.

22. Assertion (A): Swinging-bucket rotors provide better gradient resolution.

Reason (R): They allow tubes to align perpendicular to centrifugal force.
Answer: a
Explanation: Correct orientation gives sharper horizontal layers.

23. Assertion (A): Differential centrifugation cannot separate proteins.

Reason (R): Proteins require very high g-forces.
Answer: d
Explanation: Both false — proteins can be separated using ultracentrifuges.

24. Assertion (A): Refrigerated centrifuges prevent sample degradation.

Reason (R): High-speed spinning generates heat.
Answer: a
Explanation: Cooling is essential for temperature-sensitive biomolecules.

25. Assertion (A): Fixed-angle rotors do not allow sharp band formation.

Reason (R): Tubes are angled, causing smearing of gradient interfaces.
Answer: a
Explanation: Angled orientation does distort gradient layers.

📙 SECTION 4 — Match the Following

26. Match organelle with rpm (approx)

A. Nuclei — 1. 100,000 rpm
B. Mitochondria — 2. 5,000 rpm
C. Microsomes — 3. 20,000 rpm
D. Ribosomes — 4. 80,000–100,000 rpm

Correct Match:
A → 2
B → 3
C → 4
D → 1

Explanation: Increasing rpm pelleting corresponds to decreasing size.

27. Match centrifuge type with application

A. Ultracentrifuge — 1. Blood plasma separation
B. High-speed centrifuge — 2. Virus purification
C. Refrigerated centrifuge — 3. DNA/protein isolation
D. Table-top centrifuge — 4. Routine sample spin

Correct Match:
A → 2
B → 3
C → 1
D → 4

28. Match rotor type with advantage

A. Swinging bucket — 1. Fast pelleting
B. Fixed angle — 2. Best gradient separation
C. Vertical rotor — 3. Ultrahigh speed resolution

Correct Match:
A → 2
B → 1
C → 3

📘 SECTION 5 — Higher-Order / Mixed MCQs

29. After centrifugation, a researcher observes blurred gradient interfaces. The probable cause is:

a) Wrong sample
b) Using fixed-angle rotor
c) Using swinging-bucket rotor
d) Excess sucrose
Answer: b
Explanation: Fixed angles distort layering.

30. During virus purification, the sample fails to band at the expected location. The best correction is:

a) Reduce rpm
b) Increase sucrose concentration
c) Use a longer gradient
d) Increase temperature
Answer: c
Explanation: Longer gradients increase resolution.

31. Microsomes are typically isolated from:

a) 800 × g
b) 10,000 × g
c) 100,000 × g
d) 500 × g
Answer: c
Explanation: Microsomes require ultracentrifugation.

32. Which factor does NOT influence the sedimentation rate?

a) Particle density
b) Particle size
c) Viscosity of medium
d) Color of the sample
Answer: d
Explanation: Color has no physical role.

33. Which sample requires immediate cooling during centrifugation?

a) Soil
b) Mitochondria
c) DNA
d) RBC pellet
Answer: b
Explanation: Mitochondria are heat-sensitive.

34. In sucrose density centrifugation, particles settle until they reach:

a) Tube bottom
b) Their buoyant density
c) Highest sucrose layer
d) Air interface
Answer: b
Explanation: Density equilibrium principle.

📓 SECTION 6 — Practical / Lab-Based MCQs

35. If RPM is doubled, centrifugal force becomes:

a) Doubled
b) Four times
c) Half
d) Unchanged
Answer: b
Explanation: g ∝ (rpm)².

36. Which factor most strongly affects g-force?

a) Rotor radius
b) Tube color
c) Sample label
d) Brand of tubes
Answer: a
Explanation: g = 1.118 × 10⁻⁵ × r × (rpm)².

37. A sample is not pelleting well. Best solution:

a) Decrease rpm
b) Increase time
c) Shake tubes
d) Lower temperature
Answer: b
Explanation: Longer run enhances pelleting.

38. Purpose of balancing tubes is to prevent:

a) Sample mixing
b) Rotor vibration and mechanical damage
c) Temperature rise
d) Gradient collapse
Answer: b
Explanation: Imbalance may break rotor.