Overview
A centrifuge is equipment, generally driven by an electric motor, that rotates an object around a fixed axis while a perpendicular force acts on that axis. Particles in a mixture separate according to their size, shape, density, the viscosity of the medium, and the rotor speed.
This lesson covers the principle of sedimentation and the Svedberg equation, the different types of centrifugation (moving boundary, density gradient, rate zonal and isopycnic), and the safety and maintenance procedures every laboratory must follow when operating a centrifuge.
Learning Objectives
After this lesson you will be able toβ¦- Describe the principle of centrifugation and sedimentation
- Explain the Svedberg equation and the meaning of the Svedberg (S) unit
- Differentiate moving boundary, density gradient, rate zonal and isopycnic centrifugation
- Calculate relative centrifugal force using F = MrΟΒ²
- Apply safety measures and maintenance schedules when operating a centrifuge
Clinical Story
Why This MattersA research technologist needs to separate the 40S and 60S ribosomal subunits from a cell lysate to study protein synthesis. Simply spinning the tube at maximum speed would pellet everything into an uninterpretable mixture. Choosing the correct centrifugation technique β density gradient rate-zonal separation β is what makes the difference between a clean, publishable separation and a ruined sample.
Core Concepts
Centrifugation works on the principle of sedimentation: centripetal acceleration causes denser substances to move outward toward the bottom of the tube, while lighter particles move toward the top (or toward the center in the rotating frame). Particles denser than the solvent sink; particles lighter than the solvent float. The greater the density difference, the faster the particle moves; if there is no density difference (isopycnic conditions), the particle stays still.
A particle in a centrifugal field experiences three forces: the centrifugal force (FC), the buoyant force (FB), and the frictional force (Ff) between the particle and the liquid. Settling is the falling of suspended particles through the liquid; sedimentation is the termination of the settling process.
In the 1920s, T. Svedberg and J.W. Williams coupled mechanics, optics and mathematics to prove that proteins are large molecules that can be 'weighed' in a centrifuge. The sedimentation velocity of a molecule in a centrifugal field is its Svedberg constant (S value), where 1 Svedberg = 10β»ΒΉΒ³ seconds.
The Svedberg equation, s = M(1 β vΟ) / Nf, relates the sedimentation coefficient (s) to the molecular weight (M, a size term), the frictional coefficient (f, a shape term), the partial specific volume (v), the solvent density (Ο) and Avogadro's number (N).
Important: Svedberg coefficients are not additive β a 40S small ribosomal subunit and a 60S large subunit combine to form an 80S ribosome, not a 100S complex, because sedimentation rate depends on shape as well as mass.
Moving boundary (differential) centrifugation: the whole tube is filled with sample; particles distribute between pellet and supernatant depending on size, shape and density. The pellet is a mixture of everything sedimented; only the slowest-sedimenting fraction is purified, often with low yield.
Density gradient centrifugation: separates macromolecules differing only slightly in size or density using a stabilizing gradient of carbohydrate or colloidal silica that prevents convection currents. Two sub-types exist β zonal centrifugation (sample layered on top, particles move through the gradient by mass) and isopycnic centrifugation (particles move until they reach a position matching their own buoyant density).
Rate zonal centrifugation: the sample is applied as a thin zone at the top of a density gradient; particles separate into zones by size, shape and density, and the run is stopped before any particle reaches the bottom of the tube.
Laboratory Principle
Relative centrifugal force is calculated as F = MrΟΒ², where M is the mass of the particle, r is the radius of rotation in cm (distance from the axis), and Ο is the average angular velocity in radians per second (Ο = 2Ο Γ revolutions per 60 minutes). The sedimentation coefficient (S) depends on the particle's mass, its frictional coefficient (shape), and its partial specific volume (density); S values are usually corrected to water at 20Β°C (S20W) so results from different media can be compared.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| Sucrose Gradient Solution | 5β20% (w/v) linear gradient | Stabilizes rate zonal separations and linearizes particle motion | 4Β°C, prepare fresh before each run |
| Cesium Chloride (CsCl) Solution | Self-generating density range up to ~1.7 g/cmΒ³ | Isopycnic (equilibrium) banding of DNA, RNA or viruses | Room temperature, protect from contamination |
| Colloidal Silica Gradient Medium | Manufacturer-specified density range | Alternative density gradient medium to carbohydrate gradients | 2β8Β°C, away from light |
Step-by-Step Procedure
Weigh opposing tubes and balance them by mass (not just volume), since liquids of different density will not balance by volume alone.
Choose a fixed-angle or swing-out rotor and the appropriate technique β moving boundary, density gradient, rate zonal, or isopycnic β based on the separation needed.
For zonal or rate-zonal runs, carefully layer the sample as a thin zone on top of the pre-formed density gradient without disturbing it.
Centrifuge at the calculated RCF (g-force) and duration; for rate zonal runs, stop before any separated particle reaches the bottom of the tube.
Recover the separated bands or pellet β by drilling and draining the tube, decanting the supernatant, or careful pipetting from the top down β for further analysis.
Flow Diagram
Quality Control
Rotor-life management tracks total run hours or cycles per rotor serial number so aging rotors are derated (speed reduced) and eventually retired before they fail under high gravitational stress.
Automatic rotor ID and over-speed protection prevent a non-specified rotor from being run, and confirm the programmed speed never exceeds the maximum rpm rated for that specific rotor.
Reference Values
Normal Rangesβ οΈ Reference ranges may vary between laboratories. Always apply your laboratory's established reference intervals.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| Visible wobbling or shaking during a run | Unbalanced tubes or a failing rotor bearing | Stop the centrifuge immediately, re-check balance; if wobbling persists, remove from service and contact a technician |
| Isopycnically banding DNA in a self-generating CsCl gradient | Takes 36β48 hours; increasing rotor speed only redistributes the gradient, it does not shorten run time | Plan run schedules accordingly; do not attempt to shorten isopycnic runs by raising speed |
| Cracked or leaking centrifuge tube with bio-hazardous material | Contamination risk to the rotor chamber and operator | Inspect all tubes for cracks before use; discard damaged tubes; open safety buckets only inside a biological safety cabinet |
Common Errors & How to Avoid Them
Cause: Loading tubes of unequal mass (or balancing by volume instead of mass when densities differ) opposite each other in the rotor
Prevention: Always balance opposing tubes by mass on an analytical balance before starting the run; use a matched water-filled tube if needed
Cause: Assuming higher rotor speed shortens equilibrium banding time
Prevention: Understand that isopycnic banding is time-, not speed-, dependent β increasing speed only shifts the gradient position, it does not accelerate equilibrium
Cause: Using a rotor beyond its rated speed or its derated lifespan due to metal fatigue
Prevention: Follow rotor-life management logs; never exceed a rotor's maximum rated rpm; retire rotors per the manufacturer's schedule
Laboratory Tips from the Bench
Never open the centrifuge lid while the rotor is still spinning β the 'safety shutoff' only stops power to the motor; the rotor continues spinning under its own inertia for some time afterward.
When separating particles of similar size but different shape (e.g. rod-shaped vs globular), remember that the particle with the greater frictional coefficient moves slower β shape matters as much as mass.
Remember 'S is for Shape and Size, not just Speed' β the Svedberg coefficient depends on molecular weight (size) AND frictional coefficient (shape), which is why Svedberg values are never simply additive.
Important Notes
A 40S and a 60S ribosomal subunit combine to form an 80S ribosome, not a 100S complex β sedimentation coefficients reflect shape as well as mass and cannot simply be added together.
A centrifuge must never be operated on an uneven or slanted surface, as this increases the risk of vibration, tube breakage, and rotor damage.
Interactive Quiz
Test Your KnowledgeFlashcards
Tap to flipClick or tap any card to reveal the answer. Use arrow keys to navigate in single-card mode.
Clinical Case Study
Apply Your KnowledgeDr. Sharma is preparing a cell lysate sample to study translation efficiency and needs to isolate intact 40S and 60S ribosomal subunits without cross-contamination between the two populations for a downstream sequencing assay.
By applying the lysate as a thin zone on top of a sucrose gradient and centrifuging just long enough to separate the two subunits into distinct bands (rate zonal centrifugation), Dr. Sharma avoided the classic error of over-centrifuging, which would have pelleted both subunits together at the bottom of the tube.
- βRate zonal centrifugation separates particles of similar size but different shape or density into distinct bands
- βThe run must be stopped before particles reach the bottom of the tube, or the separation is lost
- βSvedberg values (40S, 60S, 80S) describe sedimentation behavior, not simple additive mass
Frequently Asked Questions
Because the sedimentation coefficient depends on both molecular weight (size) and the frictional coefficient (shape), not mass alone. When two particles combine, their combined shape changes the frictional drag, so the resulting S value is not the simple sum of the two original values.
Rate zonal centrifugation separates particles by their sedimentation rate (a function of size, shape and density) and is time-dependent β the run must be stopped before particles reach the bottom. Isopycnic centrifugation separates particles purely by matching buoyant density and is independent of time once equilibrium is reached.
If the liquids in opposing tubes have different densities, equal volumes will have different masses, creating an imbalance that can cause dangerous vibration or rotor damage β so tubes must always be balanced by weighing them on an analytical balance.
Quick Revision
10-Minute ReviewKey Takeaways
- Centrifugation exploits differences in particle density, size and shape under a powerful artificial gravitational field.
- The Svedberg (S) unit describes sedimentation velocity; 1S = 10β»ΒΉΒ³ seconds.
- Different centrifugation techniques (moving boundary, density gradient, rate zonal, isopycnic) suit different separation goals.
- Ultracentrifuges can reach up to 500,000 g under refrigeration and vacuum.
- Rotor-life management and over-speed protection are essential safety systems in modern centrifuges.
- Never open a spinning centrifuge lid, and always balance tubes by mass before starting a run.
Competency Checklist
Track Your MasteryReferences
- National Institute of Open Schooling (NIOS). Biochemistry β Module: Centrifugation (Lesson 28).
- Rickwood D. Centrifugation: A Practical Approach. 2nd ed.
- Boyer RF. Biochemistry Laboratory: Modern Theory and Techniques.