Ultracentrifugation is a crucial technique in scientific research, enabling the precise separation of components from a solution based on their size and density. To accomplish this, ultracentrifuges operate at high velocities, often exceeding 100,000 RPM. However, this can pose risks to lab safety and sample integrity if not managed correctly. The best way to mitigate these risks is by ensuring that all users have a comprehensive understanding of ultracentrifuge best practices, including precise sample preparation, appropriate rotor and vessel selection, and rigorous maintenance protocols.
For a rotor to run smoothly and safely, the samples within it must be precisely balanced. Any imbalances beyond the allowable limits—specified by the rotor type—can lead to poor separation and critical rotor damage. Selecting rotors and vessels based on sample characteristics and throughput requirements is also vital for preventing chemical incompatibilities and protecting rotor and sample integrity. After selection and before each ultracentrifugation run, rotors and vessels should be thoroughly inspected. Additionally, implementing a proactive maintenance plan can help prevent critical equipment failures, ensuring consistent and reliable results.
This resource provides a deep dive into the dos and don’ts of ultracentrifugation to enhance research, including:
- Rules for balancing samples
- Key considerations for rotor and vessel selection
- Best practices for ultracentrifuge operation
- Maintenance and sterilization tips for extending the life of your ultracentrifuge and ensuring sample integrity
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Dos and Don’ts in Ultracentrifugation
Recommendations for an optimal ultracentrifugation experience
Rotor & Vessel selection
What sample type? Determines chemical compatibility (i.e.) phenols, bases, best used in PPCO** / PP tube
What volume? Total volume and tube volume
Which parameters? RCF, speed (rpm), time
Separation method? Pelleting / Differential or Gradient
Other consideration? Sterile, Autoclavable, Re-usable, Puncturable, Clear etc…
PP
PP
PC
PP-GF
PP-GF
PPO
PPO
PP
AL
Screw Cap
Inner Lid
O-ring
Bottle
Si
NBR
Rotor and vessel sterilization
Rotors
Vessels
Sterilization Conditions
Aluminium/ PPCO** Titanium /PP
*
PC
PET PE SST
Autoclave
(121 °C 20 min)
Boiling (15 – 30 min)
Ultraviolet ray (200 ~ 300 nm)
Gas
Ethylene Oxide
Formaldehyde
70 % Ethanol Chemical 3 % Hydrogen
Peroxide
3 % Formalin
* Depending on supplier’s recommendations
Rotor cover
Thread part
Rotor packing
No scratches or corrosion
Not dirty or worn
No broken parts
Sufficiently greased
Visual check before centrifugation
Vessels
No scratches or cracks
No deformation
All caps and inner are lids undamaged and in good shape
Rotor parts
Swing-bucket rotor:
No deformation of bucket or hooks
No scratches or corrosion
Rotor body
boreholes
Drive shaft mount
No scratches or corrosion
Not dirty
Any adhesion
EIL-1529-32021
Preparative ultracentrifugation allows the separation of components of a solution based on their size and density, as well as the density of the medium the components are contained. Depending on the type of rotors and the method selected, this technique offers the opportunity to separate macromolecules or nanoparticles, as well as the fractionation of subcellular components or the purification of vesicles or viruses. With speeds of up to 1,050,000xg, ultracentrifuges and their associated rotors are impressive equipment, which is, due to the extreme centrifugal forces that are applied to the samples, often associated with some concerns, especially among new users. Considering the following recommendations will allow all users to experience a smooth and exciting subcellular exploration.
Rotor Type | Features | Separation Method | ||
Pelleting | Density gradient | |||
Fixed-angle |
| |||
Neo-angle |
| |||
Vertical |
| |||
Swing-bucket |
|
PPCO**, PP | PC | PET | PE | SST | Ti | |
Centrifugal force resistance | + + | + + | + | + | – | + |
Heat resistance | – | – | – | – | + + | + + |
Chemical resistance | + | – | – | + | + + | + + |
Acetic acid 5% | * | * | ||||
Acetic acid 60% | * | * | ||||
Nitric acid 10% | ||||||
Nitric acid 50% | * |
Store rotors clean and dry
At room temperature
Avoid humidity, high T °, UV radiation and chemical vapor
Surface: Silicone grease
Thread portion: Lubricant for screw
Packing/ O-ring:
Silicone grease
Wipe off water drops with soft cloth
Dry the rotor upside down
Wash with tap water and diluted detergent (pH 5~9)
Rinse with distilled water
STORAGE
COATING
DRYING
WASHING
Centrifuge
Remove the rotor from the centrifuge
Wipe any condensation in the centrifuge chamber with a soft cloth
Keep the centrifuge door open to allow the centrifuge chamber to dry
Clean the drive shaft mount of the rotor and the drive shaft of the centrifuge once per month
Rotor
After centrifugation
PP: PolyPropylene
(PPCO: Polypropylene-copolymer)
**PPCO is the material of PA tube
PC: PolyCarbonate
PET: PolyEthylene Terephtalate
PE: PolyEthylene
SST: Stainless Steel
Ti: Titanium
PP-GF: Polypropylene-glass fiber
PPO: Polyphenylene oxide
AL: Aluminum
NBR: Nitrile rubber
Si: Silicon rubber
*under specific conditions
++ Highly resistant
+ Resistant
- Not recommended Can be used
Drive shaft
Sample application
Balancing rules
Correct
Wrong
Cap
Tube
Density Sample
Loading the rotor
Keep the weight difference of any 2 tubes which are arranged symmetrically within:
0.2 g for Swing-bucket rotors
0.3 g for Fixed-angle rotors
Installing the rotor
cap’s number
rotor
cap’s mark bucket’s mark
bucket’s number
bucket
Swing-bucket rotor:
Match the bucket’s number with the cap’s number
Tighten the bucket cap until the cap’s mark lines up with the bucket’s mark
Place the bucket in the same numbered rotor position
Make sure that all buckets are set
Never set an empty tube in the bucket
Set a rotor carefully and vertically on the centrifuge shaft
In case of prechilled rotor, please precool the centrifuge before setting the rotor and carefully check and wipe any humidity that may appear during preparation
Carefully set the rotor the drive shaft vertically and silently
Lift the rotor up and down for 2-3 times to ensure right positioning
Eppendorf Offering & Contact
www.eppendorf.com/your-centrifuge-solution
Can’t be used
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