SlideShare a Scribd company logo
1 of 33
CENTRIFUGATION
lseidman@matcmadison.edu
OUTLINE
 Basic theory
 Applications
 Instrumentation
lseidman@matcmadison.edu
SEPARATIONS
 This begins new topic: separations
 Prior to this, talked about measurements and
solution making
lseidman@matcmadison.edu
BIOSEPARATIONS
 Separating and purifying biological materials
 Filtration and centrifugation
 Chromatography and electrophoresis also
common methods
lseidman@matcmadison.edu
PRINCIPLE
Rate of settling of a particle, or the rate of
separation of two immiscible liquids, is
increased many times by the application of
a centrifugal field (force) many times that
of gravity.
lseidman@matcmadison.edu
MANY APPLICATIONS
 Separate two immiscible liquids
 Isolate cellular organelles
 Isolate DNA, RNA, and proteins
 Isolate small particles including
 Bacteria
 Viruses
 Cells
lseidman@matcmadison.edu
SUPERNATANT AND A PELLET
 Supernatant is the liquid at the top
 Pellet is particles at the bottom
lseidman@matcmadison.edu
FORCE IN A CENTRIFUGE IS
PROPORTIONAL TO TWO
THINGS
 First, it depends on how fast the centrifuge
spins
 Second, it depends on the radius of
rotation – think about “crack the whip”
lseidman@matcmadison.edu
lseidman@matcmadison.edu
RELATIVE CENTRIFUGAL
FORCE, RCF
 Also = Xg
 RCF = 11.17(r)(n/1000)2
 Where r = radius in cm from centerline
 n = rotor speed in RPM, revolutions/minute
lseidman@matcmadison.edu
CALCULATING RCF
 Suppose rmin = 3.84 cm
 raverage = 6.47 cm
 rmax = 9.10 cm
 N = 30,000RPM
 Then, what is the RCF
on a particle at
rmin, rave, and rmax?
lseidman@matcmadison.edu
ANSWERS
 38,600 X g
 65,043 X g
 91,482 X g
 Don’t report RPM, report RCF because
everyone’s centrifuge is different
lseidman@matcmadison.edu
HOW FAST DOES A PARTICLE
SEDIMENT?
 It depends on:
 RCFs in the centrifuge
 Size of particle
 Particle density
 Liquid density
 Liquid viscosity
lseidman@matcmadison.edu
 It turns out that if:
 A particle is the same density as the liquid around
it, the particle doesn’t move
 A particle is more dense than the liquid, it moves
down the tube
 A particle is less dense than the liquid, it moves
up!
lseidman@matcmadison.edu
TWO BASIC MODES OF
CENTRIFUGATION
 Most familiar is differential centrifugation
 Also density gradient centrifugation
lseidman@matcmadison.edu
lseidman@matcmadison.edu
lseidman@matcmadison.edu
 On the next slide there is an excerpt from a
research article. Explain how centrifugation
is used in this research project.
lseidman@matcmadison.edu
How Did Scientists Find Cytochrome C? Preparation of
Mitochondria from mouse liver
The mouse livers were removed after sacrifice and dounce homogenized in ice-cold
mitochondria isolation buffer (MIB) containing 250 mM mannitol, 0.5 mM EGTA, 5
mM HEPES, and 0.1% (w/v) BSA (pH 7.2) supplemented with the protease inhibitors
of leupeptin (1 mg/ml), pepstatin A (1 mg/ml), antipain (50 mg/ml), and PMSF (0.1
mM). Unbroken cells and nuclei were pelleted by centrifugation at 600g for 5 min at
4oC. The supernatants were further centrifuged at 10,000g for 10 min at 4oC to pellet
the mitochondria. The mitochondria pellet was resuspended in 4 ml MIB and loaded
onto a continuous Percoll gradient consisted of 30% (v/v) Percoll (Sigma), 225 mM
mannitol, 25 mM HEPES, 0.5 mM EGTA, and 0.1% (w/v) BSA (pH 7.2). The
suspension/gradient was centrifuged at 40,000g for 1 hr. The mitochondria were
removed from the brownish band at 1.10 g/ml with a transfer pipette. The mitochondrial
pellets were washed with MIB by centrifuging for 10 min at 6300g at 4oC. The
mitochondria were then resuspended gently in mitochondria resuspension buffer
containing 400mM mannitol, 10 mM KH2PO4, and 50 mM Tris-HCl (pH 7.2) with 5
mg/ml BSA and stored on ice for up to 4 hr.
http://www.swmed.edu/home_pages/wanglab/Wanglab-pic/protocols.htm
lseidman@matcmadison.edu
INSTRUMENTATION
 Lots of types
 Some go faster and some slower
 Some can take a lot of volume, others little
 Some allow temperature control
 Some allow you to add sample as centrifuge is
running
 Some are specific for pathogens or whenever
aerosols must be avoided
lseidman@matcmadison.edu
TERMINOLOGY
 Desktop, or clinical centrifuges <10,000 RPM
 Superspeeds, 10,000 – 30,000 RPMs
(around 50,000 Xg)
 Ultracentrifuges up to 80,000 RPM and
500,000 Xg
lseidman@matcmadison.edu
INSTRUMENT DESIGN
lseidman@matcmadison.edu
SAFETY!!
 Centrifuges look sturdy, sort of like washing
machines
 But, they are probably the most dangerous
instrument any of you will use
 Also surprisingly easy to damage
 BE CAREFUL!!!!!
lseidman@matcmadison.edu
TWO MAIN SAFETY
CONCERNS
 First is rotor coming off shaft - disaster
lseidman@matcmadison.edu
ROTORS ARE FRAGILE
 Must withstand huge forces
 In an ultracentrifuge, a 1 gram particle
“weighs” 0.65 tons
 Any imperfection will weaken rotor
 Therefore:
lseidman@matcmadison.edu
 Expert design
 Proper use
 Retire at correct time
 Derate (run slower) when necessary
lseidman@matcmadison.edu
PROPER USE OF ROTORS
 Every rotor has a maximum speed
 As high speed rotors age, derate
 With ultrarotors retire them after certain age
or number of revolutions
 Log books record every use and revolution
 Overspeed discs on bottom
lseidman@matcmadison.edu
 Purchase the correct rotors for your
application
 Derate them as necessary – follow
manufacturer’s directions
 Balance, balance , balance
 Check your textbook, p. 561 for guidelines
lseidman@matcmadison.edu
BIGGEST CHALLENGES IS TO
READ THE CATALOGS
 Rotor and tubes must match application
 Rotor and tubes must match centrifuge
 Rotor and tubes each have maximum RPMs
at which can use them – and may be different
 Use the slower value
lseidman@matcmadison.edu
 May need adaptors to fit certain tubes into
certain rotors
 This is because rotors are engineered to take
varying size and styles of tubes
 Makes them versatile, but also requires complex
combinations of adaptors and tubes
lseidman@matcmadison.edu
GENERAL RULES
 Protect the rotors from:
 Scratches
 Moisture
 Spills
 Alkaline detergents (like Countoff, for
radioisotopes)
lseidman@matcmadison.edu
 Follow manufacturer’s directions and NEVER
NEVER NEVER NEVER! spin faster than is
supposed to go
 Select tubes that can handle the samples and
speeds you are using
 Keep your hands and hair out of centrifuges!
 Keep your hands out of rotors
 Wear glasses
 Make sure you know what you are doing
lseidman@matcmadison.edu
MUCH MORE SUBTLE, BUT
ALSO DANGEROUS,
 Aerosols
 Inevitable with normal centrifuge and very
high levels are released if accident occurs
 Special centrifuges use special rotors, caps,
and seals that prevent leakage
 Containment

More Related Content

What's hot

Types of centrifuges
Types of centrifugesTypes of centrifuges
Types of centrifuges
Shilpa Bhat
 
1957992 635169139917595000
1957992 6351691399175950001957992 635169139917595000
1957992 635169139917595000
MANJU461988
 
6 centrifugation (1)
6 centrifugation (1)6 centrifugation (1)
6 centrifugation (1)
alem010
 

What's hot (20)

Ultra centrifuge
Ultra centrifugeUltra centrifuge
Ultra centrifuge
 
Centrifugation principle and types by Dr. Anurag Yadav
Centrifugation principle and types by Dr. Anurag YadavCentrifugation principle and types by Dr. Anurag Yadav
Centrifugation principle and types by Dr. Anurag Yadav
 
Types of centrifuges
Types of centrifugesTypes of centrifuges
Types of centrifuges
 
1957992 635169139917595000
1957992 6351691399175950001957992 635169139917595000
1957992 635169139917595000
 
tissues
tissuestissues
tissues
 
Manorma negi
Manorma negi Manorma negi
Manorma negi
 
Types of centrifuges
Types of centrifugesTypes of centrifuges
Types of centrifuges
 
Centrifugation
Centrifugation Centrifugation
Centrifugation
 
Centrifugation techniques by kk sahu
Centrifugation techniques by kk sahuCentrifugation techniques by kk sahu
Centrifugation techniques by kk sahu
 
Centrifugation 1 (1)
Centrifugation 1 (1)Centrifugation 1 (1)
Centrifugation 1 (1)
 
Centrifugation by Dr. Anurag Yadav
Centrifugation by Dr. Anurag YadavCentrifugation by Dr. Anurag Yadav
Centrifugation by Dr. Anurag Yadav
 
differential centrifugation
differential centrifugationdifferential centrifugation
differential centrifugation
 
Centrifugation
CentrifugationCentrifugation
Centrifugation
 
Centrifugation sunwei 2011.3.17
Centrifugation sunwei 2011.3.17Centrifugation sunwei 2011.3.17
Centrifugation sunwei 2011.3.17
 
centrifuge principle and application
centrifuge principle and applicationcentrifuge principle and application
centrifuge principle and application
 
Density gradient centrifugation
Density gradient centrifugationDensity gradient centrifugation
Density gradient centrifugation
 
Centrifuge_Report
Centrifuge_ReportCentrifuge_Report
Centrifuge_Report
 
centrifuge
centrifugecentrifuge
centrifuge
 
Prvn ppt
Prvn pptPrvn ppt
Prvn ppt
 
6 centrifugation (1)
6 centrifugation (1)6 centrifugation (1)
6 centrifugation (1)
 

Similar to Centrifugation

Membrane filtration
Membrane filtrationMembrane filtration
Membrane filtration
Hana Gates
 
Genomic dna from different biological materials
Genomic dna from different biological materialsGenomic dna from different biological materials
Genomic dna from different biological materials
CAS0609
 

Similar to Centrifugation (20)

Membrane filtration
Membrane filtrationMembrane filtration
Membrane filtration
 
Centrifuge.pptx
Centrifuge.pptxCentrifuge.pptx
Centrifuge.pptx
 
dialysis, ultrafiltration and lyophilization
dialysis, ultrafiltration and lyophilizationdialysis, ultrafiltration and lyophilization
dialysis, ultrafiltration and lyophilization
 
Protein/DNA Sample preparation methods
Protein/DNA Sample preparation methodsProtein/DNA Sample preparation methods
Protein/DNA Sample preparation methods
 
Centrifugation
CentrifugationCentrifugation
Centrifugation
 
Use of Centrifuge
Use of CentrifugeUse of Centrifuge
Use of Centrifuge
 
Centrifugation
CentrifugationCentrifugation
Centrifugation
 
Fractionation of cells
Fractionation of cellsFractionation of cells
Fractionation of cells
 
Genomic dna from different biological materials
Genomic dna from different biological materialsGenomic dna from different biological materials
Genomic dna from different biological materials
 
Lysate Preparation Technical Tips
Lysate Preparation Technical TipsLysate Preparation Technical Tips
Lysate Preparation Technical Tips
 
Centrifugation- Techniques in Biochemistry
Centrifugation- Techniques in BiochemistryCentrifugation- Techniques in Biochemistry
Centrifugation- Techniques in Biochemistry
 
Ultracenrifugation by kanchana sivabalan
Ultracenrifugation by kanchana sivabalanUltracenrifugation by kanchana sivabalan
Ultracenrifugation by kanchana sivabalan
 
Final Centrifugation Lyophilization and UF.pptx
Final Centrifugation Lyophilization  and UF.pptxFinal Centrifugation Lyophilization  and UF.pptx
Final Centrifugation Lyophilization and UF.pptx
 
protei purification slide share mani upadhyay
protei purification slide share mani upadhyayprotei purification slide share mani upadhyay
protei purification slide share mani upadhyay
 
Equipment related to molecular biology
Equipment related to molecular biologyEquipment related to molecular biology
Equipment related to molecular biology
 
Analytical Ultracentrifugation of protein.
Analytical Ultracentrifugation of protein.Analytical Ultracentrifugation of protein.
Analytical Ultracentrifugation of protein.
 
Downstream processing - industrial microbiology
Downstream processing - industrial microbiology Downstream processing - industrial microbiology
Downstream processing - industrial microbiology
 
Chap9 downstream processing
Chap9 downstream processingChap9 downstream processing
Chap9 downstream processing
 
Recovery of protein from animal tissues by aqueous two phase system
Recovery of  protein from animal tissues by aqueous two phase systemRecovery of  protein from animal tissues by aqueous two phase system
Recovery of protein from animal tissues by aqueous two phase system
 
Protein recovery by atps
Protein recovery by atpsProtein recovery by atps
Protein recovery by atps
 

More from christanantony

C dna and genomic libraries copy
C dna and genomic libraries   copyC dna and genomic libraries   copy
C dna and genomic libraries copy
christanantony
 

More from christanantony (20)

Pcr molecular diagnosis
Pcr molecular diagnosisPcr molecular diagnosis
Pcr molecular diagnosis
 
Pcr and its application
Pcr and its applicationPcr and its application
Pcr and its application
 
Gene silencing amirtham
Gene silencing   amirthamGene silencing   amirtham
Gene silencing amirtham
 
Cloning pcr amirtham
Cloning pcr   amirthamCloning pcr   amirtham
Cloning pcr amirtham
 
C dna and genomic libraries amirtham
C dna and genomic libraries   amirthamC dna and genomic libraries   amirtham
C dna and genomic libraries amirtham
 
Spectroscopy amirtham
Spectroscopy   amirthamSpectroscopy   amirtham
Spectroscopy amirtham
 
Electrophoresis amirtham
Electrophoresis   amirthamElectrophoresis   amirtham
Electrophoresis amirtham
 
Crystallography amirtham
Crystallography   amirthamCrystallography   amirtham
Crystallography amirtham
 
Chromatography amirtham
Chromatography   amirthamChromatography   amirtham
Chromatography amirtham
 
Centrifugation amirtham
Centrifugation   amirthamCentrifugation   amirtham
Centrifugation amirtham
 
Calorimeter
CalorimeterCalorimeter
Calorimeter
 
C dna and genomic libraries copy
C dna and genomic libraries   copyC dna and genomic libraries   copy
C dna and genomic libraries copy
 
Daisy gene 123
Daisy gene  123Daisy gene  123
Daisy gene 123
 
Presentation
Presentation Presentation
Presentation
 
Cloning pcr
Cloning pcrCloning pcr
Cloning pcr
 
Labelling of dna
Labelling of dnaLabelling of dna
Labelling of dna
 
Spectroscopy
SpectroscopySpectroscopy
Spectroscopy
 
Electrophoresis wps office
Electrophoresis wps officeElectrophoresis wps office
Electrophoresis wps office
 
Crystallography
CrystallographyCrystallography
Crystallography
 
Centrifugation by akasthiya
Centrifugation  by akasthiyaCentrifugation  by akasthiya
Centrifugation by akasthiya
 

Recently uploaded

The basics of sentences session 4pptx.pptx
The basics of sentences session 4pptx.pptxThe basics of sentences session 4pptx.pptx
The basics of sentences session 4pptx.pptx
heathfieldcps1
 
Neurulation and the formation of the neural tube
Neurulation and the formation of the neural tubeNeurulation and the formation of the neural tube
Neurulation and the formation of the neural tube
SaadHumayun7
 

Recently uploaded (20)

REPRODUCTIVE TOXICITY STUDIE OF MALE AND FEMALEpptx
REPRODUCTIVE TOXICITY  STUDIE OF MALE AND FEMALEpptxREPRODUCTIVE TOXICITY  STUDIE OF MALE AND FEMALEpptx
REPRODUCTIVE TOXICITY STUDIE OF MALE AND FEMALEpptx
 
Dementia (Alzheimer & vasular dementia).
Dementia (Alzheimer & vasular dementia).Dementia (Alzheimer & vasular dementia).
Dementia (Alzheimer & vasular dementia).
 
Capitol Tech Univ Doctoral Presentation -May 2024
Capitol Tech Univ Doctoral Presentation -May 2024Capitol Tech Univ Doctoral Presentation -May 2024
Capitol Tech Univ Doctoral Presentation -May 2024
 
2024_Student Session 2_ Set Plan Preparation.pptx
2024_Student Session 2_ Set Plan Preparation.pptx2024_Student Session 2_ Set Plan Preparation.pptx
2024_Student Session 2_ Set Plan Preparation.pptx
 
How to the fix Attribute Error in odoo 17
How to the fix Attribute Error in odoo 17How to the fix Attribute Error in odoo 17
How to the fix Attribute Error in odoo 17
 
....................Muslim-Law notes.pdf
....................Muslim-Law notes.pdf....................Muslim-Law notes.pdf
....................Muslim-Law notes.pdf
 
Navigating the Misinformation Minefield: The Role of Higher Education in the ...
Navigating the Misinformation Minefield: The Role of Higher Education in the ...Navigating the Misinformation Minefield: The Role of Higher Education in the ...
Navigating the Misinformation Minefield: The Role of Higher Education in the ...
 
Post Exam Fun(da) Intra UEM General Quiz 2024 - Prelims q&a.pdf
Post Exam Fun(da) Intra UEM General Quiz 2024 - Prelims q&a.pdfPost Exam Fun(da) Intra UEM General Quiz 2024 - Prelims q&a.pdf
Post Exam Fun(da) Intra UEM General Quiz 2024 - Prelims q&a.pdf
 
The Benefits and Challenges of Open Educational Resources
The Benefits and Challenges of Open Educational ResourcesThe Benefits and Challenges of Open Educational Resources
The Benefits and Challenges of Open Educational Resources
 
slides CapTechTalks Webinar May 2024 Alexander Perry.pptx
slides CapTechTalks Webinar May 2024 Alexander Perry.pptxslides CapTechTalks Webinar May 2024 Alexander Perry.pptx
slides CapTechTalks Webinar May 2024 Alexander Perry.pptx
 
The Ultimate Guide to Social Media Marketing in 2024.pdf
The Ultimate Guide to Social Media Marketing in 2024.pdfThe Ultimate Guide to Social Media Marketing in 2024.pdf
The Ultimate Guide to Social Media Marketing in 2024.pdf
 
TỔNG HỢP HƠN 100 ĐỀ THI THỬ TỐT NGHIỆP THPT VẬT LÝ 2024 - TỪ CÁC TRƯỜNG, TRƯ...
TỔNG HỢP HƠN 100 ĐỀ THI THỬ TỐT NGHIỆP THPT VẬT LÝ 2024 - TỪ CÁC TRƯỜNG, TRƯ...TỔNG HỢP HƠN 100 ĐỀ THI THỬ TỐT NGHIỆP THPT VẬT LÝ 2024 - TỪ CÁC TRƯỜNG, TRƯ...
TỔNG HỢP HƠN 100 ĐỀ THI THỬ TỐT NGHIỆP THPT VẬT LÝ 2024 - TỪ CÁC TRƯỜNG, TRƯ...
 
Telling Your Story_ Simple Steps to Build Your Nonprofit's Brand Webinar.pdf
Telling Your Story_ Simple Steps to Build Your Nonprofit's Brand Webinar.pdfTelling Your Story_ Simple Steps to Build Your Nonprofit's Brand Webinar.pdf
Telling Your Story_ Simple Steps to Build Your Nonprofit's Brand Webinar.pdf
 
Pragya Champions Chalice 2024 Prelims & Finals Q/A set, General Quiz
Pragya Champions Chalice 2024 Prelims & Finals Q/A set, General QuizPragya Champions Chalice 2024 Prelims & Finals Q/A set, General Quiz
Pragya Champions Chalice 2024 Prelims & Finals Q/A set, General Quiz
 
Word Stress rules esl .pptx
Word Stress rules esl               .pptxWord Stress rules esl               .pptx
Word Stress rules esl .pptx
 
How to Manage Notification Preferences in the Odoo 17
How to Manage Notification Preferences in the Odoo 17How to Manage Notification Preferences in the Odoo 17
How to Manage Notification Preferences in the Odoo 17
 
[GDSC YCCE] Build with AI Online Presentation
[GDSC YCCE] Build with AI Online Presentation[GDSC YCCE] Build with AI Online Presentation
[GDSC YCCE] Build with AI Online Presentation
 
An Overview of the Odoo 17 Discuss App.pptx
An Overview of the Odoo 17 Discuss App.pptxAn Overview of the Odoo 17 Discuss App.pptx
An Overview of the Odoo 17 Discuss App.pptx
 
The basics of sentences session 4pptx.pptx
The basics of sentences session 4pptx.pptxThe basics of sentences session 4pptx.pptx
The basics of sentences session 4pptx.pptx
 
Neurulation and the formation of the neural tube
Neurulation and the formation of the neural tubeNeurulation and the formation of the neural tube
Neurulation and the formation of the neural tube
 

Centrifugation