Teknik Laboratorium Nutrisi dan Pakan (NTP392) Sentrifugasi. 1

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Sentrifugasi

D E PA R T E M E N I L M U N U T R I S I D A N T E K N O L O G I PA K A N FA K U LTA S P E T E R N A K A N I P B

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Teknik Laboratorium Nutrisi dan Pakan

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http://intp.fapet.ipb.ac.id

Sentrifugasi

 Sentrifugasi ialah proses pemisahan partikel berdasarkan berat partikel tersebut terhadap densitas layangnya (bouyant density).

 Proses pemisahan solid dari liquid dengan prinsip grafitasi. Densitas solid harus lebih besar dari densitas liquid

 Dengan adanya gaya sentrifugal maka akan terjadi perubahan berat partikel dari keadaan normal pada 1 xg (sekitar 9,8 m/s2) menjadi meningkat seiring dengan kecepatan serta sudut kemiringan perputaran partikel tersebut terhadap sumbunya

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Prinsip Pemisahan :

 Partikel yang densitasnya lebih tinggi daripada pelarut turun (sedimentasi), dan partikel yang lebih ringan mengapung ke atas.

 Perbedaan densitas yang tinggi, membuat partikel bergerak lebih cepat. Jika tidak terdapat perbedaan densitas (kondisi isoponik), partikel tetap setimbang.

 Pemisahan sentrifugal menggunakan prinsip dimana objek diputar secara horizontal pada jarak tertentu.

 Apabila objek berotasi di dalam tabung atau silinder yang berisi campuran cairan dan partikel, maka campuran tersebut dapat bergerak menuju pusat rotasi, namun hal tersebut tidak terjadi karena adanya gaya yang berlawanan yang menuju kearah dinding luar silinder atau tabung, yang disebut gaya sentrifugasi.

 Gaya sentrifugal menyebabkan partikel – partikel menuju dinding tabung dan terakumulasi membentuk endapan

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Fungsi Sentrifugasi

1. Pemisahan (padat/cair, padat/cair/cair dan padat/padat/cair)

 Sentrifugasi dapat digunakan untuk pemisahan padat – cair menyediakan padatan berat dari cairan.

 Centrifuge juga dapat digunakan untuk memisahkan fase berat, dan dua fasa cair ringan, dengan salah satu fase ringan yang lebih ringan dari lainnya.

2. Klasifikasi urutan berdasarkan ukuran dan densitas

 Centrifuge digunakan untuk mengklasifikasikan padatan dengan ukuran yang berbeda.

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Types of Centrifuges

1. Small Benchtop – with or without refrigeration – slow speed (eg up to 4000 RPM)

– common in clinical labs (blood/plasma/serum separation) – can take approx (up to) 100 tubes, depending on diameter 2. Microcentrifuges (“microfuge”, “Eppendorf”)

– take tubes of small vols (up to 2 mL)

– very common in biochemistry/molecular biology/biological labs – can generate forces up to ~15,000 x g

– with or without refrigeration

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Types of Centrifuges

3. High Speed centrifuges

– 15,000 – 20,000 RPM

– large sample capacity depending on rotor – normally refrigerated

– research applications

4. Ultracentrifuges → 65,000 RPM (100,000’s x g) – limited lifetime

– expensive

– require special rotors

– care in use – balance critical! – research applications

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Centrifuge Containers

 Bottles – large vols

• Screw-top / snap-top tubes • Open tubes

• Flip-Tops (“Eppendorfs”)

• May be reusable or disposable, depending on application

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Types of Rotors

1. Swing-out (swinging bucket) • Common in low speed centrifuges • Also high speed, ultracentrifuges

• Tubes accommodated in a pivoted bucket which rotates from a vertical to a horizontal position during acceleration

• Bucket returns to vertical as centrifuge decelerates

• Meniscus of sample always remains at right angles to axis of tube

• Six-place rotor (6 buckets) most useful – can spin 2,3,4 or 6 samples (or sets of samples)

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2. Fixed-Angle Rotors

Tubes in pocket at fixed angle in rotor

• Angle 10 to 50 degrees from vertical – at rest and during spin • Use up to 600,000 x g

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3. Vertical Rotors

First introduced in 1970’s – high-speed andultracentrifuges

• Solution re-orientates below 800 rpm, no disruption to gradient • Good for isopycnic and rate-zonal centrifugation

• Not used for pelleting – pellet would be along length of tube and would fall off as liquid decanted

• Also – “near-vertical” rotors – tube angle = 8 degrees

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a. Inspect centrifuge bottles/tubes for cracks before use

b. Cap tubes with the proper lid

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1. Preparing and Loading Samples

c. Clearly label tubes for identification

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1. Preparing and Loading Samples

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1. Preparing and Loading Samples

e. Make a counterbalance for the centrifuge tube you want to put in the centrifuge

◦ Unbalanced tubes can lead to permanent damage of the centrifuge and can be hazards if the rotor breaks free from the rest of the centrifuge.

◦ Balancing is extremely important at higher centrifugation speeds.

◦ Many centrifuges will automatically turn off if it senses an unbalanced load, but older ones may not have this feature

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1. Place the centrifuge on a firm, level surface

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Because of the high speeds the centrifuge spins at, it needs to be housed on a solid, flat surface.

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2. Choose the proper rotor to use at the

speed you need

• Rotors are designated for specific tubes and speeds.

• Use a rotor that fits the tubes of your sample and is within the limit of

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3. Load the tubes opposite each other in

the centrifuge

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• If you are only spinning one sample, you will need to make a balance to load directly opposite the sample tube.

• If you are spinning more than two tubes, only the ones directly opposite each other have to be equal in mass.

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4. Enter the centrifugation speed

• Centrifugation speed is often given in rotations per minute (rpm), or how many times the rotor completes a full rotation in one minute.

• Speed can also be given in relative centrifugal force (RCF) or the G-force on the rotor.

• Modern day centrifuges are digital and have settings for both.

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5. Keep a safe distance while the

centrifuge is running

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• Bumping or moving the centrifuge during use can cause an imbalance and lead to injury.

• Larger centrifuges can spin at very high speeds and are extremely dangerous if they become imbalanced.

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6. Turn off the centrifuge if it is wobbling

• After starting the centrifuge, stay close until it gets up to full speed in case it needs to be turned off. • When a centrifuge is off balance it may shake or

wobble.

• This can damage the equipment and can also lead to injury in severe cases.

• Many centrifuges will automatically turn off if it senses an unbalanced load, but older ones may not have this feature.

• If the centrifuge begins to shake or wobble, it is off balance and you should stop it immediately.

• A small amount of vibration is normal, but excessive wobbling is dangerous.

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7. Open the lid only after the rotor has

completely stopped

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• Many modern centrifuges have a lock on them that will not unlock until the rotor has come to a stop.

• Older centrifuges may not have this feature so make sure the rotor is no longer moving before opening the lid. • Keep power cords away from the ground

to avoid trip hazards that could move the centrifuge.

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8. Remove the tubes carefully after the

centrifuge has completely stopped spinning

• You want to gently remove the tubes so

that the separated suspensions do not mix again.

• You also want to check to see if any of the samples have leaked or tubes have broken. • If a spill occurs, clean rotor and buckets

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9. Wipe down rotor and centrifuge after

each use

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• To keep the centrifuge clean and running smoothly, lightly clean the rotor and wipe down the centrifuge at the end of a run.

• When you are finished, leave the lid open so it can air out and remain dry

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