Centrifugation techniques

1. 1
Instrumental Analysis
◘ The primary purpose of clinical Chemistry laboratory is :-
♦ facilitate the correct performance of analytic procedures .
♦ yield accurate and precise information
♦ aiding patient diagnosis and treatment
♦ Achieve reliable results …. How ?
- The clinical laboratory scientist be able to correctly use :
• Basic supplies and equipment
• possess an understanding of fundamental concepts critical to any
analytic procedure .
◘ An Instrument For Chemical Analysis :
♣ Converts Information about the physical or chemical Characteristics of
analyte to information that can be manipulated and interpreted by human .
♣ any Analytical instrument can be viewed as a communication device
between the system under study and the investigator .
♠ Stimulus :-
• It is necessary to provide a stimulus .
• It is usually in the form of electromagnetic , Electrical , Mechanical , or nuclear
energy .
• It elicits a response from the system under study whose nature and magnitude are
governed by the fundamental laws of chemistry and physics .
• the resulting information is contained in the phenomena that result from the
interaction of the stimulus with the analyte .
☻ It is important to understand the relationships among instrument components and
the flow of information from the characteristics of the analyte through the
components to the numerical or graphical output produced by the instrument .

2. 2
Centrifugation techniques
◘ Centrifugation :-
• Is carried out by spinning a biological sample at high rate of speed , thus
subjecting it to an intense force ( Artificial gravitation field )
• particles or cells in a liquid suspension, will settle at the bottom of tube due to
gravity (1x g) during long period to time.
• However, the length of time required for such separations is impractical.
• Other particles which are very small in size, will not separate at all
• So, high centrifugal force though method called centrifugation must be used in
these cases.
♦ Sedimentation
• Is the motion of molecules in solutions or, particles in suspensions, in response to an
external force such as gravity, centrifugal force or electric force.
• Centrifugation increase the rate of sedimentation by spinning the samples and creating a
centrifugal force acts on particles in an instrument called a centrifuge.
◘ A centrifuge :-
• found in every biochemistry laboratory
• Used primary for the preparation of biological samples
• use for the analysis of the physical properties of biomolecules ,
Organelles , and cells .
• Most centrifuge techniques fit into one of two categories .

3. 3
1- Preparative Centrifugation :-
- Is one that can be applied to the separation or purification of biological
samples ( cells , organelles , macromolecules , etc. ) by sedimentation .
2- Analytical Centrifugation:
- Analytical procedures are used to measure physical characteristics of
biological samples .
EX : the Purity , Size , Shape , and density of macromolecules may be
defined by centrifugation .

4. 4
◘ Basic principles of centrifugation :-
♦ A particle whether it is ( a floating solid , a precipitate , a macromolecule ,
or a cell organelle ) is subjected to a centrifugal force when it is rotated at a
high rate of speed .
♦ Centrifugal force depends on three variable :
- Mass - Speed - radius .
♦ The speed is expressed in revolutions per minute ( rpm ) .
♦ The generated centrifugal force can be measured in terms of earth’s
gravitational force ( g ) as relative centrifugal force ( RCF )
• Relative centrifugal force (RCF) is the measurement of the force applied to a
sample within a centrifuge.
• Centrifugal force is measured as X g gravity (number times gravity or × g).
• An RCF of 500Xg indicates that the centrifugal force applied is 500 times
greater than earth gravitational force
• r : the radius in centimeters , measured from the center of the centrifuge axis to
the bottom of the test tube shield .
• This can be calculated from the speed (RPM) and the rotational radius (cm)
using the following calculation.
RCF = 1.119 × 10-5
× rpm2
× r
♦ Example: calculation of RCF
◘ Spins at 20 000 revolutions per minute. Distance from the centre of the liquid to axis of rotation is 35
mm (remember to convert this to centimetres = 3.5 cm).
RCF = 1.119 x r x (revolutions per minute)2
x 10 -5
RCF = 1.119 x 3.5 x (20000)2
x 10 -5
= 1.119 x 3.5 x (4 x 108
) x 10 -5
= 1.119 x 3.5 x 4 x 103
RCF = 15666 g [where g means times the force of gravity]

5. 5
♦ we can determine RCF by use of nomogram , since RCF varied with r .
♦ Often an average RFC is determined using a value for r midway between
the top and bottom of the sample container .

6. 6
Instrumentation for centrifugation
♦ The basic centrifuge consists of two components :-
1- An electric motor with drive shaft to spin the sample .
2- A rotor to hold tubes or other container of the sample .
♦ Different classes of centrifuge are available based on several criteria ,
including :-
- refrigeration , rotor head ( eg : Fixed or swinging-bucket or vertical )
- and maximum speed attainable ( i.e. : low-speed , high speed or
ultracentrifuge )

7. 7
1- Low –speed ( clinical ) centrifuges :-
• used for routine sedimentation of relative heavy particles .
• Maximum speed in the range of ( 4000-5000 rpm ) .
• RCF averaging ( 3000 xg ) .
• Useful for the rapid sedimentation of coarse precipitates or red blood cells.
• The sample is centrifuged until the particles are tightly packed into a pellet
at the bottom of the tube .
• Operate at room temp. with no means of temp. control of the sample .
• Two type of rotor ( Fixed angle and swing-bucket ) may be used in the
instrument .

8. 8
2- High-speed Centrifuge :-
• Higher speed and temp. control of the rotor chamber are essential .
• The operator of the high- speed centrifuge can carefully control speed and
temp , which is especially important for carrying out reproducible
centrifugations of temperature-sensitive biological sample .
• Rotor chambers in most instruments are maintained at or near 40
C .
• Instruments are equipped with a brake to slow the rotor rapidly after
centrifugation .
• Rotor were construct from metal such as Aluminum and Titanium .
♠ Although metal rotor have great strength , they do have several
disadvantages :
♦ they are very heavy to handle .
♦ they are not corrosion-resistant .
♦ they become fatigued with use .
- Rotor are now available that are fabricated from carbon-fiber
composite materials .
♠ They have several advantages over heavy metal rotor :
♦ they are 60 % lighter than comparable aluminum and titanium rotor .
♦ Acceleration and deceleration times are reduced .
♦ centrifuge run time are shorter .
♦ Lower service and maintenance costs .
• three types of rotor are available
- Fixed angle - the swinging-bucket - the vertical rotor

9. 9
the swinging-bucket The Fixed angle Rotor the vertical rotor
◘ In swinging-bucket
rotors , the sample tubes
move to a position
perpendicular to the axis of
rotation during
centrifugation .
◘ These are used most
often for density gradient
centrifugation .
◘ Its’ Operation :-
(a) loading of sample
(b) sample at start of
centrifugation
(c) sample during
centrifugation separates
into two components .
(d) Rotor at rest .
◘ Is most commonly used,
consist of a solid piece of metal
with 6-12 holes at angle
between 20o
-45o
.
◘ tubes are fixed at the angle
of rotor cavity.
◘ It can hold larger volumes
than swinging bucket rotor .
◘ Useful for differential
pelleting particles .
◘ Its’ Operation :-
(a) loading sample .
(b) sample at start of centrifugation .
(c) Bands form as molecules
sediment .
(d) Rotor at rest showing separation
of two components.
◘ Vertical rotors hold tubes
parallel to the axis of rotation;
therefore, bands separate
across the diameter of the
tube rather than down the
length of the tube.
◘ The sample tubes remain in
an upright position .
◘ These rotors are used often
for gradient centrifugation .
◘ Its’ Operation :-
(a) loading sample .
(b) Beginning of centrifugation .
(c) & (d) During centrifugation .
(e) Deceleration of sample .
(f) Rotor at rest .

10. 10
3- Micro-centrifuge: (Eppendorf Micro-centrifuge)
(microfuge centrifuges or mini centrifuge or bench top centrifuge)
◘ Used for rapid pelleting of small sample .
Ex : separation of DNA, RNA (eg: Viruses ) and proteins that need
isolation.
◘ Fixed –angle rotor are able to hold up to 18 ( eighteen ) 1.5 or 0.5 ml
tubes .
◘ Speeds: up to 12000 - 15000 rpm. (low to medium).
◘ Force ( RCF ) : ( 11000 – 12000 xg )
◘ - Accelerate to full-speed in 6 seconds .
- Decelerate within 18 second .
◘ Most instruments have a variable speed control and a momentary pulse
.

11. 11
4-Ultra-centrifuges :-
◘ It is the most sophisticated instrument , because of high speeds attainable
◘ have typical fixed-angle rotor which hold six ( 0.2 – 2.2 ) ml samples .
◘ Speeds: up to 150,000 rpm (high speed).
◘ Force ( RCF ) : up to ( 1500000 xg ).
◘ Intense heat is generated in the rotor .
◘ The spin chamber must thus be refrigerated and placed under a high
vacuum to reduce friction .
◘ The sample in a cell or tube is placed in a rotor , which is then driven by
an electric motor .
◘ Although it is rare , metal rotors sometimes break into fragments when
placed under high stress .
◘ The rotor chamber covered with a protective steel armor plate .
◘ The drive shaft of the ultracentrifuge is constructed of a flexible material
to accommodate any wobble of the rotor due to imbalance of the samples .
It is still important to counterbalance samples carefully as possible before
inserting them in the rotor .

12. 12
◘ Types of ultra-centrifuge :-
♠ Two types of ultracentrifuges are available :
• Preparative model :- ( 90 % )
- Primary used for separation and purification of samples for further analysis
• Analytical model :-
- Which are designed for performing physical measurements on the sample during
sedimentation .
◘ Analytical Ultracentrifuge :-
• Have the same basic design as preparative
models , except that they are equipped with
optical system to monitor directly the
sedimentation of the sample during
centrifugation .
• sample :- nucleic acid or protein ( 0.1 -1.0 ml )
is sealed in a special analytical cell and rotated
light .
• light is directed through the sample parallel to
the axis of rotation , and measurements of
absorbance by sample molecules are made .
• is sample molecules have no significant
absorption bands in the wavelength range , then
optical systems that measure changes in refractive index may be used .
• Optical systems aided by computers are capable of relating absorbance changes or index of
refraction changes to the rate of movement of particles in the sample .
• The optical system actually detects and measures the front edge or moving boundary of the
sedimenting molecules .
• These measurements can lead to an analysis of concentration distribution within the centrifuge
cell .

13. 13
♣ Rotor Balance :-
• balancing the centrifuge load is critical .
• Many newer centrifuge will automatically decrease their speed if the load
is not evenly distributed
• but more often , the centrifuge will shake and vibrate or make noise than
expected .
• A centrifuge needs to be balanced based on equalizing both the volume
and weight distribution across the centrifuge head
• A good rule of thumb is one of even placement and one of opposition .