BS- Techniques in Biochemistry

1. CENTRIFUGATION
Technique in Biochemistry

2.  A centrifuge is a device for separating particles from a
solution according to their size, shape, density, viscosity
of the medium and rotor speed.
 Action of centrifugal force is used to promote accelerated
settling of particles in a solid-liquid mixture.
 Biological centrifugation is a process that uses centrifugal
force to separate and purify mixtures of biological
particles in a liquid medium. It is a key technique for
isolating and analysing cells, subcellular fractions,
supramolecular complexes and isolated macromolecules
such as proteins or nucleicacid.

3.  In a solution, particles whose
density is higher than that of the
solvent sink (sediment), and
particles that are lighter than it
float to the top. The greater the
difference in density, the faster
they move. If there is no
difference in density (isopyknic
conditions), the particles stay
steady. To take advantage of
even tiny differences in density
to separate various particles in a
solution, gravity can be replaced
with the much more powerful
“centrifugal force” provided by
a centrifuge

4. PRINCIPLE OF CENTRIFUGATION
Basis of separation:
 -Size
 -Shape
 -Density
Methodology:
 -Utilizes density difference between the
particles/macromolecules and the medium in
which these are dispersed
 -Dispersed systems are subjected to artificially
induced gravitational fields

6. RELATIVE CENTRIFUGAL FORCE
 the ratio of the centrifugal acceleration at a
specified radius and the speed to the standard
acceleration of gravity
 RCF =42 (rev min -1 ) 2 r/ 3600 x981= G/g
 RCF units are therefore dimensionless
(denoting multiples of g) and revolutions per
minute are usually abbreviated as r.p.m.:
RCF=1.12 x 10-5 r.p.m2 r.

7. CENTRIFUGAL FIELD

8. GENERAL STEPS IN BIOCHEMICAL SEPARATION

9. SEPARATION OF MACROMOLECULES

10. DENSITIES OF BIOLOGICAL MATERIAL

11. CELL FRACTIONATION

12. CLINICAL LABORATORY USE OF CENTRIFUGATION
 Remove cellular components from blood to provide
cell free plasma or serum for analysis.
 Concentrate cellular elements and other components
of biological fluid for microscopic or chemical
analysis.
 Remove chemically precipitated protein from an
analytical specimen.
 Separate protein bound or antibody bound ligand
from free ligand in immunochemical assay.
 Extract solute in biological fluids from aqueous to
organic solvents.
 Separate lipids compounds such as chylomicrons
from other compounds in plasma or serum or
lipoprotein from one another.

13. Types of
Centrifuge
 Fixed Angle Rotor
 Swinging Buckcet Rotor
 Ultra Centrifugation
 Axial Centrifugation

14. FIXED ANGLE ROTOR SWINGING BUCKET ROTOR
 Sediment particles have only
short distance to travel before
pelleting at the angle of 25 or 40
degree.
 Shorter run time.
 The most widely used rotor type.
 This is used to separate nuclei,
mitochondria, nucleosome and
isopycnic separations. Isopycnic
separation is when biological
particle and surrounding medium
attain the equal level at the zero
sedimentation rate.
 Longer distance of travel may
allow better separation, such as
in density gradient centrifugation.
 The particles sediment uniformly
along the horizontal tube.
 Easier to withdraw supernatant
without disturbing pellet.

15. ULTRACENTRIFUGATION AXIAL CENTRIFUGATION
 A very high speed
centrifuge with fixed head
rotors.
 Most important application
is in separating
lipoproteins.
 This is available both in
analytical and preparative
models.
 Allows tubes to spun in
vertical orientation which is
used in traditional
centrifugation.

16. THANK YOU