BIOCHEMISTRY NOTES

DAB 3- TERM 3, 2021 (MARCH)

LESSON THREE

BASIC BIOCHEMICAL TECHNIQUES

SEPARATION TECHNQIUES

(a) Cell fractionation

It is the process used to separate cellular components while preserving individual

functions of each component. Cell fractionation allows you to study the different parts
of a cell in isolation.

Step 1

(a) Breaking the cell

-In order to maintain the biological activity of organelles and biomolecules, the
tissues must be extracted in mild conditions called cell- free systems, whereby the
cellular tissues are suspended in a solution of appropriate pH and salt content usually
isotonic sucrose at temperature of 400C.

-Cell membrane is then disrupted by addition of detergent known as sodium dodecyl

sulphate (SDS). SDS can interact with both membranes and parts of the cell that are
soluble in water. Since SDS can interact with both lipid (membrane) and soluble
(cytoplasmic) parts of the cell, they allow cellular components to be mixed or
homogenized.

-When the cellular components are mixed, a cell homogenate or cell lysate is formed.
Lysing or opening the cells in SDS is usually combined with a physical method that
breaks the cell further, such as machines that are similar to blenders, glass beads, or
breaking the cell using sound energy (sonication). Using physical methods in
combination with SDS ensures that all parts of the cells in the sample get broken and
you can isolate as much of your cellular fraction as possible.
Step 2

(b) Separation

-Cell homogenates are separated into fractions by spinning them superfast in a process
called centrifugation, it is carried out in an instrument called preparative
ultracentrifuge. Centrifugation applies enough force to cell homogenates to allow
different cellular fractions to separate based on properties such as mass, density and
shape. The smaller components stay homogenized in the liquid (supernatants) i.e.
upper layer and the larger components will move to the bottom to form pellets.
Repeating the centrifugation of the supernatants with increasing force allows smaller
cellular components to be separated. Presents day ultracentrifuge rotate at speeds up
to 80,000 rpm (rpm= rotations per minute) and generates a gravitational pull of about
500,000g, so that even small molecules like, tRNA, enzymes can sediment and
separate from other components. The chamber of ultracentrifuge is kept in a high
vacuum to reduce-friction, prevent heating and maintain the sample at -40C.

-The homogenate is first filtered to remove unbroken cell clumps and collected in a
centrifuge tube.

-The filtered homogenate is centrifuged in a series of steps at successively greater

speeds, each step yields a pellet and a supernatant. Separation occur due to differences
in their size, brought about by velocity centrifugation.

-The supernatant of each step is removed to a fresh tube for centrifugation. For
instance, at low speed (600g for 10min) nuclear sediment, at medium speed (15,000g
for 5min) mitochondria sediment and at high speed (80,000g for 5min) micro-somal
fraction sediment. The final supernatant is soluble fraction or cytosol.

-The organelle fractions (pellets) obtained in velocity centrifugation is purified by

equilibrium density- gradient centrifugation. In this method organelles are separated
by their density not by their size.

-The impure organelle fraction is layered on top of a gradient solution (either sucrose
or glycerol solution). The solution is more concentrated (dense) at the bottom of the
centrifuge tube, and decreases in concentration gradually toward the top.
 -When the tube is centrifuged at high speed the various organelles migrate at an
equilibrium position where their density is equal to the density of the medium.

-Remove each layer carefully using specialized pipette.

Diagram 1

Diagram 2
 Purification of organelles by density-gradient centrification

MEMBRANE SEPARATION

A membrane is a selective barrier that permits the separation of certain components

in a fluid by combination of sieving and diffusion mechanism. Separation is achieved
by selectively passing one or more components of a stream through the membrane
while retarding the passage of one or more other components.
Membranes can selectively separate components over a wide range of particle sizes
and molecular weights, from macromolecular materials such as starch and protein to
monovalent ions. Membranes have gained an important place in chemical technology
and are used in a broad range of applications. The key properties determining
membrane performance are high selectivity and fluxes, good mechanical, chemical
and thermal stability under operating conditions, good compatibility with the
operating environment, and cost effective and defect- free production. Although the
major uses of membranes are in the production of potable water and separation of
industrial gases, they can be used for many other important applications such as
filtration of particulate matter from liquid suspensions, and air. Most specialized
applications include ion separation in electrochemical processes, dialysis of blood and
urine, artificial lungs, controlled release of therapeutic drugs, membrane- based
sensors e.t.c. Membrane processes are characterized by the fact that a feed stream is
divided into 2 streams: retentate and permeate.

The retentate is that part of the feed that does not pass through the membrane, while
the permeate is that part of the feed that does pass through the membrane. The
optional ‘sweep’ is a gas or liquid that is used to help remove the permeate. It is
important to note that there are 3 different mechanism by which membrane can
perform separations;

 By having holes or pores which are of such a size that certain species can
pass through and others cannot. This mechanism is called size exclusion.
  By selective retardation by the pores when the pores diameters are close or
bigger to molecular sizes. This mechanism is called pore flow. The bigger
molecules pass faster while smaller molecules retarded.

 By dissolution into the membrane and migration by molecular diffusion

across the membrane and re-emergence from the other side. This is called
solution diffusion.

Advantages of membrane separations

1. Can be used where large volume of substance is separated

2. This method requires low energy

3. Very simple flow sheet is required with no complex control schemes.

4. Membranes used have high selectivity thus enhancing higher separations

Disadvantages of membrane separations

1. Most of the time the separation lead to two pure products, thus there is contamination

2. Due to few stages of separation the membrane used must be perfect

3. Membranes can be incompatible with chemical used in the process

4. Membranes work best at a certain temperature range