VEER NARMAD SOUTH GUUJARAT UNIVERSITY, SURAT

Department of Biosciences
Ph. D. Course Work 2022-23
Assignment Submission
Ph. D Registration number: 1785
Name: Patel ShitalKumari Arvindbhai
Subject: CW 102: Advance in research techniques
Topic Name: Unit: 1 (1.2) Separation Techniques
Date: 25/12/2023
Signature: _____________
Separation Techniques
Separation techniques in instrumentation are essential for analysing
complex mixtures of substances and isolating individual components, These
techniques are widely used in various scientific and industrial fields, including
chemistry, biochemistry, environmental science, and pharmaceuticals,
Separation techniques are those techniques that can be used to separate two
different states of matter such as liquids and solids, Separation is an important
asset to purify component of interest from a mixture,
Separation process or a separation method or simply a separation is
methodology to attain any mass transfer phenomenon that convert a mixture of
substances into two or more distant product mixtures, Separation is an important
asset to purify component of interest from a mixture,
By separating the constituents of the mixtures, we are able to find out the
properties of the known/unknown substances from mixtures and possibly use
them for the production of useful substances such as medicines,
• Types of Separation Techniques
Separation techniques are classified based on types of mixtures,
1. For solid in liquid mixtures
a. Homogeous
• Evaporation
• Distillation
• Centrifugation
b. Heterogenous
• Sedimentation
• Filtration
• Magnetic Separation
• Fractional distillation
2. For liquid in liquid mixtures
a. Homogeous
• Simple or fractional distillation
• Chromatography
b. Heterogenous
• Partition separation using funnel

Evaporation:
In the case which we do not need to collect the solvent, The solvent is boiled off
and escape into the air while the solute is left behind in the holding container,
Note that this method is not suitable for use on solutes which can decomposed by
heating,
Distillation: To separate and collect solvent from a solution of solutes, or in a
mixture of two different liquids (with different boiling points), with the use of
heat,
The logic behind how simple distillation works is actually the same as that of
evaporation, The only difference is that a closed neck container (distillation flask)
is used to hold the mixture to be heated, with a opening/tube by the side (of the
container) connected to a condenser, The setup for simple distillation should look
something like this:
As the sea water mixture is heated, water boil and changes into water vapour gas,
since hot air rises and cold air sinks, the hot water vapour moves to the top of the
flask and passes into the condenser,
The tubes on the condenser are attached to a water source, with the water flowing
in through the lower end and flowing out through the higher end of the condenser,
This creates a cooler surface for the hot water vapour to condense on, As the
condenser is tilted downwards, towards the collecting container at the end of the
setup, the condensed water flows and drips into the collecting container,

Centrifugation:

Introduction
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 nucleic acids, The development
of the first analytical ultracentrifuge by Svedberg in the late 1920s and the
technical refinement of the preparative centrifugation technique by Claude and
colleagues in the 1940s positioned centrifugation technology at the centre of
biological and biomedical research for many decades, Today, centrifugation
techniques represent a critical tool for modern biochemistry and are employed in
almost all invasive subcellular studies, While analytical centrifugation is mainly
concerned with the study of purified macromolecules or isolated supramolecular
assemblies, preparative centrifugation methodology is devoted to the actual
separation of tissues, cells, subcellular structures, membrane vesicles and other
particles of biochemical interest, Most undergraduate students will be exposed to
preparative centrifugation protocols during practical classes and might also
experience a demonstration of analytical centrifugation techniques To aid in the
understanding of the basic principles of centrifugation, the general design of
various rotors and separation processes is diagrammatically represented,
Traditionally, marker enzyme activities are used to determine the overall yield
and enrichment of particular structures within subcellular fractions following
centrifugation, As an example, the distribution of key enzyme activities in
mitochondrial subfractions from liver is given, Miniature gel and blotting
equipment.
Centrifugation is a separation method which uses the action of centrifugal
medium, to the bottom of the container, according to their density and
Sedimentation is the deposition of particles suspended in a liquid size, For
example, when water with suspended particles of varying sizes and densities (e,
sand) is left to stand undisturbed in a container, say, a measuring cylinder, the
process of sedimentation due to gravitational force would occur, As a result,
particles of higher density would settle first to the bottom, then particles of lesser
density, and so on, thus forming a gradient according to density , If a medium
other than water is used, such x glycerol or castor oil, the rate of sedimentation
(i.e., the speed with which the suspended particle descend to the bottom) will be
reduced process is dependent on because of the higher viscosity of the medium ,
Thus, the sedimentation process is dependent on
i) the density and size of the suspended particles,
ii) the viscosity of the medium in which the particles are suspended, and
iii) the gravitational pull, the gravitational force under normal conditions is about
980 cm s - 2 (1 g unit),
This sedimentation process due to the force of gravity takes very long time
and the process is generally incomplete because very minute particles of the order
of macromolecules are insensitive to gravitational setting , However, if the liquid
with the suspended particle is subjected to centrifugal force, which is several
thousand times greater than the gravitational force , the sedimentation process
can be completed in a short time , The instrument that is used to apply the
centrifugal force to a solution with suspended material is a centrifuge.
CENTRIPETAL AND CENTRIFUGAL FORCES
The force that maintains an object in circular motion is called centripetal
force (force seeking to move towards the centre ) , If no force is exerted on an
object, it tends to move in a straight line at a constant speed , In order to centripetal
force must be applied at right angles to the object's velocity make the object to
deviate from that straight - line path to a circular path, a The centripetal force
causes a corresponding centripetal acceleration, which is also towards the centre.
In general, the centripetal force that needs to be exerted to an object of density m
to keep it moving in a circle path of radius r at a constant velocity v is Mv2/r.
CENTRIFUGE A centrifuge is a mechanical device used for separating
substances of different densities using the principle of centrifugal force. A
commis centrifuge is a container that is revolved at high speeds. The principle of
separation is similar to that of sedimentation by gravity. But in a centrifuge the
driving force is much higher because the force results from the rotation of the
liquid in the container. In the case of sedimentation, the driving force comes from
the difference in density between the solid particles and the medium in which the
particles are suspended. In a centrifuge, on the other hand, separation is effected
by a force that is 1000 to 100,000 times that of gravity. The principal components
of a centrifuge are a rotor and motor. The rotor is connected to a central shaft and
has, at the distal end, provision for holding the container. The shaft is connected
to a motor, which can be operated at different speeds. There are four types of
rotors i) swinging (swing - out) bucket ii) fixed angle iii) vertical tube iv) zonal
In the swinging bucket rotor, the centrifuge tube holders, the buckets, are hinged
to rods extending from the shaft such that they can swing freely. During
centrifugal rotation, the buckets swing out to different angles proportional to the
speed of the rotation, ultimately attaining a horizontal orientation. In the
horizontal position, the medium in the centrifuge tube orients to a plane
perpendicular to the axis of rotation, and returns back to the original plane as the
bucket swings back to its vertical position. Because of the relatively long path
length, the swinging bucket rotors are used for most preparative rate - zonal
separations. The fixed angle rotors have sockets for placing the centrifuge tubes
at a fixed angle, which is about 30 ° (14 °-40 °) to the axis of rotation. During
centrifugation, the particles in the solution travel radially outward, moving only
a short distant before striking the wall all of the centrifuge tube, and settle as a
pellet at the bottom.
GRADIENT MEDIA
There are several gradient materials used in centrifugation and include
1) sucrose and Ficoll
2) cesium chloride
3) Potassium bromide
4) Percoll
5) Metrizamide
6) Nycodenz
7) Renografin
The most common media is the 54 % (wt / wt) sucrose solution, prepared 54 g of
sucrose in 46 mL of water (assuming water has a density of 1 g / ml. Ficoll (Ficoll
400) is a commercial preparation of a hydrophilic polymer of sucrose, having a
molecular weight of 400 000. The advantage of Ficoll over sucrose is its lower
osmotic pressure, which helps in preserving the morphology and activity of
subcellular fractions.
Cesium chloride and potassium bromide are useful in isopycnic density gradient
centrifugation technique. Percoll is again a commercial preparation of density
gradient medium containing colloidal silica particles (30 nm) coated with
polyvinylpyrrolidine. This medium, because of its low osmolarity, low viscosity,
and large particle size, is suitable for separating cells, bacteria, viruses and
subcellular organelles. Metrizamide and Nycodenz are useful for the isolation of
membrane fractions by floatation. Renografin is also used for cell fractionation.

BASIC PRINCIPLES OF SEDIMENTATION

From everyday experience, the effect of sedimentation due to the influence of the
Earth’s gravitational field (g ¼ 981 cm s–2) versus the increased rate of
sedimentation in a centrifugal field (g > 981 cm s–2) is apparent, Biological
structures exhibit a drastic increase in sedimentation when they undergo
acceleration in a centrifugal field, The relative centrifugal field is usually
expressed as a multiple of the acceleration due to gravity, Below is a short
description of equations used in practical centrifugation classes, When designing
a centrifugation protocol, it is important to keep in mind that: the more dense a
biological structure is, the faster it sediments in a centrifugal field; the more
massive a biological particle is, the faster it moves in a centrifugal field the denser
the biological buffer system is, the slower the particle will move in a centrifugal
field; the greater the frictional coefficient is, the slower a particle will move; the
greater the centrifugal force is, the faster the particle sediments; the
sedimentation rate of a given particle will be zero when the density of the particle
and the surrounding medium are equal, Biological particles moving through a
viscous medium experience a frictional drag, whereby the frictional force acts in
the opposite direction to sedimentation and equals the velocity of the particle
multiplied by the frictional coefficient, The frictional coefficient depends on the
size and shape of the biological particle, As the sample moves towards the bottom
of a centrifuge tube in swing-out or fixed-angle rotors, its velocity will increase
due to the increase in radial distance, At the same time the particles also encounter
a frictional drag that is proportional to their velocity, The frictional force of a
particle moving through a viscous fluid is the product of its velocity and its
frictional coefficient, and acts in the opposite direction to sedimentation,

Types of centrifuges
Centrifugation techniques take a central position in modern biochemical, cellular
and molecular biological studies, Depending on the particular application,
centrifuges differ in their overall design and size, However, a common feature in
all centrifuges is the central motor that spins a rotor containing the samples to be
separated, Particles of biochemical interest are usually suspended in a liquid
buffer system contained in specific tubes or separation chambers that are located
in specialised rotors, The biological medium is chosen for the specific centrifugal
application and may differ considerably between preparative and analytical
approaches, As outlined below, the optimum pH value, salt concentration,
stabilising cofactors and protective ingredients such as protease inhibitors have
to be carefully evaluated in order to preserve biological function,
The most obvious differences between centrifuges are: the maximum speed at
which biological specimens are subjected to increased sedimentation; the
presence or absence of a vacuum; the potential for refrigeration or general
manipulation of the temperature during a centrifugation run; and the maximum
volume of samples and capacity for individual centrifugation tubes, Many
different types of centrifuges are commercially available including: large-
capacity low-speed preparative centrifuges; refrigerated high-speed preparative
centrifuges; analytical ultracentrifuges; preparative ultracentrifuges; large-scale
clinical centrifuges; and small-scale laboratory microfuges, Some large-volume
centrifuge models are quite demanding on space and also generate considerable
amounts of heat and noise, and are therefore often centrally positioned in special
instrument rooms in biochemistry departments,

Types of rotors
To illustrate the difference in design of fixed-angle rotors, vertical tube rotors and
swinging-bucket rotors, depending on the use in a simple low-speed centrifuge, a
high-speed centrifuge or an ultracentrifuge, different centrifugal forces are
encountered by a spinning rotor, Accordingly, different types of rotors are made
from different materials, Low-speed rotors are usually made of steel or brass,
while high-speed rotors consist of aluminium, titanium or fibre-reinforced
composites, The exterior of specific rotors might be finished with protective
paints,
Sedimentation: The tendency of particles in suspension to settle down in the
fluid due to certain forces like gravity, centrifugal acceleration, or
electromagnetism is called as sedimentation, The solid that gets settled down is
called as sediment, In laboratory it can be done in test tubes, To enhance
productivity test tubes should be placed at 45 ° angle to allow the sediments to
settle at the bottom of the apparatus, A decanter centrifuge may be used for
continuous solid - liquid separation,
Filtration: Separation of solids or groups of solids from the liquid in a mixture,
using a medium through which the liquid can pass,
The medium which we are using over here is the filter paper, The filter paper is
folded and placed onto the filter funnel,
The liquid-solid mixture is poured onto the filter paper, Using a filter paper with
pores of a smaller size than the solid particles (and is larger than the size of the
liquid molecules), the liquid (or solvent) should pass through the filter paper, and
is collected by a collection container placed at the bottom of the filter funnel,
The liquid that passes through the filter paper is called the filtrate while the solid
left on the filter paper is called the residue,
Magnetization or Magnetic Attraction: This method involves the
separation of magnetic substances from non-magnetic substances by means of a
magnet, so, as Takes advantage of physical property of magnetism, it useful only
for certain substances such ferromagnetic (materials strongly affected by
magnetic fields) and paramagnetic (materials that are less affected, but the effect
is still noticeable), This method involves the separation of magnetic substances
from non - magnetic substances by means of magnet,

Fractional Distillation: Used to separate miscible liquids with different but

very close boiling point, This method is more efficient than simple distillation,
A fractionating column is introduced between the distillation flask and the
condenser, The upper portion of the column, which is closer to the condenser, is
cooler than the lower portion and hence, only gases with the same temperature as
the upper portion are allowed to pass on to the condenser, On the other hand, the
gases with higher boiling points will condense and flow back to the bottom into
the distillation flask, and is heated into a gas again, At the end, liquid with the
lowest boiling point will be the first to boil and hence the first to be distilled out
and collected,
Sublimation: To separate a mixture of solids containing one which sublimes
and one (or more than one) which does not, by heating the mixture, An cotton-
stoppered inverted funnel is placed over the mixture, When the mixture is heated,
the heat-liable solid sublime and turn into a gas, and travel to the top of the
inverted funnel, Once the hot gas touches the cooler funnel, it solidifies back into
a solid, The solid can then be scrapped off and collected in another container from
the funnel,
Decanting: A crude way of separating insoluble solids from liquids, as the
liquid is poured away and collected in another container, Note that the insoluble
solid should be able to settle down on standing and this method is not effective
for obtaining clear liquid from the mixture especially when the insoluble solid is
very fine and light,
Crystallisation: Used to separate a dissolved heat-liable (will decompose
upon heating and hence can sublime) solid (solute) from a solution,
You will need a saturated solution to being with, A saturated solution is a solution
that contains the maximum amount of solute dissolved in a given volume of
solvent at a particular temperature, Do not mix this up with a concentrated
solution, which is a solution that contains lots of solute dissolved in it, The
amount of solute in a concentrated solution may/may not be the maximum
amount which can be dissolved in the solution,
First, you will need to heat to evaporate off most of the solvent from a solution to
make a hot and nearly saturated solution, Else, if you already have a saturated
solution, heat it up slightly such that the solution becomes hot,
After which, allow the hot solution to cool naturally, The solubility of the solute
decreases as the solution is cooled, and the excess solute which can no longer be
dissolved in the saturated solution crystallizes out of the solution, The crystals
which are formed can be separated from the remaining solution by filtration,
Seeding: Using a small crystal of salt (the “seed”) to collect the solid solute
crystals in a saturated solution, No heat is required but will take a long time,
As the saturated solution continues to evaporate overtime, there is an increasing
amount of solute which will crystallise out from the solution, These solid solute
crystals will attach itself to the “seed”, resulting in a grow in size of the “seed”
overtime, The enlarged “seed” can then be removed from the solution at the end
of the procedure (or when it is large enough for use in other applications),
Chromatography
➢ INTRODUCTION
Chromatography is a wide range of physical methods used for the Exploiting one
or more of the differences in these properties, it is possible to physically separate
the components, For example, differences in the size or molecular weights of the
constituent proteins of a sample such as a tissue or body fluid would enable us to
separate these proteins using appropriate chromatographic techniques, The basic
principle of all types of chromatography is the differential movement of a mixture
over or through an adsorptive material, The differential movement is related to
the differences in the physical, chemical, and other properties of the components
of the mixture, The mixture itself does not move but is carried by another material
which may be a liquid or a gas, The material carrying the mixture is referred to
as the mobile phase, The material over or through which the mobile phase is
flushed is called the stationary phase, The stationary phase may be solid, gel,
liquid, or a mixture of liquid and solid, This phase is immobilized, hence called
the stationary phase, The stationary and mobile phases are mutually immiscible,
The components of the mixture interact with both the stationary phase and the
mobile phase differentially according to the differences in their properties, The
differential interaction determines the rate of the migration of the components
over or through the stationary phase, Thus, different components migrate at
different rates, Those components that react more (i,e,, have higher affinity)
towards the mobile phase and less towards the stationary phase migrate faster, On
the other hand, components reacting less with mobile phase and more with
stationary phase migrate slowly, The faster or slower migration of the different
components is reflected in the distances they have travelled over or through the
stationary phase in a given time, The differential migration of the different
components results in their separation, The ratio of the concentration of the solute
(a component of the sample mixture) in the stationary phase (C) to the
concentration of the same in the mobile phase (C) of a chromatographic system
is called distribution coefficient (K) or partition coefficient, K = Cs/Cm
CLASSIFICATION OF CHROMATOGRAPHIC METHODS
We can classify the different chromatographic methods on the following bases:
1. Nature of the mobile and stationary phases
2. Principle of separation of the components
3. Geometry of the stationary
4. phase Mode of operation
Nature of the Phases
On the basis of the nature of the mobile phase used in a chromatography
system, we can classify it as gas chromatography or liquid chromatography,
Gas chromatography employs a gaseous fluid as the mobile phase which is
called the carrier gas, The carrier gas is generally an inert g The stationary phase
of gas chromatography is generally a solid adsorbent or a liquid distributed over
the surface of a porous, inert support, Depending on the nature of the stationary
phase of a gas chromatographic system, we can further classify it as gas - solid
chromatography and gas-liquid chromatography,
Liquid chromatography employs a liquid as mobile phase and a solid stationary
phase, and therefore is described as liquid - solid chromatography, The liquid has
a low viscosity and flows through the solid stationary phase bed, The stationary
phase may be an immiscible liquid coated onto a porous support, a thin film of
liquid phase bonded the surface of a sorbent, or simply a sorbent of specified pore
size,
Principle of Separation
When the mobile phase flows through the stationary phase, the different solutes
are retained at different distances from the origin, On the basis of the specific
process of retention, which leads to the separation of the different solutes, we can
classify the chromatographic methods a adsorption, partition, ion - exchange,
molecular exclusion, and affinity,
In adsorption chromatography the components of a sample are separated on the
basis of the differences in their adsorption desorption behaviour between the
mobile and the stationary phases,
Partition chromatography is one in which the separation is based on differences
between the solubility of the sample components in the stationary phase (gas
chromatography), or on differences phases (liquid chromatography), between the
solubilities of the components in the mobile and stationary,
Ion-exchange chromatography separates the sample components on the basis of
differences in their ion - exchange affinities, For example, anions like SO3-, or
cations like N (CH3)3+, " attached to a stationary phase, such as resin,
Molecular exclusion chromatography or gel - filtration or gel permeation
chromatography is a technique in which retention of sample components is
according to their molecular size, The stationary phase is a gel consisting of
porous beads with strictly controlled pore size, The sample components with
molecular size smaller than that of the pore size, get trapped in the pores and as a
result migrate slowly, On the other hand, molecules larger than the pore
dimension migrate faster without any hindrance, This chromatographic technique
is also useful in the determination of the relative molecular weight of a
macromolecule, purpose,
Affinity chromatography uses column packing that has been chemically altered
by attaching a compound with a specific affinity for the desired molecules (e,g,
biological compounds such as an enzyme), The packing substance used, called
the affinity matrix, is inert and easily modified, Agarose is the most familiar
substance used for this The ligands (or " affinity tails ", are substances capable of
forming complexes with specific molecules) that have been genetically
engineered to possess a specific affinity are inserted into the matrix, The desired
molecules in the mobile phase adsorb to the ligands on the matrix because of the
unique biological specificity of the analyte and ligand interaction,
PAPER CHROMATOGRAPHY
Principle
It is a method suitable for separation of small amounts of low molecular weight
compounds that are soluble in liquid phase, The paper used is a purified cellulose,
Cellulose is a polysaccharide composed of glucose high quality filter paper (e,g,
Whatman filter paper), which is made of molecules which have a large number
of hydroxyl groups, The cellulose fibres in the filter paper hold moisture tightly
by the formation of hydrogen bonds between their hydroxyl groups and water
molecules, Even a sheet of ' dry ' paper contains about 10 % water by mass, Thus,
water acts as a liquid stationary phase, The mobile phase is any solvent consisting
of an aqueous solution or an organic liquid such as ethanol that is partially
miscible with water, Choice of the solvent depends on the nature of the solutes to
be separated, A mixture of two or three solvents may also be required, The mobile
phase moves along the paper by a mechanism of capillary action resulting from
the forces between the solvent and the solid fibres of the paper,
The mode of separation of sample components is partition, Solutes are separated
based on the interactions between two non - miscible liquid phases according to
their relative solubility, However, cellulose itself assumes a negative charge in
the presence of water, Therefore, the paper exhibits ion exchange and adsorptive
properties, Specialized chromatographic papers that are impregnated with
alumina, silica gel, ion - exchange resin, etc, are also available, The separation
method in these modified papers may vary according to the nature of the
stationary phase,
The solutes that are highly water - soluble or have the greatest hydrogen bonding
capacity move slower along the paper, while less polar compounds will travel
faster with the solvent,
Mobile Phase of Paper Chromatography
As already seen, unless modified, the stationary phase of a paper
chromatographic system is water which is strongly adsorbed to the cellulose, The
mobile phase of a paper chromatography is generally a mixture of solvents such
as alcohols, acids, esters, ketones, phenols, amines, hydrocarbons, etc, The
factors which determine the choice of the mobile phase for paper chromatography
are:
I. It must yield high degree of separation of the sample components, mixture,
II. It should contain fewer number of constituents in the solvent i
III. The constituents of the solvent mixture should have more or less equal
degree of evaporation,
IV. Solvent phase should be immiscible with the stationary phase,
V. The sample components should possess distinctly different solubilities in
the mobile and the stationary phase,
VI. Partition coefficient of the sample components should have a range
between 1 and 100, with a higher affinity towards the stationary phase, i,e,,
water,
VII. It should have lower boiling point so that it can be easily removed from the
paper, so that another solvent can be used for second development as in
two - dimensional chromatography,
VIII. It should be stable and should not get oxidized during migration,
Some of the commonly used solvent mixtures of the paper chromatographic
system are:
1. butyl alcohol + acetic acid + water
2. butyl alcohol + pyridine + water
3. methyl alcohol + pyridine + water
4. propyl alcohol + petroleum ether
5. chloroform + petroleum ether
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