Programme :Msc Nutraceticals I

Sem
Course:131P20C104 -
Spectroscopy
 Course outcome:
1. Understand the techniques of cellular fractionation, including
extraction procedures.
2. Familiarize with centrifuge techniques, such as zonal, density
gradient, and ultracentrifugation, and their applications in
biological and chemical separations.
3. Gain knowledge of various electrophoresis techniques.
4. Comprehend the basic concepts of spectroscopy and the Beer-
Lambert law.
5. Gain practical skills in using spectroscopic techniques for
qualitative and quantitative analysis.
6. Develop proficiency in selecting and applying appropriate
instrumentation techniques for specific analytical and research
purposes.
 131P20L104 – Spectroscopy

1. Introduction to Centrifuge
2. AGE/PAGE Electrophoresis
3. Extraction and estimation of total sugars from food
products by Anthrone method.
4. Estimation of proteins using Biuret
5. Estimation of protein by A280 method.
6. Estimation of lycopene by spectrophotometry
7. Estimation of total phenols using spectrophotometry.
Programme :Msc Nutraceticals I Sem
Course:131P20C104
Topic: Introduction to instrumentation
techniques
Credits: 1 Lectures: 15
Unit-1- Techniques of cellular fractionation,
DD 11-08-23
 Introduction to instrumentation techniques

Chapter -1 Techniques of cellular fractionation,

Chapter 2- Centrifuge techniques: zonal, density gradient and ultracentrifugation techniques

and their applications.

Chapter 3- Electrophoresis: zonal, paper, gel electrophoresis and isoelectric focusing and their
application.
Techniques of cellular fractionation
 What is Cell Fractionation?
• Cell fractionation is a procedure for rupturing cells, separation and suspension of cell
constituents in isotonic medium

• Why ?

• In order to study their structure, chemical composition and function.

• i.e

• Individual study of an organelle

• Study of biochemical functions
• To understand molecular basis of disease
• Food Science – Understand the function of each part of the cell

• Cell fractionation involves 3 steps:

• Extraction,
• Homogenization and
• Centrifugation.
 Extraction:

• It is the first step toward isolating any sub-cellular structures.

• In order to maintain the biological activity of organelles and bio-molecules,

they must be extracted in mild conditions called cell-free systems.

• For these, the cells or tissues are suspended in a solution of appropriate pH

and salt content, usually isotonic sucrose (0.25 mol/L) at0-40°C.

https://www.biologydiscussion.com/cell/cell-fractionation-extraction-homogenization-and-
centrifugation/5848
• The suspended cells are
then disrupted by the
process of homogenization.
Homogenization
• i) Grinding

• (ii) High Pressure (French

Press or Nitrogen Bomb)

• (iii) Osmotic shock,

• (iv) Sonication (ultrasonic

vibrations).

• Grinding is done by pestle

and mortar or potter
homogenizer (a high-speed
blender). The later consists
of two cylinders separated
by a narrow gap.
 Mortar & Pestle Method
• Also Called Homogenization. Mortar & Pestle are a set of
two tools using since the Stone Age.
• Used for Grinding/Crushing the Substances into a
fine paste or powder in the Kitchen, Pharmacy and
Laboratory Practices.
• Mortar is a bowl, typically made of hard wood, metal,
ceramic or granite & the Pestle is a blunt, club shaped
object.
• While disruption, the Biological material first need to be
submerged in the Liquid Nitrogen (-196 C ̊ ) for Freezing
Purpose. The Frozen Biological Material finely ground
with Mortar & Pestle & Made into Powder.
• The Powdered Material then Dissolve in Suitable Solvent &
Extract the Desired Component.
 Blending Method

• It is a physical method for rupturing the

mammalian and plant cells by shear force.
• The process is usually carried out in a typical
domestic kitchen blender.
• In this method, the tissue is cut into small pieces
and blended with buffer for about one minute
to disrupt the cell.
• The process is usually carried out in a typical
domestic kitchen blender.
• A blender with small beads can also be
used for microorganism
 Bead Beating
• It is also called Grinding with Abrasives.
• It is a physical method to rupture cell in presence of
abrasives like sand, alumina, glass beads, etc.
• It can be carried out in Mortar- Piston or Dynomill.
• Grinding in mortar and piston with sand or alumina in
presence of buffer cause rupturing of the cell wall.
• This method is useful for disrupting bacterial or plant cells.
• However, this is applicable to the relatively small amount of
sample.
• Dynomill is a large scale version of grinding in mortar and piston.
• It comprises a chamber containing glass beads and numbers of
rotating impeller discs.
• It is an electrical process of rupturing cells.
• Cells get ruptured when they are caught between colliding beads.
• It can process up to 5kg cells per an hour.
• This method helps in the production of industrially important
proteins and enzymes
 Ultra Sonication
• This method is suitable for a small volume of the suspension of
cultured or microbial cells.
• A Sonicator probe generates high-frequency sound waves (<20 KHz)
for 30-60 Sec.
• These sound waves cause disruption of cells by shear force
and cavitations.
• Cavitation refers to an area of alternate compression
and rarefaction which causes tension in a cell that causes rupturing
of the cell.
• In Cavitation, gas bubbles are formed which explodes as they
decompress after applying pressure that produces a local shock
wave.
• The cells are disrupted by pressure change. Since a considerable
amount of heat is generated during this process, the sample must be
kept on ice during treatment.
 Cell breaking

• Before Extraction cell walls

should be ruptured

• Detergents allow cell wall lysis –

opening of cell membrane -
Contents inside the cell
membrane can obtained after
cell lysis
• Another strategy for cell lysis is to use digestive Enzymatic
enzymes which decompose the cell wall. Disruption
• Different cell types possess different kind of cell
walls and membranes, and thus the used enzyme of Cells
depends
• For Eg. Lysozyme is commonly used enzyme to
digest cell wall of gram positive bacteria.
• Lysozyme hydrolyzes β-1-4-glucosidic bonds in
the Peptidoglycon. The cell wall of gram negative
bacteria differs from the cell wall of gram
positive bacteria so Lysozyme is not very efficient
in the case of gram negative cell wall.
• The Cell wall of Yeast and Fungi differs and
commonly used enzyme mixture for degradation
of its cell wall Zymolyase. It has β-1, 3 glucanase
and hydrolase activities.
 Microwave / Thermolysis
• Thermolysis is potential in isolation of Periplasm proteins in G (-) bacteria which
are released when the cells are heated up to 50ºC.
• Cytoplasmic proteins can be released from E. coli within 10min at 90 ºC.
 Freeze Thaw

• This can cause the cells to burst due to the formation and melting
of ice crystals.

• Gradual freezing, leading to the formation of larger crystals, can

cause an extensive damage to the cell.

• By combining this method with cell grinding offers great results.

 Osmotic shock
This is a technology which can be utilized in biotechnical applications to cause cell lysis.

In this technology, cells are first exposed to either high or low salt concentration.

Then the conditions are quickly changed to opposite conditions which lead to osmotic
pressure and cell lysis
 Electric Discharges

• It is also possible to achieve cell disruption via electrical discharges

in mammalian cells, which are cells that are bounded by plasma
membranes and, unlike plant cells.

• This method allows researchers to examine secretion by

exocytosis.
Centrifugation
 Principle
Rate of settling of a particle, or the rate of separation of two immiscible
liquids, is increased many times by the application of a centrifugal field
(force) many times that of gravity.
 Supernatant and Pellet

 Supernatant is the liquid at the top

 Pellet is particles at the bottom

 Force in a centrifuge

• First, it depends on how fast the

centrifuge spins

• Second, it depends on the radius of

rotation

• A) The longer the string being whirled,

the greater the radius of rotation and
more is the force experienced by the
stone
• b) In a centrifuge further, the particle
is from center of rotation the more
force it experience
 Relative centrifugal force, RFC
 RCF = 11.17(r)(n/1000)2
 = Xg, 100xg, 500xg [ Can be expressed in multiples of g]
 Where r = radius in cm from centerline
 n = rotor speed in RPM, revolutions/minute
 RPM indicates the speed at which the rotor is rotating.
 RCF is the term used to describe the amount of accelerative force applied to a sample in a centrifuge.
 RCF is measured in multiples of the standard acceleration due to gravity on the Earth’s surface (x g).
 This is why RCF and “x g” are used interchangeably in centrifugation protocols.
 The two variables that describe RCF are the radius and the angular velocity of the rotor, i.e., how wide the
rotor is and how fast it is moving.
 RCF is more precise than RPM because the rotor size might differ, and RCF will be different, while the
revolutions per minute stay the same.
t
 Sedimentation speed
It depends on:  It turns out that if:
 RCFs in the centrifuge
 A particle is the same density as the liquid
 Size of particle around it, the particle doesn’t move
 Particle density  A particle is more dense than the liquid, it

 Liquid density moves down the tube

 Liquid viscosity  A particle is less dense than the liquid, it

moves up!
 Types of centrifugation

• Most familiar is

• Differential centrifugation

• Density gradient centrifugation

 Differential Centrifugation
• Separation is achieved primarily based on the size of the
particles in differential centrifugation.
• This type of separation is commonly used in simple pelleting
and in obtaining partially-pure preparation of subcellular
organelles and macromolecules.
• For the study of subcellular organelles, tissue or cells are first
disrupted to release their internal contents.
• This crude disrupted cell mixture is referred to as a
homogenate.
• During centrifugation of a cell homogenate, larger particles
sediment faster than smaller ones and this provides the basis
for obtaining crude organelle fractions by differential
centrifugation.
• A cell homogenate can be centrifuged at a series of
progressively higher g-forces and times to generate pellets of
partially-purified organelles.
 RCF or Xg of DC

• When a cell homogenate is centrifuged at 1000 x g for 10 minutes, unbroken

cells and heavy nuclei pellet to the bottom of the tube.
• The supernatant can be further centrifuged at 10,000 x g for 20 minutes to
pellet subcellular organelles of intermediate velocities such as mitochondria,
lysosomes, and microbodies.
• Some of these sedimenting organelles can obtained in partial purity and are
typically contaminated with other particles.
• Repeated washing of the pellets by resuspending in isotonic solvents and re-
pelleting may result in removal of contaminants that are smaller in size
• Obtaining partially-purified organelles by differential centrifugation serves as
the preliminary step for further purification using other types of centrifugal
separation (density gradient separation).
 Density gradient centrifugation.
• Density gradient centrifugation is the preferred method to
purify subcellular organelles and macromolecules.
• Density gradients can be generated by placing layer after
layer of gradient media such as sucrose in a tube with the
heaviest layer at the bottom and the lightest at the top in
either a discontinuous or continuous mode.
• The cell fraction to be separated is placed on top of the layer
and centrifuged.

• Density gradient separation can be classified into two

categories.

• 1. Rate-zonal (size) separation.

• 2. Isopycnic (density) separation.

 Rate zonal (size) or Zonal separation
• Rate-zonal separation takes advantage of particle size and
mass instead of particle density for sedimentation.

• Figure 2 illustrates a rate-zonal separation process and the

criteria for successful rate-zonal separation.

• Examples of common applications include separation of

cellular organelles such as endosomes or separation of
proteins, such as antibodies.

• For instance, Antibody classes all have very similar

densities, but different masses.

• Thus, separation based on mass will separate the different

classes, whereas separation based on density will not be
able to resolve these antibody classes.
 Criteria for successful rate-zonal
centrifugation

• Density of the sample solution must be less than that of the lowest
density portion of the gradient.
• Density of the sample particle must be greater than that of the
highest density portion of the gradient.
• The pathlength of the gradient must be sufficient for the separation
to occur.
• Time is important. If you perform too long runs, particles may all
pellet at the bottom of the tube.

https://www.masterflex.com/tech-article/basics-of-centrifugation
 Isopycnic separation

• In this type of separation, a particle of a particular density

will sink during centrifugation until a position is reached
where the density of the surrounding solution is exactly the
same as the density of the particle.
• Once this quasi-equilibrium is reached, the length of
centrifugation does not have any influence on the
migration of the particle.
• A common example for this method is separation of
nucleic acids in a CsCl gradient. Figure illustrates the
isopycnic separation and criteria for successful separation.
 Criteria for successful isopycnic
separation
• Density of the sample particle must fall within the limits of the
gradient densities.
• Any gradient length is acceptable.
• The run time must be sufficient for the particles to band at their
isopycnic point. Excessive run times have no adverse effect.
Applications of density gradient media for
isopycnic separations
 Ultracentrifugation

• Ultracentrifuge is a sophisticated and advanced centrifuge that operates

at an extremely high speed and separates smaller molecules that
cannot be separated from the traditional centrifuges.
• The speed of the rotors in ultracentrifuge can range from 60,000 rpm to
150,000 rpm.
• Ultracentrifuges are mostly operated in more facilitated laboratories to
perform more advanced operations.
• These are larger in size and can operate samples either in batches or as a
continuous flow system.
• Most ultracentrifuges are refrigerated in order to control the heat that
might be generated due to the excessive speed.
 Principle of Ultracentrifuge
•The ultracentrifuge works on the same principle as all other centrifuges.

•The working of an ultracentrifuge is based on the sedimentation principle, which states that the denser
particles settle down faster when compared to less dense particles under gravity.

•However, the sedimentation of particles under gravity would take a larger amount of time, and that is why
an additional force is applied to aid the sedimentation process.

•In an ultracentrifuge, the sample is rotated about an axis, resulting in a perpendicular force, called
centrifugal force, that acts on different particles on the sample.

•The larger molecules move faster, whereas the smaller molecules move slower.

•At the same time, denser molecules are moved outwards to the periphery of the tubes whereas the less
dense molecules are rotated towards the center of the tube.

•Once the process is completed, the larger and more dense particles settle down, forming pellets at the
bottom of the tube. In comparison, the smaller and less dense particles remain either in the suspended in
the supernatant or float on the surface.
Chapter -1 Techniques of cellular fractionation, Extraction procedures, Batch extraction,
continuous extraction and counter current extraction.

Chapter 2- Centrifuge techniques: zonal, density gradient and ultracentrifugation

techniques and their applications.

Chapter 3- Electrophoresis: zonal, paper, gel electrophoresis and isoelectric focusing and
their application.
 Electrophoresis

• Electrophoresis is the migration of

charged particles or
molecules in a medium under the
influence of an applied
electric field.
 Electrophoresis
• Electrophoresis is useful in identification and structure
determination of big molecules like polymers and proteins .

• Simple, rapid and highly sensitive Separation technique

• It is used for separation of

• Proteins in body fluids: serum, urine, CSF

• Proteins in erythrocytes: hemoglobin

• Nucleic acids: DNA, RNA

• Food Science – Purify large quantitates of Proteins

 Definition
• Electro means Electricity

• Phoresis means Separation

• Separation of serum proteins by the effect of an electric current.

• Electrophoresis is a physical method of analysis which involves separation of the

compounds that are capable of acquiring electric change in conducting
electrodes.

• Electrophoresis may be defined as the migration of the charged particle through

a solution under the influence of an external electrical field.

• Ions that are suspended between two electrodes tends to travel towards the
electrodes that bears opposite charges
Uses

• Macromolecules can be characterized by their

rate of movement in an electric field.

• This property is used to determine protein

molecular weights, to distinguish molecules by
virtue of their net charge or their shape and to
separate different molecular species
quantitatively.

• Rate of movement of macromolecules in an

electric field is useful parameter to know any
change in amino acid regarding its charge.
 Principle
• Electrophoresis is similar to chromatography.
• Electric field is used as a dragging force.

• Any charged ion or molecule migrates when placed in an electric field.

The rate of migration depends upon

• 1. Net charge of molecule

• 2. Size and shape of particle

• 3. Strength of electrical field

• 4. Properties of Supporting medium

• 5. Temperature of operation
 Principle
The law of electrostatics states :
F electric = qE
where F electric is electrical force on an ion, q is the charge on the ion and E is the electric field strength
The resulting electrophoretic migration of the ion though the solution is opposed by a frictional force
F friction = Vf
where V is velocity (rate of migration) of the ion and f is its ‘frictional coefficient’.
The frictional coefficient is a measure of the drag that the solution exerts on the moving ion and is dependent on the size,
shape and state of the ion as
well as on the viscosity of the solution.

v= E * q / f
v = velocity of migration of the molecule.
E = electric field in volts per cm
q = net electric charge on the molecule
f = frictional coefficient
The movement of charged particle in an electric field is expressed in terms of electrophoretic mobility, denoted
by μ.
where, μ = v/E OR μ = q/f
For molecules with similar conformation f varies with size but not with shape.
Thus electrophoretic mobility (μ) of a molecule is directly proportional to charge density (charge/mass ratio)
 Factors affecting Electrophoresis

Electrophoresis velocity v depends up on

Inherent factors External environment

Magnitude of analyte charge Solution pH
Charge density Electric field
Molecular weight Solution Viscosity
Tertiary or quaternary Temperature
structure ie shape
 Factors effecting electrophoresis

• Strength of the electric field

• Charge of the sample used
• Size of the biomolecule
• Binding strength of the molecule
• Hydrophobicity of the sample taken
• Shape of the biomolecule
• Ionic strength of the buffer ( solution)
 Mobility
Under the electrical field, the mobility of the particle is determined by two factors:

Its charge – higher the charge greater the electrophoretic mobility.

Frictional coefficient - Size and shape of the particle decide the velocity with which the
particle will migrate under the given electrical field and the medium.

Bigger the molecule greater are the frictional and electrostatic forces exerted on it by the
medium.

Consequently, larger particles have smaller electrophoretic mobility compared to smaller

particles
Rounded contours elicit lesser frictional and electrostatic retardation compared to sharp
contours.

Therefore, globular protein move faster than fibrous protein

Schematic Representation of
Electrophoresis
 Strength of electrical field
• it determined by the force
exerted on the particle,
and the charge the particle
carrying.
• F=QV
• when force is exerted on
the particle it start moving,
however the moment is
restricted by the
experience of the frictional
force because of the
viscosity
 Effect of pH on Mobility

• As the molecule exist as amphoteric , they will carry the charges based on the
solvent pH.

• Their overall net charge is NEUTRAL when it is at zwitter ion state. And hence the
mobility is retarded to zero.

• Mobility is directly proportional to the magnitude of the charge, which is

functional of the pH of solvent.

• The pH is maintained using Buffers of different pH.

 Power supply
Drives the moment of ionic species in the medium and allow the adjustment and
control of the current or voltage.

Constant delivery is required.

Pulsed power can also be applied.

 Buffer
• The buffer in electrophoresis has two fold purpose:

• Carry applied electrical current

• They set the pH as which electrophoresis is carried out.

• Thus they determine;

• Type of charge on solute.

• Extent of ionization of solute

• Electrode towards which the solute will migrate.

• The buffer ionic strength will determine the thickness of the

ionic cloud.
 Commonly buffers used
• Buffer pH value
• Phosphate buffer around 7.0
• Tris-Borate-EDTA buffer (TBE) around 8.0
• Tris-Acetate EDTA buffer (TAE) above 8.0
• Tris Glycine buffer (TG) more than 8.5
• Tris -Citrate-EDTA buffer (TCE) around 7.0
• Tris -EDTA buffer (TE) around 8.0
• Tris -Maleic acid -EDTA buffer (TME) around 7.5
• Lithium Borate - buffer (LB) around 8.6
 Supporting medium

• Supporting medium is a matrix in which the protein separation takes place.

• Various type has been used for the separation either on slab or capillary
form.

• Separation is based on to the charge to mass ratio of protein depending on

the pore size of the medium, possibly the molecular size.
 Properties of the supporting medium
Chemical nature inert

Availability easy

Electrical conductivity high

Adsorptivity low

Sieving effect desirable

Porosity controlled

Transparency high

Electro-endosmosis (EEO) low

Rigidity moderate to high

Preservation feasible

Toxicity low

Preparation easy
zonal, paper, gel electrophoresis and
isoelectric focusing and their application.
 Types of Electrophoresis

• 1) Zone Electrophoresis
• a) Paper Electrophoresis
• b) Capillary Electrophoresis
• c) Gel Electrophoresis
• d) Thin Layer Electrophoresis
• e) Cellulose acetate Electrophoresis

• 2) Moving Boundary Electrophoresis

• a) Capillary Electrophoresis
• b) Isotachophoresis
• c) Isoelectric Focusing
• d) Immuno Electrophoresis
 ZONE Electrophoresis
Paper Electrophoresis

It is the form of electrophoresis that is carried

out on filter paper. This technique is useful for
separation of small charged molecules such as
amino acids and small proteins.

Filter paper: It is the stabilizing medium. We

can use whatman filter paper, cellulose acetate
filter paper or chromatography paper.

Apparatus: Power pack, electrophoretic cell

that contains electrodes, buffer reservoirs,
support for paper
 Paper Electrophoresis
Sample application : The sample may be applied as a spot (about 0.5
cm in diameter) or as a uniform streak.

Electrophoretic run: The current is switched on after the sample has been
applied to the paper and the paper has been equilibrated with the buffer.

The types of buffer used depends upon the type of separation.

Once removed, the paper is dried in vacuum oven.

Detection and quantitative assay: To identify unknown components

in the resolved mixture.

The electrophoretogram may be compared with another electrophoretogram on which standard

components have been electrophoresced under identical conditions.

Physical properties like fluorescence, ultraviolet absorption or radioactivity

are exploited for detection.
 Gel Electrophoresis
It is a technique used for the separation of Deoxyribonucleic acid, Ribonucleic
acid or protein molecules according to their size and electrical charge using an
electric current applied to a gel matrix.

▪ What is a gel?
Gel is a cross linked polymer whose composition and porosity is chosen based
on the specific weight and porosity of the target molecules.

Types of Gel:

▪ Agarose gel.

▪ Polyacrylamide gel.
 Agarose Gel Electrophoresis
 A highly purified uncharged polysaccharide derived from agar.

 Used to separate macromolecules such as nucleic acids, large proteins and protein
complexes.

 It is prepared by dissolving 0.5% agarose in boiling water and allowing it to cool to

40°C.

 It is fragile because of the formation of weak hydrogen bonds and hydrophobic

bonds

Gel structure of agarose

 ADVANTAGES: DISADVANTAGES:
Easy to prepare and small Electro osmosis is high.
concentration of agar is required.
Resolution is less compared to polyacrylamide
gels.
Resolution is superior to that of filter paper.

Large quantities of proteins can be Different sources and batches of agar

separated and recovered. tend to give different results and
purification is often necessary
Adsorption of negatively charged protein molecule
is negligible.

It adsorbs proteins relatively less when compared

to other medium.

Sharp zones are obtained due to less

adsorption.

Recovery of protein is good, good method for

preparative purpose
 Polyacrylamide gel Electrophoresis
• Frequently referred to as PAGE.

• Cross-linked polyacrylamide gel are formed from the polymerization of the

monomer in presence of small amount of N,N”-methylene- bisacrylamide.

• Bisacrylamide –two acrylamide linked by the methylene group.

• The polymerization of the acrylamide is an example for free radical

catalysis.

• They are defined in terms of total percentage of acrylamide present, and

pore size vary with conc.
 Free radical polymerization reaction of
polyacrylamide.

• The polyacrylamide gel matrix

is formed by polymerizing
acrylamide and a small
quantity (usually 5% or less) of
the cross-linking reagent, N,N’-
methylenebisacrylamide, in
the presence of a catalyst,
tetramethylethylenediamine
(TEMED), and source offree
radicals, ammonium
persulfate,
 Polyacrylamide gel Electrophoresis
• Commonly used components: Acrylamide monomers, Ammonium persulphate,
Tetramethylenediamine (TEMED), N,N’-methylenebisacrylamide.

• These free radicals activate acrylamide monomers inducing them to react with other
acrylamide monomers forming longchains.

• Used to separate most proteins and small oligonucleotides because of the presence
of small pores.

• Made in conc. between 3-30% acrylamide.

• Thus low % has large pore size and vice versa.

• Proteins are separated on the basis of charge to mass ratio andmolecular size, a
phenomenon called Molecular sieving.
 ADVANTAGES:

▪Gels are stable over wide range of pH and temperature.

▪Gels of different pore size can be formed.

▪Simple and separation speed is good comparatively.

 Gel electrophoresis in food science

• Protein separation

• Native Electrophoresis – (non denatured protiens, separated in their

native form based on charge, size, and shape of the molecule.)

• Denaturing electrophoresis - electrophoresis commonly used for

separating proteins in denatured form
 PAGE for protein separation
Polyacrylamidegel electrophoresis (PAGE) with an anionic detergent sodium
dodecyl sulfate (SDS), is used to separate protein subunits by size.

Proteins are solubilized and dissociated into subunits in a buffer containing SDS
and a reducing agent.

Reducing agents, such as mercaptoethanol or dithiothreitol, are used to reduce

disulfide bonds within a protein subunit or between subunits.

Proteins bind SDS, become negatively charged, and are separated based on
size alone.
A discontinuous gel matrix is usually used to improve
resolution of proteins within a complex mixture. Procedure
The discontinuous matrix consists of a stacking gel with a
large pore size (usually 3–4% acrylamide) and a resolving
gel of a smaller pore size.

To perform a separation, proteins in a buffer of the

appropriate pH are loaded on top of the stacking gel.

Bromophenol blue tracking dye is added to the protein

solution.
This dye is a small molecule that migrates ahead of the
proteins and is used to monitor the progress of a
separation.
After an electrophoresis run, the bands on the gels are
generally visualized using a protein stain such as
Coomassie Brilliant Blue or silver stain.

Specific enzyme stains or antibodiescan be used to detect

a protein.
 Applications
• Electrophoretic techniques can be used as one step in a purification
process or to aid in the biochemical characterization of a protein.

• Electrophoresis is often used to determine the protein composition of

a food product.
• For example, differences in the protein composition of soy protein
concentrates and whey protein concentrates produced by different
separation techniques can be detected.

• SDS-PAGE is used in characterization protocols to determine subunit Separation of molecular mass standards and the unknown
composition of a protein and to estimate subunit molecular mass. protein.
• Molecular mass can usually be estimated within an error of ±5%,
although
• highly charged proteins or glycoproteins may be subject to a larger
error. Molecular mass is determined by comparing Rm of the protein
subunit with Rm of protein standards of known molecular mass.

• Commercially prepared protein standards are available in several

molecular mass ranges.
• To prepare a standard curve, logarithms of protein standard molecular
• mass are plotted against their corresponding Rm values.
• The molecular mass of the unknown protein is determined from its Rm
value using the standard curve. Standard curve for estimating protein molecular mass.
 Types of Electrophoresis

• 1) Zone Electrophoresis
• a) Paper Electrophoresis
• b) Capillary Electrophoresis
• c) Gel Electrophoresis
• d) Thin Layer Electrophoresis
• e) Cellulose acetate Electrophoresis

• 2) Moving Boundary Electrophoresis

• a) Capillary Electrophoresis
• b) Isotachophoresis
• c) Isoelectric Focusing
• d) Immuno Electrophoresis
 Moving boundary electrophoresis-

• In this method, the electrophoresis is carried

in without a supporting media.

• The sample is dissolved the buffer and

molecules move to their respective counter
charge electrodes.

• Moving boundary electrophoresis is carried

out in a U shape tube with platinum
electrodes attached to the end of both arms
• this method, the electrophoresis is carried in
solution,
 Procedure
• At the respective ends, tube has
refractometer to measure the change in
refractive index of the buffer during
electrophoresis due to presence of molecule.

• Sample is loaded in the middle of the U tube

and then the apparatus is connected to the
external power supply.

• Charged molecule moves to the opposite

electrode as they passes through the
refractometer, a change can be measured.

• As the desirable molecule passes, sample can

be taken out from the apparatus along with
the buffer
 Disadvantages of Moving Boundary
electrophoresis

• The resolution of the technique is very low due to the mixing of the
sample as well as over-lapping of the sample components.

• The electrophoresis technique is not good to separate and analyze

the complex biological sample instead it can be used to study the
behavior of the molecule in an electric field.
 Capillary Electrophoresis
• Capillary electrophoresis is an analytical technique that separates ions based on their electrophoretic
mobility with the use of an applied voltage.

• The electrophoretic mobility is dependent upon the charge of the molecule, the viscosity, and the atom's
radius. The rate at which the particle moves is directly proportional to the applied electric field the greater
the field strength, the faster the mobility.

• Neutral species are not affected, only ions move with the electric field.
• If two ions are the same size, the one with greater charge will move the fastest.
• For ions of the same charge, the smaller particle has less friction and overall faster migration rate.

• Capillary electrophoresis is used most predominately because it gives faster results and provides high
resolution separation.

• It is a useful technique because there is a large range of detection methods available.

Capillary Electrophoresis

• Employing a capillary in electrophoresis

had solved some common problems in
traditional electrophoresis.

• For example, the thin dimensions of the

capillaries greatly increased the surface to
volume ratio, which eliminated
overheating by high voltages.

• The increased efficiency and the amazing

separating capabilities of capillary
electrophoresis spurred a growing interest
among the scientific society to execute
further developments in the technique.
 Instrumental Setup
• A typical capillary electrophoresis system consists of a high voltage power supply, a sample introduction system, a
capillary tube, a detector and an output device.
• Some instruments include a temperature control device to ensure reproducible results. This is because the separation of
the sample depends on the electrophoretic mobility and the viscosity of the solutions decreases as the column
temperature rises.
• Each side of the high voltage power supply is connected to an electrode.
• These electrodes help to induce an electric field to initiate the migration of the sample from the anode to the cathode
through the capillary tube.
• The capillary is made of fused silica and is sometimes coated with polyimide.
• Each side of the capillary tube is dipped in a vial containing the electrode and an electrolytic solution, or aqueous buffer.
Before the sample is introduced to the column, the capillary must be flushed with the desired buffer solution.
• There is usually a small window near the cathodic end of the capillary which allows UV VIS light to pass through the
analyte and measure the absorbance.
• A photomultiplier tube is also connected at the cathodic end of the capillary, which enables the construction of a mass
spectrum, providing information about the mass to charge ratio of the ionic species.
 Types- of Capillary electrophoresis
• There are six types of capillary electroseparation available:

• 1 Capillary zone electrophoresis (CZE)

• 2 Capillary gel electrophoresis (CGE)

• 3 Micellar electrokinetic capillary chromatography (MEKC)

• 4 Capillary electrochromatography (CEC )

• 5 Capillary isoelectric focusing (CIEF)

• 6 Capillary isotachophoresis
 Applications

• May be used for the simultaneous determination of the ions NH4 +, Na +, K +, Mg2+ and Ca 2+ in
saliva.

• One of the main applications of CE in forensic science is the development of methods for
amplification and detection of DNA fragments using polymerase chain reaction.

• Capillary electrophoresis (CE) has become an important, cost effective approach to do DNA
sequencing.

• A specialized type of CE, affinity capillary electrophoresis (ACE ), utilizes intermolecular binding
interactions to understand protein ligand interactions.

• A major use of CE by forensic biologists is typing of STR from biological samples to generate a
profile from highly polymorphic genetic markers which differ between individuals.
 Application in food science
• There are three variations of capillary electrophoresis commonly used for protein separations.
• Capillary zone electrophoresis or free solution electrophoresis is much like native PAGE,
except proteins are separated in free solution inside capillary tubes filled with buffer of the desired pH.
• Diffusion is prevented within the narrow diameter of the capillaries, so a gel matrix is not required.
• In capillary zone electrophoresis, electroosmotic flow also influences the separation of proteins within
capillary tubes.
• The negatively charged fused silica capillary wall [containing silanol groups (SiO−)] attracts positively
charged ions (cations) from the buffer to form a double ion layer at the interface between the capillary
column wall and the buffer.
• When the electric field is applied, the cations forming the double layer are attracted toward the cathode
and “pull” other molecules (independent of charge) in the same direction.
• Thus, in free solution capillary electrophoresis, cations, anions, and uncharged molecules can be
separated in a single run.
• Electroosmotic flow can be controlled by changing the pH or ionic strength of the buffer to alter the
charge on the capillary wall and change the rate of protein migration.
• Capillary zone electrophoresis is used for a variety of applications, including the fractionation of milk,
cereal, soybean, and muscle proteins
SDS capillary gel electrophoresis techniques can be used to separate proteins
by size and to determine molecular mass.

In this technique proteins are denatured and dissociated in the presence of

SDS and a reducing agent, then fractionation occurs in polyacrylamide gel-
filled capillaries of specific pore sizes.

Alternatively, linear polymers, such as methyl cellulose, dextrans, or

polyethylene glycol, are added to the buffer within the capillary in a technique
called dynamic sieving capillary electrophoresis.

These entangled polymers act like the pores of the polyacrylamide gel to slow
migration of the larger proteins and allow separation by size.
 Capillary isoelectric focusing.

• Proteins also can be separated on the basis of their isoelectric points, in a

technique called capillary isoelectric focusing.

• Ampholytes are used to form a pH gradient within the capillary tube. A gel
matrix is not needed.

• In this technique, electroosmotic flow is minimized by coating the capillary walls

with buffer additives to prevent undesirable effects caused by surface charge.
 Isoelectric focusing
• Electrophoretic method that separates proteins according to the iso electric points.

• Is ideal for separation of amphoteric substances.

• Separation is achieved by applying a potential difference across a gel that contain a pH

gradient.

• Isoelectric focusing requires solid support such as agarose geI and poly acrylamide gel

• Isooelectric focusing gels contain synthetic buffers called ampholytes that smooth the
pH gradients

• Ampholytes are complex mixtures of synthetic polyamino polycarboxylic acids

• Commercially available ampholytes are BIO-LYTE PHARMALYTE

 Principle
• Isoelectric focusing is a modification of electrophoresis, in which
proteins are separated by charge in an electric field on a gel matrix in
which a pH gradient has been generated using ampholytes.
• Proteins are focused or migrate to the location in the gradient at
which pH equals the pI of the protein.

• Resolution is among the highest of any protein separation technique

and can be used to separate proteins with pIs that vary less than 0.02
of a pH unit.
 Procedure
• A pH gradient is formed using ampholytes, which are small polymers (molecular mass of
about 5000 Da) containing both positively and negatively charged groups.

• An ampholyte mixture is composed of thousands of polymers that exhibit a range of pH

values.

• Ampholytes are added to the gel solution prior to polymerization.

• After the gel is formed and a current applied, the ampholytes migrate to produce the pH
gradient; negatively charged ampholytes migrate toward the anode while positively
charged ampholytes migrate toward the cathode.

• Ampholyte mixtures are available that cover a narrow pH range (2–3 units) or a broad
range (pH 3– 10) and should be selected for use based on properties of the proteins to
be separated.
 Advantages
• It gives good separation with a high resolution compared to any other method
• Resolution depends on

• 1.ThepH gradient,

• 2.The thickness of the gel

• 3.Time of electrophoresis

• 4.The applied voltage

• 5.Diffusion of the protein into the gel

 Applications
• Isoelectric focusing is the method of choice for determining the isoelectric point of a protein
and is an excellent method for determining the purity of a protein preparation.
• For example, isozymes of polyphenol oxidase and other plant and animal proteins are identified
using isoelectric focusing.
• Isoelectric focusing is used to differentiate closely related fish species based on protein
patterns.
• Isoelectric focusing and SDS-PAGE can be combined to produce a two-dimensional
electrophoretogram that is extremely useful for separating very complex mixtures of proteins.
• This technique is called two-dimensional electrophoresis.
• Proteins are first separated in tube gels by isoelectric focusing.
• The tube gel containing the separated proteins is then placed on top of an SDS-PAGE slab gel,
and proteins are separated.
• Thus, proteins are separated first on the basis of charge and then according to size and shape.
Over 1000 proteins in a complex mixture have been resolved using this technique.