BCH 410

INTRODUCTION TO BIOCHEMICAL RESEARCH

Electrophoresis

Dr. Aliyu A.Turaki

turakialiyu@yahoo.com
 Principle
 Charged ion or molecule migrates when placed in an electric field

 Rate of migration depends on its net charge, size, shape and the
applied electric current
v = μeE
where, v = velocity of an ion
E = electric field strength (Vcm-1)
μe = electrophoretic mobility
= distance migrated in a certain time period

 The electrophoretic mobility is given by

q
μe = (when electric force = frictional drag)
6πηr
showing that small highly charged species have high mobility and vice
versa.
 Basic of electrophoresis
 Differential rate of migration of ion molecule in an electrolyte
solution under the influence of an applied electric field in a
support medium (e.g. paper, gel or capillary tube)
 A useful method to separate substances based on their charge –
to – mass ratios.

Motion of a charged particle by electrophoresis

Driving force of migration
Resultant of the electrostatic force of attraction
between the electric field and the charged molecule,
and the retarding forces due to friction and electrostatic
repulsion from molecules of the transport medium.

Illustration of electrophoresis retardation

 Factors affecting electrophoretic mobility
 Charge – higher the charge greater the mobility.
 Size – bigger the molecule greater the frictional and electrostatic
forces exerted on it by the medium i.e. larger particles have
smaller electrophoretic mobility compared to smaller particles.
 Electric field – increase of migration with the increase of voltage
gradient.
 Buffer – dependence of migration on pH of the buffer.
 Ionic strength – greater the ionic strength of the buffer solution
higher proportion of the current hence electrophoretic mobility
Types of electrophoresis

Electrophoresis

Frontal
Zone electrophoresis
Electrophoresis

Micro Moving Paper Cellulose acetate Gel

electrophoresis boundary electrophoresis electrophoresis electrophoresis

 Paper (filter paper such as Whatman no.1 and no.3MM, Used to

good effect).
 Cellulose acetate (containing 2 to 3 acetyl groups, to give sharper
bands, more easily rendered transparent, low solvent capacity,
enhancing the resolution).
 Gels (3 dimensional semisolid colloids, resolving power enhanced
due to sieve effect operating, prepared from starch, agar, or
polyacrylamide).
• Gel electrophoresis: is a laboratory method used to
separate mixtures of charged molecules in solution
primarily, DNA, RNA and proteins, migrate in response to
an electrical field.
• The molecules to be separated are pushed by an electrical
field through a gel that contains small pores in a buffer
solution.
• Their rate of migration through the electrical field,
depends on the strength of the field, on the net charge,
size, and shape of the molecules, and also on the ionic
strength, viscosity, and temperature of the medium in which the
molecules are moving.
• As an analytical tool, electrophoresis is simple, rapid and highly
sensitive.
• It can be used analytically to study the properties of a single charged
species or mixtures of molecules. It can also be used preparatively
as a separating technique
• A buffer solution is a mixture of chemical compounds in a
given proportion to give a specific PH. There are a number
of The most common buffers used for electrophoresis of
nucleic acids are Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA
(TBE).
• Buffer solutions are used to provide ions that carry a
current and maintain the pH at a relatively constant value.
Why do scientists use gel electrophoresis?
One of the advantages of gel electrophoresis is
that scientists can separate several samples side by side so
they can compare them. The comparison of separated DNA
molecules is the basic method behind the DNA fingerprints
that forensic scientists use to compare samples from crime
scenes with those of suspects.
 Electrophoresis Techniques

Low voltage (LVE)

High voltage (HVE)

SDS polyacrylamaide gel (SDS-PAGE)

Techniques Isoelectric focusing

Immunoelectrophoresis

Denaturing Gradient Gel Electrophoresis (DGGE)

Discontinuous electrophoresis
Electrophoresis
Electrophoresis is usually done with gels formed in
tubes, slabs, or on a flat bed.
In many electrophoresis units, the gel is mounted
between two buffer chambers containing separate
electrodes, so that the only electrical connection
between the two chambers is through the gel.
 Applications of electrophoresis
 DNA analysis
 RNA analysis
 Protein analysis
 Antibiotic analysis
 Vaccine analysis
 Detection of damaged genes by gel electrophoresis
 Forensic research
Electrophoresis Gel apparatus

Tube Gel Units Flat Bed Unit

Slap Gel Unit

 General procedure for electrophoresis

Immersion of two electrodes in

two separate buffer chambers but
not fully isolated from each other

Migration of charged particles

from one chamber to the other
by using an electric field

Separation of different ions

migrating at different speeds

Fundamental of electrophoresis
 What is the Role of the Solid Support Matrix?
• It inhibits convection and diffusion, which would otherwise impede separation of
molecules
• It allows a permanent record of results through staining after run
• It can provide additional separation through molecular sieving
Agarose and Polyacrylamide
• Agarose and polyacrylamide differ greatly in their physical and chemical
structures, they both make porous gels.
• A porous gel acts as a sieve by retarding or, in some cases, by completely
obstructing the movement of macromolecules while allowing smaller molecules
to migrate freely.
• This operator can take advantage of molecular size differences among proteins
• The pores of an agarose gel are large, it is used to separate macromolecules such
as nucleic acids, large proteins and protein complexes
• Polyacrylamide, which makes a small pore gel, is used to separate most proteins
and small oligonucleotides.
• Both are relatively electrically neutral
 Agarose Gels
• Agarose is a highly purified uncharged polysaccharide derived from
agar with a repeating Unit of Agarose, 3,6-anhydro-L-galactose.
• Agarose dissolves when added to boiling liquid. It remains in a liquid
state until the temperature is lowered to about 40° C at which point
it gels
• The pore size may be predetermined by adjusting the concentration
of agarose in the gel
• Agarose gels are fragile, however. They are actually hydrocolloids,
and they are held together by the formation of weak hydrogen and
hydrophobic bonds.
 Polyacrylamide Gels

• Polyacrylamide gels are tougher than agarose gels

• Acrylamide monomers polymerize into long chains that are
covalently linked by a cross linker.
• Polyacrylamide is chemically complex, as is the production and use
of the gel.
 Electrophoresis Equipment
Horizontal or submarine gel Vertical gel

Combs are used to put wells in the cast gel for sample loading.

Regular comb: wells separated by Houndstooth comb: wells

an “ear” of gel. immediately adjacent
 Running a Gel
• Use the proper gel concentration for sample size range.
– 0.5–5% agarose
– 3.5–20% polyacrylamide
• Use the proper comb (well) and gel size.
• Load sample mixed with tracking dye (dye + density agent).
 Running a Gel

• Detect bands by staining during or after electrophoresis

• Ethidium bromide: for double-stranded DNA
• SyBr green or SyBr gold: for single- or double-stranded
DNA or for RNA
• Silver stain: more sensitive for single- or double-stranded
DNA or for RNA and proteins
• Red safe: for single- or double-stranded DNA or for RNA
Protein Gel Electrophoresis

1. Native PAGE
2. Native Gradient PAGE
3. Urea PAGE
4. SDS PAGE
5. SDS Gradient PAGE
6. IEF
7. 2D PAGE
8. Western Blot
 Principle

Proteins move in the electric field. Their relative speed depends

on the charge, size, and shape of the protein.
 Protein visualization on gels

 Immediately after electrophoresis proteins in the gels

are precipitated by either adding alcohol containing
solutions or strong acids (e.g. TCA).
 Protein are often stained by Coomassie Blue dye or by
photography-like treatment with AgNO3 (silver
staining)
 There are many other stains available (e.g. Stains-all,
fluorescence probes etc.)
Silver and coomsassie blue stained protein gel

• Silver staining is usually 10-100 times more sensitive than Coomassie

Blue staining, but it is more complicated.
• Faint but still visible bands on this gel contain less than 0.5 ng of protein!
 Native PAGE
 Separates folded proteins and protein-protein or
protein-ligand complexes by charge, size, and shape.
Useful for
• Examining protein-protein protein-ligand interactions
• Detecting protein isoforms/conformers
 WHAT IS CHROMATOGRAPHY?

Chromatography is a physical process of separation in which the

components to be separated are distributed between 2 immiscible
phases a stationary phase which has a large surface area and
mobile phase which is in constant motion through the stationary
phase.
According to the force of separation:
• Adsorption chromatography
• Partition chromatography
• Ion exchange chromatography
• Gel filtration chromatography
• Affinity chromatography
Invention of Chromatography by Mikhail Tswett
What type of chromatography did he invent?

Ether Chromatography

Chlorophyll
Colors
CaCO3

Adsorption chromatography
Chromato-graphy / -graph / -gram / -grapher

• Chromatography: Analytical technique

• Chromatograph: Instrument
• Chromatogram: Obtained “picture”
• Chromatographer: Person
 THIN LAYER CHROMATOGRAPHY
• One of analysis method that is used to identify the unkonwn
compounds and to determine the purity of mixture.
• This method is simple, rapid and cheap.
• Used to separate non-volatile compounds in a mixture.
• Involves the distribution of the compounds (or ions) between two
phases, one of which is stationary and the other moving.
• Performed on a sheet of glass, plastic, or aluminum, which is coated
with a thin layer of adsorbent material, usually silica gel, aluminum
oxide (alumina), and is referred as the stationary phase.
• The separation process depends on differences in how strongly the
components in the mixture (analytes) are adsorbed to the stationary
phase and how soluble they are in the moving phase.
• The differences depend on the relative polarities of the components.
• It is similar to solvent extraction, which depends on the relative
solubilities of the compounds in two solvents.
Interaction Between Solutes, Stationary Phase,
and Mobile Phase

• Differences in the interactions between the solutes and

stationary and mobile phases enable separation.
Solute
Degree of adsorption,
solubility, ionicity, etc.

Stationary
Mobile phase
phase
 STATIONARY PHASE

• Silica is commonly used as stationary phase

• The separation of sample mixture will be depent on the polarity
of sample.

• Some modified silica is also used in certain purposes.

• A plate of TLC can be made from aluminum or glass which is
coated by a solid matter as a stationary phase.

- The coated material has 0.1-0.3mm in thickness

-some of them has been added by fluorescent indicator that will

make it florescence during the UV light exposure.
 Stationery phase Description Application

Silica gel G Silica gel with average Used in wide range

particle size 15µm pharmacopoeial test
containing ca 13% calcium
sulfate binding agent
Silica gel G254 Silica gel G with Same application with
fluorescence added Silica gel G where
visualization is to be
carried out under UV
light.
Cellulose Cellulose powder of less Identification of
than 30µm particle size. tetracyclines
 MOBILE PHASE
• The ability of mobile phase to move up is depend on the polarity itself
• Volatile organic solvents is preferably used as mobile phase.

SOLVENT POLARITY INDEX

Heksana 0
Butanol 3.9
Chloroform 4.1
Methanol 5.1
Ethanol 5.1
Acetonitrile 5.8
Air 9.0
 MATERIALS

• TLC plate
• ‘Developing container’
- chamber/ jar/ glass beaker
• Pencil
• Ruler
• Capillary pipe
• Solvents / mobile phase
- organic solvents
• UV lamp
 methodology

1.Developing Container Preparation 2. TLC Plate Preparation

Solvent is transferred into the container

with 0.5-1cm in dept from the bottom  Commercially obtained with 5cm
x 20cm in size
 Prepare your size when necessary
 Line 1 cm from the bottom with a
pencil as a part should be spotted.
 3.Spotting’ TLC plates 4.‘Develop the plate’

 Make sure that your sample is  after spotting, put the plate inside the
liquified already. chamber in the ascendant position
 stick it using capillary pipe & spot  Make sure that the depth of solvent doesn't
onto the line you have made touch the spots
 Let it develop up to the 1cm from the top of
plate
 After that, pull out the plate from the
chamber and let the solvent be vaporized
 5. Detection of spots

- The color samples are easy

to be seen and no need to
use UV lamp to detect them

6. DETECTION OF SPOT
1) Iodination-put the plate in which the spots face to the iodine crystal
and see what is the spot color changing
2) Ninhydrin:
-specific identification of amino acid compounds.
- Ninhydrin solution will show a purple spot when it is sprayed to the
amino acid spot.
3) KMnO4
used to identify a reducing agent such as glucose, fructose, vitamin C and
others.
4) Alkaline tetrazolium blue
specifically used for corticosteroid identification
 Determination of Rf Values
• The distance taken through by the solvent to move up will be assigned as
solvent front
• The distance taken trough by the sample to move up will be assign as sample
front
• Rf value is obtained by dividing the sample front toward solvent front

solvent front

component B

dS
Less polar!
dB

Rf of component A = dA dS
component A

dA More polar!
Rf of component B = dB dS origin

The Rf value is a decimal fraction, generally only reported to two decimal places
 Calculation of Rf’s
2.0 cm
Rf (A) = = 0.40
5.0 cm
Solvent Front

Rf (B) = 3.0 cm = 0.60

5.0 cm
Distance solvent
migrated = 5.0 cm
4.0 cm
Distance A
migrated = 3.0 cm Rf (C) = 0.8 cm = 0.16
5.0 cm

Distance B
migrated = 2.0 cm 3.0 cm Rf (D) = 4.0 cm = 0.80
5.0 cm
Distance C
migrated = 0.8 cm
0.8 cm
Rf (U1) = 3.0 cm = 0.60
Origen
x x x x x 5.0 cm
A B U C D
0.8 cm
Rf (U2) = = 0.16
5.0 cm

The Rf is defined as the distance the center of the spot moved divided by the
distance the solvent front moved (both measured from the origin)
 Rf value
Rf values can be used to aid in the identification of a substance
by comparison to standards.
The Rf value is not a physical constant, and comparison should
be made only between spots on the same sheet, run at the
same time.
Two substances that have the same Rf value may be identical;
those with different Rf values are not identical.

A substance whose structure resembles the stationary phase

will have a low Rf
A substance that has a similar structure to the mobile phase will
have a high Retention Factor
The Retention Factor will change whenever either the mobile or
stationary phase changes 40
 Comments
• Do not let the plate sit in the solvent chamber after the
solvent front reaches the top of the plate. Why?
• When spotting, the solution typically adsorbs very quickly and
the spot can easily get larger than desired (1 mm diameter).
• Make 3-4 lanes on each plate, marked with pencil and ruler
and experiment with large and small spots to find which
works best.
• Record the Rf values for each substance.
• Follow lab handout for all experimental procedures.
• Keep the layer of liquid with the most color.
• Do not evaporate the dichloromethane solution
– Solvents used are flammable, use caution
• Separation of components should be easy to distinguish.
HIGH PERFORMANCE THIN LAYER CHROMATOGARPHY(HPTLC)

Introduction

 HPTLC is the improved method of TLC which utilizes the

conventional technique of TLC in more optimized way.
 HPTLC takes place in highspeed capillary flow range of the mobile
phase.
There are three main steps HPTLC procedure
 Sample preparation, volume precision and exact position are
achieved by use of suitable instrument.
 Solvent (mobile phase) migrates the planned distance in layer
(stationary phase) by capillary action. In this process sample
separated into it’s components.
 Separation tracks are scanned in densitometer with light
beams in visible or uv region
Dr. A.R.Bekhradnia 43
 Selection of HPTLC plates

• Previously hand made plates is used in TLC for both qualitative and
quantitative work. Certain drawbacks with that is nonuniform layer,
formation of thick layer, paved for advent of precoated plates.

• Nowadays precoated plates are available in different format and thickness

by various manufactures. Precaoted plates can be used for both
qualitative and quantitative work in HPTLC, they are

• GLASS PLATES
• POLY ESTER/POLYETHYLYNE
• ALUMINIUM PLATES
 Glass Plates:
 Offers superior flat and smooth surface.
- fragile
- high weight
- higher production cost

Polyester/polyethylene plates:
Thickness of plate is 0.2mm.
- It can be produced in roll forms.
- Unbreakable.
- Less packing material is required.
- Development of plate can not be above temperature
1200 c loses its shape.
Aluminium plates:
- Thickness of plate is 0.1mm.
- It can be produced in roll forms.
- Unbreakable.
- Less packaging material is required.

SORBENTS USED IN HPTLC PLATES:

sorbents which are used in convential TLC are also used in HPTLC
with or without modification.
- silica gel 65F
- highly purified silicagel 60
- aluminium oxide
- cellulose microcrystalline
- silica gel
- reversed stationary phase
 Layer thickness :
The layer thickness in HPTLC is around 100-200cm,in conventional it is 250mm.
Layer prewashing:
- Ascending method
- Dipping method
- Continuous method

ACTIVATION OF PRECOATED PLATES

• The plates are activated by placing in an oven at 110-1200 C for 30 min, this step will
removes water that has been physically absorbed on surface at solvent layer.

• Freshly opened box of HPTLC plates usually does not require activation.

• Activation at higher temp and for longer time is avoided which leads to very active
layer and there is risk of sample being decomposed.
 Solvents used in HPTLC
- Methanol (commonly used)
- Chloroform:methanol:ammonia(90:10:1)
- Chloroform:methanol(1:1)
- Methylene chloride:methanol(1:1)
- Ammonia(1%)solution

Application of sample and standard

• Usual concentration range is 0.1-1µg / µl,above this causes poor
separation.

• Linomat IV (automatic applicator) - nitrogen gas sprays sample

and standard from syringe on TLC plates as bands.

• Band wise application - better separation - high response to

densitometer.
 Chromatographic development and drying
• After development, remove the plate and mobile phase is
removed from the plate - to avoid contamination of lab
atmosphere.

• Dry in vacuum desiccator - avoid hair drier because essential oil

components may evaporate.

Detection and visualization

• Detection under UV light is first choice - non destructive.
• Spots of fluorescent compounds can be seen at 254 nm (short wave
length) or at 366 nm (long wave length).
• Spots of non fluorescent compounds can be seen - fluorescent
stationary phase is used - silica gel GF.
 AUTOMATIC TLC SAMPLER

• pre-equilibration with solvent vapor

• low solvent consumption

Start of development
LIGHT WEIGHT TWIN TROUGH CHAMBER
 NANOMAT

Automatic Developing Chamber ADC

UV –INSPECTION -UV Lamps.

TLC Scanner 3 with “CATS” Soft Ware.
Video Scan.
 HPTLC TLC
 100µm • 250µm
 High due to smaller particle size • Less
generated
• 10 - 15 cm
 3 - 5 cm
 Shorter migration distance and the
• Slower
analysis time is greatly reduced
 Wide choice of stationary phases like
silica gel for normal phase and C8 , C18 • Silica gel , Alumina
for reversed phase modes
 New type that require less amount of • More amount
mobile phase
 Auto sampler • Manual spotting
 Use of UV/ Visible/ Fluorescence scanner
scans the entire chromatogram
qualitatively and quantitatively and the
• Not possible
scanner is an advanced type of
densitometer
 APPLICATIONS
• Pharmaceutical Researches
• Biomedical Analysis
• Clinical Analysis
• Environmental Analysis
• Food Industry
• Therapeutic drug monitoring to determine concentration of drug
and it’s metabolite in blood, urine etc
• Analysis of environmental pollutions levels
• Quantitative determination of prostaglandin’s and thromboxanes
in plasma
• Analysis of nitrosoamines in food and body fluids
• Determination of ascorbic acid in wine
• Characterization of hazards in industrial waste
Thank you