Chapter12.

Electrophoresis
Introduction:

 Electrophoresis is the motion of dispersed particles relative to a fluid under the influence
of a spatially uniform electric field.
 Electrophoresis of positively charged particles (cations) is sometimes
called cataphoresis, while electrophoresis of negatively charged particles (anions) is
sometimes called anaphoresis.
 The electrokinetic phenomenon of electrophoresis was observed for the first time in 1807
by Russian professors Peter Ivanovich Strakhov and Ferdinand Frederic Reuss
at Moscow State University, who noticed that the application of a constant electric field
caused clay particles dispersed in water to migrate.
 It is ultimately caused by the presence of a charged interface between the particle surface
and the surrounding fluid. It is the basis for analytical techniques used in chemistry for
separating molecules by size, charge, or binding affinity.
 Electrophoresis is used in laboratories to separate macromolecules based on size. The
technique applies a negative charge so proteins move towards a positive charge.
Electrophoresis is used extensively in DNA, RNA and protein analysis.

Principle:

Electrophoresis is a method used to separate charged particles from one another based on
differences in their migration speed. In the course of electrophoresis, two electrodes (typically
made of an inert metal, e.g. platinum) are immersed in two separate buffer chambers.

The two chambers are not fully isolated from each other. Charged particles can migrate from one
chamber to the other (Figure-1). By using an electric power supply, electric potential (E) is
generated between the two electrodes. Due to the electric potential, electrons move by a wire
between the two electrodes. More specifically, electrons move from the anode to the cathode.
Hence, the anode will be positively charged, while the cathode will be negatively charged. As
mentioned above, the two electrodes are immersed in two buffer chambers.
Electrons driven to the cathode will leave the electrode and participate in a reduction reaction
with water generating hydrogen gas and hydroxide ions. In the meantime, at the positive anode
an oxidation reaction occurs.

Electrons released from water molecules enter the electrode generating oxygen gas and free
protons (which immediately form hydroxonium ions with water molecules).

The amount of electrons leaving the cathode equals the amount of electrons entering the cathode.
As mentioned, the two buffer chambers are interconnected such that charged particles can
migrate between the two chambers.

These particles are driven by the electric potential between the two electrodes. Negatively
charged ions, called anions, move towards the positively charged anode, while positively
charged ions, called cations, move towards the positively charged cathode.

Figure: 1 The principle of electrophoresis

Types of Electrophoresis:

There are numerous applications of electrophoresis. Routine protein electrophoresis performed in

clinical laboratories is the oldest method and therefore the most frequently used method. With
the advent of molecular diagnostics, several other electrophoresis methods have become very
important, highly automated, and have several important applications.

Types of electrophoresis that will be discussed are:

 Routine electrophoresis
 High resolution electrophoresis
 Polyacrylamide gel electrophoresis
 Capillary electrophoresis
  Isoelectric focusing
 Immunochemical electrophoresis
 Two-dimensional electrophoresis
 Pulsed field electrophoresis

Applications:

 Electrophoresis is a technique used in laboratories in order to separate macromolecules

based on size.
 Gel electrophoresis is a biochemistry technique for separating protein molecules of
varying sizes in a mixture by moving them through a block of gel by means of an electric
field, with smaller molecules moving faster and farther than larger ones.
 Electrophoresis units are research tools designed for DNA, protein, and nucleic acid
applications, and they are constructed for molecular biology and bioresearch laboratory
and classroom settings.
 Gel electrophoresis technique is used in DNA fingerprinting, for instance, and other
processes in which large molecules are to be identified. Fragments of DNA are placed in
a gel and an electrical field is turned on.
 Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology &
biochemisty.
 The fragments move in response to the field, with smaller fragments generally moving
faster. After a time, the fragments have separated enough to form a series of separated
lines that become like a barcode that characterizes the DNA.

Gel Electrophoresis:

Gel electrophoresis is a method for separation and analysis of macromolecules

(DNA, RNA and proteins) and their fragments, based on their size and charge. It is used in
clinical chemistry to separate proteins by charge or size (IEF agarose, essentially size
independent) and in biochemistry and molecular biology to separate a mixed population of DNA
and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate
proteins by charge.

Gel electrophoresis uses a gel as an anticonvective medium or sieving medium during

electrophoresis, the movement of a charged particle in an electrical field. Gels suppress the
thermal convection caused by application of the electric field, and can also act as a sieving
medium, retarding the passage of molecules; gels can also simply serve to maintain the finished
separation, so that a post electrophoresis stain can be applied. DNA Gel electrophoresis is
usually performed for analytical purposes, often after amplification of DNA via polymerase
chain reaction (PCR), but may be used as a preparative technique prior to use of other methods
such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for
further characterization.

Types of gel electrophoresis:

1. Agarose gel electrophoresis

2. Polyacrylamide gel electrophoresis
3. Starch gel electrophoresis

Application of gel electrophoresis:

 Estimation of the size of DNA molecules following restriction enzyme digestion, e.g.
in restriction mapping of cloned DNA.
 Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
 Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior
to Northern transfer.
 Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology &
biochemisty.

 The results can be analyzed quantitatively by visualizing the gel with UV light and a gel
imaging device.
 The image is recorded with a computer operated camera, and the intensity of the band or
spot of interest is measured and compared against standard or markers loaded on the
same gel.
 The measurement and analysis are mostly done with specialized software.
 Depending on the type of analysis being performed, other techniques are often
implemented in conjunction with the results of gel electrophoresis, providing a wide
range of field-specific applications.

Laser:
 The word Laser is actually an acronym for Light Amplification by Stimulated Emission
of Radiation.
 In 1960, the first fully functional laser was completed, but the technology of the laser
goes back to Einstein's study of blackbody radiation in 1917.
 Blackbody radiation refers to a cavity that absorbs all the radiation that falls upon it and
re-emits part of this radiation in a proportion of quantized energy.
 Max Planck made this discovery. It was one of the founding discoveries in quantum
physics (the physics of the subatomic world). This study of blackbody radiation led
Einstein to discover the phenomenon of stimulated emission.
 Stimulated emission is when electrons absorb energy from an electric current or other
source of electromagnetic waves and become 'excited,' meaning they jump from a lower
energy level to a higher energy level.
 When the electrons return to their original energy state they give off a photon
(electromagnetic radiation). This light is different from the normal light spectrum that we
can see because the photons emitted are all the same wavelength, focused, and
directional. This stimulated emission of photons from excited electrons is the main
principle of how lasers work.
 Laser is the abbreviation for “Light amplification by stimulated emission of radiation”.
 Laser light is emitted when many atoms undergo similar energy transitions at the same
time.
 This is achieved by promoting a large number of atoms to an energy level above the
ground state. As an electron in one of the excited atoms jumps down from its higher
energy level it emits a photon.
 As this photon travels past another atom in an excited state, it causes the electron in this
atom to jump down to the lower level.
 The passage of light thus stimulates the emission of radiation from other atoms –
producing the intense beam of light characteristic of the laser.
 The laser was first demonstrated by the American Theodore Maiman in 1960 using a
ruby laser. The stimulated emission of radiation was initially postulated by Einstein in
1917.
 The laser’s counterpart in the microwave part of the spectrum paved the way for the
production of laser light after two American scientists Schawlow and Townes made a
theoretical paper proposing how the maser technology could be widened to fit the visible
part of the spectrum.
 A laser consists of an active medium which is placed between two mirrors. The
arrangement of the mirrors is called a laser cavity. On of the mirrors is semi-transparent
to release the laser that is generated in the cavity.
 Energy must be supplied from ex. flash bulb. The supplement of energy in the form of a
for instance flash bulb is called pumping.
 The active medium consists of atoms or molecules. Normally almost all of the atoms are
in their lowest energy level, the so called ground state.
 From there they can be transferred to a higher, excited energy level through absorption of
a light quantum (a photon) with the energy ∆E = hv.
 The upper state is often very short-lived (microsecond to nanosecond) and the atom
returns to the ground state during the emission of radiation.
 Two radiation processes can happen; spontaneous emission or stimulated emission. The
spontaneous emission consist of a random emission of photons in random direction is
responsible for the excited state being so short-lived.
 The stimulated emission can happen if the atom is shone with radiation that has the
frequency that is equal to the transition (v). The stimulated photons are emitted with the
same frequency as that of the stimulating photons.
 The probability for the decay of an excited atom is equal to the probability for absorption
of a photon by an atom in its ground state and being excited.
 The stimulated emission can be regarded as a negative absorption.
 Lasers can be modified to have an enormous sharp in frequency, effect in pulse and
length of pulse. This can’t be obtained in the same laser system.
 According to Heisenberg’s uncertainty relationship an ultra short pulse can not be sharp
in frequency.
 Different types of laser:
1. Gas laser (ex. argon laser)
2. Carbon dioxide laser
3. Diode laser Dye laser

 The technique of laser has a vast number of adaptations for example in:
1. Measuring in the industry workshop.
2. Astronomy (ex. measuring the distance from the earth to the moon)
3. Welding, cutting, hardening of metals etc.
4. Analysis techniques (analytic chemistry, remote analysis of pollutants)
5. Military use - Information technology IT (transmissions in fiber optic communication
systems, data processing)
6. Medicine (treatment of diseases)
__________________________________________________________________
 Maser
 The word Maser an acronym as well, stands for Microwave Amplification
by Stimulated Emission of Radiation. Masers were invented in 1953 by two scientists,
Charles Townes and Arthur Schawlow.

 Masers use microwaves, hence the M in its acronym. Microwaves are a type of
electromagnetic radiation that fall on the long wavelength side of the electromagnetic
spectrum.

 Microwaves have a shorter wavelength than radio waves but a longer wavelength than
the colors we see in visible light.

 Since microwaves have very long wavelengths, they have a low frequency and therefore
are low energy. Microwaves are not nearly as dangerous as ultraviolet or gamma rays
which have very short wavelengths and high frequencies.

 Masers, like lasers, also use stimulated radiation emission as the essential principle of
how they work. The electrons in masers are hit by some form of electromagnetic waves
which excite the electrons to a higher energy level, and when the electrons move down to
a lower energy level, they emit a photon with a longer wavelength and lower frequency
(less energy) than a laser. These are known as microwaves.

 Maser Maser is the abbreviation for “Microwave amplification by stimulated emission of

radiation”. Maser is an amplifier or generator for electromagnetic radiation usually
microwaves. The laser is an optical maser.
 Einstein showed in 1917 how atoms, ions or molecules can emit radiation in the form of
energy quanta (photons) through spontaneous (disordered photon emission) or photon
emission stimulated through a signal.
 It was unclear for a long time if stimulated emission happens orderly (amplify the signal)
or if it also add together the photos disorderedly (increasing the noise). It wasn’t until
1954 when C. H. Townes showed that it amplifies the signal.
 Some gases (ex. ammonium) and solid materials (ex. ruby) can be used in a maser. An
example of adaptations is sensitive amplifier for microwaves.