Electrophoresis

Download as docx, pdf, or txt
Download as docx, pdf, or txt
You are on page 1of 10

Electrophoresis

2022

A Seminar on

BASIC INFORMATION ON ELECTROPHORESIS

Subject Name: Modern Pharmaceutical Analytical Techniques

Subject Code: MPH 101T

Name of Student: Rathod Mansi Prakashbhai

Semester: 1

Course: M.Pharm (Pharmaceutics)

Seminar Mentor: Dr. Sharddha J. Parmar

Designation: Assistant Professor

Department of Pharmaceutical Sciences

Sardar Patel University

Vallabh Vidhyanagar

Rathod Mansi Page 1


Electrophoresis
2022

INDEX

Sr. No. Details of Content Pg No.

1 Overview on Electrophoresis 3

2 Principle of Electrophoresis 4

3 Instrumentation 4

4 General Operational Procedures 5

5 Detection 5

6 Types of Electrophoresis 6

7 Application 10

8 Reference 10

Rathod Mansi Page 2


Electrophoresis
2022

Overview on Electrophoresis
Introduction

Many substances exist in solution as electrically charged species. Once ionized, cations (+) or anions
(-) can be separated in an electrical field where they will migrate toward the cathode or anode
depending on their charge. ‘Electro’ means electric and ‘phoresis’ means to carry.
The rate at which ions move toward the attracting electrode is dependent on the balance between the
impelling force of the electric field on the charged ion and the frictional and electrostatic retarding
effects (i.e., the drag) between the sample and the surrounding medium. The degree of the drag
depends on the size and shape of the molecule and on the viscosity of the matrix. An estimate of the
velocity of a molecule in an electric field, in a solution, is given by the following equation:
v = Eq/d6πrn
where, v is the velocity at which the molecule is moving, E the electric field, q the net charge on the
molecule, d the distance between the electrodes, r the radius of the molecule, and Z the solution
viscosity.
The movement of an ion is proportional to its charge and the electric field, and inversely proportional
to its size and the viscosity of the surrounding medium.
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 tend to travel
towards the electrodes that bear opposite charges.
“Electrophoresis” literally means running in the electric field. The charged molecule moves to their
counter charge electrodes but electric field is removed before it reaches the electrode. Movement of
charged species in an electric field gives differential mobility to the sample based on the charge and
consequently resolve them. Movement of the charged particle is retarded with the addition a
polymeric gel so that a sufficient time is available for resolving the sample. The polymeric gel is inert,
uncharged and does not cause retardation by binding the molecule. Instead it, forms pores of different
size and sample pass through these pores and as a result their electrophoretic mobility is reduced.

Figure 1: Movement of the charged particle in an external field.

Rathod Mansi Page 3


Electrophoresis
2022

Principle of Electrophoresis

Mobility and Conductivity

When a charged particle of an ith species with charge number z i is placed in the electric field with an
intensity E (in Vm-1), the Coulombic force Fc = z ieE acting on the particle causes its movement; e
=1.602 * 10-19 C is the elementary charge. As in electrophoresis the particles to be separated move
in liquid or gel form, they face obstacles during the movement – mostly molecules of the solvent or
polymer chains of the gel. The collisions and/or interactions with them causes the friction force F f,
which is proportional to the velocity of the moving particle, and which acts against the Coulombic
force. After a very short time (10-13–10-10 s) both forces are balanced, Fc = Ff, and a mean velocity of
the particle attains a constant value. The net movement of charged particles in the electric field is
called electromigration and the velocity of such movement is called electrophoretic velocity. Various
species have, under given conditions, a different electrophoretic velocity. When moving for a certain
time, they are spatially separated. This is a separation principle of the majority (but not all)
electrophoretic separation techniques.

Instrumentation
The supporting medium where electrophoresis takes place is called the electrophoretic system or the
separation system. The separation systems are solutions of electrolytes that are filled into a separation
column or a separation channel. Both ends of the column are connected with vessels, which are also
filled with the solution of electrolytes and serve as a stock of the solution. The electric voltage (500–
30,000 V), which is called driving voltage, is provided by two electrodes in the two vessels and rises
the driving electric field. The analyzed species (analytes) are introduced through a narrow sample
plug, which then migrate across the longitudinal axis of the column in the liquid, the negatively
charged analytes (anions) in the direction of the anode and the positively charged ones (cations) in the
direction of the cathode. As various species have different velocities in the electric field, they are
spatially separated from each other and are detected by a suitable detector, which is located at a
certain position of the column or, sometimes, at its end.

The separation column is often a very thin tube, with an inner diameter of 10–500 mm, which is
called the capillary; the electrophoretic methods that utilize such a capillary are called the capillary
electrophoretic methods. The separation column can also be formed by a channel in various materials
(glass, plastic, silica, etc.), which is fabricated by means of technologies normally used for the
production of electronic microchips. These approaches are called ‘lab-on-a-chip’ technologies and
electrophoresis that uses this approach is called chip or microchip electrophoresis.

A hydrophilic cross-linked gel (mostly polyacrylamide or polysaccharide gel), which is soaked with a
solution of electrolytes, or a viscous solution of a linear polymer with an electrolyte, can also serve as
the separation medium. Electrophoresis using such gels or polymeric media is called gel
electrophoresis. The polymeric chains of the gel serve in most cases not only as a supporting medium
but also influence to a great extent the movement of the separated species.

Rathod Mansi Page 4


Electrophoresis
2022

Figure 2: Schematic diagram of an electrophoretic instrument

General Operational Procedures


Sample Application:

Samples are generally applied using a micropipette or syringe. The position of application will depend
on whether the sample contains components that will migrate to both electrodes. In this case the
sample is usually applied to the center of the support medium as a spot or narrow streak and
components will migrate in both directions. For samples where the components are all positively or
negatively charged, sample application should be at one end of the medium farthest away from the
attracting electrode. For a gel support medium the sample is usually applied to a well cast in the gel.
For capillaries the sample is applied at one end furthest from the detector. This is usually achieved by
dipping the capillary into the sample vial, the vial is then pressurized causing a volume to be forced
into the capillary. Sample volumes used in electrophoresis can vary from 1*10 -5 cm3 (in CE) to 5 cm3
for some preparative separations.

Detection
Detection of unknown compounds or molecules after electrophoresis is usually in situ. Most
biological molecules are colorless and need to be treated to produce stable colored compounds.
Examples are the visualization of proteins in polyacrylamide gel with Coomassie Blue or silver stain.
The medium can also be treated with compounds that bind to the sample causing them to fluoresce
under ultraviolet (UV) light. Enzymes can be detected by histochemical methods where substrates are
converted into insoluble colored products. It is possible to detect many biological compounds by
immunological methods; for example, in western blotting the separated components are transferred
from a polyacrylamide gel onto another suitable medium (e.g., nitrocellulose) that can then be treated
with antisera and the conjugated product detected by various methods. If a sample contains
radioactive components then the radioactive element can be detected by several different methods,
e.g., phosphor imaging, fluorography. In CE, there are detectors built into the instrument to detect the
peaks as they are resolved. The most frequently used is a UV absorbance detector. There are also

Rathod Mansi Page 5


Electrophoresis
2022
fluorescence, conductivity, and indirect detectors available. It is possible to combine CE and mass
spectrometry to obtain structural information about the resolved peaks, making this a very powerful
technique.
A number of methods are used for recovering separated compounds from the support medium.
Cellulose acetate will dissolve in acetone leaving the separated compound in solution.
Macromolecules can be recovered from starch and polyacrylamide gels by electrodialysis. In some
forms of preparative electrophoresis the sample is run until the components ‘run off the end’ and can
be collected.

Types of Electrophoresis
Paper Electrophoresis :

Paper electrophoresis is the simplest and cheapest form of electrophoresis. A strip of commercially
available chromatography paper is soaked in buffer and placed with one end in each buffer reservoir
(connecting wicks may be used). It is important that the paper is saturated with the buffer since it is
the buffer that conducts the majority of the current. A spot of sample is placed in the center of the
strip. When the current is applied ions separate out and migrate toward the attractive electrodes
Paper electrophoresis has several limitations. Only small charged molecules can be reliably separated
since many macromolecules adsorb on to the paper, although it is possible to reduce the adsorption by
using a buffer that is more alkaline than the isoelectric point of the sample. Paper systems are
associated with high electrical resistance causing heating that dries out the paper. Although this
method has been largely superseded by gel methods for many samples it is still useful for separation
of small molecules such as amino acids, small peptides, nucleotides, and inorganic ions. An important
limitation to this technique is that considerable diffusion of small molecules occurs at low voltage.

Figure 3: Paper electrophoresis

Zone Electrophoresis:

This mode is characterized by two features: (1) the whole separation system together with electrode
vessels is filled with a homogeneous solution of electrolytes, which is called the background
electrolyte (BGE), (2) the mixture of analyzed species to be separated is introduced or injected in a
rather low amount as a short plug at one end of the separation column. The mode of zone
electrophoresis is often used in capillary columns, when it is called capillary zone electrophoresis
(CZE). The BGE is mostly formed by a suitable buffer to maintain a certain pH, which is optimal for
the separation of a given mixture. This mode is also almost exclusively used for gel electrophoresis:
here the whole separation system is formed by a buffer-soaked hydrophilic slab gel and the mixture to

Rathod Mansi Page 6


Electrophoresis
2022
be separated is introduced in little amounts at one end. After applying the driving electric field the
analytes migrate in the column or in the slab gel with mutually different velocities so that they are
spatially separated. After some time they are detected with a suitable detector located near the second
end of the separation column; in the case of the slab gel electrophoresis, the plate with the gel is
removed from electricity and the separated analytes are visualized as spots by a suitable reagent. The
conductivity of the BGE in the separation system is only slightly influenced by the presence of the
separated analytes, if their amount is low enough. The individual zones of analytes, although narrow
at the beginning, become broader when moving in the column as diffusion causes their dispersion and
their longitudinal profile attains a Gaussian shape. If, however, the amount of an analyte is rather
high, it influences conductivity in its own zone, so it is no longer constant. Then, the nonlinearity
arising in this way causes the electromigration dispersion – another type of broadening of the zones
that further deforms zones to the triangular shape. The quantitative determination of a particular
analyte is performed by integration of its detector signal.

Figure 4: Zone electrophoresis

Isotachophoresis:

Isotachophoresis is a technique based on the principles of moving boundary electrophoresis. Two


buffer systems are used: a leading electrolyte and a trailing electrolyte. The leading electrolyte has a
higher mobility than the fastest sample component; likewise the trailing electrolyte has a slower
mobility than the slowest component. When an electric field is applied the leading electrolyte moves
quickly toward the appropriate electrode with the sample ions following, creating zones in order of
their mobilities. This method can only be used for either anions or cations, not both at the same time.
Any charged substance can be separated by isotachophoresis. Isotachophoresis is usually carried out
in capillaries. This method has the advantages of no sample preparation, speed, and is applicable to a
wide sample range. Isotachophoresis has been used for analysis of organic acids in silage, anions in
urine and serum, inorganic ions in water, proteins, and amino acids.

Rathod Mansi Page 7


Electrophoresis
2022

Figure 5: Isotachophoresis

Isoelectric Focusing

IEF is an electrophoretic technique for separation of amphoteric species, mostly proteins. It is a


technique based on differences in isoelectric points (pI). The species are separated in the pH gradient
that is formed in the separation system. The pH gradient is generated in stabilizing matrices of special
gels with proteolytic groups, mostly in slabs. Alternatively, it can be generated in a free solution
without the gel by passing the electric current through the mixture of a series of ampholytes having
isoelectric points in close proximity to each other. The analyte migrates in the separation system by
electrophoretic movement according to its net charge. When it reaches a position where the pH is
equal to its pI, the net charge becomes zero and the movement stops. The separated species are in this
way focused at pH positions corresponding to their pI. When the slab gel is used, they are visualized
by a suitable reagent; when focused in free solution in the column, the content of the column is
pushed out by pressure or by changing electrolytes in the electrode vessels and detected by a detector
located at the end.

Rathod Mansi Page 8


Electrophoresis
2022

Figure 6: Isoelectric Focusing

Capillary Electrophoresis:

CE separates samples by applying voltage across buffer-filled capillaries. The capillaries are usually
made of fused silica with an external polyimide coating for increased mechanical strength. Capillaries
are between 25 and 100 cm long with an internal diameter of 25–100 mm. After passing through a
detector the separated ions are seen as peaks. The area of each peak is proportional to the
concentration of the molecule, which enables quantitative studies to be carried out. Advantages of CE
are that analysis time is rapid: 1–30min and operating costs are low. Detection can be by a variety of
methods depending on the sample. CE has become a range of techniques that include: capillary zone
electrophoresis where separation is based on size and charge differences; capillary gel electrophoresis
analogous to SDS-PAGE where separation is based solely on size; capillary isoelectric focusing,
separation of neutral compounds using surfactant micelles and capillary isotachophoresis. Using these
methods it is possible to use CE for separation of many different sample types, e.g., inorganic ions
such as metal ions and anions (chloride, sulfate, and nitrate), DNA, serum proteins. CE is routinely
used in a clinical setting, in the pharmaceutical industry, and also in forensic laboratories.

Rathod Mansi Page 9


Electrophoresis
2022

Figure 7: Capillary Electrophoresis

Application
 For estimation of molecular weight of proteins and nucleic acids
 Separation of organic acids, alcohols, insulin, alkaloid, carbohydrates, amino acids, phenol,
nucleic acids
 Determination of structure of proteins
 Purification of isolated proteins
 Identifying disulfide bond between proteins
 Agriculture testing
 In food industry
 In forensic science
 Isolation of targeted DNA as well as to estimate size of DNA

Reference
1. R.J. Fritsch, I. Krause, in Encyclopedia of Food Sciences and Nutrition (Second Edition), 2003

2. B. Gas, in Encyclopedia of Analytical Science (Second Edition), 2005

3. R. Stringer, ELECTROPHORESIS | Overview, Editor(s): Paul Worsfold, Alan Townshend, Colin Poole,
Encyclopedia of Analytical Science (Second Edition), Elsevier, 2005, Pages 356-363

Rathod Mansi Page 10

You might also like