View
311
Download
5
Category
Tags:
Preview:
Citation preview
ELECTROPHORESIS
sudheerkumar kamarapu 1
Sudheer kumar kamarapu
Assistant professor
Sri Shivani college of Pharmacy
CONTENTS
sudheerkumar kamarapu 2
Introduction
Principle And Theory
Factors
Classification
Capillary electrophoresis
Zone Electrophoresis
INTRODUCTION
sudheerkumar kamarapu 3
The word electrophoresis is derived from a Greek word, which means borne by electricity.
It is a separation technique in which the components are separated due to their varying behaviour under the influence of applied electric field.
It is defined as the migration of charged molecules under the influence of external electric field.
The major requirement of the component to be subjected to electrophoresis is that the component should be charged.
INTRODUCTION (cont..,)
sudheerkumar kamarapu 4
It is mostly used for the separation of complex biological substances such as:
Proteins
Polysaccharides
Nucleic acids
Peptides
Aminoacids
Oligosaccharides
Nucleosides
Organic acids
Small anions and cations in body fluids
Cont...
sudheerkumar kamarapu 5
Separation Scientists work in many different disciplines, some of these include:
Analytical Chemistry
Biochemistry
Biotechnology
Forensic Science
Food Science
Clinical Science
Neuro-Science
Medical Research and Production
Pharmaceutical Science
Other Disciplines
PRINCIPLE
sudheerkumar kamarapu 6
This technique is mainly used for separation of complex mixtures of biological substances which possess ionisable functional groups.
Therefore they can be made to exist as electrically charged species, either as cation/anion.
Molecules with similar charges will have different m/e ratio.
This forms basis for differential migration when these ions in solution are subjected to an electric field.
PRINCIPLE (cont..,)
sudheerkumar kamarapu 7
Therefore electrophoresis can be applied to any mixture in which the components carry a charge & have differential mobilities in an electrical field.
The migration in an electrophoretic system depends on properties of particle as well as instrumental system
Based on Stoke’s law the mobility of particle (µ) can be calculated from
µ = Q/π r η
Where,
Q= charge of particle
µ= mobility of ion
r= radius of particle in cm
η = viscosity of medium
TECHNIQUES OF ELECTROPHORESIS
sudheerkumar kamarapu 8
It can be carried out by using either:
1. Low voltage
2. High voltage
Low voltage electrophoresis:
• It consists of two compartments to hold the buffer & electrodes & a suitable carrier for support medium, such that its ends are in contact with buffer compartments
sudheerkumar kamarapu 9
TECHNIQUES OF ELECTROPHORESIS
(cont..,)
sudheerkumar kamarapu 10
• The design of carrier depends on the medium
• The medium doesn’t dip into electrode compartments, but into separate compartments connected by wicks with anode & cathode cells
• The apparatus is enclosed to avoid evaporation
• LVE can be used in principle to separate any ionic substances
• Its main application is examination of biological and clinical specimens for aminoacids and proteins
TECHNIQUES OF ELECTROPHORESIS
(cont..,)
sudheerkumar kamarapu 11
High voltage electrophoresis:
• The construction of apparatus is similar to that of LVE except that it contains additional cooling system.
• It was found that much reduced analysis time could be achieved by using high voltage gradient
CLASSIFICATION
sudheerkumar kamarapu 12
A. Capillary electrophoresis
B. Zone electrophoresis 1. Paper electrophoresis
2. Cellulose acetate electrophoresis
3. Thin layer electrophoresis
4. Gel electrophoresis
Capillary
Electrophoresis
sudheerkumar kamarapu 13
sudheerkumar kamarapu 14
In practical terms, a positive (anode) and negative (cathode) electrode are placed in a solution containing ions.
Then, when a voltage is applied across the electrodes, solute ions of different charge, i.e., anions (negative) and cations (positive), will move through the solution towards the electrode of opposite charge.
Capillary electrophoresis, then, is the technique of performing electrophoresis in buffer-filled, narrow-bore
capillaries, normally from 25 to 100 pm in internal diameter (ID).
sudheerkumar kamarapu 15
Definition: The differential movement for migration of ions by attraction or repulsion in an electric field.
Separation of components of a mixture using an electric field
v=Eq/f v = velocity of molecule
E = electric field
q = net charge of molecule
f = friction coefficient
Capillary Electrophoresis – The Basics
Of Instrumentation
sudheerkumar kamarapu 16
Electrophoresis in a buffer filled, narrow-bore capillaries
Each capillary is about 25-100 μm in internal diameter
When a voltage is applied to the solution, the molecules move through the solution towards the electrode of opposite charge
Depending on the charge, the molecules move through at different speeds Separation is achieved
Basics cont.
A photocathode is then used to
measure the absorbencies of the
molecules as they pass through
the solution
The absorbencies are analyzed
by a computer and they are
represented graphically
sudheerkumar kamarapu 17
Equipment
sudheerkumar kamarapu 18
Capillary tube
Varied length but normally 25-
50 cm
Small bore and thickness of the
silica play a role
Using a smaller internal diameter
and thicker walls help prevent
Joule Heating, heating due to
voltage
Equipment Cont….
sudheerkumar kamarapu 19
Detector
UV/Visible absorption
Fluorescence
Radiometric (for radioactive
substances)
Mass Spec.
sudheerkumar kamarapu 20
Capillary Electrophoresis Apparatus
sudheerkumar kamarapu 21
The Electropherogram
sudheerkumar kamarapu 22
The data output from CE is presented in the form of an electropherogram, which is analogous to a chromatogram.
An electropherogram is a plot of migration time vs. detector response.
The detector response is usually concentration dependent, such as UV-visible absorbance or fluorescence.
The appearance of a typical electropherogram is shown in Figure for the separation of a three component mixture of cationic, neutral and anionic solutes.
ZONE
Electrophoresis
sudheerkumar kamarapu 23
ZONE ELECTROPHORESIS
sudheerkumar kamarapu 24
It involves migration of charged particles, which are supported on relatively inert and homogeneous solid or gel framework.
In this method the separated components are distributed into discrete zones on stabilizing media.
The zones are heterogeneous and physically separated from one another.
It is classified based on supporting material used.
They are:
1. Paper electrophoresis
2. Cellulose acetate electrophoresis
3. Thin layer electrophoresis
4. Gel electrophoresis
sudheerkumar kamarapu 25
Principle :
Basically a supporting media is saturated with buffer solution and a small amount of sample solution is applied as narrow band.
On application of potential difference between the ends of strip, each component migrates at a rate determined by its electrophoretic mobility.
ADVANTAGES AND DISADVANTAGES
sudheerkumar kamarapu 26
Useful in biochemical investigation.
Very small quantity of samples can be analysed.
Useful to study both simple and complex mixtures equally.
Equipment cost is low and maintenance is easy.
Unsuitable for accurate mobility and isoelectric point determination.
Complications such as capillary flow, electro osmosis, adsorption and molecular sieving are introduced.
GENERAL METHOD OF OPERATION
sudheerkumar kamarapu 27
Saturation of medium: the supporting medium other than gel must be saturated with a buffer so that it can conduct current.
Sample application: sample is applied as spot or streak.
Electrophoretic separation: the power is switched on at required voltage.
After completion of separation the power is switched off before supporting media is removed.
Removal of supporting medium: paper, cellulose acetate strips and thin layer plate are removed and air dried or in oven. The gels are removed by forcing water from hypodermic syringe.
INSTRUMENTATION
sudheerkumar kamarapu 28
1. Electrophoretic chamber: It contains buffer solution.
2. Electrodes : Ag/AgCl reversible electrodes can be used.
3. Diffusion barriers: The electrode should be separated from the electrophoretic bed by a barrier such as gel, filter paper, sponge.
4. Supporting media: It should have low resistance to electric current, inert to sample, electrolyte and developing reagents.
PAPER ELECTROPHORESIS
sudheerkumar kamarapu 29
One of the simplest process in electrophoresis involves spotting a mixture of solute in middle of paper , moistening the paper with some electrolyte and placing it between two sheets of glass.
The ends of paper strip extending beyond glass plate are immersed in beakers of electrolyte.
A potential of 5V/cm of paper length is placed from a DC source.
It is allowed to continue for a period of several hours.
ADVANTAGES AND DISADVANTAGES
sudheerkumar kamarapu 30
It is economical and also easy to use.
Some compounds such as proteins can not be adequately resolved.
There are three types of paper electrophoresis:
1. Horizontal
2. Vertical and
3. Continuous
sudheerkumar kamarapu 31
sudheerkumar kamarapu 32
CELLULOSE ACETATE
ELECTROPHORESIS
sudheerkumar kamarapu 33
1. Cellulose acetate strips, which are used widely in clinical laboratories produce excellent separations of 7 to 9 protein fractions in a few hours.
2. this material is exceedingly fine and homogeneous, and little tailing is encountered due to adsorption.
3. It is especially useful for separating alpha immunoglobulins from albumin.
4. It contains 2 to 3 acetyl groups per glucose unit and its absorption capacity is less than that of paper.
GEL ELECTROPHORESIS
sudheerkumar kamarapu 34
The separation here is brought about through molecular sieving technique, based on molecular size of substances
THIN LAYER ELECTROPHORESIS
sudheerkumar kamarapu 35
1. Electrophoretic studies can be also carried out on thin layers of silica, keisulghur,alumina.
2. The studies with these materials offer advantages of speed and resolution when compared with paper.
3. They have greatest application in combined electrophoretic-chromatography studies in two-dimensional study of proteins and nucleic acid hydrolysates.
THANK
YOU
sudheerkumar kamarapu
36
Recommended