electro phoresis sud mpharm pdf

ELECTROPHORESIS

sudheerkumar kamarapu 1

Sudheer kumar kamarapu

Assistant professor

Sri Shivani college of Pharmacy

CONTENTS


Introduction

Principle And Theory

Factors

Classification

Capillary electrophoresis

Zone Electrophoresis

INTRODUCTION


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..,)


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...


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


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..,)


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


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


(cont..,)


• 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


(cont..,)


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


A. Capillary electrophoresis

B. Zone electrophoresis 1. Paper electrophoresis

2. Cellulose acetate electrophoresis

3. Thin layer electrophoresis

4. Gel electrophoresis

Capillary

Electrophoresis



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).


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


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


Equipment


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….


Detector

UV/Visible absorption

Fluorescence

Radiometric (for radioactive

substances)

Mass Spec.

Capillary Electrophoresis Apparatus


The Electropherogram


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


ZONE ELECTROPHORESIS


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


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


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


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


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


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


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

CELLULOSE ACETATE

ELECTROPHORESIS


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


The separation here is brought about through molecular sieving technique, based on molecular size of substances

THIN LAYER ELECTROPHORESIS


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

electro phoresis sud mpharm pdf

Technology

word electrophoresis

layer electrophoresis

paper electrophoresis

high voltage electrophoresis

electric field q

charge of particle

separation technique

migration of ions