mpharm 1st year modern pharmaceutical analaysis

1. paper electrophorosis
P.S.Vijinkumar
MPHARM IST YEAR
Pharamceutics

2. ELECTROPHORESIS & ITS PRINCIPLE
 Electrophoresis is a method of separation where in charged molecules
migrate in differential speeds in an applied electricfield.
 when electricity is applied to the medium containing biologicalmolecules,
depending on their net charge and molecular size, they migrate differentially,
thus different proteins/DNA can be separated.
 Depending on the kind of charge the molecule carry, they movetowards
either to
 To cathode
 or anode

4. FREE ELECTROPHORESIS
 In this type of electrophoresis a free electrolyte is taken in place of
supporting media.
 It is mostly of two types-
1. Micro Electrophoresis: It is mostly used in calculating Zeta potentials(a
colloidal property of cells in a liquid medium) of the cells.
2. Moving boundary Electrophoresis which for many years had been used
for quantitative analysis of complex mixtures of
macromolecules, esp. Proteins.

5. ZONE ELECTROPHOROSIS
 It involves the migration of the charged particle on the supporting media can
be Paper, Cellulose acetate membrane, Starch Gel, Polyacrylamide.
 Components separated are distributed into discrete zone on the support media.
 Supporting media is saturated with buffer solution, small volume of the sample
is applied as narrow band.
ADVANTAGES:
 Useful in biochemical investigations.
 Small quantity of sample can be analysed.
 Cost is low and easy maintenance.
DISADVANTAGES:
 Unsuitable for accurate mobility and isoelectric point determination. Due to
thepresence of supporting medium, technical complications.

6. PAPER ELECTROPOROSIS

7. PAPER ELECTROPHOROSIS
 Paper Electrophoresis is one of the type of zone electrophoresis.
Principle:
 When charged molecules are placed in an electric field, they migrate
toward either the positive or negative pole according to their charge.
 In contrast to proteins, which can have either a net positive or net negative
charge, nucleic acids have a consistent negative charge imparted by their
phosphate backbone, and migrate towards the anode.

8. EQUIPMENTS:
The equipment required for electrophoresis consist a basically of two items, a
 POWER PACK and
 ELECTROPHORETIC CELL.
1. Power pack: Power pack provides a stabilized direct current & has controls
for both voltage & current output, I which have an out put of 0 to 500V and 0
to 150mA are available.
2. The Electrophoretic cell: It contains: the electrodes, buffer reservoirs, a
support for paper and a transparent insulating cover. The electrodes are
usually made of platinum.

9. WORKING
1) A long strip of filter paper is moistened with a suitable buffer solution
of the desired p H and the sample is applied transversely across the
central part of the strip.
2) Ends are fixed to dip in buffer solutions in two troughsfitted with
electrodes.
3) Electric field of about 20 volts/cm is established.
4) The charged particles of sample migrate along the strip towards
respective electrodes of opposite polarity, according to net charges,
sizes and interactions with the solid matrix.

10. 5)Homogeneous group of particles migrate as a separate band
6) The electrophoresis is carried out for 16-18 hours.
7)Proteins are stained (bromophenol blue) to make them visible
8) The separated proteins appear as distinct bands.

11. Paper electrophoresis ( one dimensional )

14. FACTORS AFFECTING SEPARATION
1. The Sample-
 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.
 Shape- The globular protein will migrate faster than
the fibrous protein
3+
2+
MORE MOBILITY
MORE MOBILITY

15. 2. Electric field-
 Increase of migration with the increase of voltage gradient.
3. Buffer-
 Migration of charge particle depend on of the buffer.
Composition
 Commonly used buffers are Formate", "Acetate", Citrate, Phosphate",
"EDTA“
 The choice of buffer depends upon the type of sample being electrophoresed.
20 V
10 V
MORE MIGRATION

16. b) pH:
 The extent of ionization depends on pH, especially in organic
compounds.
 The ionization increases with increase in pH of an organic acids and its
just reverse for the organic bases therefore affecting its rate of migration.
3.TheMedium:
 The inert medium can exert adsorption,molecular sieving effects &
electro-osmosis - processes that affect the electrophoretic rate.
Adsorption:
 It means retention of a component on the surface of supporting medium.
 The rate and resolution of the electrophoretic separation can be
efficiently reduced by adsorption.

17. b) Molecular sieving:
 Media such as "Polyacrylamide", "Agar", "Starch" & "Sephadex have
cross-linked structures giving rise to pores within the gel beads.
4) Heat generation in electric fields
 One of the practical problems encountered in electrophoresis is
generation of heat from resistance in the electrophoretic medium.
 Heating not only changes viscosity and density of the electrophoretic
media, it also damages equipment.

18. APPLICATIONS:
1) Paper electrophoresis has emerged as a simple, inexpensive, and accurate
laboratory procedure for various research and clinical studies.
2) Clinical applications of paper electrophoresis include study of sickle cell
disease, hemoglobin abnormalities, and separation of blood clotting factors
and serum plasma proteins from blood sample.

19. 4) It has also been used in separation and identification of alkaloids.
5)PE can also be used for testing water samples, toxicity of water, and other
environmental components.
6)Drug-testing industry uses paper electrophoresis to determine presence of
illegal drugs crime suspects.