This presentation includes the main applications of the paper electrophoresis on the basis of the principle given

1. APPLICATION OF PAPER
ELECTROPHORESIS
SUBMITTED TO –
DR. B.M GURUPADYYAY
M.PHARM, PH.D
DEPARTMENT OF PHARMACEUTICAL
CHEMISTRY
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SUBMITTED BY-
SHALIN SHIKHAR
M.PHARM(1ST YEAR)
DEPARTMENT OF
PHARMACEUTICS

2. CONTENTS
 PRINCIPLE OF ELECTROPHORESIS
 PRINCIPLE OF PAPER ELECTROPHORESIS
 APPLICATION OF PAPER ELECTROPHORESIS
 SEPERATION OF AMINO ACIDS
 SEPERATION OF PROTEIN SERUMS
 STUDY OF SICKLE CELL ANAEMIA IN HEMOGLOBIN
 SEPERATION OF ANTIBIOTICS CAN BE CARRIED OUT
 OTHER USES
REFERENCES
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3. ELECTROPHORESIS
PRINCIPLE-
Under the influence of an electric field charged molecules and
particles migrate in the direction of the electrode bearing the
opposite charge.
Because of their varying charges and mass, the different
molecules and particles of a mixture will migrate at different
velocities and thus will thus be separated into single fractions.
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4. PAPER ELECTROPHORESIS
PRINCIPLE
A mixture of ions or ionisable substance is applied on the centre
of a paper, previously immersed in a buffer of known ionic strength.
This paper is placed across two trays, filled with buffer , into
which two electrodes are immersed.
When a voltage is applied across these electrodes, the ions or
ionisable substance migrate towards anode and cathode, based on
their charges.
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5. Applications-
Separation of amino
acids into acidic or basic
type
Separation of proteins in
serum (into albumin
alpha1, alpha2, beta and
gamma globulins).
Study of Sickle Cell
Anaemia in Hemoglobin
Separation of enzymes in
blood.
Separation of antibiotics
in different samples can
be carried out.
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6. SEPERATION OF AMINO ACIDS
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7. SEPARATION OF AMINO ACIDS
AT NEUTRAL SOLUTION:
 The acidic amino acids will have a net positive charge and will migrate toward the cathode(-ve)
 The basic amino acids will have net –ve charge and migrate toward the anode(+ pole).
 Electrically neutral amino acids or the zwitter ion will not migrate and they will stay at the
origin.
 Ninhydrin is added on the spot of amino acids.
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9. EFFECT OF INCREASE AND DECREASE OF THE PH
IN THE AMINO ACID
Amino acid in basic solution
 If we increase the pH of a solution of an
amino acid by adding hydroxide ions, the
hydrogen ion is removed from the -
NH3
+ group.
 The amino acid would be found to travel
towards the anode the positive electrode.
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10. MECHANISM
Amino acid in acidic solution
 If we decrease the pH by adding an acid to a solution of an
amino acid, the -COO- part of the zwitter ion picks up a
hydrogen ion.
 This time, during electrophoresis, the amino acid would
move towards the cathode (the negative electrode).
Amino acids as zwitterions
An amino acid has both a basic amine group and an acidic
carboxylic acid group.
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11. SEPERATION OF
PROTEINS IN SERUM
 The serum proteins move through the fine
cellulose mesh of the paper under the influence of
an electric current
 The separation of the various fractions being
brought about by differences in their
electrophoretic mobility at a selected Ph
 After separation, the protein bands are fixed in
position and stained with a protein-specific stain,
such as Amido Black, and destained.
 Usually five separate fractions are seen: one
albumin and four globulin fractions, designated
ALPHA 1,ALPHA2, GAMMA and BETA.
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13. STUDY OF SICKLE CELL ANAEMIA
 All varieties of hemoglobin moved to the anode with varying degrees of mobility.
 An adult hemoglobin migrates with greater mobility than any other hemoglobin as it
has the highest number of positive charge.
 Sickle cell(trait) hemoglobin has fewer positive charge as compared to the normal
adult hemoglobin, but more positive charge as compared to sickle thalassemia, so it
is intermediate.
 NORMAL HEMOGLOBIN> SICKLE CELL(TRAIT)> SICKLE
THALASSEMIA>SICKLE CELL
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14.  a- Normal Adult Hemoglobin(A*:100 & S*:0)
 b- Sickling Trait( A :65%& S: 35%)
 c- Sickle thalassemia ( A:18% & S: 82%)
 d- Sickle Cell Anemia(A:0 % S: 100%)
 A- NORMAL HEMOGLOBIN , S- SICKLING HEMOGLOBIN
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15. SEPARATION OF ANTIBIOTICS IN DIFFERENT
SAMPLES
 Paper Electrophoresis is useful in separating water-soluble antibiotics, although it takes more time and
mobility of the sample may be influenced by the inorganic salts.
 It is found that high voltage paper electrophoresis for 15 minutes, give a good separation of antibiotics
especially water soluble basic or amphoteric antibiotics even with crude preparations.
 Antibiotics on the are detected by the process of bioautography* using test organisms such as
Straphylococus aureus, Micrococus flavus, Bacillus subilis or Eschericha coli.
 Ninhydrin and Sakagauchi Reaction* and ultraviolet light is used for the detection of the antibiotics.
 *Bioautography is a laboratory technique to detect substances affecting the growth rates of test organisms
in complex mixtures and matrices .
 * Sakagauchi Reaction is a chemical test used for detecting the presence of arginine in proteins.
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16. OTHER USES
 Paper electrophoresis is also used in the separation and identification of
alkaloids.
 P.E can also be used for testing the suitability of Municipal water supplies, toxicity
of water and other environmental components.
 Drug-testing industry uses paper electrophoresis to determine presence of
illegal or recreational drugs in job applicants and crime suspects.
 PE has been used since 1950s by the investigators and in forensics to analyze
inks used in currency to check the counterfeiters.
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17. REFERENCE -
 Text Book of Pharmaceutical Analysis, DR.S.RAVI SANKAR
 Electrophoresis In Practice, REINER WESTERMEIER
 Analytical Separation Of Amino Acids By Paper Electrophoresis(Research
Article), GLADYS N. ATFIELD and C.J.O. R. MORRIS
 Paper Electrophoresis Of Abnormal Hemoglobins and Its Clinical
Applications(Research Article, ARNO G. MOTULSKY, MILTON H PAUL, And
E.L.DURRUM
 Lawrence Chasin and Deborah Mowshowitz Department of Biological
Sciences Columbia University New York, NY
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