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AssignmentFile 867 01122023143532

The document discusses different electrophoresis techniques used for separating chemical constituents. It describes how electrophoresis works by separating charged particles in an electric field based on their migration rate. Key techniques discussed include paper electrophoresis, gel electrophoresis, and capillary electrophoresis. Gel electrophoresis is commonly used to separate DNA, RNA, and proteins by applying a voltage across a gel that separates particles by size as they migrate through pores in the gel. Capillary electrophoresis separates particles within a thin capillary tube using an electric field.

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48 views

AssignmentFile 867 01122023143532

The document discusses different electrophoresis techniques used for separating chemical constituents. It describes how electrophoresis works by separating charged particles in an electric field based on their migration rate. Key techniques discussed include paper electrophoresis, gel electrophoresis, and capillary electrophoresis. Gel electrophoresis is commonly used to separate DNA, RNA, and proteins by applying a voltage across a gel that separates particles by size as they migrate through pores in the gel. Capillary electrophoresis separates particles within a thin capillary tube using an electric field.

Uploaded by

Prince Dogra
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© © All Rights Reserved
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Download as PDF, TXT or read online on Scribd
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Pharmacognosy and Phytochemistry - II 14.

18 Isolation and Characterization Techniques

14.3 ELECTROPHORESIS
Like chromatography, electrophoresis is another very important technique which is used
for the separation of constituents. It is also known as cataphoresis. The separation in
electrophoresis is based on the different migration rate of charged ion in an electric field. The
Swedish chemist in 1930 named Arne Tiselius performed the serum protein study by this
technique and got noble prize in 1948. Electrophoresis use extensively for the separation of
various types of constituents like Vitamins, Inorganic anions and cations, amine drugs,
catechol, nucleic acid, polynucleotide, nucleotide, proteins, carbohydrates, drugs etc.
Electrophoresis is one of the most common tools for the separation of protein
(antibodies, enzymes, hormones) and nucleic acid (RNA and DNA). In the electrophoresis
separation process a small amount of sample is introduced (in the form of band) into
aqueous buffer solution and applied high voltage, entire length of buffer by the help of a
pair of electrodes (present at the end of buffer). Due to the field the charged sample will
move towards the electrodes (depending upon their charges) and the separation will take
place.
The movement of the sample ions depends upon the different factors like size of the
ions, voltage applied, charge of ions, viscosity of the medium, ionic strength and pH of the
buffer media etc.
(a) Size of the ions: Mobility of the sample is inversely proportional to their size. It
means larger the size of the sample particle will give the lower the rate of separation.
(b) Voltage applied: If we have to obtain the sharp band we should apply higher
voltage which also speed up the separation process. But due to high voltage the
problem of evaporation of solvent or buffer may arise.
(c) Charge of ions: Higher the charge on the ions the mobility of the sample will be
higher or rate of the separation will be faster.
(d) Viscosity of the medium: Mobility is proportional to viscosity of medium.
(e) Adsorption: Sample is retained over the supporting medium is known as adsorption
which causes tailing of sample and this reduces the resolution and rate of separation.
(f) Temperature: The migration time decreases with increase in the temperature.
Type of Electrophoresis:
1. Zone electrophoresis: In zone electrophoresis the charged molecule or ion moves
on the supporting media like gel, paper etc. Example of zone electrophoresis are:
(a) Paper electrophoresis
(b) Gel electrophoresis
(c) Thin layer electrophoresis
(d) Cellulose acetate electrophoresis
2. Moving boundary electrophoresis: In the moving boundary electrophoresis the
charged molecule can move freely in a free moving solution. There is no supporting
media like gel or paper required. Examples are:
(a) Capillary electrophoresis
(b) Isotacto electrophoresis
(c) Isoelectric focusing
(d) Immuno electrophoresis
Pharmacognosy and Phytochemistry - II 14.19 Isolation and Characterization Techniques

14.3.1 Paper Electrophoresis


This technique can be useful for the separation of amino acids, small proteins or small
charged molecules. The paper strips moist with buffer and the end of this paper strip dipped
into buffer solution which contain electrode. The sample should be applied in the centre of
the paper and the high voltage applied. The sample will migrate depending on their charges.
After electrophoresis the isolated constituents can be identified by various techniques of
staining depending upon their chemical structure.
14.3.2 Gel Electrophoresis
Just like the other electrophoresis techniques it also involves the electric field. Those
phytoconstituents which have to be separated are introduced into the gel under electric field.
The gel contains the pore through which phytoconstituents have to move. The small
molecules of the phytoconstituents will travel the larger distance (because it can penetrate
small pore of the gel) through the gel compare to the larger molecules. Generally the DNA
and RNA (contain negative charge) can be easily separated by gel electrophoresis. If protein
have to be separated by this technique they are first treated with sodium dodecyl sulphate
which unfold the protein and makes them linear and coated with negative charge. Currently
agarose gel or polyacrylamide gel are used for gel electrophoresis.
Side view: Sample loaded
into well

Plastic gel box

Gel Buffer

Electric field and


direction of migration

Negative (-) electrode Positive (+) electrode


Fig. 14.13: Diagrammatic representation of Gel electrophoresis
14.3.3 Capillary Electrophoresis
In the capillary electrophoresis separation takes place inside the capillary having internal
diameter 10 to 100 µm. The ends of fused silica capillary tube dip into the buffer solution. It
contain platinum electrode (cathode and anode). Capillary tube contain optical window for
detection which attached with UV detector. To introduce the sample the inlet buffer should
be replaced with sample and sample can be injected by pressure, electro kinetically, capillary
action and siphoning. On applying the voltage the migration of the sample starts which is
known as electrophoretic migration but this electrophoretic migration is also effected by
electron osmotic flow of the buffer solution. Generally the electro osmotic flow is towards the
cathode (or negative charged). Though the electrophoretic mobility is lower than electro
osmotic flow all the analytes are carried towards cathode with different speed. These can be
detected by detector.

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