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ELECTROPHORESIS

                        Beckmann column
                        chromatography instrument




Guided by                        By
Mr. Saurabh Pandey               Saurav Ghoshal
                                 Gulam Rafey
                                 M.Pharm (Pharmaceutics)
                                 PSIT
                                                       1
Electrophoresis
                          2


    The term literally means “migration with
electricity.”
    It involves the separation of components of a
sample by the differential rate of migration of
ions by attraction or repulsion in an applied dc
electric field.
    The technique was first developed by Arne
Tiselius in 1930s for the study of serum proteins.
Why Electrophoresis
                         3

     The process has exhibited unparalleled
resolution in the separation of charged species.
     Electrophoresis may separate between the
polynucleotides which may differ by may be a single
or just a few nucleotides.
     The Human Genome Project was possible only
as a result of electrophoresis.
Types of Electrophoretic techniques
                           4

Electrophoretic techniques may be of two types
A) Electrophoresis
-Slab electrophoresis-TLC
-Capillary electrophoresis-Column

B) Electrochromatography
Principle
                            5

     The principle involved in both slab and capillary
electrophoresis     are    same      which       involves
electrophoretic mobility of the ions.
     It has been found that the migration velocity
(v)(cms-1)of a molecule in an electric field is given by
           v=µeE
where E=strength of electric field(Vcm-1)
        µe=electrophoretic mobility (cm2V-1s-1)
6

The value of (E) depends upon
 The charge of the analyte ion
 The frictional retarding forces
       which includes

         Size and shape of the ion
         Viscosity of the medium in which migration
         occurs
Plates
                             7

The plate count in electrophoresis is given by the formula
N=µeV/2D
Where V=applied voltage
   D=diffusion coefficent of the solute(cm2s-1)
 Because the capillary has a small cross sectional area and
 longer length when compared with that of a slab and
 hence has much higher resistance when compared to that
 of a slab.
      Thus in the capillary format much higher voltages
 may be applied than in the case of slab which is of greater
 advantage.
Electroosmotic Flow (EOF)
                                 8

 An important        feature of CE is the bulk flow of liquid
    through the capillary this is called the electroosmotic flow
    and is caused as follows.
   Silica capillary walls contain surface silanol(Si-OH) groups
    and these are ionized at pH values higher than 3 and the wall
    becomes (-)vely charged.
   Wall attracts cations and double-layer forms
   Cations in the diffuse layer are attracted towards cathode
    and migrate in that direction as they are in solution the
    buffer fluid is also dragged along with them.
   The direction of flow of EOF may be reversed by treatment
    with cetyl trimethylammonium bromide.
Formation of double layer for electroosmotic flow
                        9
Net result of electrophoretic and electroosmotic
                      flow
                       10
Flow Profile in Electroosmotic flow

                                           11
A key feature of EOF is that it has flat flow profile, which is shown in Figure
alongside the parabolic flow profile generated by an external pump, as used for
HPLC.

 EOF has a flat profile because its driving force (ie., charge on the capillary wall)
is uniformly distributed along the capillary, which means that no pressure drops
are encountered and the flow velocity is uniform across the capillary.

          In HPLC, in which frictional forces at
 the column walls cause a pressure drop across
 the column, yielding a parabolic or laminar
 flow profile.



The flat profile of EOF is important because it minimizes zone
broadening, leading to high separation efficiencies that allow separations
on the basis of mobility differences as small as 0.05 %.
Slab electrophoresis
                               12

1. porous layer 2-10 cm long - paper, cellulose acetate,
   polymer gel soaked in electrolyte buffer
2. Slow
3. simple but difficult to automate
4. poor quantitation
5. large quantities (mL)
6. large cross-sectional area, short length leading to low
   electrical resistance, high currents
7. Vmax=500 V
8. N=100-1000 low resolution
Slab Electrophoresis
         13
Slab Electrophoresis
         14
Capillary Electrophoresis
                            15

 Electrophoresis in buffer filled, narrow-bore
    capillaries.
   Each capillary is about 25-100 μm in internal
    diameter.
   Small cross-sectional area, long length leading to
    high resistance, low currents
   Vmax=20-100 kV
   N=100,000-10,000,000 high resolution
Contd...
                                16

 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.
 Thus separation is achieved.
 A suitable detector is then used to detect the solute
  as it comes out from the end of the capillary.
 The data obtained are analyzed by a computer and
  represented graphically.
Line Diagram of instrumentation
               17
Instrumentation
       18
Instrumentation - Capillary
                          19

The length varies but normally 30-100 cm.
Internal diameter (10-100µm).
Small bore and thickness of the silica play a role.
Using a smaller internal diameter helps prevent Joule
  Heating, heating due to voltage.
Capillaries (Column)
                           20

The capillaries are normally made of fused silica as in
 the case of Gas chromatography Open Columns.
The column is coated with polyimide to improve the
 durability.
Glass and Teflon have also been used but silica
 provides best functionality.
These may be made hydrophobic by the incorporation
 of alkyl groups which would shield the silanol groups
 and thus reduce electroosmotic flow.
Injection
                          21

1.Remove anode end capillary from buffer.
2.Place end of capillary in sample.
3.Injection volume ranges from 5-50nl. While values
  as low as 100pl have been reported.
3.Apply field for short time (electrokinetic
  injection)discriminates against low migration rate
  analytes or apply pressure for short time (pressure
  injection) or place the sample chamber at a higher
  level (Siphoning).
4.Replace in buffer.
Detector
                                  22
   UV/Visible absorption
   Fluorescence
   Radiometric (for radioactive substances)
   Mass Spectrometry
   Electrochemical
     Conductivity
     Amperometry
     Potentiometry

   Indirect Detection
Detectors used and their limits of detection
                      23

Type of detector       LOD (attomoles)
Absorption             1-1000
Fluorescene            1-0.01
Mass                   1-0.01
Conductivity           100
Potentiometry          1
Amperometry            0.1
Detector Cells
      24




                 a) 3 mm Z cell
                 b) 150 µm bubble cell
                 c) Multireflection cell
Variants of CE
                         25

Capillary Zone Electrophoresis
Capillary Gel Electrophoresis
Capillary Isoelectric Focusing
Capillary Isotachophoresis
Micellar Electrokinetic Chromatography
Capillary Zone Electrophoresis
                          26

     In this the buffer composition is constant
throughout the region of the separation.
     Here the anions/cations when allowed to migrate in
the field towards the anode and cathode respectively
tend to be separated into bands which may be separate or
may overlap depending upon the migration velocity.
     The method may be used for both ions as well as
derivatized molecules.
     Frequently used for separation of mixtures of
cations and anions. Synthetic herbicides, Pesticides,
pharmaceuticals may be separated and analyzed after
derivatization.
Zone Electrophoresis
        27
Capillary Gel Electrophoresis
                                     28

        It is generally performed in porous gel formed by a polymer matrix.
        The polymer used is normally Polyacrylamide ( monomer Acrylamide
  CH2=CH-CO-NH2)
        The gel used adds an additional component of sieving (size) into the
  separation process.
        Other gels used include
Agarose
Polyethylene Glycol
Methyl Cellulose
Polyvinyl Alcohol
Dextran
Polydimethylacrylamide, etc..
        The method finds most frequent application in DNA sequencing.
The columns used may be of two types
 Low viscosity gels-replaceable
 High viscosity gels-fixed
Sieving Action
      29
Capillary Isotachophoresis
                              30

     In this process all analyte bands ultimately migrate at the
same velocity.
     In this the sample is injected between two buffers , a
leading one containing ions of higher mobility than any of the
analyte ions and a terminating one with ions of mobilty lower
than the analyte ions.
     At the beginning of the separation procedure all the ions
migrate at the different velocities and then as there is
separation the faster moving electrons experience a weaker
electric field as compared to that of the slow moving electrons
and thus are retarded. As this process of retardation based on
charge differences reaches equilibrium each of the bands are
separated on the basis of their migration rates.
     Markers may be added between bands to separate them.
Isotachophoresis
       31
Capillary Isoelectric Focussing
                                   32

 In this technique the separation of amphiprotic substances can be
  performed in a buffer soln. whose pH varies along its length. In this
  procedure one end of the capillary bearing the cathode is dipped in
  a soln. of strong base (NaOH) and the end bearing anode is dipped
  into a soln. of a strong acid (H3PO4).
                                  +
 When the process starts the H ions migrate towards the cathode
               -
  and the OH migrates towards the anode, thus establishing a pH
  gradient, with higher pH towards anode.
 The analyte in combination with some other ampholytes (contain
  carboxylic and amine gp.) is also introduced in the sample and these
  migrate on the basis of their charge and during migration they may
  be protonated or lose proton depending on the direction of
  migration due to the existing pH gradient, the migration continues
  till the pH region where the ion has reached is equal to the pI and at
  this point the analyte or ampholytes becomes neutral. Once this
  occurs several sharp bands are formed and no further migration
  occurs.
Contd..
                           33

The bands may then be mobilized by adding NaCl to
                                                      -
 base end and then this leads to less migration of OH
 and then the pH gradient is disturbed leading to
 migration of the soln. towards the cathode leading to
 the detection of the bands.
Capillary Isoelectric Focussing
              34




                            Theoretical
                            separation of
                            compounds with
                            varying pI
Micellar Electrokinetic Chromatography
                                            35
• Earlier, neutral molecules were not considered to be separated by electrophoresis
• In 1984, Terabe and his group reported a technique that enabled capillary
    electrophoresis (CE) instrumentation to be used in the separation of neutral (as well
    as ionic) species=MEKC
            MEKC is primarily used for separation of neutral analytes but is also used for
    separation of ionic analytes
•   In this system, micelles are described as “pseudo-stationary phase” and the buffer as
    “mobile phase”=system for partitioning of analytes
•   When micelles are not present, neutral molecules will migrate with the
    electroosmotic flow and no separation will occur.
•   MEKC has the capability of separating, within a single run, anionic, neutral, and
    cationic species.
•   The micellar solution is divided into 2 flow preferences. The aqueous buffer has a
    strong flow toward the cathode (EOF) and the micelles are slowed down because
    they have anionic character and due to electrophoretic mobility they are attracted
    towards the anode.
Contd..
                                           36


•   The EOF is much stronger than the electrophoretic mobility, the solution is all
    headed toward the cathode, but the micelles head at a much slower rate
    because they are attracted to the anode on the opposite side.
•   The negatively charged species are repelled by the anionic micelle while the
    positively charged species are attracted to the micelle; because the micelle
    elutes later than the buffer, so do the positively charged species which are
    attracted to it
   The more negatively charged species - more repelled by micelle than the less
    negatively charged species (elute earlier)
   The more positively charged species - more attracted (strong electrostatic
    interaction) to the micelle than the less positively charged species (elute later
    with the micelle)
   The neutral species migrate between the two extremes and their separation is
    based on hydrophobicity
PACKED COLUMN
         ELECTROCHROMATOGRAPHY
                            37

 Electrochromatography is a hybrid of HPLC & CE
  that factor offers some of the best features of the two
  methods. Like HPLC & MEKC, it is applicable to the
  separation of neutral species or charged species.
 In electrochromatography, a polar solvent is usually
  driven by electroosmotic flow through a capillary
  packed with a reversed-phase HPLC packing.
  Separation depend on distribution of the analyte
  species between the mobile phase and the liquid
  stationary phase held on the packing.
Field Flow Fractionation
                           38

This separation technique involves the separation of
 the components of a mixture during its flow through
 a ribbon like channel by the application of a thermal,
 electrical, centrifugal or flow field.
      As the components migrate through the channel
 the channel the applied field in perpendicular
 direction leads to the accumulation of the particles at
 the bottom known as accumulation wall.
Field Flow Fractionation
           39
Contd..
                                40
The ribbon like channel has following dimensions
 Length 25-100 cm
 Width 1-3 cm
 Thickness 50-500 µm
       Depending on the type of applied field it may be of four
  types:
Thermal
Electrical
Sedimentation
Flow

Application- Mainly for particles of high mol. mass and
 neutral particles.
Applications
                               41

         The vast applications of electrophoresis include
   Vaccine analysis.
   Protein and DNA analysis are also important
    electrophoresis applications.
   It has allowed researchers to map and see the differences
    in the genetic code of species on earth.
   Electrophoretic DNA analysis also provides a reliable tool
    in forensic investigations.
   Determination of impurities.
   Chiral Analysis.
   Analysis of carbohydrates and other macromolecules.
   Analysis of inorganic anions/metal ions.
Vaccine Analysis
                           42

Vaccine analysis is one of the many important
 applications of electrophoresis. There are several
 vaccines that have been purified, processed and
 analyzed through electrophoresis, such as the
 influenza vaccine, hepatitis vaccine and polio
 vaccine.
Manufacturers such as Wyeth, Merck and Sanofi-
 Aventis have been using electrophoresis as an
 effective vaccine analysis method.
DNA Analysis
                                       43

       Electrophoresis is one way of analyzing DNA, which is the unique code of
every individual.
       Through electrophoresis, specific DNA sequences can be analyzed,
isolated and cloned. The analyzed DNA may be used in forensic investigations
and paternity tests.
       For this wells are formed at one end of an agarose gel for the loading of
the DNA sample. The slab is then placed horizontally into the electrophoresis
buffer chamber The DNA migrates in bands toward the positive electrode. The
smaller molecules pass through the matrix more rapidly than the larger ones,
which are restricted. The DNA bands are then stained using a fluorescent dye
such as ethidium bromide. The stained gel is then viewed directly under
ultraviolet light and photographed.
       Another technique for detection involves the use of different fluorescent
dyes which tend to bind to the bases of nucleotides and thus give unique staining
profile
Protein Analysis
                          44

Electrophoresis has advanced our understanding on
  the structure and function of proteins. These
  molecules are needed by our body cells and may be
  analyzed, for instance, by getting blood and urine
  samples. Then through electrophoresis, the amount
  of proteins in your blood or in your urine is
  measured and compared to established normal
  values---lower or higher than the normal levels
  usually indicates a disease.
Assay of Drugs
                          45

Atropine sulphate i.v. soln.
Codeine phosphate syrup
Ketamine HCl i.v.soln.
Phenylepherine HCl i.v. soln.
 have sucessfully been assayed by this process.
46

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Electrophoresis ppt.

  • 1. ELECTROPHORESIS Beckmann column chromatography instrument Guided by By Mr. Saurabh Pandey Saurav Ghoshal Gulam Rafey M.Pharm (Pharmaceutics) PSIT 1
  • 2. Electrophoresis 2 The term literally means “migration with electricity.” It involves the separation of components of a sample by the differential rate of migration of ions by attraction or repulsion in an applied dc electric field. The technique was first developed by Arne Tiselius in 1930s for the study of serum proteins.
  • 3. Why Electrophoresis 3 The process has exhibited unparalleled resolution in the separation of charged species. Electrophoresis may separate between the polynucleotides which may differ by may be a single or just a few nucleotides. The Human Genome Project was possible only as a result of electrophoresis.
  • 4. Types of Electrophoretic techniques 4 Electrophoretic techniques may be of two types A) Electrophoresis -Slab electrophoresis-TLC -Capillary electrophoresis-Column B) Electrochromatography
  • 5. Principle 5 The principle involved in both slab and capillary electrophoresis are same which involves electrophoretic mobility of the ions. It has been found that the migration velocity (v)(cms-1)of a molecule in an electric field is given by v=µeE where E=strength of electric field(Vcm-1) µe=electrophoretic mobility (cm2V-1s-1)
  • 6. 6 The value of (E) depends upon  The charge of the analyte ion  The frictional retarding forces which includes Size and shape of the ion Viscosity of the medium in which migration occurs
  • 7. Plates 7 The plate count in electrophoresis is given by the formula N=µeV/2D Where V=applied voltage D=diffusion coefficent of the solute(cm2s-1) Because the capillary has a small cross sectional area and longer length when compared with that of a slab and hence has much higher resistance when compared to that of a slab. Thus in the capillary format much higher voltages may be applied than in the case of slab which is of greater advantage.
  • 8. Electroosmotic Flow (EOF) 8  An important feature of CE is the bulk flow of liquid through the capillary this is called the electroosmotic flow and is caused as follows.  Silica capillary walls contain surface silanol(Si-OH) groups and these are ionized at pH values higher than 3 and the wall becomes (-)vely charged.  Wall attracts cations and double-layer forms  Cations in the diffuse layer are attracted towards cathode and migrate in that direction as they are in solution the buffer fluid is also dragged along with them.  The direction of flow of EOF may be reversed by treatment with cetyl trimethylammonium bromide.
  • 9. Formation of double layer for electroosmotic flow 9
  • 10. Net result of electrophoretic and electroosmotic flow 10
  • 11. Flow Profile in Electroosmotic flow 11 A key feature of EOF is that it has flat flow profile, which is shown in Figure alongside the parabolic flow profile generated by an external pump, as used for HPLC. EOF has a flat profile because its driving force (ie., charge on the capillary wall) is uniformly distributed along the capillary, which means that no pressure drops are encountered and the flow velocity is uniform across the capillary. In HPLC, in which frictional forces at the column walls cause a pressure drop across the column, yielding a parabolic or laminar flow profile. The flat profile of EOF is important because it minimizes zone broadening, leading to high separation efficiencies that allow separations on the basis of mobility differences as small as 0.05 %.
  • 12. Slab electrophoresis 12 1. porous layer 2-10 cm long - paper, cellulose acetate, polymer gel soaked in electrolyte buffer 2. Slow 3. simple but difficult to automate 4. poor quantitation 5. large quantities (mL) 6. large cross-sectional area, short length leading to low electrical resistance, high currents 7. Vmax=500 V 8. N=100-1000 low resolution
  • 15. Capillary Electrophoresis 15  Electrophoresis in buffer filled, narrow-bore capillaries.  Each capillary is about 25-100 μm in internal diameter.  Small cross-sectional area, long length leading to high resistance, low currents  Vmax=20-100 kV  N=100,000-10,000,000 high resolution
  • 16. Contd... 16  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. Thus separation is achieved.  A suitable detector is then used to detect the solute as it comes out from the end of the capillary.  The data obtained are analyzed by a computer and represented graphically.
  • 17. Line Diagram of instrumentation 17
  • 19. Instrumentation - Capillary 19 The length varies but normally 30-100 cm. Internal diameter (10-100µm). Small bore and thickness of the silica play a role. Using a smaller internal diameter helps prevent Joule Heating, heating due to voltage.
  • 20. Capillaries (Column) 20 The capillaries are normally made of fused silica as in the case of Gas chromatography Open Columns. The column is coated with polyimide to improve the durability. Glass and Teflon have also been used but silica provides best functionality. These may be made hydrophobic by the incorporation of alkyl groups which would shield the silanol groups and thus reduce electroosmotic flow.
  • 21. Injection 21 1.Remove anode end capillary from buffer. 2.Place end of capillary in sample. 3.Injection volume ranges from 5-50nl. While values as low as 100pl have been reported. 3.Apply field for short time (electrokinetic injection)discriminates against low migration rate analytes or apply pressure for short time (pressure injection) or place the sample chamber at a higher level (Siphoning). 4.Replace in buffer.
  • 22. Detector 22  UV/Visible absorption  Fluorescence  Radiometric (for radioactive substances)  Mass Spectrometry  Electrochemical  Conductivity  Amperometry  Potentiometry  Indirect Detection
  • 23. Detectors used and their limits of detection 23 Type of detector LOD (attomoles) Absorption 1-1000 Fluorescene 1-0.01 Mass 1-0.01 Conductivity 100 Potentiometry 1 Amperometry 0.1
  • 24. Detector Cells 24 a) 3 mm Z cell b) 150 µm bubble cell c) Multireflection cell
  • 25. Variants of CE 25 Capillary Zone Electrophoresis Capillary Gel Electrophoresis Capillary Isoelectric Focusing Capillary Isotachophoresis Micellar Electrokinetic Chromatography
  • 26. Capillary Zone Electrophoresis 26 In this the buffer composition is constant throughout the region of the separation. Here the anions/cations when allowed to migrate in the field towards the anode and cathode respectively tend to be separated into bands which may be separate or may overlap depending upon the migration velocity. The method may be used for both ions as well as derivatized molecules. Frequently used for separation of mixtures of cations and anions. Synthetic herbicides, Pesticides, pharmaceuticals may be separated and analyzed after derivatization.
  • 28. Capillary Gel Electrophoresis 28 It is generally performed in porous gel formed by a polymer matrix. The polymer used is normally Polyacrylamide ( monomer Acrylamide CH2=CH-CO-NH2) The gel used adds an additional component of sieving (size) into the separation process. Other gels used include Agarose Polyethylene Glycol Methyl Cellulose Polyvinyl Alcohol Dextran Polydimethylacrylamide, etc.. The method finds most frequent application in DNA sequencing. The columns used may be of two types  Low viscosity gels-replaceable  High viscosity gels-fixed
  • 30. Capillary Isotachophoresis 30 In this process all analyte bands ultimately migrate at the same velocity. In this the sample is injected between two buffers , a leading one containing ions of higher mobility than any of the analyte ions and a terminating one with ions of mobilty lower than the analyte ions. At the beginning of the separation procedure all the ions migrate at the different velocities and then as there is separation the faster moving electrons experience a weaker electric field as compared to that of the slow moving electrons and thus are retarded. As this process of retardation based on charge differences reaches equilibrium each of the bands are separated on the basis of their migration rates. Markers may be added between bands to separate them.
  • 32. Capillary Isoelectric Focussing 32  In this technique the separation of amphiprotic substances can be performed in a buffer soln. whose pH varies along its length. In this procedure one end of the capillary bearing the cathode is dipped in a soln. of strong base (NaOH) and the end bearing anode is dipped into a soln. of a strong acid (H3PO4). +  When the process starts the H ions migrate towards the cathode - and the OH migrates towards the anode, thus establishing a pH gradient, with higher pH towards anode.  The analyte in combination with some other ampholytes (contain carboxylic and amine gp.) is also introduced in the sample and these migrate on the basis of their charge and during migration they may be protonated or lose proton depending on the direction of migration due to the existing pH gradient, the migration continues till the pH region where the ion has reached is equal to the pI and at this point the analyte or ampholytes becomes neutral. Once this occurs several sharp bands are formed and no further migration occurs.
  • 33. Contd.. 33 The bands may then be mobilized by adding NaCl to - base end and then this leads to less migration of OH and then the pH gradient is disturbed leading to migration of the soln. towards the cathode leading to the detection of the bands.
  • 34. Capillary Isoelectric Focussing 34 Theoretical separation of compounds with varying pI
  • 35. Micellar Electrokinetic Chromatography 35 • Earlier, neutral molecules were not considered to be separated by electrophoresis • In 1984, Terabe and his group reported a technique that enabled capillary electrophoresis (CE) instrumentation to be used in the separation of neutral (as well as ionic) species=MEKC MEKC is primarily used for separation of neutral analytes but is also used for separation of ionic analytes • In this system, micelles are described as “pseudo-stationary phase” and the buffer as “mobile phase”=system for partitioning of analytes • When micelles are not present, neutral molecules will migrate with the electroosmotic flow and no separation will occur. • MEKC has the capability of separating, within a single run, anionic, neutral, and cationic species. • The micellar solution is divided into 2 flow preferences. The aqueous buffer has a strong flow toward the cathode (EOF) and the micelles are slowed down because they have anionic character and due to electrophoretic mobility they are attracted towards the anode.
  • 36. Contd.. 36 • The EOF is much stronger than the electrophoretic mobility, the solution is all headed toward the cathode, but the micelles head at a much slower rate because they are attracted to the anode on the opposite side. • The negatively charged species are repelled by the anionic micelle while the positively charged species are attracted to the micelle; because the micelle elutes later than the buffer, so do the positively charged species which are attracted to it  The more negatively charged species - more repelled by micelle than the less negatively charged species (elute earlier)  The more positively charged species - more attracted (strong electrostatic interaction) to the micelle than the less positively charged species (elute later with the micelle)  The neutral species migrate between the two extremes and their separation is based on hydrophobicity
  • 37. PACKED COLUMN ELECTROCHROMATOGRAPHY 37  Electrochromatography is a hybrid of HPLC & CE that factor offers some of the best features of the two methods. Like HPLC & MEKC, it is applicable to the separation of neutral species or charged species.  In electrochromatography, a polar solvent is usually driven by electroosmotic flow through a capillary packed with a reversed-phase HPLC packing. Separation depend on distribution of the analyte species between the mobile phase and the liquid stationary phase held on the packing.
  • 38. Field Flow Fractionation 38 This separation technique involves the separation of the components of a mixture during its flow through a ribbon like channel by the application of a thermal, electrical, centrifugal or flow field. As the components migrate through the channel the channel the applied field in perpendicular direction leads to the accumulation of the particles at the bottom known as accumulation wall.
  • 40. Contd.. 40 The ribbon like channel has following dimensions  Length 25-100 cm  Width 1-3 cm  Thickness 50-500 µm Depending on the type of applied field it may be of four types: Thermal Electrical Sedimentation Flow Application- Mainly for particles of high mol. mass and neutral particles.
  • 41. Applications 41 The vast applications of electrophoresis include  Vaccine analysis.  Protein and DNA analysis are also important electrophoresis applications.  It has allowed researchers to map and see the differences in the genetic code of species on earth.  Electrophoretic DNA analysis also provides a reliable tool in forensic investigations.  Determination of impurities.  Chiral Analysis.  Analysis of carbohydrates and other macromolecules.  Analysis of inorganic anions/metal ions.
  • 42. Vaccine Analysis 42 Vaccine analysis is one of the many important applications of electrophoresis. There are several vaccines that have been purified, processed and analyzed through electrophoresis, such as the influenza vaccine, hepatitis vaccine and polio vaccine. Manufacturers such as Wyeth, Merck and Sanofi- Aventis have been using electrophoresis as an effective vaccine analysis method.
  • 43. DNA Analysis 43 Electrophoresis is one way of analyzing DNA, which is the unique code of every individual. Through electrophoresis, specific DNA sequences can be analyzed, isolated and cloned. The analyzed DNA may be used in forensic investigations and paternity tests. For this wells are formed at one end of an agarose gel for the loading of the DNA sample. The slab is then placed horizontally into the electrophoresis buffer chamber The DNA migrates in bands toward the positive electrode. The smaller molecules pass through the matrix more rapidly than the larger ones, which are restricted. The DNA bands are then stained using a fluorescent dye such as ethidium bromide. The stained gel is then viewed directly under ultraviolet light and photographed. Another technique for detection involves the use of different fluorescent dyes which tend to bind to the bases of nucleotides and thus give unique staining profile
  • 44. Protein Analysis 44 Electrophoresis has advanced our understanding on the structure and function of proteins. These molecules are needed by our body cells and may be analyzed, for instance, by getting blood and urine samples. Then through electrophoresis, the amount of proteins in your blood or in your urine is measured and compared to established normal values---lower or higher than the normal levels usually indicates a disease.
  • 45. Assay of Drugs 45 Atropine sulphate i.v. soln. Codeine phosphate syrup Ketamine HCl i.v.soln. Phenylepherine HCl i.v. soln. have sucessfully been assayed by this process.
  • 46. 46