What Factors are Affecting on Electrophoresis Methods

Electrophoresis is one of the important tool in Diagnostic labs, Pharma labs, Forensic labs and many more labs. First of all we should know What is Electrophoresis? Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field.

This electro-kinetic phenomenon was observed for the first time in 1807 by Ferdinand Frederic Reuss (Moscow State University), who noticed that the application of a constant electric field caused clay particles dispersed in water to migrate. This method was developed by “Arne.W.K.Tiselius” in 1937

It is ultimately caused by the presence of a charged interface between the particle surface and the surrounding fluid.

^{Factors affecting on Electrophoresis:}

The rate of migration (Separation of particles) during electrophoresis will depend on the following factors:

1.  The Sample
2.  The Electric Field
3.  The Medium
4.  The Buffer
 

1. The Sample:
 
Charge/mass  ratio  of  the  sample  dictates  its  electrophoretic  mobility.  The  mass  consists  of  not
only the size (molecular weight) but also the shape of the molecule. 

a) Charge: The  higher  the  charge,  greater  is  the  electrophoretic  mobility.  The  charge  is  dependent  on  pH  of  the medium. 
b) Size: The bigger molecules have a small electrophoretic mobility compared to the smaller particles. 
c) Shape: The globular protein will migrate faster than the fibrous protein
 

2. The Electric Field:
The rate of migration under unit potential gradient is referred to as “Mobility of the ion”.  An increase in potential gradient increases the rate of migration.
 
3. The Medium:
The  inert  medium  can  exert  adsorption  &  molecular  sieving  effects  on  the  particle,  influencing  its  rate  of migration. 

a) 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.

b) Molecular sieving: Media  such  as  “Polyacrylamide”,  “Agar”,  “Starch”  & “Sephadex”  have  cross-linked structures giving rise to pores within the gel beads.

• Sephadex, molecules larger than the pores are excluded from entering the gel beads & these molecules migrate faster.  
• Polyacrylamide,  Starch  &  Agarose  the  larger  molecules  also  are  made  to  squeeze through the pores. The smaller molecules pass through the pores easily, but the larger molecules are retarded.
  

 
4. The Buffer: The buffer can affect the electrophoretic mobility of the sample in various ways.
A) Composition:  Commonly  used  buffers  are  “Formate”,    “Acetate”,  “Citrate”,  “  Phospahate”,  “EDTA”
“Acetate”  “Pyridine”,  “Tris”  (2-amino,  2-hydroxymethyl,  1,3-diol  pentane)  and “Barbitone”  etc.   

The  choice  of  buffer  depends  upon  the  type  of  sample  being electrophoresed. 
 
b) Ionic Strength: “Ionic Strength (I) is a measure of the electrical environment of ions in a sol”.  When increase ionic strength of the buffer means a larger share of the current being carried by the buffer ions & meager (small quantity) proportion carried by the sample ions.

When decrease ionic strength, a larger share of the current being carried by the sample ions leading to a faster separation.

Note: The ionic strength used is usually between 0.05 to 0.1M.

c) pH:
  The pH determines the degree of ionization of organic compounds; it can also affect the rate of migration of these compounds. When increase pH, increases ionization of organic acids. Decrease in pH, increases ionization of organic bases. E.g.: an Ampholyte (Amino acid) - The amino acid has both acidic & basic properties

Read More: Chromatography, types, Principle and Applications.

Paper Chromatography

Paper chromatography has proved to be very successful in the analysis of chemical compound and lipid sample in particular.

Nature of the paper:

The paper commonly used consists of highly purified cellulose. Cellulose, a homopolysaccharide of glucose. Contains several thousand anhydro-glucose units-linked through oxygen atoms. The paper exhibits weak ion exchange and adsorptive properties. Modified forms of paper have been produced in which the paper has been impregnated with alumina, silica gel, and ion-exchange resin etc.

The chemical composition of whatmann filter paper no: 1 is: a-cellulose (98 to 99%), b-cellulose (0.3 to 1%), Pentosans (0.4 to 0.8%), Ash (0.07 to 0.1%) & ether soluble matter (0.015 to 0.1%).

Apparatus:

The apparatus required for paper chromatography are

1) Support for paper

2) Solvent trough

3) Airtight chamber

4) Whattmann filter paper number 1

5) Capillary tubes

6) Samples – Amino acids (or) Pigments

7) Solvents

8) Platinum loop

Paper development

There are two main techniques, which may be employed for the development of paper Chromatograms.

1) Ascending techniques

2) Descending techniques

3) Radial development

4) Two-dimensional chromatography

1) Ascending techniques:

The filter paper is then dried and equilibrated by putting it into on airtight cylindrical jar, which contains an aqueous solution of a solvent. The most widely applicable solvent mixture is n-butanol: acetic acid: Water (4:1:5), which is abbreviated as BAW.

The sheet of paper is supported on a frame with the button edge in contact with a trough with solvent. The arrangement is contained in an airtight tank lined with paper saturated with the solvent to prove a constant atmosphere and separations are carried out in a constant temperature room. Thus, the solvent will ascend into the paper this process is, therefore, termed “Ascending Chromatography”

2) Descending techniques:

The end of the filter paper may be put into the solvent mixture contained in a narrow trough mounted near the top of the container. In this chromatography, the solvent will descend into the paper and this process is then termed “Descending Chromatography”.

This method is convenient for compounds, which have similar Rf values since the solvent drips off the bottom of the paper, thus giving a wider separation.

3) Two dimensional chromatography (3D):

The mixture is separated then the first solvent, which should be volatile: then after drying, the paper is turned through 90⁰ and separation is carried out in the second solvent.

After locating the migrated unknown sample along with standard known sample, a map is obtained and comparing their position with a map of known compounds can identify compounds.

Locating the compounds:

Strip is removed when the solvent has migrated over most of the available space. The distance to which the solvent has run is marked. In most cases, the completed Chromatogram is colorless with no indication of the presence of any compounds. Such a chromatogram is said as “Undeveloped” for locating the various compounds. The filter paper strip is first dried, then sprayed with 0.5% Ninhydrin in acetone and at least heated for a few minutes at 80 to 100⁰ C. the reaction occurs and the colored spots appear at the sites of the amino acids, such as Chromatogram is now called “Developed”.

In paper chromatography, the stationary cellulose phase is more polar than the mobile organic phase.

Identifying the compounds:

The ratio of the distance travelled by a component (i.e. amino acid) to that travelled by the solvent front, both measured from the marked point of the application of the mixture, is called the “Resolution front (Rf)” value for that component.

   Distance from origin run by the compound

Rf = -----------------------------------------------------------------------------

Distance from origin run by the solvent

 

Detection:

The filter paper strip may be sprayed with ninhydrin and heated so that the colored spots indicating the location of amino acids may develop. The color densities of these spots may be measured with a recording transmittance (or) reflectance photometer device.

Ninhydrin test:

• Amines (including α-amino acids) react with ninhydrin to give a coloured product.
• It can be used qualitatively (e.g. for chromatographic visualisation) or quantitatively (e.g. for peptide sequencing).
• The α-amino acids typically give a blue-purple product.
• Proline, a secondary amine, gives a yellow-orange product.
• The test is sensitive enough that ninhydrin can be used for the visualisation of fingerprints.

Applications:

By using this technique

1) To check the control of purity of pharmaceuticals,

2) To the detection of adulterants,

3) To detect the contaminants in foods and drinks,

4) To the study of ripening and fermentation,

5) To the detection of drugs and dopes in animals & humans

6) To the analysis of cosmetics

7) To the analysis of the reaction mixtures in biochemical labs.