What is the Principle, Procedure and Applications of Paper Electrophoresis?

Paper Electrophoresis is one of the zone electrophoresis. This is very important method in all laboratories. In this article let us learn the details of the paper chromatography with suitable notes. I have given the info about this in Notes.

Principle:
 
“The charge carried by a molecule depends on the pH of the medium. Electrophoresis at low voltage is not usually to separate low molecular weight compounds because of diffusion, but it is easier to illustrate the relationship between charge and pH with amino acids than with proteins (or) other macromolecules”.
 
Filter paper:
Paper of good quality should contain at least 95% α-cellulose and should have only a very slight adsorption
capacity.
 
Apparatus:

• The  equipment  required  for  electrophoresis  consists  basically  of  two  items,  a POWER PACK and an ELECTROPHORETIC CELL. 
• Power  pack  provides  a  stabilized  direct  current  &  has  controls  for  both voltage  & current out put, which have an out put of 0 to 500V and 0 to 150mA are available.
• The  Electrophoretic  cell contains  the  electrodes,  buffer  reservoirs,  a  support  for paper and a supporting transparent insulating cover. The electrodes are usually made of platinum. 
• The  two  arrangements  of  the  filter  strips  are  commonly  used.  The  horizontal  & vertical arrangements. Both the arrangements are equally viable & the choice usually depends upon personal preferences. 

  

Sample application:
 
The  sample  may  be  applied  as  a  spot  (about  0.5cm  in  diameter)  or  as  a  narrow  uniform  streak.
Special  devices  are  available  commercially  for  this  purpose.  The  sample  can  be  applied  before  the  paper has been equilibrated with buffer (or) after it.
 
Procedure:
 
After the sample has been applied to the paper and the paper has been equilibrated with the buffer.
The current is switched on. Commonly used buffers are,

The device providing stable voltage (or) current is available. Frequent observation is necessary to run  electrophoretic  apparatus.  Overheating  can  be  avoided  by  placing  the  entire  equipment  in  the  cold
room. The process does not take longer than two hours. After 2 hours switched off the power and paper is
removed. Once removed, the paper is dried in hot oven at 110⁰C.

Detection & Quantitative assay: 
To  identify  the  unknown  electrophorogram,  compare  the  Unknown  electogram  with  standard electrogram  under  standard  conditions.Individual  compounds  are  usually  identified  by  physical  properties by the following methods. 
 

i) Fluorescence: 
 
a)  Staining  with  “Ethidium  bromide”  and  subsequent visualization  of  the  electrophoreticgram  under  UV light makes DNA & RNA fluoresce and thus facilitates their detection.
 
b) Flourescamine staining is utilized for detecting amino acids, amino acid derivatives, peptides & proteins.
 
c) DANSYl chloride may be used in place of fluorescamine.
 
ii) UV absorption:
Proteins, Peptides & nucleic acids absorb in the range of 260 to 280nm, this property these can be detect.

iii)) Staining:

iv)  Detection of Enzymes in situ:
 

• If the component  to be separated is an enzyme. Special techniques  may be  used to detect it.
• The paper strip, which  have separated enzyme, is impregnated with the substrate  for the enzyme desired to be separated. 
• The  paper  strip  is  now  placed  in  a  suitable  buffer  along  with  electrophoretogram.  The  color bands will appear which indicates the position of enzyme.

v) Quantitative estimation:
 
The color density of the zone may be multiplied with the area of the zone and the resulting value would be a rough estimate of the concentration of the component. 

 

Applications: 
 

• Serum analysis for diagnostic purpose is routinely carried about by paper electrophoresis.
• Muscle proteins, egg white proteins, milk proteins & snake, insect venom analysis done by this technique.

How to classify the Electrophoresis

Electrophoresis is working on the basic principle of migration of charged particles under the influence of electric field. The electrophoresis are mainly TWO types of electrophoresis,

1)  Free Electrophoresis (or) Electrophoresis without stabilizing media
2)  Zone Electrophoresis (or) Electrophoresis in Stabilizing media 
 

1) Free Electrophoresis:
In this Electrophoresis, Stabilizing media (Agar, Starch, Polyacrylamide)is not using. It has TWO main techniques:
 
A)  Micro electrophoresis:
 
It  involves  the  observation  of  motion  of  small  particles  in  an  electric  field  with  a microscope. In modern days this technique is applied only for measuring the Zeta Potentials of cell such as RBCs, Neutrophils and Bacteria etc.

What is this, I want more? See the Details-

• Micro electrophoresis is the best-known method for determination of zeta potentials of fiber fines and filler particles in paper machine white water.
• The apparatus includes a capillary cell, two chambers that include electrodes, and a means of observing the motion of particles. Because the capillary diameter is usually smaller than the length of a cellulosic fiber, these may be removed by filtration through a screen before the test.
• The apparatus is filled with very dilute suspension and the chambers are closed. A direct-current voltage is applied between electrodes in the respective chambers.
• One uses a microscope to determine the velocity of particles.
• The ratio or the velocity to the electrical field strength is known as the electrophoretic mobility.
• By making reasonable assumptions about the size of the observed particles and the electrical conductivity it is possible to calculate the value of the zeta potential.
• Zeta potential values near to zero indicate that the particles in the mixture are likely to stick together when they collide, unless they also are stabilized by non-electrical factors.
• Particles having a negative zeta potential are expected to interact strongly with cationic additives.

Read More: What are the factors affecting on Electrophoretic methods

B)  Moving Boundary Electrophoresis (m.b.e.):
 
The  technique  was  first  developed  by  A.Tiselius  of SWEDEN  in  the  1937.  this technique  is  conducting  in  U-shaped  observation  cell.  It  is  very  popular  for  quantitative analysis of complex mixtures of macromolecules, especially Proteins.

What is this, I want more? See the Details-

• Moving-boundary electrophoresis or free-boundary electrophoresis is electrophoresis in a free solution. It was developed by Arne Tiselius in 1937.
• Tiselius was awarded the 1948 Nobel Prize in chemistry for his work on the separation of colloids through electrophoresis, the motion of charged particles through a stationary liquid under the influence of an electric field.
• The apparatus includes a U-shaped cell filled with buffer solution and electrodes immersed at its ends.
• The sample applied could be any mixture of charged components like a protein mixture.
• On applying voltage, the compounds will migrate to the anode or cathode depending on their charges.
• The change in the refractive index at the boundary of the separated compounds is detected using Schlieren optics at both ends of the solution in the cell.

2) Zone Electrophoresis: 
 
KONIG published  the  first  experiment  on  the  use  of filter  paper  as  a  stabilizing  medium  in electrophoresis.  In  this,  several  stabilizing  media  are  using  like  Agar,  Starch  &  Polyacrylamide.  In  this, “Microlitres” of sample can also analyze.

General techniques of Zone Electrophoresis:

1. Paper Electrophoresis
2. SDS-PAGE
3. Iso-electric focusing

Read More: Electrophoresis principle, types, Procedures and Applications.

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.