MadSci Network: Molecular Biology

Re: Separation techniques including electrophoresis

Date: Wed Oct 4 13:44:52 2000
Posted By: Chris Neale, Undergraduate, Biology, University of Waterloo
Area of science: Molecular Biology
ID: 970061020.Mb

There are many ways to separate the parts of a heterogeneous solution.

A heterogeneous solution is a solution consisting of non-identical parts.  
A solution here is taken to be fluid and so must be a liquid or a gas.

A measurable quality of the things to be separated must exist and have 
values that can be resolved as different.  We also need a mechanism to sort 
these different values.  White pebbles can be picked by hand out of a 
garden.  The difference is the colour, the mechanism is the eye (detector) 
and the hand (effector.)

Measurable qualities that can be used for separation and their 
corresponding sorting mechanism include

density --> centrifugation
boiling point --> distillation
size --> filtration
solubility --> solvation
melting point --> crystallization

Chromatography is a very important set of separation techniques.  It is 
also very diverse.  Chromatography is based on differential migration rates 
of components of a solution as they move through some stationary phase.  
Since the solution moves, we call it the mobile phase.  We are required to 
select some form of stationary phase, a mobile phase in which to dissolve 
our materials, and a mechanism to make the solution move.  While this is 
rather complicated, it is this freedom of choice that makes chromatography 
such a powerful technique with many applications.

An examples follows:

Stationary phase: paper
Mobile phase: water
material to be separated: black marker ink

The paper is marked with ink and one end is dipped into the water.  The ink 
mark must not directly touch the water.  Water, the mobile phase, is pulled 
up the stationary phase by capillary action.  Capillary action can be 
observed when you wet the end of a paper towel.  Notice that the water is 
"sucked" up.  As the water moves, it pulls the ink with it.  Black ink is 
composed of many colours, each from a different molecule.  These molecules 
adsorb the paper differently and those that adsorb well spend more time on 
the paper and less time in the water moving up the column.  After 10 
minutes, we observe a colour smear.  After 1 hour some bands of colour 
resolve themselves and separation is accomplished.  Note that many solvents 
work better than water for this experiment.  

* If the ink touches the water basin, it will dissolve directly and the 
separation will not work.

Practically any soluble or volatile substance can be purified by 
chromatography. Some combination of stationary phase, mobile phase, and 
conditions will achieve separation.

Another example.

Stationary phase: column packed with beads that have very small pores.
Mobile phase: variable
material to be separated: things too small to see.

Gravity pulls the mobile phase through the column.  If the particles are 
small enough to move through the pores they travel a much larger distance 
before reaching the bottom.  Since the speed of particles is not related to 
their size in an important way, larger particles come out the bottom first, 
and smaller particles later.

* The materials to be separated should enter the column at approximately 
the same time.  We use a very small volume to do this.  Once they enter, we 
add extra mobile phase to push our sample through.  This is the reason we 
didn't want the ink to touch the water basin earlier ... so that it would 
all begin the journey through the stationary phase at the same time.

Sorry to keep you waiting.  Now that we have the background information...

Electric charges migrate in an electric field.  Electrophoresis is the 
movement of electrically charged particles through a stationary phase when 
a voltage is applied across that stationary phase.  The points of voltage 
application are called electrodes.

In our first two examples, we used capillary action and gravity to effect 
the flow of the mobile phase, and the mobile phase carried our particles.  
Now we will charge the particles and directly pull them through the 
stationary phase.  This is electrophoresis.

Now all we require is a charged group of particles.  Electrophoresis is 
incapable of separating uncharged particles, unless it is possible to 
induce a charge on particles to allow separation.


DNA is negatively charged.  This negative charge resides in the phosphate 
backbone.  If we apply an electric field to DNA, the DNA will migrate 
toward the positive end.  The positive electrode is called the cathode and 
the negative electrode is the anode.  DNA is negatively charged and so 
will migrate toward the cathode.

When attempting DNA electrophoresis, we require a stationary phase.  The 
stationary phase in common use is agarose gel.  Agarose is the starchy 
ingredient in agar and is derived from an algae.  When agarose cools, 
it gels like Jello® as the individual molecules interact.  These 
interactions form a 3 dimensional lattice (or mesh) structure with small 
pores that DNA can fit through.  This does not work like the column example 
given before, instead the DNA has to "find its way" through the agarose.  
Smaller particles migrate faster than larger DNA's and reach the cathode first.  
We could use many other stationary phases, but agarose melts easily, 
so we have an easy technique for DNA recovery after separation.

If we want to separate proteins based on size, we have to give the proteins a net 
charge using the detergent SDS which surrounds the protein.  Proteins can 
also be separated according to their isoelectric point.  The isoelectric 
point of a protein is the pH at which that protein's net charge is zero.  
Individual amino acids (protein building blocks) have groups that can be 
negatively ionized and other groups that can be positively ionized.  To further 
complicate matters, some amino acids have individual side chains with specific 
properties.  Each different protein has different amino acids and, therefore, 
a different isoelectric point.  If we make a gel that has a pH gradient, 
the protein will migrate until the point at which its electric charges cancel 
out and electrophoresis no longer has any means to move the proteins.

The mobile phase used in electrophoresis is a buffer containing ions so that 
it can more easily carry a current.  This current-carrying capacity reduced 
overheating of the mechanism.

I hope this background information is useful.  Unfortunately, 
electrophoresis is hard to perform and rather dangerous without the proper 
equipment.  All universities and hospitals with research laboratories will 
certainly have such equipment.  The required voltage is easily supplied by 
several small batteries, but these will not supply much amperage.  In addition, 
we can't directly see DNA at these quantities, and the visualizing agents are 
highly carcinogenic and involve UV light.


DNA and proteins may not be your thing.  Anything with a charge will 
work.  During an electric current, electrons move through a wire by 
electrophoresis, although you won't hear this explanation often.

Perhaps you could construct a model or demonstrate the technique with some 
other form of chromatograhy.

Please contact MadSci if you have any further questions.


Moderator's note:
You might also check out one of the previous posts regarding various forms and 
applications of chromatography:

Is it possible to filter or separate colour pigments dissolved in oil?

How to separate colors in leaves using chromatography

Why do certain colors of dye to move through a flower's stem more quickly?

Water soluble pigment extraction

What do eluting agents do for the process of chromatography?

How does one differentiate (the fat content) between full-cream milk and skimmmed 

Why does adenine have more mobility in a chromatograph then cytosine?

Current Queue | Current Queue for Molecular Biology | Molecular Biology archives

Try the links in the MadSci Library for more information on Molecular Biology.

MadSci Home | Information | Search | Random Knowledge Generator | MadSci Archives | Mad Library | MAD Labs | MAD FAQs | Ask a ? | Join Us! | Help Support MadSci

MadSci Network,
© 1995-2000. All rights reserved.