1. What is Electrophoresis?
The speed of migration of molecules in an aqueous medium under an applied electric field
depends on the size and charge of the molecule. This phenomenon, which is called electrophoresis,
may be used to separate a mixture of molecules into its components.
2. What are its applications?
Electrophoresis has a wide range of applications. Gel electrophoresis is a standard procedure in
molecular biology. The separation medium is a porous gel permeated by an electrolyte (a
salt solution). A drop of the sample is placed on the gel and an electric voltage is
applied. The gel may be stained later to make specific components visible. In this way
biologists “run a gel” to discover for example which proteins are present or absent
when certain genes are expressed. We have all heard of “DNA fingerprinting” or the
“Human Genome Project”. These applications use electrophoresis to sort DNA by
size
(see The figure on left, from Agilent technologies).
3. What is Capillary Electrophoresis?
Capillary Electrophoresis (CE) is the modern way of “running a gel”. In CE instead of using a
porous network permeated by an electrolyte as the molecular race track one uses a single
micro-capillary filled with an electrolyte that connects reservoirs at either end. The capillary
must be very narrow 25-75 micron internal diameter is commonly used. Larger diameters result
in poor separation due to excessive Joule heating, convective mixing of the fluid and other effects.
A UV light source and photodetector near one end of the capillary picks up the signal as a series
of peaks and troughs that correspond to modulation of the UV intensity due to adsorption by
sample components.
4. What are the different modes of CE?
The simplest kind of separation in CE is Zone Electrophoresis. Here molecules migrate in
response to an applied electric field and separate into zones by virtue of their different
electrophoretic mobilitiies. Usually the migration of the molecules is accompanied by a
bulk flow of the fluid in the capillary (electroosmotic flow) because of electrostatic
charge on the capillary wall. The electroosmotic flow causes both positive and negative
components in the sample to drift in the same direction and therefore pass through a
single detector near the capillary exit. Another kind of separation that is used for
proteins is known as isoelectric focussing. Here an electric field and a pH gradient
are simultaneously applied across the capillary. Proteins have the property that their
charge depends on the pH of the surrounding medium. Therefore they move to the
location where the pH is such that the charge is neutral (the iso-electric point of the
protein). In iso-tachophoresis all sample components actually move at the same velocity
but arrange themselves in layers with the ordering depending on the mobility of the
ions.
5. What are the advantages of CE?
The main advantage of CE is that wet chemistry can be done via fluidic circuits on glass or
silicon chips in the same way that digital electronics is done today. Many biochemical protocols
(such as genome sequencing) call for a series of chemical operations repeated a vast number of
times. Therefore, miniaturization provides the usual advantages of scalability and parallelization
that drove the semiconductor revolution in the last century. However, such a “Lab on a Chip”
that performs complex biochemistry in a miniaturized automated setting is in its infancy
when compared to the vastly developed semiconductor chip. Practical, albeit quite
simple on chip CE systems are sold commercially by some biotech companies such as
Caliper.
6. What is the role of mathematical modeling in CE?
The design issues in CE are similar to the questions that arise in optical systems such as the
microscope. Just as for a microscope one is interested in the minimum achievable angular
resolution, in a CE system we would like to know the smallest difference in mobilities of
molecules that may be detected. An ideal microscope should be “diffraction limited”, that is, it
has the best optical performance that is possible under the constraint that light is a
wave and undergoes diffraction. Likewise, an ideal CE system is “diffusion limited”
that is, the resolution is as good as it can be, given the constraint that concentration
peaks spread due to the diffusivity of molecules. In a microscope, there are a host of
phenomena: spherical aberration, chromatic aberration etc. that stands in the way
of designing a diffraction limited instrument. Likewise in CE, there are phenomena
such as Taylor dispersion, Electromigration Dispersion etc. that stand in the way of
achieving a diffusion limited performance. The only difference is that in designing a
microscope one needs to understand the physics of light propagation through media
whereas in CE the relevant physics has to do with fluid flow and transport in an ionic
medium.
7. Where can I learn more about this?
