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BIO-CHEMISTRY
Capillary Electrophoresis
Madam Taliha
Submitted By:
DEPARTMENT OF BIO-
CHEMISTRY
UNIVERSITY OF SARGODHA
INTRODUCTION
Capillary electrophoresis, or CE,
describes a family of techniques used to
separate a variety of compounds. These
analyses, all driven by an electric field ,
are performed in narrow tubes and can
result in the rapid separation of many
hundreds of different compounds. The
versatility and number of ways that CE
can be used means that almost all
molecules and even whole organisms
can be separated using the powerful
methods. The components of a CE instrument
The mobility for an ion in a particular medium is constant and characteristic of that
ion. The ions mobility is a result of two factors. The ion is attracted to the electrode
of opposite charge, pulling it through the medium. At the same time however
frictional forces try to prevent the ion moving. The balance of these determines the
actual overall mobility.
A small ion will have less frictional drag and hence move through the medium faster
than a large one. Similarly a multiply charged ion will experience more attraction to
the electrode and also move through the medium faster.
It is the difference in solute velocities that is responsible for the separating effect in
electrophoresis.
The zeta potential is determined by the surface charge of the capillary wall which is
strongly pH dependant. As a result the magnitude of the EOF is very dependent on
pH, being large at high pHs where the silanols are extremely ionized.
The effect of the capillary material on the magnitude of the EOF is illustrated in the
following figure.
The magnitude of the EOF is usually much larger than the mobility of the species
under analysis. As a result of the EOF all species are moved through the capillary,
even if they are attracted to the electrode at the injection end.
If you would like to learn some more about the EOF and its contribution to the
migration of the analytes please.
EOF Control
Although the EOF is beneficial, it often needs to be controlled. At high pHs the flow
may be too great resulting in little time for separation of analytes. Also certain modes
of capillary electrophoresis require reduction or elimination of the EOF.
To control the EOF one must alter the capillary surface or the buffer viscosity. There
are various methods to accomplish this, as detailed in table:
Another way to control the EOF is to modify the wall with coatings. These can be
permanently bonded to the wall, or alternatively present as a dynamic layer
replenished by a buffer additive. The use of wall coatings is described in and
illustrated in figure given below:
Joule Heating
When the fields are applied in electrophoresis the system tends to heat up due to
the extra energy. Performing efficient electrophoresis involves finding a way to
dissipate this heat. In classical gel electrophoresis a thin slab of anti-convective
polymer is used. Heat is an issue because it caused temperature gradients within
system leading to extra dispersion and can change the viscosity of the system. The
large surface area and heat capacity of the gel help prevent the build up of too much
heat. However it is still a problem and this limits the voltages that can be used in gel
electrophoresis.
When using capillaries the build up of heat is not a major problem because the large
surface area - to - volume ratio of the capillaries helps dissipate any extra energy. If
the temperature across the whole capillary were to rise it would not be a major
problem because the viscosity effects etc. would be across the whole system.
However localized increases in temperature cause fluctuations in conditions
throughout the whole system and it these that mostly lead to dispersion. The narrow
heat dispersing nature of the capillary helps avoid these localized increases in
temperature.
Table given below illustrates methods to reduce Joule heating. The most effective
way is probably to use an external method of temperature control. This can simply
be achieved by blowing air around the capillary to help remove the heat. Indeed
many instruments now come fitted with pumps to achieve this. A more effective
method involves passing coolant around the capillary to remove the heat. This
method is extremely effective at removing excess energy but does involve
complicated engineering.
Injection
The injection process is also an important factor within the separation. It is important
that the plug of sample is kept to a minimum length otherwise dispersion will become
significant reducing the efficiency of the separation. Practically injection plug lengths
are kept to 1 or 2% of the capillary length, leading to the injection of nanolitre
amounts of sample.
Figure: Effect of injection plug length on resolution
Sample can be injected either using pressure or an electrical force. Pressure can be
applied by applying an external force or alternatively by raising the sample vial
above the outlet causing siphoning. Electrical forces are simply applied by switching
the power source on for a few seconds. The injection processes are illustrated in the
figure below.
Figure: Injection methods onto the capillary and how they occur
Pressure, or hydrodynamic, injections are generally used as they are they are not
influenced by the conductivity of the sample buffer or the mobility of the analytes.
Pressure injections are also generally more reproducible and quantifiable. An
advantage of electrokinetic injections however is that they can be used to
concentrate samples in the capillary prior to analysis. Using isotachophoresis effects
sample can be concentrated in a narrow zone at the tip of the capillary prior to
analysis.
Sample Concentration
In order to increase the sensitivity of capillary electrophoresis it is possible to
concentrate sample bands in the capillary. These methods are based on differences
in the strength of the electric field experienced by sample bands. This effect is
similar to the electro dispersion described in the flow and dispersion section. If the
conductivity of the sample is lower than that of the buffer then when a voltage is
applied a field will develop across the sample zone causing ion to migrate faster
than the sample buffer. Once the ions reach the running buffer boundary the field
decreases and they migrate slower. This continues until all the sample zone reaches
the boundary with a resultant increase in concentration here. Stacking is
demonstrated in figure 10a.
Figure10: Field amplified sample injection a) sample dissolved in buffer, b) sample dissolved
in water, c) short plug of water injected before sample in (b)
Solute - Wall Interactions
The capillary provides an excellent support for an electrophoretic separation but
interactions between the solutes and the wall can lead to loss of resolution in the
separation. Depending on the extent of any interactions peak tailing can occur or the
solute may be completely adsorbed to the wall. The causes of these interactions are
described later but both result in the efficiency of the separation being lowered. Any
loss of efficiency is undesirable as it may cause different analytes to migrate
together leading to poor resolution and difficulty in quantify analytes.
Most of the solute - wall interaction occurs because of ionic attraction between
cationic solutes and negatively charged wall. The use of narrow capillaries also
means that there is a relatively large surface area - volume ratio increasing the
chance of interaction. Adsorption is especially prevalent with large molecules such
as peptides and proteins which contain many charged groups and are slow to
diffuse. This means they have a lot of time to interact with the wall and a lot of sites
to do it.
Wall Coatings
Coating the wall is also another method of decreasing solute adsorption. The
coatings can be used in different ways, either as a temporary layer built up from a
buffer additive, or as a permanent chemical bound to the silica wall. These methods
can be used to eliminate or reverse the wall's charge, they can alter its
hydrophobicity and they limit any adsorption.
Permanent Modifications
Permanent modification can be achieved with covalently bound or physically
adhered phases. These are described in table given below
PEG
LC phases • Can increase protein adsorption
The most widely used approach is silylation coupling as it is cheap, can be used with
a variety of different ligands and is easily performed in the lab. Unfortunately the
siloxane bond is only stable between pH 4 & 7, hydrolysis limiting the long term
stability.
As with covalent coatings, additives added to the running buffer interact with the wall
and alter charge and hydrophobicity. These modifiers are easier to use and easier to
optimize than covalent coatings as they are simply prepared by dissolving the
appropriate additive in the buffer.
• Can be used in
conjunction with
reversed phase LC
surface
Quaternary amines • Decreases or reverses EOF • Also act as ion-pairing
agents
Selectivity
The order with which the solutes migrate is determined by the mechanism of the
separation. The different mechanisms present different selectivities to the
separation, and by controlling the selectivity it is possible to improve the resolution of
the separation. The concept of selectivity applies to most chromatographic and
electrophoretic separations, the method of control being particular for the technique
being used. In CE the selectivity is altered through changes in running buffer pH or
by the use of buffer additives.
Table below lists some common additives which are used to affect the separation.
The overall gain by altering selectivity is an increase in resolution for the entire
separation.
• EOF reversal
References
1. Skoog, D.A.; Holler, F.J.; Crouch, S.R "Principles of Instrumental Analysis" 6th ed. Thomson
Brooks/Cole Publishing: Belmont, CA 2007.
2. Skoog, D.A.; Holler, F.J.; Crouch, S.R "Principles of Instrumental Analysis" 6th ed. Chapter 30
Thomson Brooks/Cole Publishing: Belmont, CA 2007.
3. Lin H.; Natan, M.; Keating, C. Anal. Chem. 2000, 72, 5348-5355.
4. Skoog, D.A.; Holler, F.J.; Nieman, T.A. "Principles of Instrumental Analysis, 5th ed." Saunders
college Publishing: Philadelphia, 1998.
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• Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 113.
• Foley, J.P. Anal. Chem. 1990, 62, 1302.
• Carretero, A.S.; Cruces-Blanco, C.; Ramirez, S.C.; Pancorbo, A.C.; Gutierrez, A.F. J.
Agric. Food. Chem. 2004, 52, 5791.
• Cavazza, A.; Corradini, C.; Lauria, A.; Nicoletti, I. J. Agric. Food Chem. 2000, 48, 3324.
• Rodrigues, M.R.A.; Caramao, E.B.; Arce, L.; Rios, A.; Valcarcel, M. J. Agric. Food
Chem. 2002, 50, 425.
• CE animations