ASSIGNMENT

BIO-CHEMISTRY

Capillary Electrophoresis
Madam Taliha

Submitted By:

Saud Ahmad Asif 125

Naveed Shahzad 114
Muhammad Haris Bilal 121

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

There are a number of different ways of

performing CE separations. This makes the technique especially useful as it
optimized for the separation you are interested in whether this be ensuring purity
during manufacture, or diagnosing illness in a hospital. Separations driven by
electrophoresis also have a novel separating mechanism. This makes them useful in
situations where other liquid phase separation techniques are limited or impractical.

The advantages of capillary electrophoresis are it:

• has very high efficiencies, meaning hundreds of components can be

separated at the same time
• requires minute amounts of sample
• is easily automated
• can be used quantitatively

Consumes limited amounts of reagents

Movement in an Electric Field
Separation by electrophoresis is based on differences in solute velocity in an electric
field. The velocity of an ion is a function of its electrophoretic mobility and the applied
voltage.

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.

Electro Osmotic Flow

When a current is applied to the capillaries used in CE there is a bulk flow of
movement through the system. This is known as electro osmosis and is a result of
the surface charge on the inside of the capillary wall. Possess a negative charge
which results from the ionization of the surface or the adsorption of ionic species. In
fused silica capillaries both processes occur and this result in the inside wall being
extremely negatively charged. As the walls of the capillary are negatively charged, a
layer of cations builds up near the surface to maintain the charge balance. This
creates a double layer of ions near the surface and a potential difference. This is
known as the zeta potential. When the voltage is applied across the capillary the
cations forming the double layer are attracted to the cathode. They therefore move
through the capillary, and as they are solvated, drag the bulk solution behind them.

The double layer of ions is illustrated in the figure

Figure: The build up of ions at the capillary wall

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.

Figure: The rate of EOF in different materials at different pHS

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:
Methods to control EOF
Variable Result
Electric field Proportional change in EOF
Buffer pH EOF decreased at low pH,
increased at high pH
Ionic strength or buffer Decreases zeta potential and
concentration EOF when increased
Temperature Changes viscosity
Organic modifier Changes zeta potential and
viscosity (usually decreases
EOF)
Surfactant Adsorbs to capillary wall via
hydrophobic and/or ionic
interactions
Neutral hydrophilic polymer Adsorbs to capillary wall via
hydrophobic interactions
Covalent coating Chemical bonding to capillary
wall
Comment
Efficiency and resolution may
decrease when lowered
Joule heating may result when
increased
• Best method to change EOF
• May change charge or structure
of solute
• High ionic strength generates
high currents and Joule heating
• Low ionic strengths may result
in sample adsorption
• May distort peak shape if
conductivity different from
sample conductivity
• Limits sample stacking if
reduced
• Often useful as temperature is
controlled instrumentally
• Complex changes, effects need
to be determined experimentally
• May alter selectivity
• Anionic surfactants can
increase EOF
• Cationic can reverse or
decrease EOF
• Can significantly alter selectivity
• Decreases EOF by shielding
surface charge and increasing
viscosity
• Many modifications possible
• Stability often problematic
The EOF can be controlled using the electric field, but often this results large change
in resolution and analysis time leading to poor separations By altering the pH of the
buffer it is easy to control the separation, and this can also be used to affect solute
charge and mobility leading to better separations. Another alternative is to alter the
concentration of the buffer. High buffer concentrations decrease the effective charge
of the walls, but this can lead to large amounts of Joule heating which is detrimental
to the separation.

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:

Figure 4: EOF can be modified by including additives, such as surfactants

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.

Methods to control Joule heating and temperature gradients

Variable Effect
Decrease electric field • Proportional decrease in heat generated

• Reduces efficiency and resolution

Reduce capillary inner diameter • Dramatic decrease in current
• Decreases sensitivity

• May cause increased sample adsorption

Decrease buffer ionic strength or
concentration
Active temperature control
• Proportional decrease in current
• May cause increased sample adsorption
• Thermostats and removes heat from
capillary

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.

To reduce solute - wall interactions a number of different approaches can be taken.

The simplest approaches involve modification of the running buffer. For example an
increase in concentration decreases solute interactions by shielding the analytes
from the capillary wall's charge. However a high ionic strength decreases the EOF
increasing the amount of time solutes spend in the capillary. It also leads to an
increase in current and subsequent Joule heating which reduces the efficiency of the
separation. Another simple approach is to use an extreme of pH. At a low pH the
silanols are all protonated and uncharged which prevents any ionic interactions.
Conversely at high pH the walls are extremely negative, but any ionic analytes will
also tend to be negative. Thus they will repel one another preventing any interaction
occurring.

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.

Mathematically one expects large molecules to be efficiently analyzed by CE. This is

because they have large diffusion coefficients so they do not diffuse out of the bands
they are injected in. However it is often found that these large molecules experience
significant wall interactions leading to significant peak tailing and even total
adsorption. As protein analysis is an important part of analytical chemistry many
approaches have been taken to overcome this problem. Many of these are based on
wall coatings.
One can minimize these interactions by careful choice of buffer; however capillary
wall modifications are an alternative to limit solute adsorption. Two different
approaches have been taken to these modifications, either permanent modification
of the wall or dynamic deactivation.

Permanent Modifications
Permanent modification can be achieved with covalently bound or physically
adhered phases. These are described in table given below

Table 4: Bonded or adhered phases

Type Comment
Silylation coupling (Si - O - Si - R) • Numerous usable functional groups
• Simple to prepare
R = Polyacrylamide, sulfonic acid, aryl penta fluro • Siloxane bond stable between pH 4 & 7

• Limited long term stability

Direct Si - C coupling
Polyacrylamide via Grignard
Adsorbed polymers
Cellulose, PEG, PVA
Adsorbed, crosslinked polymer
Cellulose, polyvinyl alcohol
GC phases
• Si - C binding eliminates silylation
• pH stable between pH 2 & 10
• Difficult to prepare
• Poor long term stability
• Low pH range (2 - 4)
• Relatively hydrophobic
• Reversed EOF
• Useful for basic proteins
• Stable at physiological pH
• Hydrolytically unstable

PEG
LC phases • Can increase protein adsorption

C2, C8, C16

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.

Depending on the deactivation the EOF can be eliminated or reversed. Neutral

deactivation with polyacrylamide eliminates EOF for example. This results from an
decreased effective wall charge and increased viscosity at the wall. Deactivation
with cationic groups reversed the EOF, whilst deactivation with amphoteric
molecules allows one to control the direction of the EOF by altering the pH.
Dynamic Deactivation
Dynamic deactivation is achieved by adding modifiers to the running buffer. Dynamic
coatings are more stable than permanent ones as they are continuously
regenerated.

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.

Some common dynamic additives are listed in table given below

Table 5: Dynamic deactivation methods

Type Result Comment
Extremes of pH • Reduce columbic interactions • pH range from 2 to 12
by eliminating wall and solute • EOF nearly eliminated
charge differences at low Ph
• EOF very fast at high
pH
• May denature proteins

• Can decrease peak

capacity by decreasing
charge differences
High buffer ionic • Reduce columbic interactions • Decrease EOF
strength/concentration
• Often limited by Joule
heating
Hydrophilic polymers • Mask wall charge and reduce • Increase viscosity
EOF
• Can separate by size if
used at high
concentrations (CGE)
Surfactants • Deactivate capillary surface • Wide variety of
through hydrophobic and/or surfactants
ionic interaction • Easy to use
• MEKC above CMC
• Can decrease or
reverse EOF
• May irreversibly
denature protein

• 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.

Table 6: Additives in CZE

Additive Example Use
Surfactants SDS, CTAB, BRIJ, TWEEN • EOF mobilization
• Solubilise hydrophobic solutes
• Ion pairing

• MEKC above CMC

Zwitterionic substances MES, Tris, CHAPS, CHAPSO • Increase ionic strength without
increasing conductivity

• Affect selectivity of proteins

Linear hydrophilic polymers Methyl cellulose, • Reduce EOF
polyacrylamide, PEG, PVA • Minimize sample adsorption at
low concentrations

• CGE at high concentrations

Organic modifiers Methanol, acetonitrile • Alter EOF (generally reduce)

• Change selectivity in MEKC and

chiral analyses
Chiral selectors Cyclodextrins, crown ethers, • Chiral separations
bile salts
• Solubilisation of hydrophobic
solutes
Metal ions K, Na, Cu, Li • Alter selectivity in MEKC and
CGE
Hydrogen Urea • Melt double stranded DNA in
bonding/solubilising agents CGE
• Solubilise proteins

• Alter selectivity in MEKC

Complexing buffers Borate • Carbohydrate separations
Quaternary amines Diaminopropane • Ion pairing

• 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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• CE animations