Electrophoresis

Electrophoresis

• Electrophoresis is a separation method based on the difference in mobility that

analyte exhibit in an applied electric field. Electrophoretic methods can be applied
to the separation of a wide variety of samples, including proteins, nucleic acids,
amino acids and carbohydrates. Separation can be carried out on an analytical as
well as preparative scale. The main advantage over liquid chromatography
methods is the higher efficiency of electrophoretic separations. This is especially
true for large molecules.
• In general, electrophoresis can be categorised according to their functionalities:
– Gel-electrophoresis
– Capillary electrophoresis
– Electrofocusing
– Micellar electrokinetic chromatography
– Dielectrophoresis
 Principle and Theory of Electrophoresis

• Electrophoresis is the movement of electrically charged particles or molecules in a

conductive medium under the influence of an applied electric field. The
conductive medium is usually an aqueous buffer, also referred to as an electrolyte
or run buffer. The mixture of analytes is introduced into the medium containing
the run buffer and an electric field is applied.

Upon application of the electric field, the anions start moving towards the
positive electrode (anode). Differences in charge and size lead to different
mobilities and thus separation of the different sample component.
 Electrophoretic separation

• Electrophoresis separations can be performed in free solution or in a solution

containing a non-conductive matrix such as an agarose or polyacrylamide gel. For
free solution based separations, two analytes can only be separated if they have
different charge to size ratio. Joule heating can interfere with the separation and
cause band broadening.

• The separation of analytes in a gel is based on difference in mobility. Additionally,

the gel has a sieving effect. Larger compounds are retarded more than smaller
compounds. This means that in gel electrophoresis, two compounds with the same
charge to size ratio can be separated as long as they are different in size.

• The efficiency of an electrophoretic separation is governed by two main factors the

electrophoretic mobility (µep) of the analytes and the electroosmotic flow (EOF) of
the bulk solution.
 Electrophoretic Mobility

The electrophoretic mobility, µep, determines the velocity of a compound in an applied

electric field, compound with different µep can b separated from each other.
• When an ion of charge q is placed into an electric field E, it experiences an electric
force Fef:

• The movement of the ions is opposed by the frictional

force, Ffr, of the medium molecule. This force is directly
proportional to the radius of the ion r, and its electrophoretic migration
velocity, vep, as well as the viscosity of the medium, η

• In a constant electric field, when the equilibrium is reached between the frictional
and the electric force:

Hence, µep describes charge/ratio (q/r)

 Joule Heating

• Joule heating refers to electrical current partially converted to heat as it passes

through a capillary
• To minimise Joule heating, two approaches can be used. (1) by applying a low
electric field and by decreasing the conductivity of the separation buffer. (2)
improve the disspation of heat by using small diameter capillary or gels. These
have a large surface-to-volume ratio, allowing heat to dissipate more quickly.
• Proper capillary cooling will restore a flat temperative profile which allows for
more efficient, highly resolved and stable separations.

Inner Wall Temperature

Diameter temperature Difference
25 µm 26º C 0.53º C
50 µm 28.2º C 1.39º C
75 µm 31.2º C 3.14º C
100 µm 34.7º C 5.58º C
 Electroosmotic Flow
• Electroosmotic Flow (EOF) describes the movement of ions through a solute under the
control of an applied potential. In CE, the capillary columns consist of silica with silanol
groups exposed on the inner surface. The exposed silanol groups are ionized above pH 3,
therefore creating a negatively charged inner capillary surface. Cations present in ionic
solutions will migrate toward the negatively charged wall forming an electric double
layer. Generation of an electrical potential across the column now causes cations to
migrate towards the cathode. Electroosmotic flow results as the solvated cations
clustered at the capillary walls drag the bulk solution in tow towards the cathode.

The electric double layer

consisting of the rigid
Stern layer in proximity to
the capillary surface and the
diffuse layer into the bulk
Solution.
 Electroosmotic Flow

• The velocity of the EOF, vEOF, is directly proportional to the dielectric constant ε
and the zeta potential ζ of the buffer as well as the strength of the applied electric
field, E. vEOF is inversely proportional to the viscosity η, of the separation buffer.

• The electroosmotic mobility, µEOF , is defined as:

• The flow profile of the EOF has the form of a plug. This homogeneous velocity
distribution minimises brand broadening and thus, increases separation efficiency

A parabolic flow profile occurs in pressure

driven flow such as in liquid chromatography,
wehreas the EOF flow profile has the form of
a plug.
 Control of the EOF
• In capillaries, the EOF can be controlled:
– Operating at lower pH: surface charges are neutralized by operating at a pH low
enough to protonate the silanol groups. This however only occurs at pH < 4, a pH at
which many biomolecules are not stable.

– Chemical modification: a hydrophobic surface can be obtained by treatment with

sulfonic acid, maintains a constant, high EOF. Hydrophobic functional groups
attached to the surface lead to suppression of the EOF. A problem with chemical
modifications is that their long-term stability is often very poor.

– Dynamic coating: A polymeric viscous layer is formed by the addition of polymer

such as polyethylene glycol (PEG) to the run buffer, which masks the charges and
suppresses the EOF.

– Additives: cellulose or polyvinyl alcohol increase the viscosity and thus reduce the
vEOF. Furthermore, they suppress analyte surface interactions. Organic solvents
such as methanol or acetonitrile can reduce or increase the viscosity respectively.
Cationic surfactants adsorb onto the capillary walls and thus change the surface
charge. This reverses the direction of the EOF. Surfactants must be used at low
concentration to avoid the formation of micelles and deteriorate sample stability.
 Separation Efficiency and Resolution

• Efficiency and resolution of an electrophoretic separation are influenced by the

electrophoretic flow as well as the EOF. The apparent mobility, µapp, of an analyte
is determined by the sum of its electrophoretic mobility, µep, and the
electroosmotic mobility, µEOF:

Apparent mobilities of cations, neutral

molecules and anions during
Electrophoresis with predominant EOF.
 Separation Efficiency and Resolution

• The migration velocity, v, of an analyte is defined as the product of its apparent

mobility, µapp, and the applied electric field strength, E. the field E is the ratio of
applied, V, over capillary length, L. Hence, v can be expressed as:

• The migration time, t, of an analyte, is defined as the following:

• Hence, the large the applied field strength E, the faster the velocity and the
shorter the migration time. This however, is restricted by Joule heating.
 Separation Efficiency and Resolution

• The number of theoretical plates, N, in electrophoresis can be approximated by:

• The plate number is independent of the capillary length and the migration time.
Large voltages lead to an increase in plate numbers. Large molecules, with low
diffusion coefficients, give high number of theoretical plates

• In theory, millions of theoretical plates per meter can be achieved with

electrophoresis making this technique superior to LC, where the number of plates
per column is typically in the order of tens of thousands.

• In practice, lower plate numbers are observed in electrophoresis. Band broadening

is caused by sample injection, Joule heating and adsorption of analytes to the
separation matrix leading to plate numbers in the order of hundreds of thousands
rather than millions.
 Gel Electrophoresis

• In gel electrophoresis, separation takes place

in an electrically non-conductive hydrogel
medium such as agarose or polyacrylamide,
containing an electrolyte buffer. The pores of
the gel function as a molecular sieve, which
retards the migrating molecules according to
their size. The gel acts as an anti-convective
support medium which minimises the
diffusion of sample molecules and thus,
reduces band broadening. As EOF is
suppressed in gel electrophoresis, only
analytes with a net charge can be separated.

• Instrumentation for Gel electrophoresis. (a) in

an upright tube, or flat rectangular slab gels
can be used which are positioned (b)
horizontally, (c) vertically.
 Sodium Dodecyl Sulfate-Polyacrylamide Gel
Electrophoresis (SDS-PAGE)

• The separation principle of SDS-PAGE is solely based on the difference in protein

size. The proteins are totally denatured under heating and in the presence of the
anionic detergent SDS. This detergent binds to proteins in a constant ratio of 1.4 g
SDS per 1g of protein (or one SDS with two amino acid residues). The large
negative charge of SDS masks the intrinsic charge of proteins, so that all SDS
treated proteins have approximately a constant net charge per unit mass. Protein
separation depends only on the molecular sieving effect of the gel pores.

SDS

• Sample preparation usually involves heating the proteins to 95°C, in the presence
of excess SDS and a thiol-reducing agent such as β-mercapto ethanol. This results
in complete unfolding of the tertiary and secondary structure. The molecules are
stretched, sulfide bridges are cleaved and SDS binds to the amino acids.
 SDS-PAGE Gel Media

• Polyacrylamide (PA) gels are prepared by co-

polymerisation of acrylamide and the cross-
linking agent N,N’-methylene-bisacrylamide in
the chosen electrophoresis buffer. The pore
size and the molecular sieving properties of PA
gels depends on the total gel concentration
T% and the degree of cross-linking C%.

• T% values between 5% and 20% are

commonly used. The higher the T% the more
restrictive the gel. For example. At 5% T and
3% C the pore size is about 5 nm, whereas at
20% T pore size are much smaller.
 SDS-PAGE Gel Media

(A) Effect of various concentrations of

polyacrylamide on the electrophoretic
mobility of various proteins

(B) Protein and peptide molecular

weight separation guidelines
for polyacrylamide gels
 Discontinuous Multiphasic Gel-Buffer Electrophoresis

• The high resolution of protein separation is achieved by using a stacking gel with
multiphasic buffer systems. The stacking gel concentrates proteins from very dilute
solutions. This process significantly improves resolution of the subsequent
separation by shrinking the original sample in to a very thin, high concentrated
starting zone so that all of the protein molecules begin the separation at very
nearly the same point.

Stacking Gel:
large pore, 3 % acrylamide)

Resolving Gel:
small pore, 5-7% acrylamide
 Optimization of Protein Separation

• Buffer Composition: Tricine (pKa 8.15), for example, instead of glycine (pKa 9.6) in the
running buffer facilitates the resolution of small peptide at high acrylamide
concentration.

• pH: The sieving properties of SDS-PAGE, can be altered by a subtle change of pH of the
resolving gel. For instance, by increasing the pH from 8.9 to 9.2, the linear calibration
range of a 10% gel can be extended to include low Mr proteins of 10-20K without
scarifying the resolution of high Mr proteins as occurs with high percentage gels.

• Gel additives: SDS can cause protein aggregation and precipitation as well as abnormal
protein migration leading to poor resolution, and it crystallizes at low temperatures.
These limitation of SDS have led to the development of cationic detergent such as CTAB.

• Gradient acrylamide gels: gradient gel whose pore size changes from the top to
bottom, enable the separation of proteins over a larger molecular-wegith range and
with higher resolution than uniform concentration gel (see slide 16, table 3.1).
 Visualization and Detection

• Detection with organic dyes: conventional Coomassie brilliant Blue R250 in

methanol and acetic acid can stain proteins. The detection limits are 30-300 ng
proteins.
• Fluorescent probes: SYPRO dyes and Nile Red can detect proteins in SDS-PAGE
using a simple, one step staining procedure. The fluorophore are virtually non-
fluorescent in aqueous solutions, but they become highly fluorescent in non-polar
solvents or upon association with SDS-protein complexes. Detection limits are in
the range of 2-10 ng.
• Silver staining: protein detection depends on the binding of silver ions to the
amino acid side chains, primary the sulfhydril and carboxiyl groups of proteins,
followed by reduction to free metallic silver. The protein bands are visualized as
spots where the reduction occurs. The sensitivity is in the low ng range.

Coomassie staining
of proteins
Coomassie brilliant
Nile Red
Blue R250
 Agarose gel electrophoresis
• Agarose gels are easy to cast and is particularly suitable for separating larger DNA. The
separated DNA can be stained with ethidium bromide and viewed under UV light. Most
agarose gels used are between 0.7 - 2% dissolved in a suitable electrophoresis buffer.
• An agarose is a polysaccharide polymer material, generally extracted from seaweed.
Agarose gel has large pore size for the electrophoresis of DNA and large protein
molecules. The pore size of a 1% gel has been estimated from 100 nm to 200-500 nm,
and its gel strength allows gels as dilute as 0.15% to form slab for gel electrophoresis.
Agarose gel has lower resolving power than polyacrylamide gel for DNA but has a
greater range of separation, and is therefore used for DNA fragments of usually 50-
20,000 bp in size. The limit of resolution for standard agarose gel electrophoresis is
around 750 kb.
• A number of factors can affect the migration of nucleic acids: the dimension of the gel
pores (gel concentration), size of DNA being electrophoresed, the voltage used, the
ionic strength of the buffer, and the concentration intercalating dye such as ethidium
bromide if used during electrophoresis.

Digital image of 3 plasmid restriction digests

run on a 1% w/v agarose gel, 3 volt/cm,
stained with ethidium bromide. The DNA size
http://en.wikipedia.org/wiki/
marker is a commercial 1 kbp ladder.
Agarose_gel_electrophoresis
 The Principle of Isoelectric Focusing

• Isoelectric focusing (IEF) allows the separation of zwitterionic analytes such as

proteins or peptides according to the isoelectric point, pI.
• Instead of using a buffer with a constant pH over the whole gel, in IEF a pH
gradient is generated in which the pH value increases smoothly from anode to
cathode. When a zwitterionic compound such as a protein is placed into this pH
gradient , it migrates until it reaches a point where its net charge equal zero. This is
the position in the gradient, where the pH equals the protein’s pI.

When a protein is placed in a pH gradient and a voltage is applied,

It migrate towards its isoelectric point.
 Formation of pH Gradients

• For good separation, a stable pH gradient with constant conductivity is required.

This can be achieved by carrier ampholytes or immobilized pH gradients (IPG).

A pH gradient with increasing pH over the gel IEF instrumentation and

length formed by a mixture of hundreds of IPG drug strips
ampholytes each with a different pH. The
concentration of each ampholytes is the same
to ensure a homogeneous conductivity.
 2D Gel Electrophoresis for Protein Separation

• In 2D-GE, two electrophoresis modes are combined on a single gel. One separation
is performed by IEF in the first dimension, followed by another separation
perpendicular to the IEF by SDS-PAGE. With this method, mixtures with thousands
of proteins or nucleic acids can be separated with high resolution.

2D-GE of proteins from the soil

bacterium Burkholderia cepacia. The
separation in the first dimension was
achieved by IEF. In the second
dimension, the proteins were
separated according to their
molecular weight in a polyacrylamide
gel with a pore size gradient.
 Capillary Electrophoresis (CE)

• CE separations are carried out in a small diameter capillary containing a free

solution of electrolyte rather than on a slab gel. Convective flows due to Joule
heating occur more easily in a free solution than in the gel. Electroosomotic flow is
often part of the separation process.
 Capillary Electrophoresis Instrumentation

• The instrumentation needed to perform capillary electrophoresis is relatively

simple. The system's main components are a sample vial, source and destination
vials, a capillary, electrodes, a high-voltage power supply, a detector, and a data
output and handling device. The source vial, destination vial and capillary are filled
with an electrolyte such as an aqueous buffer solution.
 Capillary Electrophoresis Instrumentation

• The power supply: high voltage is applied over the capillary by two platinum
electrodes, which are dipped into the buffer reservoirs at each end of the capillary.
Typically, voltage up to 30 kV are applied, with electrical currents up to about 300
µA. For a 100 cm long capillary, this results in an electric filed of E = 300 Vcm-1.

• The capillary: typically, it has the internal diameters of 20 to 100 µm and outer
diameters of about 400 µm. They are typically between 10 to 100 cm long. The
most popular material is fused silica, i.e. amorphous quartz, which is transparent
to UV and visible light and can be coated with a polyimide layer of about 10 µm
thickness to increase flexibility.

• The source and destination vials: the vials and capillary are filled with electrollyte
buffer which typically has concentration in the order of 10-100mM. A controlled
pH is crucial for maintaining a constant net charge on the biomolecules and thus
the electrophoretic mobility µep.
 pH, Silanol Population, and the rate of EOF flow.

20
• At very low pH, not many silanols 18
are ionized and the EOF is slow.
16
14
• As pH increases the number of
ionized sites also increases. The 12
EOF speed rises steadily. 10 EOF
8
• At very high pH values, a maximum 6
number of ionized sites is reached.
The EOF speed also reaches a 4
maximum. 2
0

11
2

8
Separation Efficiency (Y) and Diffusion Coefficient (X)

• Note the very dramatic drop in

separation efficiency with
increasing diffusion coefficient.

• This means that in some cases,

there is no real advantage over
conventional HPLC for smaller
molecules.
 CE Injection System

There are two principle methods:

• Pressure differential works by applying a pressure across the capillary while it it is

dipping into the sample solution.

• Electrokinetic injection works by applying a voltage and allowing ions to migrate

into the capillary because of their charge.

Injection volumes are typically very small:

• Typically if injection volumes exceed 1% of the column volume, separation

efficiency severely suffers.

• Sample volume can be increased by focusing the ions inside the capillary. This
technique uses a combination of additives to the medium and selectively applied
charges.
 CE Detection Scheme

• The path length of the detection cell in capillary electrophoresis (~ 50 micrometers)

is far less than that of a traditional UV cell (~ 1 cm). To improve the sensitivity, the
path length can be increased by expanding at the detection point, creating a
"bubble cell" with a longer path length or additional tubing can be added at the
detection point. Besides UV, fluorescence, refractive index, electrochemical,
conductivity, radioisotopes and etc. can be used to detect analytes.

Techniques for increasing the path length of the capillary: a.) a bubble cell and b.) a
z-cell (additional tubing).
 Micellar Electrokinetic Chromtography (MEKC)

• MEKC is a modification of capillary electrophoresis (CE), where the samples are

separated by differential partitioning between micelles (pseudo-stationary phase)
and a surrounding aqueous buffer solution (mobile phase).
• In CE, the solution contains a surfactant at a concentration that is greater than the
critical micelle concentration (CMC). Above this concentration, surfactant
monomers are in equilibrium with micelles.

(A) (B)
 Surfactants for MEKC

• A suitable detergent for MEKC must have a good solubility in the buffer, low UV
absorption, a low viscosity and not to high a CMC to avoid extensive Joule.
• The aggregation number AN, is defined as the number of molecules that the
micelle consists of. AN for Sodium cholate is 2-4 molecules. For SDS, AN is 62.
• Cationic surfactants, adsorb strongly to the negatively charged capillary walls in
which the charge of the capillary surface is reversed and consequently, the
direction of the EOF is reversed.
 Analytes Separate in MEKC

• Sodium dodecyl sulfate (SDS) is the most commonly used surfactant in MEKC
applications. The anionic character of the sulfate groups of SDS cause the
surfactant and micelles to have electrophoretic mobility that is counter to the
direction of the strong electroosmotic flow. As a result, the surfactant monomers
and micelles migrate quite slowly, though their net movement is still toward the
cathode. During a MEKC separation, analytes distribute themselves between the
hydrophobic interior of the micelle and hydrophilic buffer solution.

• In MEKC, hydrophilic analytes

elute at T0,
• Hydrophobic analytes
elute at Tr,
• more hydrophobic
analytes elute at Tmc.
 Parameters Influencing MEKC Separations

• pH: the pH can have dramatic effects on the EOF and the charge of the micelles
might also be altered resulting in a different tmc.

• Organic Modifiers: acetonitrile, DMF and tetrahydrofuran can alter the viscosity of
the buffer and thus the velocity of the electroosmotic flow. Non-ionic surfactants
such as triton X-100 can combine with the ionic surfactatns to form co-micelles.
These can have different mobilities and thus a different tmc than the original
micelles.

• Surfactants: changing the surfactants is similar to changing the stationary phase in

liquid chromatography. The micelle concentration has an influence on analyte
retention time. The CMC for SDS is 8 mM, and typical concentrations used for
MEKC are between 25 and 150 mM. A high concentration of SDS molecules results
in a large number of micelles being generated. The probability of an analyte
partitioning into a micelle is thus much larger.
 Capillary Electrophoresis
Summary

• Capillary electrophoresis provides much greater separation efficiencies (N

values) than HPLC.

• Capillary electrophoresis also is very poor for preparative separations.

• Very small volumes are injected; concentration sensitivity is poor vs. HPLC
but mass sensitivity is good.
 Some interesting video of capillary electrophoresis

• http://www.youtube.com/watch?v=L4Q8fski6
48

• http://www.youtube.com/watch?v=CXenfe4l
MxQ