TOPIC 5

CAPILLARY
ELECTROPHORESIS

1
 Course Learning Outcomes
Students should be able to:
1.Understand the concepts & principles of
separation in CE.
2.Compare the separation occurs in CE with
chromatographic separation.

3.Differentiate different modes of CE & the

application of each mode.

2
 Introduction

Electrophoresis is the migration of charged species

or ions in a solution which has been applied an
electric field.

Electrophoresis as a separation method based on the

differential rates of migration of charged species in a
buffer solution towards the electrode of opposite
charge.
Cations migrate toward the negatively charged electrode
(cathode).

Anions are attracted toward the positively charged electrode

(anode).

Neutral solutes are not attracted to either electrode.

+ -

4
 Capillary Electrophoresis
Is a new separation technique to separate minute
quantities of ionic species (based on charge) in
relatively short time with high resolution.

It offers the ability to analyze a nanoliter (10-9 L) & over

1 million theoretical plates.
✔ CE, N (100,000-200,000)
✔ HPLC, N (5,000-20,000)

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Basic design of CE instrumentation

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CE instrument

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 Main components in CE :
✔ A fused silica capillary.
✔ 2 buffer reservoirs; source vial & destination
vial.
✔ 2 electrodes.
✔ A high-voltage power supply.
✔ A detector.
✔ A data output & handling device.

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 Capillaries
The capillaries used are normally narrow bore (10-100
µm I.D., 30-100 cm long) fused silica capillaries
covered with an external polyimide.

A small portion of this coating is removed to form a

window for detection purposes.

The window is aligned in the optical centre of the

detector.

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Capillaries are typically
30-100 cm long with 10-100
µm I.D..

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Capillary cartridge
Detection
window

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 Sample introduction

The capillary are fill with a buffer solution.

To introduce the sample (0.1-10 nL injection volume),

the capillary inlet is placed into a vial containing the
sample & then returned to the source vial.

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Basic design of CE instrumentation

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The migration of the analytes is then initiated by an electric
field that is applied between the source & destination vials
& is supplied to the electrodes by the high-voltage power
supply (5-30 kV dc).

Electrodes made of an inert material such as platinum are

inserted into the electrolyte reservoirs to complete the
electrical circuit.

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The analytes separate as they migrate due to their
electrophoretic mobility & are detected near the outlet
end of the capillary.

In CE, nothing is retained, so the analogues term to

retention time is migration time.

The migration time is the time it takes for a solute to

migrate from the point of introduction to the detector.

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Detector are similar to HPLC (spectrometry :
fluorescence, UV or UV-Vis absorbance;
electrochemical : conductivity, amperometry).

The output of the detector is sent to a data output &

handling device such as an integrator or computer.

The data is then displayed as an electropherogram.

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 Electropherogram

Detector response as a function of migration time

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Movement of ions in the capillary as a result of :

1. Electrophoretic flow
2. Electroosmosis flow (EOF)

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 Electrophoretic flow

Electrophoretic flow caused by the presence of an

electric field that has been applied to the buffer
solution across the capillary column.

Charged molecules migrate in the direction of the

electrode bearing the opposite charge.

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 Cations migrate toward the negatively charged
electrode (cathode).

Anions are attracted toward the positively charged

electrode (anode).

Neutral solutes are not attracted to either electrode.

+ -
#
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Electrophoretic flow velocity is proportional to :

✔ 1. Applied electric field

Highest voltages will result in the shortest times
for the separation.

✔ 2. Charge to mass ratio

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 Migration rates
The migration of solutes is determined by their charge
to mass ratio (the larger the ratio, the faster an
ion migrate in the electric field).
✔ Small highly charged solutes will migrate more quickly then
large less charged solutes.

✔ If 2 ions are the same size, the one with greater charge will
move the fastest.

✔ For ions of the same charge, the smaller particle has less
friction & overall faster migration rate.

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 The length of the arrow indicates the magnitude of the
velocity; the direction of the arrow indicates of the
motion. The positive electrode in the right & the negative
electrode in the left.

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Electroosmotic flow (EOF)

EOF is a net flow (or bulk flow) of buffer solution in

the capillary towards one single direction under an
electric field.

All analytes flow in the same direction by EOF.

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EOF caused by the electric double layer that develops at
the silica/solution interface.

This occurs when the buffer running through the silica

capillary has pH>3, cause the inside wall of the silica
capillary negatively charged (Si-O-) due to the
ionization of the Si-OH groups.

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Buffer cations congregate in an electric double layer
adjacent to the negative surface of the silica capillary
(i.e. fixed & mobile layer).

The inner layer (fixed layer) results from cations being

tightly bound to the wall.

The outer layer (mobile layer) is farther from the Si-O-,

only loosely bound.

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The cations in the mobile layer are attracted to
cathode.

Since the cations in the mobile layer are solvated, they

drag the bulk solution along with them, causing the
EOF of the buffer solution.

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Depiction of the interior of a fused silica
capillary in the presence of buffer
solution

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Solvated cations drag solvent molecules during the
migration, hence there is net solution movement from
anode to cathode.

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Since the EOF of the buffer solution is generally greater
than that of the electrophoretic flow of the analytes, all
analytes are carried along with the buffer solution
towards the cathode.

Even though analyte migrate according to their charges

within the capillary, the EOF rate is usually sufficient to
sweep all positive, neutral & negative analytes toward
the same end of the capillary.

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The order of migration will be cations, neutrals &
anions.

None of the neutral molecules will be separated

(single band) since the net charge is zero.

The anions will still migrate toward the cathode

because the EOF is greater than the electrophoretic
migration.

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Diagram of the separation of charged & neutral
analytes (A) in capillary according to their respective
electrophoretic & EOF mobilities.
 Electroosmotic flow

EOF

Electrophoretic flow

+ -
 Electrophoretic flow + EOF

EOF
+ -

tm (min)
0
 Sample problem

Analyte species (A:polar neutral, B:non polar neutral,

20 + 25 + 20 - 25 -
X , X , Y , Y ) in an aqueous sample were
separated by CE in 15 mM borate buffer (pH 5.8). The
CE instrument was set up with the detection end at
cathode.

Describe the order of elution for the above separation.

35
Cations eluted first, the greater the charge to mass ratio, the
greater the EOF, the faster the elution for 20X+ compared to
25 +
X.

Followed by neutral species which eluted together as they

were not separated.

Anions eluted last, even though 20Y- has greater charge to

mass ratio & greater the EOF but towards opposite direction.
Thus it eluted later than 25Y-.
20 + 25 +
Order of elution: X , X , A&B, 25Y-, 20Y-

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The significance of conducting electrophoresis at high
pH is to establish EOF.

At pH>3, silica capillary is negatively charged.

Buffer cations congregate in an electrical double layer
adjacent to the negative surface of the silica capillary.
The cations in the diffuse outer double layer are
attracted to the cathode, & since the cations are
solvated, they drag the bulk solvent along with them.

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Effect of pH on the EOF

Large charge density

Low charge density

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 Velocity of EOF depends on :
1. Charge density of the capillary wall (charge density α
pH of the buffer solution).
The degree of ionization of silica is controlled by the
pH of the buffer.
EOF will increase with pH until all the available Si-OH
lining the wall of the capillary are fully ionized.
If the pH is increases, charge density increases,the
magnitude of EOF increased & thus decrease the
migration time.

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2. Ionic strength of buffer.

3. Electric field strength.

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Viscosity of the running buffer effect on the migration
time :

✔ The more viscous the buffer, the slower the

EOF. Thus migration time increased.

✔ Less viscous buffer, the higher the EOF. Thus

migration time decreased.

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 To reduce velocity of EOF :

1. Lowering the pH, capillary wall less

ionized, so that the charge on the capillary
wall is reduced.

2. Adding cations that adhere to the capillary

wall & effectively neutralize its charge.

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Advantages of EOF :

✔ The EOF makes possible the simultaneous

analysis of cations, anions & neutral species in a
single analysis.

✔ The EOF generally stronger than electrophoretic

migration, hence all species are swept towards
the negative electrode.

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 Sample problem 2
EOF is pH dependent. Explain why & how does this affect
the migration time.

EOF mobility proportional to the surface charge density on

the silica. The degree of ionization of silica is controlled by
the pH of the buffer. If the pH is increases, charge density
increased, the magnitude of EOF increased & thus
decrease the migration time.

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 Sample problem 3
Consider a CE expt. designed to separate 5
components with similar masses. At pH 6.7, the
components are A+, B2+, C-, D & E (neutral species).
The electrophoresis was run with the injection end
positive & the detection end negative. The EOF was
greater than the electrophoretic flow.

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i. With reasons, describe the order of elution.

Since the EOF was greater than electrophoretic flow,

the EOF is sufficient to sweep all the ions towards the
detector. The larger charge to mass ratio, the faster an
ion migrates.

The order of elution; first B2+, A+ (cations), then D & E

(neutral species), finally C- (anion).

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ii. Explain what would happen to the separation if the
pH is decreased to 4 (assume that the charges of
the components do not change).

At pH 4.0, the silanols on the capillary wall were less

ionized compared to pH 6.7, therefore the EOF was
reduced & this decreased the migration rate of
analytes but the elution order was remained.

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iii. Explain what would happen to the separation if the
silanols on the capillary wall were partially ionized.

The EOF is reduced, therefore decreased the

migration rate but the elution order is not changed.

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iv. The pH was decreased to 2.0 (assume that the
charges of the components do not change).

When the pH<3, the capillary wall was not

ionized. Therefore, migration based on
electrophoretic flow, whereby the ions migrated
toward electrode with opposite charge & the
neutrals were not attracted to either electrode.
Since detector was at cathode (-ve), only
cations were separated & detected based on
charge to mass ratio.

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 Efficiency of CE
The efficiency of CE separations is typically
much higher than other separation techniques
like HPLC.
Cross-section of a capillary :

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 In CE, a very narrow open-tubular capillary is
used : Lower H

✔ No A term (multipath) because tube is open.

✔ No C term (no mass transfer between phases (Cm &

Cs)) because there is no stationary phase (hence no
Cs).

✔ Only the B term (longitudinal diffusion) remains:

H = B/u. Rapid separation reduce B term.

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 Flow profile
In electrically driven systems (CE), the driving force
of the EOF is uniformly distributed along the entire
length of the capillary (except right at the wall where
the double layer is fixed).

As a result, the flow profile is pluglike, the analyte

molecules are swept along at the same rate across
the capillary, which minimize sample dispersion &
generates very sharp peaks.

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Capillary flow profiles

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Result : EOF does not contribute significantly
to band broadening like pressure-driven flow
in LC & related techniques.

Therefore, contributed to the high resolution

of CE.

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 In pressure-driven flow :

✔ The flow profile is parabolic; it is fastest at the

center & slows to 0 at the walls.

✔ This result in band broadening (GC & HPLC peak

become broader the further the migrate).

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 Applications of CE
Due to its high efficiency & resolution, CE can be
used to separate & determine a wide variety of
comps :
✔ Simple inorganic ions, metal ions,
oligosacharides, nucleic acids (RNA & DNA) &
proteins.
Commonly used to analyze larger, water-soluble
biomolecules.

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 CE is a attractive technique :
✔ Requires small amounts of buffer & sample to
perform several analysis.
✔ Capable of separating comps with different size to
charge ratios.

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 Modes of capillary electrophoresis

1.Capillary zone electrophoresis (CZE)

2.Capillary gel electrophoresis (CGE)

3.Micellar electrokinetic capillary

chromatography (MEKC or MECC)

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Capillary Zone Electrophoresis (CZE)
Known as capillary electrophoresis.

The simplest form & commonly used of CE.

Separations of small molecular weight inorganic &

organic ions (small ions).

The separation mechanism is based on differences

in the charge to mass ratio.

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Fundamental of CZE are constant field strength &
buffer solution throughout the length of the capillary.

The applied potential causes the different ionic comps

of the mixture to each migrate according to its own
mobility & to separate into zones that may be
completely resolved or may partially overlapped.

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Completely resolved zones have regions of
buffer between them.

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 Separation of cations in CZE :

✔ The walls of the capillary are untreated, the

EOF & the cations movement is toward the
cathode.

How EOF can be manipulated to speed up the

separation of anions?

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 Separation of anions in CZE :
✔ The EOF is reversed by treating capillary wall with a
cationic surfactant (alkyl ammonium salt such as
ethyl ammonium bromide)..

✔ The surfactant absorbs on the capillary wall & makes

the wall positively charged.

✔ Buffer anions congregate near the wall & are swept

towards the anode, thus reversing the EOF.

✔ The detector must be placed at anode end & the

sample introduction at cathode end.

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Charge reversal created by a cationic surfactant coated
on the capillary wall. The diffuse layer contains excess
anions & EOF is in the opposite direction.

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Another approach for the separation of anions
is to operate at a low pH where the silanol
groups are not ionized & electrophoretic
migration of anions dominates towards the
anode.

The detector must be placed at anode end &

the sample introduction at cathode end.

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 IMPORTANT
NOTES
If the capillary wall is negative, EOF is toward the
cathode & the order of elution is cations >
neutrals > anions.

If the capillary wall charge is reversed by treating it

with cationic surfactant, then the order of elution is
anions > neutrals > cations.

Neither scheme separates neutral molecules

from one another.

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 Sample problem 4

Consider a CE expt. designed to separate 5 components

with similar masses. At pH 6.7, the components are A+,
B2+, C-, D and E (neutral species). The electrophoresis
was run with the injection end positive & the detection
end negative. The EOF was greater than the
electrophoretic flow.

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Describe how the original CE expt. would be different if the
capillary wall is treated with ethyl ammonium bromide.
Include the order of elution.

The ammonium cation become attached to the negatively

charged silica surface & in turn, create a negatively charged
doubled layer of solution, which is attracted toward the
anode, thus reversing the EOF. Therefore, anion, C-
migrates first followed by neutral species, D & E, & last
cations; A+, B2+, but all the analytes were not detected as
the detector was not at anode.

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 Sample problem 5
You are required to separate a sample mixture of anions at
pH4 using CE. Describe how you would position the major
components of the instrument (include treatment of column,
if required).

For the separation of anions, the wall of the capillary tubing

must be treated first with an alkyl ammonium bromide, so
that the EOF is reversed towards the anode , thus the
detector must be placed at anode end & sample introduction
at cathode end.

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Capillary Gel Electrophoresis (CGE)

The separation is carried out in a porous gel polymer

matrix filled in a capillary tube.

CGE is more suitable for large molecular weight of

ionic compounds because the porous polymer matrix
provide a molecular sieving action which retard the
migration of analyte to various extends depending
upon the pore size of the polymer & the size of the
analyte ions.

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This sieving action is particularly helpful in
separating macromolecules (large Mw) such as
proteins & DNA fragments where they have the
same charge but differ in size.

The migration of analytes depends upon the

pore size of the polymer & the size of the analyte
ions.

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+ -

Smaller molecules migrate faster than larger

molecules through the polymer matrix
 Sample problem 6
What are the function & the significance of a molecular
sieving action in CGE.

A molecular sieving action retarded the migration of

analyte to various extends depending upon the pore
size of the polymer & the size of the analyte ions.

This sieving action is particularly helpful in separating

macromolecules such as proteins & DNA fragments
that have the same charge but differ in size.

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 Micellar Electrokinetic Capillary
Chromatography (MEKC or MECC)

Used to separate neutral & ionic species.

Principle of separation based on :

Electrophoretic mobility in free solution : for ionic
species based on charge to mass ratio.
Partitioning between micelle & solution : for
neutral species based on hydrophobicities.

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MEKC is a form of chromatography in which surfactants
are added to the buffer solution at concentrations that
form micelles.
E.g. of anionic surfactant is sodium dodecyl sulfate
(SDS).

Sodium dodecyl sulfate (SDS)

Hydrophobic Hydrophilic

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Above a certain concentration, called the “critical
micelle concentration” (CMC), the surfactant
molecules will self-aggregate, forming micelles in
aqueous solution.

Micelles are formed with the hydrophobic

(hydrocarbon) tails pointing inward & the hydrophilic
(negatively charged) heads pointing outward into the
aqueous solution.

Micelles move towards cathode but slower than EOF.

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Even though these anionic micelles are attracted toward the anode
(since the micelles are negatively charged) which gives a large
electrophoretic mobility, they will still migrate toward the cathode
because of EOF.

However, the micelles move toward the cathode at a slower rate

than the bulk of the aqueous (buffer) because of their attraction
towards the anode.
--
- -
- -
Micelle
- -
- -
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The interior of the micelles is non-polar, therefore capable
to absorb non-polar (neutral) analytes into the
hydrocarbon tails of the particles & solubilizing the
non-polar analytes.

Partitioning of the neutral analytes based on their varying

hydrophobicities. More hydrophobic spend more time
inside the micelle, migrate slowly cause longer migration
time compared to less hydrophobic.

The exterior of the micelle is polar making it soluble

in water. Therefore, polar compounds will be at the
exterior of the micelle and migrate faster than non
polar compounds.

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If the concentration of SDS is too low (below the CMC
level), micelles will not form.

Hence, neutral molecules cannot be separated (eluted

as a single peak).

A hydrophilic neutral molecule will spend almost no time

inside the micelle & will therefore migrate essentially at
the same rate of the bulk flow and eluted earlier.

A hydrophobic neutral molecule will spend nearly all the

time inside the micelle and will eluted later, together
with micelle.

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MEKC is a form of chromatography because the
micelles behave as a “pseudo-stationary phase” in
the capillary because their concentration is uniform
throughout the capillary.

During electrophoresis, the micelles interact with

solutes in a similar manner to chromatography.

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Analyte will partition between the mobile phase (buffer)
& micelles (pseudo-stationary phase) as the analyte
travels through the capillary.

Migration times of cations & anions also are affected by

micelles, because ions partition between the mobile
phase & the micelles & interact electrostatically with
the negatively charged micelles.

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 E.g. for SDS, migration order will be :
1. Anions – electrostatic repulsions from micelle.
i. More negatively charged anionic species.
ii. Less negatively charged anionic species.
2. Neutrals – hydrophobicity.
(Less hydrophobic, migration time faster, more
hydrophobic, spend more time in the micelle, so
migrate slowly).
3. Cations – attraction to micelle.
ii. Less positively charged species.
iii. More positively charged species.

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 Sample problem 7
Explain how micelles able to separate neutral compounds
in MEKC.
The interior of the micelles is non-polar, therefore capable
to absorb non-polar (neutral) analytes into the
hydrocarbon interior of the particles & solubilizing the
non-polar analytes. Partitioning of the analytes based on
their varying hydrophobicities. The exterior of the micelle is
polar making it soluble in water.

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 Sample problem 8
Analyte species (A: polar neutral, B: non polar neutral,
20 + 25 + 20 - 25 -
X , X , Y , Y ) in an aqueous sample were
separated by capillary electrophoresis (CE) in 15 mM
borate buffer (pH 5.8). The CE instrument was set up
with the detection end at cathode.

The pH was 5.8 & added with high concentration of

anionic surfactant (above CMC level).

84
With the addition of anionic surfactant, micelles were
formed and moved towards cathode but slower than EOF.
Neutrals can be separated based on their varying
hydrophobicities. A was a hydrophilic neutral molecule,
thus spend almost no time inside the micelle and migrate
essentially at the same rate as the EOF and elute earlier.
Micelles are capable to absorb B as the hydrophobic
neutral molecule into the hydrocarbon interior of the
particles and move with the micelles and therefore eluted
later.

85
 Sample problem 9
Four types of water soluble compounds; A and B
(neutral compounds whereby A is slightly more
hydrophobic), C (an anionic compound) and D (a
cationic compound) are separated by micellar
electrokinetic capillary chromatography (MEKC) in
15 mM borate buffer (pH 8) with 50 mM sodium
dodecyl sulfate.

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i. With justification, predict the order of elution of the
above compounds.

1. Compound C (anion) – electrostatic repulsions

from micelle.
2. Compound B (neutral) – less hydrophobic, thus
migration time faster than A.
3. Compound A (neutral)- more hydrophobic, thus
spend more time in the micelle compared to
compound B.
4. Compound D (cation) – attraction to micelle.

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ii. What would the order of elution have been in
the absence of sodium dodecyl sulfate?

1. Compound D

2. Compound A & B

3. Compound C

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MECC is more versatile than CE because it can
separate neutral molecules & ionic species.

In MECC, micelles act as stationary phase.

Therefore, increase slightly the Cs term. This
decrease the efficiency, whereas in CE, no stationary
phase. Therefore, Cs term is zero.

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 Advantages of MECC compared to HPLC.

1. MECC has much higher column efficiency

(>100,000 plates) than HPLC due to :
▪ No packing, hence no A term.
▪ Rapid separation, reduce B term.

2. Changing the phase (pseudo-stationary phase) is

simple, involving only the changing the micelle
composition of the buffer. For HPLC, changing the
stationary phase means changing the type of
column packing.

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