CAPILLARY

ELECTROPHORESIS

TOPIC 5
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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.

 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 .

Basic design of CE instrumentation

CE instrument

 Main components in CE :
9 A fused silica capillary.
9 2 buffer reservoirs; source vial & destination
vial.
9 2 electrodes.
9 A high-voltage power supply.
9 A detector.
9 A data output & handling device.
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Capillaries
The capillaries used are normally narrow bore
(25-75 m I.D.) 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 2575 cm long with 25-75 m

I.D..
9

Capillary cartridge
Detection
window

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Sample introduction
The capillary are fill with aqueous 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.
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 retain, so the analogues term to
retention time is migration time.
The migration time is the time it takes a solute to
move from the beginning of the capillary to the
detector window.
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 The majority of commercial detection system

use is UV or UV-Vis absorbance.
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 :

9

Applied electric field.

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

Charge to mass ratio.

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Migration rates
The migration of solutes is determined by their
charge to mass ratio.
9 Small highly charged solutes will migrate more
quickly then large less charged solutes.
9 If 2 ions are the same size, the one with greater
charge will move the fastest.
9 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.

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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)

Sample problem 1
Analyte species (A:polar neutral, B:non polar
neutral, 20X+, 25X+, 20Y-, 25Y-) 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.

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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

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 Velocity of EOF depends on :

1. Charge density of the capillary wall (charge
density pH of the buffer solution).
EOF will increase with pH until all the
available Si-OH lining the wall of the
capillary are fully ionized. (The degree of
ionization of silica is controlled by the pH of
the buffer. If the pH is 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 :
9

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, 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 :
9

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.

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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 the
electrophoretic flow.

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i. With reasons, describe the order of elution.

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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).

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iii. Explain what would happen to the separation

if the silanols on the capillary wall were
partially ionized.

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iv. The pH was decreased to 2.0 (assume that the

charges of the components do not change).

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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
9 No A term (multipath) because tube is open.
9 No C term (no mass transfer between phases
(Cm & Cs)) because there is no stationary
phase (hence no Cs).
9 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 :
9 The flow profile is parabolic; it is fastest at the
center & slows to 0 at the walls.
9 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 :
9 Simple inorganic ions, metal ions,
oligosacharides, nucleic acids (RNA &
DNA) & proteins.
Commonly used to analyze larger, watersoluble biomolecules.
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 CE is a attractive technique :
9 Requires small amounts of buffer & sample
to perform several analysis.
9 Capable of separating comps with different
size to charge ratios.
CE limitation : Inefficient at separating neutral
comps.

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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.
The separation mechanism is based on
differences in the charge to mass ratio.
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 Fundamental to 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 :

9 The walls of the capillary are untreated,
the EOF & the cations movement is
toward the cathode.

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 Separation of anions in CZE :

9 The EOF is reversed by treating the walls of
the capillary with a cationic surfactant (alkyl
ammonium salt such as ethyl ammonium
bromide)..
9 The positively charged ammonium ions
become attached to the negatively charged
double layer of solution, which is attracted
toward the anode, thus reversing the EOF.
9 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 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.

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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).

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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 helpful in separating large
molecules based on 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 the 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.

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Micellar Electrokinetic Capillary

Chromatography (MEKC or MECC)
Used to separate neutral & ionic species.
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).
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Sodium dodecyl sulfate (SDS)

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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.
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Micelle

--

OSO3-Na+

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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.
The more time the neutral molecule spends
inside the micelle (retain), the longer is its
migration time.
The exterior of the micelle is polar making it
soluble in water.
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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).

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 Even though these anionic micelles are

attracted toward the anode (since the micelles
are negatively charged), in an uncoated fused
silica capillary they 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.
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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.
i. Less positively charged species.
ii. More positively charged species.
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Sample problem 7
Explain how micelles able to separate neutral
compounds in MEKC.

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Sample problem 8
Analyte species (A: polar neutral, B: non polar
neutral, 20X+, 25X+, 20Y-, 25Y-) 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).
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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.

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ii. What would the order of elution have

been in the absence of sodium dodecyl
sulfate?

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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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